MXPA06013698A - Inhibition of hiv-1 replication by disruption of the processing of the viral capsid-spacer peptide 1 protein. - Google Patents

Abstract

Inhibition of HIV1 replication is described by interrupting the processing of the viral Gag capsid (CA) protein (p24) of the precursor protein separator CApeptide 1 (SP1) (p25). Also included are amino acid sequences that contain a mutation in the Gag p25 protein, with the mutation resulting in a decrease in the inhibition of processing p25 to p24 by dimethylsuccinyl-betulinic acid or demethyl-succinylbetulin, polynucleotides encoding these mutated sequences and antibodies that selectively bind to these mutated sequences. Inhibition methods, inhibitor compounds and methods for discovering inhibitor compounds that target proteolytic processing of the HIV gag protein are included. In one embodiment, these compounds inhibit the interaction of the HIV protease enzyme by binding to Gag instead of the protease enzyme. In another embodiment, recombinant viruses or proteins that contain mutations in the region of the Gag proteolytic cleavage site can be used in detection assays to identify compounds that target proteolytic processing.

Description

INHIBITION OF IMMUNODEFICIENCY VIRUS 1 REPLICATION HUMAN (HIV-1) BY INTERRUPTION OF THE PROCESSING OF CAPSIDA-PEPTIDO PROTEIN SEPARATOR 1 VIRAL Field of the Invention The invention includes methods of inhibition, inhibitors and methods for the discovery of inhibitors of human immunodeficiency virus (HIV) infection. BACKGROUND OF THE INVENTION The human immunodeficiency virus (HIV) is a member of the lentivirus, a subfamily of retroviruses. The viral genome contains many regulatory elements that allow the virus to control its replication rate in both resting and dividing cells. More importantly, HIV infects and invades the cells of the immune system; destroys the body's immune system and makes the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate approaching 100% for several years. HIV-1 is trophic and cytopathic for T4 lymphocytes, the cells of the immune system that express the cell surface differentiation CD4 antigen, also known as OKT4, T4 and leu3. The viral tropism is due to the interactions between the viral envelope glycoprotein, REF: 177967 gpl20, and cell surface CD4 molecules (Dalgleish et al., Nature 312: 763-767 (1984)). These interactions not only mediate the infection of susceptible cells by HIV, but are also responsible for virus-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death and progressive depletion of CD4 cells in patients infected with HIV. These events result in HIV-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms. In addition to CD4 + T cells, the variety of HIV hosts includes mononuclear phagocytic lineage cells (Dalgleish et al., Supra), which include blood monoliths, tissue macrophages, skin Langerhans cells and dendritic reticulum cells within the lymph nodes. HIV is also neurotropic, capable of infecting monoliths and macrophages in the central nervous system causing severe neurological damage. Macrophages and monoliths are the main deposits of HIV. They can interact and fuse with T cells that have CD4, causing depletion of T cells and thus contributing to the pathogenesis of AIDS. Considerable progress has been made in the development of drugs for HIV-1 therapy. Therapeutic agents for HIV may include, but are not limited to, without limitation, at least one of AZT, 3TC, ddC, d4T, ddl, tenofovir, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, lopinavir, amprenavir, atazanavir and fosamprenavir, or any other anti-drug -retroviral or antibodies in combination with each other or associated with a biologically based therapeutic product, such as, for example, peptides derived from gp41, enfuvirtide (Fuzeon, Timeris-Roche) and T-1249 (Trimeris), or soluble CD4, antibodies to CD4 , and CD4 or anti-CD4 conjugates, or as are additionally presented herein. The combinations of these drugs are particularly effective and can reduce viral RNA levels to undetectable levels in the plasma and delay the development of viral resistance, with resulting improvements in the health and life span of the patient. Despite these advances, there are still problems with currently available drug regimens. Many of the drugs exhibit severe toxicities, have other side effects (eg, redistribution of fat), or require complicated dosing schedules that reduce compliance and thus limit efficacy. Resistant strains of HIV frequently appear for extended periods of time even in combination therapy. The high cost of these drugs is also a limitation to their widespread use, especially outside developed countries.

The development of additional drugs to circumvent these issues is still a major need. Ideally, these would target different stages in the viral life cycle, adding to the armament for combination therapy, and exhibit minimal toxicity, while still having lower manufacturing costs. The assembly of the HIV virion takes place in the surface membrane of the infected cell where the viral Gag polyprotein accumulates, leading to the assembly of immature virions that sprout from the cell surface. Within the virion, Gag is cleaved by the viral proteinase (PR) in the matrix (MA), capsid (CA), nucleocapsid (NC), and the C6-terminal structural proteins (Wiegers K. et al., J. Virol 72: 2846-2854 (1998)). The processing of Gag induces a reorganization of the internal virion structure, a process called "maturation". In the mature HIV particles, the MA lines the inner surface of the membrane, while the CA forms the conical nucleus that encases the genomic RNA that becomes complex with? C. Cleavage and maturation are not required for particle formation but are essential for infectivity (Kohl,? Et al., Proc. Nati, Acad. Sci. USA 85: 4686-4690, (1998)). The CA and NC as well as NC and p6 are separated in the Gag polyprotein by short separating peptides of 14 and 10 amino acids (p2), respectively (peptide separator 1 (SPI) and SP2, respectively) (Wiegers K. et al., Virol. 72: 2846-2854 (1998), Pettit, SC et al., J. Virol. 68: 8017-8027 (1994), Liang et al., J. Virol. 76: 11729-11737 (2002)). These separating peptides are released by cleavage mediated by PR in their N and C terms during the maturation of the particles. The individual cleavage sites in the HIV Gag and Gag-Pol polyproteins are processed at different rates and this sequential processing results in intermediate Gag compounds that appear momentarily before the final products. These intermediate compounds may be important for the morphogenesis or maturation of the virions but do not contribute to the structure of the mature viral particle (eigers et al., And Pettit, et al., Supra). The initial Gag cleavage event is presented at the SPI C-terminus and separates the N-terminal MA-CA-SPl intermediate from an intermediate C-terminal NC-SP2-p6 compound. Subsequent splits separating MA from CA-SPl and NC-SP2 from p6 are presented at a rate approximately 10 times lower. Cleavage of SPl from the C-terminus of CA is a late event and occurs at a rate 400 times lower than cleavage at the SPl-NC site (Weigers et al., And Pettit, et al., Supra). The non-cleaved CA-SPl intermediate protein is alternatively called "p25", while the excised CA protein is alternatively called "p24" and the cleaved SPI peptide is called alternatively "p2". Cleavage of SPI from the C-terminus of CA appears to be one of the final events in the Gag processing cascade and is required for the final condensation of capsids and the formation of mature infectious viral particles. Electron micrographs of mature virions reveal particles that have condense electrodense nuclei. On the other hand, electron microscopy studies of defective viral particles for CA-SP1 cleavage show particles that have an electron dense ribonucleoprotein core and an increasingly dense electrodensate layer located just inside the viral membrane (Weigers et al., supra). Mutations at or near the CA-SPl cleavage site have been shown to inhibit Gag processing and disrupt normal maturation processes, thereby resulting in the production of non-infectious viral particles (Weigers et al., Supra). ). Phenotypically, these particles exhibit a defect in Gag processing (which manifests itself in the presence of a p25 band (CA-SP1) in the Western blot analysis) and the abnormal morphology of particles described above which results from the defective condensation of capsids. Previously, betulinic acid and planic acid were isolated from Syzigium claviflorum and it was determined that they have anti-HIV activity. Betulinic acid and planic acid exhibited inhibitory activity against HIV-1 replication in H9 cells with EC50 values of 1.4 μM and 6.5 μM, respectively, and therapeutic index values (T.I.) of 9.3 and 14, respectively. Hydrogenation of betulinic acid produced dihydrobetulinic acid, which showed slightly more potent anti-HIV activity with an EC50 value of 0.9 and an IT value of 14 (Fujioka, T., et al., J. Nat. Prod. : 243-247 (1994)). Esterification of betulinic acid with certain substituted acyl groups, such as 3 ', 3'-dimethylglutaryl and 3', 3'-dimethylsuccinyl groups produced derivatives with improved activity (Kashiwada, Y., et al., J. Med. Chem. 39: 1016-1017 (1996)). Acylated derivatives of betulinic acid and dihydrobetulinic acid which are potent anti-HIV agents are also described in U.S. Patent No. 5,679,828. Anti-HIV assays indicated that 3-0- (3 ', 3' -dimethylsuccinyl) -betulinic acid (DSB) and the dihydrobetulinic acid analogue demonstrated both extremely potent anti-HIV activity in acutely infected H9 lymphocytes with EC50 values of less than 1.7 x 10"5 μM, respectively, these compounds exhibited remarkable TI values of more than 970,000 and more than 400,000, respectively, US Patent No. 5,468,888 describes 28-amido derivatives of lupans which is described as having a cytoprotective effect for cells infected for HIV.

R = H (Betulinic Acid) Japanese Patent Application No. JP 01 143,832 discloses that betulin and 3, 28-diesters thereof are useful in the anti-cancer field. U.S. Patent No. 6,172,110 describes betulinic acid and dihydrobetulin derivatives having the following formulas or pharmaceutically acceptable salts thereof, Derivatives of Betulin and Dihydrobetulin. wherein R. is a C2-C2 carboxycyl substituted unsubstituted, R2 is a substituted or unsubstituted C2-C20 carboxycyl and R3 is hydrogen, halogen, amino, mono- or dialkylamino optionally substituted, or -0R4, where R4 is hydrogen, C..4alkanoyl, benzoyl or carboxyacyl or C- C_0 substituted or unsubstituted; where the dashed line represents an optional double bond between C20 and C29. U.S. Patent Application No. 60 / 413,451 describes 3,3-dimethylsuccinyl-betulin and is incorporated herein by reference. Zhu, Y-M. et al., Bioorg. Chem Lett. 11: 3115-3118 (2001); Kashiwada Y. et al., J. Nat. Prod. 61: 1090-1095 (1998); Kashiwada Y. et al., Nat. Prod. 63: 1619-1622 (2000); and Kashiwada Y. et al., Chem. Pharm. Bull. 48: 1387-1390 (2000) describe dimethylsuccinyl-betulinic acid and dimethylsuccinyl-oleanolic acid. Esterification of the 3 'carbon of betulin with succinic acid produces a compound capable of inhibiting the activity of HIV-1 (Pokrovskii, AG et al., Gos Nauchnyi Tsentr Virusol. Biotekhnol. "Vector," 9: 485-491 (2001 )). International published application No. WO 02/26761 describes the use of betulin and analogs thereof to treat fungal infections. There is a need for new HIV inhibition methods that are effective against drug-resistant strains of the virus. The strategy of this invention is to provide therapeutic methods and compounds that inhibit the virus of different ways of the appropriate therapies. The compound and methods of the present invention have a novel mechanism of action and are therefore active against strains of HIV that are resistant to current inhibitors of transcriptase and reverse protease. As such, this invention offers a completely new approach to treating HIV / AIDS.

Brief Description of the Invention In general, the invention provides methods for inhibiting, inhibiting compounds and methods for identifying inhibitor compounds that target the proteolytic processing of the Gag protein of HIV-1. In a modality, these compounds can directly or indirectly inhibit the interaction of a protease enzyme with the Gag protein of HIV-1. In another embodiment, this inhibition of the interaction is presented by the binding of a Gag compound. Inhibition of protease cleavage of HIV-1 Gag CA-SPl protein by 3-0- (3 ', 3"-dimethylsuccinyl) -betulinic acid (DSB) is an example, but can be selected as targets other proteolytic cleavage sites by a similar approach using inhibitory compounds that interact with the substrate in a manner similar to that in which DSB interacts with Gag. Another aspect of the invention relates to a method to inhibit the processing of the Gag p25 viral protein (CA-SP1) to p24 (CA), but which has no effect on other Gag processing steps. In a further aspect of the invention there is still a method for identifying compounds that inhibit the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but have no effect on other Gag processing steps. In one aspect, the invention relates to a compound or pharmaceutical composition identified by the method for identifying compounds that inhibit HIV-1 replication described herein. In another aspect, the present invention relates to a polynucleotide comprising a sequence encoding an amino acid sequence containing a mutation in the p25 protein of Gag, this mutation that results in a decrease in the inhibition of p25 processing to p24 by 3-0- (3, 3-dimethylsuccinyl) betulinic acid. This aspect of the invention also relates to a vector, virus and host cell comprising this polypeptide, and a method for making this protein. A further aspect of the present invention relates to an amino acid sequence that contains a mutation of the p25 protein of Gag, the mutation that results in a decrease in the inhibition of processing from p25 to p24 by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. A further aspect of the present invention relates to an antibody that selectively binds to an amino acid sequence that contains a mutation in the p25 protein of Gag, this mutation that results in a decrease in the inhibition of p25 processing to p24 by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. Also included in this aspect of the invention is a method for making this antibody, a hybridoma that produces the antibody and a method for making the hybridoma. In a further embodiment, the invention relates to a kit comprising a polynucleotide, polypeptide or antibody described herein. The invention further relates to a method for inhibiting HIV-1 infection in cells of an animal by contacting these cells with a compound that blocks the maturation of virus particles released from infected, treated cells. In one embodiment, the released virus particles exhibit non-condensed nuclei and a distinctive thin electrodend layer near the viral membrane and have reduced infectivity. A method is included for contacting animal cells with a compound that both inhibits Gag viral p25 protein processing and disrupts the maturation of the proteins. particles of the virus. Also, a method for treating HIV-infected cells is included, wherein the HIV that infects the cells does not respond to other HIV therapies. This invention further includes a method for identifying compounds that inhibit the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but have no significant effects on other steps of Gag processing. The method comprises contacting HIV-1 infected cells with a test compound, and subsequently analyzing the viral particles that are released to detect the presence of p25. Methods for detecting p25 include western blotting of viral proteins and detection using an antibody to p25, gel electrophoresis, and imaging of metabolically labeled proteins. Methods for detecting p25 include immunoassays using an antibody to p25 or SP1 (p2) or to an epitope tag inserted in the SPI sequence. The invention further relates to a method for identifying compounds comprising contacting HIV-1 infected cells with a compound, and subsequently analyzing the virus particles released by the cells contacted, by derivative sectioning and transmission electron microscopy, and determining if viral particles with uncondensed nuclei and a thin, distinctive electrodensate layer are present near the viral membrane. The invention also relates to compounds identified by the detection methods mentioned above. In further embodiments, the invention relates to a method of treating HIV-1 infection in a patient by administering a compound that inhibits the processing of Gag's viral p25 protein (CA-SP1) to p24 (CA), but does not affect Significantly other Gag processing steps. In related modalities, this inhibition can be achieved by different observable phenotypes. For example, the inhibition can not necessarily significantly reduce the amount of virions released from infected, treated cells, and / or this inhibition may have little or no significant effect on the amount of RNA incorporation in the virions released; and / or the inhibition interrupts the maturation of the virions released from infected cells treated with the compound. In related embodiments, the structure of the virions can be affected, and a majority of the virions released from the infected, treated cells exhibit electron-dense, spherical nuclei that are acéntricos with respect to the viral particle; and / or have increasingly dense electrodense layers that are just inside the viral membrane; and / or have reduced infectivity or do not have infectivity. In additional embodiments, the invention relates to to a method for treating HIV-1 infection in a patient by administering a compound that inhibits HIV protease interaction with CA-SP1, which results in inhibition of Gag viral p25 protein processing (CA-SPl) ) to p24 (CA), but has no significant effect on other Gag processing steps. This inhibition can be direct, or alternatively, indirect; and / or may comprise the compound that binds to the Gag viral protein such that the interaction of the HIV protease with CA-SP1 is inhibited. The invention also relates to a method for treating HIV in a patient with a compound that binds at or near the cleavage site of the viral p25 protein from Gag (CA-SP1) to p24 (CA), thereby inhibiting the interaction of the HIV protease with the CA-SP1 cleavage site and results in the inhibition of p25 to p24 processing. In other embodiments, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), wherein the compound binds to a polypeptide with an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or that is identical to a selected sequence from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID O: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). In other embodiments, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), wherein the compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or that is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18 (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) approximately nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) approximately nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) about nucleotides 1858-1920 of SEQ ID NO: 19. In another aspect, the invention relates to a method for inhibiting the processing of Gag viral p25 protein (CA-SP1) by administration of a compound. In related embodiments, this compound binds to a polypeptide with an amino acid sequence that is at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or that is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (C) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118), - and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). In related embodiments, the invention relates to a method for inhibiting the processing of Gag viral p25 protein (CA-SP1) by administering a compound wherein the compound binds to a polypeptide encoded with a polynucleotide sequence having the less about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide p selected from the group consisting of: (a) about nucleotides 1243 -1435 of SEQ ID? O: 18 (b) approximately nucleotides 1729-1920 of SEQ ID? O: 19; (c) about nucleotides 1344-1435 of SEQ ID? O: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) approximately nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. The invention can be useful in the treatment of HIV in patients who are not treated adequately by other HIV-1 therapies. Accordingly, the invention also relates to a method of treating a patient in need of therapy, wherein the HIV-1 infecting cells does not respond to other HIV-1 therapies. In another embodiment, the methods of the invention are practiced in a subject infected with an HIV that is resistant to a drug used to treat HIV infection. In one application, HIV is resistant to a protease inhibitor, a polymerase inhibitor, a nucleoside analogue, a vaccine, a binding inhibitor, a immunomodulator, or any other inhibitor. In another embodiment, the methods of the invention are practiced in a subject infected with HIV that is resistant to a drug used to treat HIV infection which is selected from the group consisting of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polimannoacetate, castanospermine; contracan; pharmatex cream, CS-87, penciclovir, famciclovir, acyclovir, citofovir, ganciclovir, dextran-sulfate, D-penicillamine phosphono trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, T peptide, phenytoin, isoniazid, ribavirin , rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, and combinations thereof. The compounds of the invention are also useful as part of the combination of therapies. Accordingly, in one aspect, the combination refers to a method for treating HIV in a patient, wherein the patient is administered with the compound in combination with at least one antiviral agent. Suitable anti-viral agents include, but are not limited to: zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, ampligen, butylated hydroxytoluene; polimannoacetate, castanospermine; contracan; pharmatex cream, CS-87, penciclovir, famciclovir, acyclovir, citofovir, ganciclovir, dextran-sulfate, D-penicillamine phosphonoformate trisodium, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, T peptide, phenytoin, isoniazid, ribavirin , rifabutin, ansamycin, trimetrexate, SK-818, suramin, UA001, enfuvirtide, peptides derived from gp41, antibodies to CD4, soluble CD4, molecules containing CD4, CD4-IgG2, and combinations thereof. In another embodiment, the patient is administered with the compound in combination with an immunomodulatory agent, anticancer agent, antibacterial agent, antifungal agent or a combination thereof. The invention also relates to compounds. These compounds are useful in a method for treating patients infected with HIV; in a method for inhibiting processing of Gag viral p25 protein (CA-SP1) to p24 (CA), or in a method for treating human blood and human blood products. These compounds useful in the present invention include, but are not limited to, dimethylsuccinyl-betulinic acid or dimethylsuccinyl-betulin derivatives, or are selected from the group consisting of O- (3 ', 3' -dimethylsuccinyl) betulinic, 3-0- (3 ', 3'-dimethylsuccinyl) betulin, 3-0- (3', 3'-dimethylglutaryl) betulin, 3-0- (3 ', 3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0- (3', 3'-dimethylglutaryl) betulinic acid, (3 ', 3'-dimethylglutaryl) dihydrobetulinic acid, 3-O-diglycolyl-betulinic acid, 3-O acid -diglycolyl-dihydrobetulinic and combinations thereof. The compounds of the invention can be used alone, or administered with additional compounds, including zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, tenofovir, adefovir , atazanavir, fosamprenavir, hydroxyurea, AL-721, amplify, butylated hydroxytoluene; polimannoacetate, castanospermine; contracan; pharmatex cream, CS-87, penciclovir, famciclovir, acyclovir, citofovir, ganciclovir, dextran-sulfate, D-penicillamine phosphonoformate trisodium, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, T peptide, phenytoin, isoniazid, ribavirin , rifabutin, ansamycin, trimetrexate, SK-818, suramin, UAOOl, enfuvirtide, peptides derived from gp41, antibodies to CD4, soluble CD4, molecules containing CD4, CD4-IgG2, and combinations thereof; an antiviral agent, an immunomodulatory agent, anticancer agent, antibacterial agent, an antifungal agent, or combinations thereof. In further embodiments, the invention relates to a method of treating human blood products comprising contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA). In one aspect, the compound does not significantly affect other Gag processing steps. In related embodiment of this invention, this inhibition does not significantly reduce the amount of virions released from the treated infected cells; and / or has little or no significant effect on the amount of RNA incorporation in the virions released; and / or inhibits the release of virions released from infected cells, treated with the compound, and / or affects viral morphology. These effects on viral morphology include, but are not limited to: virions released from infected, treated cells exhibit electron-dense, spherical nuclei that are acéntricos with respect to the viral particle; and / or have increasingly electron dense layers that are just inside the viral membrane; and / or have reduced infectivity or do not have infectivity. In related embodiments, the method comprises administering the compound that inhibits the interaction of HIV protease with CA-SP1, which results in the inhibition of Gag viral p25 protein processing.

(CA-SPl) to p24 (CA) but has no significant effect on other Gag processing steps. This can be via direct or indirect inhibition of the interaction of HIV protease with CA-SP1; and / or can comprise that the compound binds to the Gag viral routine such that the interaction of HIV protease with CA-SP1 is inhibited; and / or the compound binds at or near the cleavage site of the Gag viral p25 protein (CA-SP1) to p24 (CA), thereby inhibiting the interaction of the HIV protease with the CA cleavage site. -SPl and resulting in the inhibition of p25 to p24 processing. In a further embodiment, the invention relates to a method for treating human blood products comprising contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in wherein the compound binds to a polypeptide with an amino acid sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KN MTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? ANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (c) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). In a related embodiment, the invention relates to a method for treating human blood products comprising contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), wherein the compound binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a polynucleotide selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18 (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. The invention also incorporates methods for identifying compounds that inhibit HIV-1 replication. Accordingly, the invention also includes a method for identifying compounds that inhibit HIV-1 replication in cells of an animal, comprising: contacting a Gag protein comprising an CA-SPl cleavage site with a test compound; add a marked substance that binds selectively near the excision site of CA-SPl; and measuring the competition between the binding of the test compound and the marked substance at the cleavage site of CA-SPl. In further embodiments of this method, the compounds inhibit the interaction of HIV-1 protease with a target site by binding to the target site.

These methods also include embodiments wherein the region of the CA-SP1 cleavage site is contained within a polypeptide or recombinant peptide fragment; and / or wherein the labeled substance is a labeled antibody specific for CA-SP1, and measuring the change in the amount of the labeled antibody bound to the protein in the presence of the test compound as compared to a control. The brands include, enunciatively and without limitation, an enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electrodense substance, radioisotope and a combination thereof. The method for identifying compounds that inhibit HIV-1 replication in cells of an animal also comprises, in one embodiment, measuring the change in the amount of labeled 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid bound to the protein in the presence of the test compound, compared to a control; and wherein the labeled substance is 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In an alternative embodiment, the invention comprises a method for identifying compounds that inhibit HIV-1 replication in cells of an animal comprising: contacting a polypeptide comprising a CA-SP1 cleavage site, with a protease in the presence of a test compound. Preferably, the protease it is related to HIV-1 protease, or it is HIV protease. In one embodiment, the method comprises; contacting a polypeptide comprising a wild-type CA-SPl cleavage site, with a protease in the presence of a test compound and also contacting a polypeptide comprising a mutant CA-SPl cleavage site or a protein comprising an alternative protease cleavage site with HIV-1 protease in the presence of the test compound, detecting cleavage, and comparing the amount of cleavage of the native wild-type polypeptide to the amount of cleavage of the mutant polypeptide to the amount of cleavage of the protein comprising an alternative protease cleavage site. In an aspect related to this invention, the cleavage type region of CA-SP1 wild type or CA-SP1 mutant or alternative protease is contained within a polypeptide or recombinant peptide fragment. In a further related aspect, the polypeptide is labeled with a fluorescent portion and a fluorescence quench portion, each attached to opposite sides of the CA-SP1 cleavage site, and wherein the detection comprises measuring the fluorescent portion signal . In another related embodiment, the polypeptide is labeled with two fluorescent portions, each linked to opposite sides of the CA-SP1 cleavage site, and wherein the detection comprises measuring the energy transfer fluorescent from one portion to the other in the presence of the test compound. In a further embodiment, the effect of the test compound on cleavage of the polypeptide is detected by measuring the amount of a labeled antibody that binds SPI or p24 (CA). In a related aspect, the labeled antibody that binds to CA, or the antibody that binds to SPI is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electrodensate substance, radioisotope and combinations thereof. The invention also relates to a method for identifying compounds that inhibit the replication of HIV-1 in cells of an animal. In one embodiment, the method comprises: contacting a test compound with cells infected with wild type virus isolates and with cells infected with virus isolates having significantly reduced sensitivity to 3-0- (3 ', 3'- dimethylsuccinyl) betulinic; and selecting test compounds that are more active against wild type virus isolate compared to virus isolates having reduced sensitivity to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In another embodiment, the method comprises contacting cells infected with HIV-1 with a test compound; lyse the infected cells or viral particles released to form a lysate, and analyze the lysate to determine if cleavage of the CA-SP1 protein has occurred. In this later modality, the analysis may comprise measuring the presence or absence of p25; and / or performing a western blot of viral proteins and detecting p25 using a p25 antibody; and / or performing a gel electrophoresis of viral proteins and imaging the metabolically labeled proteins, and / or performing an immunoassay. This immunoassay can be performed by methods known in the art, including but not limited to: (a) capturing p25 and p24 on a substrate using an antibody that selectively binds p24; and (b) detecting the presence or absence of p25 in the substrate by using an antibody that binds selectively to p25. The invention also includes modifications of the above test as will be obvious to one skilled in the art. In a further embodiment, the method for identifying a compound according to the invention comprises the use of an epitope tag sequence inserted in SPI and the selective detection of p25 is performed using an antibody to the epitope tag. The invention also relates to a method for identifying compounds that inhibit the replication of HIV-1 in the cells of an animal comprising: contacting cells infected with HIV-1 with a test compound and subsequently analyze the virus particles using transmission electron microscopy. This analysis includes, for example, looking for the presence of spherical nuclei that are acentric with respect to the viral particle; and / or having increasingly dense electrodense layers that are just inside the viral membrane. In further aspects, the invention relates to an isolated polynucleotide comprising a sequence encoding an amino acid sequence containing a mutation in a p25 HIV Gag protein (CA-SP1), this mutation resulting in a decrease in inhibition of the processing of p25 (CA-SP1) to p24 (CA) by 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid DSB). This inhibition of p25 processing may be due to a decrease in the inhibition of the interaction of the HIV-1 protease with Gag; and / or a decrease in the inhibition of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid to Gag; and / or a decrease in DSB binding at or near the Gag CA-SPI cleavage site. Suitable polynucleotides also include those that code for a mutation at or near the CA-SP1 cleavage site; or in the SPI domain of SP-1; and / or those which code for a mutation in or near the amino acid sequence G / SHKARV / ILAEAMSQV (SEQ ID NO: 1); and / or those that code for the sequences of amino acids GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and / or isolated polynucleotide which is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9; and / or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 4, and SEQ ID NO: 6; and / or having at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 9; and / or having at least about 95% identity to a polynucleotide selected from the group consisting of SEQ NO: 5 and SEQ ID NO: 7; and / or having at least about 80% identity to a polynucleotide of SEQ ID NO: 10. In further embodiments, the polynucleotide having more than about 40%, 50%, 60%, 70%, 80%, 90% 95%, 99% identity or that is identical to the polynucleotide sequences listed above. The invention also relates to vectors comprising these polynucleotides as described above; to a host cell comprising this vector; and a method for producing a polypeptide comprising incubating the host cell containing this vector in a medium and recovering the polypeptide from the medium. In one embodiment, the invention relates to an antibody. This antibody can bind to a polypeptide with an amino acid sequence that has at least approximately 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, or which is identical to a sequence selected from the group consisting of: (a) KN MTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM ( SEQ ID? O: 21); (b) K? MTETLLVQ? A? PDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (C) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118); and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). In a further related embodiment, the invention relates to an antibody that binds to a polypeptide encoded by a polynucleotide sequence having at least about 40%, 50%, 60%, 70%, 80%, 90%, 95 % or 99% identity, or which is identical to a polynucleotide with a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID? O: 18 (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) approximately nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) approximately nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. In one embodiment, the antibody binds to the amino acids of the CA-SP1 region of the Gag HIV-1 polypeptide wherein the amino acids comprise: SHKARILAEAMSQV ( SEQ ID NO: 25) or GHKARVLAEAMSQV (SEQ ID NO: 26). In one embodiment, the invention relates to an antibody that inhibits the binding of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid to the CA-SPl region of the Gag polypeptide. The invention also relates to mutant HIV-1 viruses. In this embodiment, the invention is a HIV-1, recombinant, mutant virus, wherein the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in the virus is not significantly inhibited by acid 3-0- (3 ', 3'-dimethylsuccinyl) betulinic. In related embodiments, this virus is not inhibited by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In another embodiment, 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid does not inhibit the interaction of the protease with the Gag polypeptide in this virus. In another, the virus does not bind to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In further embodiments, the invention relates to viruses wherein the amino acids of the CA-SP1 region are replaced with alternative amino acids, or the amino acids are added to the CA-SP1 region, or where the amino acids are deleted. In one modality; one or more amino acids are deleted from the amino acid sequence AEAMSQV (amino acids Nos. 8-14 of SEQ ID NO: 26) in the CA-SPI region. A mutant virus can be used of the methods of the invention described elsewhere herein. For example, these viruses are useful in a method to identify a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), the method comprising compare the ability of the compound to inhibit HIV-1 replication compared to the replication of a mutant virus summarized above. This inhibition can be examined in a cell, or in an animal or in vi tro. The invention also relates to retroviruses not of HIV-1 which are sensitive to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In some embodiments, the retrovirus codes for a CA-SP1 polypeptide with an amino acid sequence "comprising the EAMSQV sequence (amino acids numbers 8-14 of SEQ ID NO: 26) at or near the CA-SPl cleavage site. In another embodiment, the retrovirus codes for a CA-SP1 polypeptide with an amino acid sequence comprising the sequence VLAEAMSQV (amino acids Nos. 6-14 of SEQ ID NO: 26) at or near the CA-SPl cleavage site. In another embodiment, the retrovirus codes for a CA-SP1 polypeptide with an amino acid sequence comprising the sequence GHKARVLAEAMSQV (SEQ ID NO: 26) at or near the CA-SP1 cleavage site; in another, the retrovirus comprises the amino acid sequence having at least 60%, 70%, 80%, 90% identity or that is identical to the sequence encoded by the polynucleotide of SEQ ID NO: 26, SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96 OR SEQ ID NO: 98; in another embodiment, the retrovirus comprises the amino acid sequence having at least 60%, 70%, 80%, 90% sequence identity or which is identical to the sequence of SEQ ID NO: 91; SEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97 or SEQ ID NO: 99. In another embodiment, the retrovirus comprises the nucleic acid sequence having at least 70%, 80%, 90% or which is identical to the sequence of SEQ ID NO: 90; SEQ ID NO: 92; SEQ ID NO: 94; SEQ ID NO: 96 or SEQ ID NO: 98. The retroviruses of this embodiment of the invention include, without limitation, HIV-2., HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus (VAS), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) ), encephalitis and caprine arthritis virus (CAEV), Visna-maedi virus, or feline leukemia virus (FeLV). In a related embodiment, the invention relates to a method for making a non-HIV-1 recombinant lentivirus sensitive to DSB. This method comprises: deleting from the genome of the lentivirus the nucleotides corresponding to nucleotides 1370-1413 of SEQ ID NO: 18, in HIV-1; and inserting nucleotides 1370-1413 of SEQ ID NO: 18 or nucleotides 1857-1899 of SEQ ID NO: 19 in the non-HIV-1 lentivirus region. Examples of chimeric lentiviruses that were, are or can be constructed by this method are described in Figure 10. These viruses can be used in the methods of invention described elsewhere in the present. For example, these recombinant non-HIV-1 lentiviruses can be used in a method to identify a compound that inhibits the Gag viral p25 protein (CA-SP1) to p24 (CA) method, the method of comparing ability of the compound to inhibit the replication of a non-wild type HIV-1 virus with the recombinant DSB-sensitive variant thereof. This inhibition can occur in a cell, in an animal; or in vi tro. The invention also relates to an animal model of lentivirus infection comprising a suitable non-human animal host infected with a lentivirus sensitive to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In this modality, the lentivirus may include, without limitation and without SIV limitation; FIV; EIAV; BIV; CAEV; and Visna-Maedi virus. The invention also relates to isolated polypeptides. In one embodiment, the invention relates to a polypeptide that contains a mutation in an HIV CA-SPI protein, the mutation that results in a decrease in the inhibition of the p25 procedure by 3-0- (3 ', 3'-dimethylsuccinyl) etulinic. In a related embodiment, this polypeptide is encoded by a polynucleotide that contains a mutation located at or near the CA-SPl cleavage site or in the SPI domain encoded by SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 10 and / or is encoded by a polynucleotide selected from the group eme consists of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , and SEQ ID NO: 9; and / or comprises a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); and / or is encoded by an isolated polynucleotide that hybridizes under severe conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7 and 10; and / or is part of a chimeric or fusion protein. The invention also relates to antibodies that selectively bind to an amino acid sequence that contains a mutation in an HIV CA-SP1 protein that results in a decrease in the inhibition of the p25 procedure (CA-SP1) to p24 (CA) by 3-0- (3 ', 3' -di-ethylsuccinyl) betulinic acid. In this embodiment, the antibody selectively binds to a mutation located at or near the CA-SP1 cleavage site or SPl domain of CA-SP1; in another, the antibody selectively binds to a mutation comprising a sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) or SHKARILAEVMSQV (SEQ ID NO: 3); in another embodiment, the antibody selectively binds to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

In another embodiment, the invention relates to an antibody that selectively binds to SPI but not to CA-SP1; another that binds selectively to CA-SP1 but not CA; another that binds selectively to CA but not to CA-SPl; and an additional antibody that binds selectively at or near the CA-SPl cleavage site. The invention also relates to a compound identified by any of the methods explained above. In one embodiment, the compounds are not a compound selected from the group consisting of 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid, 3-0- (3 ', 3'-dimethylsuccinyl) betulin, 3-0 - (3 ', 3' -dimethylglutaryl) betulin, 3-0- (3 ', 3'-dimethylsuccinyl) dihydrobetulinic acid, 3-0- (3', 3'-dimethylglutaryl) betulinic acid, (3 ', 3) acid '~ dimethylglutaryl) ihydrobetulinic, 3-0-diglycolyl-betulinic acid, 3-0-diglycolyl-dihydrobetulinic acid, and combinations thereof. The invention also relates to a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises dimethylsuccinyl-betulinic acid or dimethylsuccinyl-betulin derivatives; in another, the pharmaceutical composition comprises a compound selected from the group consisting of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid, 3-0- (3', 3'-dimethylsuccinyl) betulin, 3-0- (3 ', 3' -dimethylglutaryl) betulin, 3-0- (3 ', 3' -dimethylsuccinyl) dihydrobetulinic acid, 3-0- acid (3 ', 3' -dimethylglutaryl) betulinic, (3 ', 3' ~ dimethylgutaryl) dihydrobetulinic acid, 3-0-diglycolyl-betulinic acid, 3-0-diglycolyl-dihydrobetulinic acid, and combinations thereof. In another embodiment, the pharmaceutical composition comprises one or more compounds identified according to the methods of the invention that are not listed otherwise; or any pharmaceutically acceptable salt, ester or prodrug thereof, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprising an antiviral agent which may include any of zidovudine, lamivudine, didanosine, zalcitabine, stavudine, abacavir, nevirapine, delavirdine, emtricitabine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, tenofovir, amprenavir, adenofovir, atazanavir, fosamprenavir, hydroxyurea, AL-721, amplify, butylated hydroxytoluene; polimannoacetate, castanospermine; contracan; pharmatex cream, CS-87, penciclorvir, famciclovir, acyclovir, citofovir, ganciclovir, dextran-sulfate, D-penicillamine, trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, T peptide, phenytoin, isoniazid, ribavirin, rifabutin, ansamycin, trimetrexate, SK-818, suramin, USA001, combinations thereof, and any other antiviral agent, immunomodulatory agent, anticancer agent, antifungal agent, antibacterial agent or combinations thereof.

The invention also relates to a method for determining whether an individual is infected with HIV-1 which is susceptible to a treatment by a. compound that inhibits the processing of p25. In one embodiment, the method comprises taking blood from the patient, genotyping the viral RNA and determining whether the viral RNA contains mutations in the sequences encoding the region of the CA-SPl cleavage site. The invention also relates to a method for treating a disease in a patient in need thereof, comprising: identifying a compound that inhibits the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but which has no significant effect on other Gag processing steps; obtain regulatory approval for the sale and use of the compound; packaging the compound for the sale and treatment of a disease in a patient in need thereof. Brief Description of the Figures Figure 1. DSB does not interrupt the HIV-1 protease activity at a concentration of 50 μg / mL. In samples containing DSB, recombinant Gag is correctly processed. In contrast, indinavir blocks protease activity at 5 μg / mL as evidenced by the absence of bands corresponding to p24 and the MA-CA precursor.

Figure 2. Western blots of Gag associated with virion derived from H9 / HIV-1IIIB, chronically infected H9 / (HIV-1) in the presence of DSB (1 μg / mL), indinavir (1 μg / mL) or control (DMSO). Gag proteins were visualized using HIV-Ig (HIV-1) or monkey anti-SIVmac251 serum (HIV-2 and SIV; NIH AIDS Research and Reference Reagent Program). Figure 3. Analysis by MS of HIV-infected cells treated with DSB. The MS data shows two primary differences between the samples treated with DSB and those not treated. The virions generated in the presence of DSB are characterized by an absence of mature conical nuclei. In these samples, the nuclei are uniformly spherical and often acentric. Second, many virions exhibit an electrodensade layer within the lipid bilayer but outside the nucleus (indicated by arrows in the sample panels treated with DSB). In the samples treated with DSB, no mature viral particles were observed. Figure 4 depicts the amino acid sequences in the CA-SPl cleavage site region of DSB-sensitive HIV-1 isolates NL4-3 and RF (# 1; SEQ ID NO: 1) and HIV-1 isolates resistant to DSB (# 2; SEQ ID NO: 2 (NL4-3), and # 3 SEQ ID NO: 3 (RF)). The differences between the native and DSB-resistant sequences comprise a change from alanine to valine in the first residue in the 3 'direction (# 2) and a change from alanine to valine in the third residue in the 3' direction (# 3) of the CA-SPl cleavage site (- | -). These residues are underlined and in bold for easy identification. Figure 5 depicts the consensus homosense + sequence for the NB4-3 mutant resistant to DSB A364V (SEQ ID NO: 4) < which begins with the start of gag and continues in pol, which includes the entire coding region of protease.

The nonsense mutations not found in the sequence M1991 of the GENBANK of the NL4-3 wild type are in bold and shaded in gray. The coding sequence for the region of the consensus CA-SPl cleavage site is underlined. The shaded area that includes the cleavage site denotes the SPl sequence. The first mutation is the A364V mutation. The second amino acid change (in the protease) was also found in the original clone and it has been confirmed that it corresponds to a sequencing error in the original GENBANK entry. Therefore, mutations in the protease did not actually occur. Figure 6 depicts the consensus homosense + sequence for the DSB-sensitive isolate of NL4-3 origin (SEQ ID NO: 5) that was passed in the absence of the drug in parallel with the mutant isolate A364V. Figure 7 depicts the consensus homosense + sequence for the RF mutant of HIV-1 resistant to DSB, A366V (SEQ ID NO: 6) which begins with the start of the gag and continues in pol, which includes the entire coding region of protease. The missense mutations not found in the wild type HIV-1 RF GENBANK M17451 sequence are shaded gray. The region of the CA-SP1 cleavage site is underlined. The only nonsense mutation was not found either in the identically passed DSB-isolated isolate is the A366V mutation at the CA-SPl cleavage site. Figure 8 depicts the homosense + consensus sequence for the DSB-sensitive HIV-1 RF origin isolate (SEQ ID NO: 7), which was passed in the absence of the drug in parallel with the mutant A366V isolate. Figure 9 depicts the polynucleotide sequences, SEQ ID NO: 8 and SEQ ID NO: 9, which code for the polypeptides designated herein as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. SEQ ID NO: 10 and 12 represent the nucleotide sequences coding for the origin polypeptide sequences designated SEQ ID NO: 1. SEQ ID NO: 1 is a consensus sequence based on the sequences of the NL4- region 3 and RF. FIGURE 10A: Amino acid sequences in the lentivirus CA-SP1 region (SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 30, respectively). FIGURE 10B: Amino acid sequences of the CA-SP1 region in the RF strains of HIV-1 (SEQ ID NO: 11) and NL4-3 (SEQ ID NO: 13).

FIGURES 10C-10D: Nucleotide sequences of the chimeric SVI of the gag gene. The 42 nucleotide sequence coding for the seven amino acids in the five direction with apostrophe and seven amino acids in the three direction with the apostrophe of the CA-SPl cleavage site is underlined and in bold. FIGURES 10E-10H: Nucleotide sequences of the gag gene of chimeric FIV, EIAV and BIV to be prepared according to the invention. The sequence of 42 nucleotides are encoded for the seven amino acids in the five direction with the apostrophe and the seven amino acids in the three direction with the apostrophe of the CA-SPl cleavage sites are underlined and in bold type (nucleotide sequence: SEQ ID NO: 16; amino acid sequence SEQ ID NO: 17). FIGURE 10F. Nucleotide sequence of the GAG gene of Chimeric Feline Immunodeficiency Virus (FIV) containing the CA-SP1 region of HIV; nucleotides 1-1353 of the chimeric FIV-GAG gene correspond to nucleotides 628-1980 in the chimeric FIV genome. The nucleotide sequence SEQ ID NO: 94 encoding amino acids SEQ ID NO: 95. FIGURE 10G. Nucleotide sequence of the GAG gene of Chimeric Equine Infectious Anemia Virus, (EIAV) that contains the CA-SP1 region of HIV; Nucleotides 1-1587 of the Chimeric EIAV-GAG gene correspond to nucleotides 450-1910 in the genome of Chimeric EIAV. The Nucleotide sequence SEQ ID NO: 96 encoding the amino acid sequence SEQ ID NO: 97.

FIGURE 10H. Nucleotide sequence of the GAG gene of the Bovine Immunodeficiency Virus (BIV) Chimeric containing the CA-SP1 region of HIV; nucleotides 1-1471 of the chimeric BIV-GAG gene correspond to nucleotides 316-1746 in the chimeric BIV genome. The Nucleotide Sequence SEQ ID NO: 98 o_ue encodes for the amino acid sequence SEQ ID NO: 99. Figure 11: Replication kinetics of mutants resistant to (DSB) PA-457. Figure 12: Suppressions of the sequential SPl point in the context of NL4-3 used to identify residues necessary for DSB activity. The amino acid sequence of the SPI domain in NL4-3 is shown. "?" indicates the deletion and "-" means identical residues between the point deletion mutants and NL4-3 (SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; SEQ ID NO: 85; SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 100; SEQ ID NO: 101; SEQ ID NO: 102; SEQ ID NO: 103, respectively). Figure 13. Summary of particle production and infectivity of dot deletion mutants. Figure 14. Western transfers for viruses containing point deletions in SP1, in the presence (+) and absence (-) of DSB. Figure 15. Substitution of residues VL-AEAMSQV (SEQ ID NO: 32) of HIV-1 CA-SP1 in the SIVmac239 structure that returns to SIVmac239 responsive to DSB (SEQ ID NO: 14; SEQ ID NO: fifteen; SEQ ID NO: 20; SEQ ID NO: 27; SEQ ID NO: 28; SEQ ID NO: 13; respectively). (Upper panel) amino acid sequences near the CA-SPl cleavage site (qμe including cote SPI region) are shown for SIVmac239, HIV-1 NL4-3 and a number of SIV mutants in which various NL4- residues 3 (were inserted). The ions ("-") indicate that the residues are the same as those in SIVmac239. (Bottom panel) Western blots showing CA and SP-SP proteins for this series of viruses in the presence (+) or absence (-) of DSB. Figure 16: Preservation of sequence in the CA-SPl region of Lentivirus. Cloning strategy: substitution of specific HIV-1 CA-SP1 residues in the corresponding Gag region of IVF, EIAV or BIV. Figure 17. NL4-3 SP1 of HIV-1 marked with an epitope. The sequences of the SP1 peptides with inserted peptide tags are shown. "?" indicates residue deleted and "-" indicates that the residue is identical to that in SPI of NL4-3. (Figure 17 (1); SEQ ID NO: 15; SEQ ID NO: 104; SEQ ID NO: 105; SEQ ID NO: 106; SEQ ID NO: 107, respectively); (Figure 17 (2), SEQ ID NO: 15, SEQ ID NO: 108, SEQ ID NO: 109, respectively); (Figure 17 (3); SEQ ID NO: 15;; SEQ ID NO: 110; SEQ ID NO: 111; respectively); (Figure 17 (4); SEQ ID NO: 15; SEQ ID NO: 112; SEQ ID NO: 113; SEQ ID NO: 114; respectively); (Figure 17 (5), SEQ ID NO: 15, SEQ ID NO: 115, respectively). Figures 18A-18C: Polynucleotide sequence of the HIV-1 RF strain. The nucleotide sequence of the Gag polyprotein is underlined and in bold. The sequence of 42 nucleotides encoding the seven amino acids in the 5 'and 7' directions in the 3 'direction of the CA-SP1 cleavage site is highlighted in green. 129 additional nucleotides (43 amino acid residues) in the 5 'direction of the CA-cleavage site and the remaining 21 nucleotides (seven amino acid residues) in SPI are highlighted. Figure 19A-19E: Polynucleotide sequence of strain NL4-3 of HIV-1. The nucleotide sequence of the Gag polyprotein is underlined and in bold. The sequence of 42 nucleotides ejue codes for the seven amino acids in the 5 'direction and the seven amino acids in the 3' direction of the CA-SPl cleavage site is highlighted in green. 129 additional nucleotides (43 amino acid residues) in the 5 'direction of the CA cleavage site and the remaining 21 nucleotides (seven amino acid residues) in SPI are highlighted. Figure 20: A schematic representation of the Gag protein and the CA-SPl sequences of the SHIV used in this study. The sequences flanking the cleavage site of CA-SP1 for SIV Mac239 and NL4-3 of HIV-1 are shown at the top and bottom of the sequence listing, respectively. A line of dashes (-) represents residues are identical in the SIV MAC239, a delta (?) Represents the SIV residues that are suppressed in the SHIVs Figure 21: Western transfer analysis of the Gag processing profiles for the SHIV of the panels 1-3. Figure 21A shows the Gag processing of the cell-associated virus while Figure 21B shows the Gag processing profile for the cell-free virions. Normal Gag processing indicates by a plus sign (+), while a faulty processing profile is indicated by a minus sign (-). Figure 22: Western blot analysis of the effect of DSB at 1 μg / ml in the conversion of the capsid precursor, CA-SPl to mature capsid protein. In panel A, the virus for Western blotting was obtained using a constant volume of the cell culture supernatant. This resulted in variability in the intensity of the viral protein bands due to the differences between the SHIVs in the level of virus production. Panel B shows the Gag processing profiles obtained when increased amounts of viral protein are used by Western blot analysis. Only SHIVs that exhibit normal Gag processing are included. The asterisk (*) in panel A indicates the weak band of CA-SPl for Gl of SHIV observed in the autoradiography can not see, however, the sensitivity to DSB for this virus is classical based on +/- in the results observed when the analysis is performed using increased amounts of protein (panel B). Figures 23A-23H: Alignment of the CA-SP1 region in clinical isolates of HIV-1, obtained from "HIV Sequence Compendium 2002", Kuiken et al. eds. Los Alamos National Laboratory, Los Alamos, NM. (www.hiv.lanl.gov). Detailed Description of the Invention The present invention relates to methods for inhibiting the replication of HIV-1 in the cells of an animal. More specifically, the invention comprises methods for inhibiting replication of HIV-1 in the cells of a mammal by contacting infected cells with a compound that inhibits the processing of Gag's viral p25 protein (CA-SP1) to the p24 protein (ca). More specifically, these compounds inhibit the processing of Gag's viral p25 protein (CA-SP1) to p24 (CA) without significantly affecting other Gag processing steps. "A compound that does not significantly affect other Gag processing steps" means that the compound in question predominantly inhibits the processing of p25 to p24, but does not necessarily preclude the possibility of having minor effects in other processing steps of Gag. "Significant" or "significant manner", where it is not defined otherwise in the present, means a change observable or measurable compared to the process in the absence of a compound. However, not all observable or measurable changes may be necessarily significant. Several viral phenotypes can also be observed in the practice of the method of the invention. One result of contacting an infected cell with the compounds of the invention may be the formation of non-infectious viral particles. Alternatively, or in addition, contacting infected cells with a compound that inhibits the processing of p25 to p24, results in the formation of non-infectious viral particles, but where there is no significant effect in other steps of Gag processing. . This can not significantly reduce the amount of virus released from treated cells and / or have no significant effect or no significant effect on the amount of RNA uptake in the virions released. Accordingly, the invention also relates to a method for inhibiting HIV infection in cells of an animal comprising contacting the cells with a compound that inhibits p25 processing and also affects other viral phenotypes, described above. Mutant viruses defective in CA-SP1 cleavage have been shown to be non-infectious (Wiegers K. et al., J. Virol. 72: 2846-2854 (1998)). 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) is an example of a compound which interrupts the processing of p25 to p24 and potently inhibits the replication of HIV-1. The activity of this specific compound for the processing step from p25 to p24, no other steps in Gag processing. Additionally, treatment with DSB results in the normal morphology of the HIV particle as described in Figure 1. 1. Identification of HIV-1 determinants associated with sensitivity to 3-0- (3 ', 3' - acid). dimethylsuccinyl) betulinic (a) Generation and selection of HIV-1 virus resistant to DSB mutant forms of HIV-1 have been generated in which the amino acid sequence in the region of the CA-SPl cleavage site is modified, decreasing the sensitivity of these strains to compounds that interrupt the processing of CA-SPl. Data on these mutant viruses have been used to identify the amino acid residues in wild type Gag that are involved in the antiviral activity of these compounds. In one embodiment, compounds that interrupt CA-SP1 processing directly or indirectly inhibit the interaction of the HIV-1 protease with the region of the Gag protein region containing these amino acid residues. In another embodiment, the compounds that interrupt CA-SP1 processing serve the region containing these amino acid residues. As used herein, the term "link", "attached", or "linking" refers to the link or link including, ionic interactions, hydrophobic interactions electrostatic, hydrogen bonds, etc .; and also include associations that may be covalent, for example, by chemical coupling. The covalent bonds can be, for example, ester, ether, phosphoester, thioester, thioether, urethane, amide, amine, peptide, imide, hydrazone, hydrazide, carbon-sulfur bonds, carbon-phosphorus bonds and the like. The term "joined" is broader and includes terms such as "coupled", "conjugated" and "joined". In another embodiment, compounds that interrupt CA-SP1 processing bind to another Gag region and thereby inhibit the interaction of HIV-1 protease with the region of the CA-SP1 cleavage site. In another embodiment, viruses or recombinant proteins that contain mutations in the region of the CA-SPl cleavage site can be used in detection assays to identify compounds that disrupt CA-SPl processing. In a set of experiments, amino acid residues in HIV-1 Gag that are included in the interruption of CA-SP1 processing by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) were identified at sequencing the gag-pol gene of virus isolates that have been selected for resistance to DSB. The amino acid sequences of these resistant viruses were compared to the gag-pol gene sequences of DSB-sensitive HIV-1 isolates. Two individual amino acid changes were identified in the DSB-resistant viruses, a substitution of alanine (Ala) to valine (Val) in residue 364 (SEQ ID NO: 4) and in a second isolated, at residue 366 (SEQ ID NO: 6), in the Gag polyprotein (see Figure 4). These residues are located immediately in the 3 'direction of the CA-SPI cleavage site (in the N-terminus of SPI). Alanine is highly conserved in these positions throughout all of the HIV-1 subtypes listed in the Los Alamos National Laboratory. The five amino acid residues in the 5 'direction and in the 3' direction at the CA-SPl cleavage site are also highly conserved among the various subtypes. However, isoleucine replaces valine at the position two residues in the 5 'direction of the cleavage site in several coatings (compare Figure 4, SEQ ID NO: 1). (nHIV Sequence Compendium 2002", Kuiken et al., eds. 'Los Alamos National Laboratory, Los Alamos, NM. ). In order to more extensively correlate viral genetic determinants for DSB resistance, additional experiments were performed to select in vitro viruses that are drug resistant. Transplanted shows parallel cultures of Jurkat T cells (5 x 105 each) with the clone pNL4-3 of proviral DNA in the presence or absence of 10-50 ng / ml of DSB. The cells were passed every two days, fresh drug was added in each pass. The replication of the virus by measuring the activity of inverted transcriptase in culture supernatants. The virus was isolated from culture supernatants harvested at selected time points, and genomic DNA was amplified by RT-PCR using cells spanning the coding region between the N-terminus of CA and the N-terminus of RT. The amplified product was then sequenced using the same set of cells. In one experiment, an A366V mutation was identified in the SPI region of the NL4-3 virus cultured in the presence of DSB (note: the numbering is relative to the Gag polyprotein). In the additional step, a double mutant was identified that contained a G357S mutation in CA as well as the A366V mutation in SPI. The A366V mutation was previously identified in experiments that select resistant variants of the RF isolate. Interestingly, the wild type RF sequence also contains a serine residue at position 357 in CA (Figure 4). Since serine is present in this position in the isolates (such as RF) that are sensitive to DSB, the CA G357S mutation alone is not sufficient to confer resistance to DSB. To determine the contribution of each of these mutations to drug resistance, the A366V mutation and the A366V / G357S double mutation were again managed in the structure of NL4-3 wild type by site-directed mutagenesis. The resulting constructs were transfected into Jurkat T cells and characterized in a virus replication assay as described above for resistance selection. The analysis of SDS-PAGE of the transfected cell lysates and of the virus released in the media, showed that the Gag mutant in A366V was processed and released from the cells inefficiently (data not shown) and replicated in this way very poorly yet in the absence of the drug (Figure 11). However, the double mutant A366V / G357S was efficiently replicated in the presence or absence of DSB. These data indicate that the resistant mutant, A366V, requires a serine at position 357 in the Gag CA region to compensate for a deleterious effect on virus replication (Figure 11). In a further experiment, ten different resistant isolates were generated. The sequencing of these isolates identified four additional mutations not previously seen in the resistance selection experiments. These were H358Y, L363F and L363M in CA, and A402T in the NC region of Gag. None of these mutations is present in the consensus sequence of HIV-1 A-O coatings, reflecting the extent of DSB activity against coatings genetically diverse in HIV-1. The L363M substitution in CA was found in the HIV-1 consensus sequence that may partly explain the specificity of DSB for HIV-1. These results demonstrate the presence of specific genetic determinants for DSB activity in HIV-1, and that these determinants are centered around the cleavage site of CA-SPl. (a) Suppression studies of HIV-1 NL4-3 and insertion of SIV used to identify viral genetic determinants of sensitivity to DSB. The results of the in vitro resistance selection experiments indicated that the determinants of the inhibitory activity of HIV-1 to DSB correlate to the Gag region flanking the CA-SPl cleavage site. In order to better define the viral genetic determinant for DSB, mutagenesis studies of HIV-1 point deletion and SIV insertion were undertaken to identify the specific amino acid residues associated with the activity of the compound. The study was carried out as follows. Individual deletions of residues were handled starting with residue E365 and continuing through residue M377 in the SPI domain of the infectious HIV-1 molecular clone NL4-3 (Figure 12). He effect of these deletions points or point deletions in the production of viral particles, infectivity, Gag processing and sensitivity to DSB was determined. The results of these experiments were used to identify the Gag residues in the region of the CA-SPl cleavage site that are associated with DSB activity. The residues associated with the activity were inserted into the site region of CA-SP1 of the DSB-resistant virus, SIV (Mac 239 isolate) to generate a chimeric SIV virus (SHIV) of HIV-1. Point-specific substitution of HIV-1 residues from the N-terminus of the CA protein was made in this bladder virus until the minimum sequence necessary to rescue the activity of DSB was identified. This minimum sequence necessary to gain DSB activity is considered a primary viral genetic determinant of DSB activity. The molecular determinant of DSB activity can be suggested. 1. Methods: (a) Construction of individual point deletion mutants of NL4-3. Individual point-deletion constructs were generated using the PCR-ligation-PCR (PLP) strategy as described above. Plasmid DNA NL4-3 of HIV-1 was used as the template to perform all PCR reactions for generate point deletions that encompass the entire Gag SPI domain with the exception of the first SPI residue. E365 was generated using NL4-3 as the template with Vent DNA-polymerase (NEB) by using the primer in the 3'-specific direction of suppression (Primer 1) with the primer in the universal 5'-direction (Primer 2). ) (Table 1). The fragment derived from these was called as a first flanking PCR fragment. A second flanking fragment was amplified using the deletion-specific 5 'direction primer (Primer 3) and the primer in the universal 3' direction (Primer 4) (Table 1). To generate other suppression constructs (? A366,? M367,? S368,? Q369,? V370,? T371,? N372,? P373,? A374,? T375,? 1376, and? M377). PCR procedures were similarly performed by varying the primers in the 3 'direction and in the 5' direction specific to the deletion corresponding to each specific point deletion (Table 1). Each of these two parallel, parallel PCR fragments was gel purified, phosphorylated using T4-polynucleotide kinase (NEB), and ligated using T4-DNA ligase.

(NEB). After inactivation at 65 ° C for 15 minutes, the ligation reaction was used for a subsequent amplification with the primer in the universal 5 'direction (Primer 2) the primer in the 3' direction (Primer 4). This product was purified on gel, digested with Spel and Apal, and then ligated into the Spel sites and Apal of the proviral DNA clone NL4-3. Normal PCR conditions were used for the reactions described above. These include, a denaturation cycle at 95 ° C for 1 minute 30 seconds, followed by 30 cycles of denaturation at 95 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. PCR reactions were established using the following components: 5 μL of buffer Thermophilic NEB 10 x 2 μL of 10 mM sNTPs 1 μL of 10 nM MgSO 2 1 μL of primer in the 5 '50 pmol direction 1 μL of primer in direction 3 50 pmol 1 μL template DNA 50 ng / μL 0.5 μL DNA polymerase Vent 38.5 μL ddH20 A 10 μL aliquot was run on a 1.0% randomized gel to ensure that the product was amplified in size Right. The PCR products were then isolated in gel and purified with Qiaex II gel extraction equipment (Qiagen).

The two adjacent PCR fragments purified in gel are phosphorylated individually in the next reaction when using 5 T4-polynucleotide kinase (NEB) before ligation. The reaction of phosphorylation was established as follows: 2 μL of T4 buffer-polynucleotide-kinase 10X 2 μL of 10 mM ATP 10 1 μL of T4-polynucleotide kinase 15 μL of gel-purified DNA from each of these two adjacent PCR fragments. The reaction was incubated at 37 ° C during 1 hour. After inactivation at 65 ° C for 10 minutes, the l- > adjacent phosphorylated PCR fragments were then ligated together using T4-DNA-ligase (NEB) under the following terms : 3 μL of 10X T4-DNA-ligase buffer 20 13 μL of each of the two PCR fragments adjacent 1 μL of T4-DNA ligase After incubation overnight at 16 ° C, the ligation reaction product was used in a PCR reaction of 25 second round to amplify the full-length PCR fragment encompassing these two adjacent PCR products. The second round PCR reaction was performed as described above with the exception that only the primer was used in the universal 5 'direction (Primer 2) and the primer in the 3' direction (Primer 4). Again, an aliquot of 10 μL was run on an agarose gel to ensure that the correct product was amplified. The full-length PCR fragments were then gel isolated and purified using a Qiaex II assay. The purified full-length PCR fragment, together with NL4-3, was then cut with Spel and Apal under the following conditions: 2 μL of NE buffer 10X (NEB) 1 μL of Apal (NEB) 1 μL of Spel (NEB) 16 μL of full-length PCR product (1 μg) or NL4-3 (500 ng) The digestion mixture with restriction enzyme above was incubated at 37 ° C for 2 hours. The digested DNA fragments for the full-length PCR product and the plasmid NL4-3 were individually isolated on gel and purified using a Qiaex II kit. The digested NL4-3 vector and fragment of full length PCR were ligated using T4-DNA-Iigase under the following procedures: 1 μL of 10X T4-DNA-ligase buffer 1 μL (25-50ng) of NL4-3 vector digested 7 μL of PCR fragment (700 pb) digested (200 ng-400 ng) 1 μL of T4-DNA ligase The ligation reaction was incubated at 16 ° C overnight and bound products were transformed into Escherichila coli maximum efficiency Stbl2 (Invitrogen) by heat shock from according to the instruction (Invitrogen). The proviral DNA clones were then detected by automatically sequencing using a Taq Dye Deoxy Terminator cycle sequencer (Applied Biosystems) individually using internal primers (Primer 29 and 30). After verification of mutations, proviral DNA clones were used for future studies. 1. Construction of SIV cyclimeric mutants A panel of SIV chimeric constructs having several residues of the CA-SPl boundary region of NL4-3 was generated using the SlVmac239 molecular clone using PCR and cloning procedures described above. These constructions and their amino acid sequences in the CA-SP1 boundary region are shown in Figure 15. SIV mac239 was used to generate the SIV DD and DE constructs. The SIV DD construction was used to generate SIV DM. Different SIV chimeric constructs were produced in the PCR by varying the primers in the 5 'direction and in the 3' direction, mutagenic, respectively, corresponding to each chimera (Table 1). Each of these two parallel, parallel PCR fragments was gel purified and used directly without phosphorylation treatment for a subsequent amplification with the universal 5 'steer primer (Primer 31) and the 3' steer primer (Primer 32) .

This product was gel purified, digested with BamHl and Sbfl, and then ligated into the BamHl and Sbfl sites of the proviral DNA clone SIVmac239. The proviral DNA clones were then detected by automatically sequencing using a Team t ^ Dye Deoxy Terminator cycle Sequencer (Applied Biosystems) individually using an internal primer (Primer 39). After verification of the mutations, the proviral DNA clones were used to give several future studies. 2. Cell culture and DNA transfection HeLa cells were maintained in DMEM (Invitrogen) (10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin) and they went to confluence. Jurkat cells were grown in RPMI 1640 (Invitrogen) (10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin) and passed every two or three days. To characterize the effect of suppression or substitution in the production of viral particles and processing of the Gag polyprotein, the proviral DNAs of NL4-3 or SIVmac239 of HIV-1 and respective mutants were transfected into HeLa cells by using the transfection reagent FuGÉNE 6 (Roche). Briefly, cells were seeded in a 6-well plate (Corning) at a concentration of 0.5 x 105 per well the day before transfection to reach 60 to 80% confluence on the day of transfection. For each transfection, 3 μl of FuGENE 6 in 100 μl of serum free DMEM was diluted followed by the addition of 1 μg of DNA. After gentle mixing, the DNA-lipid complex mixture was gently added dropwise into the cells containing 2 ml of complete DMEM medium. Twenty-four hours after transfection, the medium containing the DNA-FuGENE 6 complexes was removed, 2 ml of fresh DMEM was added to the transfected cells. At 48 hours after transfection, viral particles containing the medium were harvested and clarified by centrifugation at 2000 rpm at 4 ° C for 20 minutes in a Sorvall RT 600B centrifuge. The supernatants that containing the viral particles were then concentrated through a 20% sucrose cushion in a microcentrifuge at 13,000 rpm at 4 ° C for 120 minutes and the pellets were resuspended in lysis buffer (150 mM Tris-HCl, Triton X -100 to 5%, deoxycholate at 1%, pH 8.0). The level of production of viral particles were point-deletion mutants and wild-type NL4-3 was determined by p24 antigen capture ELISA (ZeptoMetrix, Buffalo, NY). To examine the effect of suppression or substitution on the processing of the Gag polyprotein (in the absence of DSB), SDS-PAGE and Western blotting were performed. In summary, viral proteins were separated in a 12% Bis-Tris NuPAGE gel.

(Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen) followed by the block in a PBS buffer < It contains 0.5% Tween and 5% dry milk. The membrane was incubated with immunoglobulin from patients infected with HIV-1 (HIV-Ig) (NIH AIDS research and reference reagent program) and hybridized with goat anti-human horseradish peroxidase (Sigma). For the membrane < In the case of SIV proteins, the membrane was incubated with a reference polyclonal immune serum from a monkey infected with SIV (NIH AIDS Research and Referent Reagent Program) and hybridized with goat anti-monkey radish peroxidase (Sigma). He Immune complex was visualized with an ECL system (Amersham Pharmaceutical Biotech) according to the instructions provided by the manufacturer. To address the effect of suppression or substitution on the ability of DSB to inhibit CA-SP1 processing, HeLa cells were transfected with wild type HIV-1 NL4-3 or SIVmac239 and the respective mutant provirales DNAs using the procedure described. previously. DSB was maintained at a concentration of 1 μg / ml and control of DMSO throughout the whole culture and SDS-PAGE / Western blot to analyze the viral proteins derived from these transfections were performed as described in the previous paragraph. The infective dose of 50% tissue culture (TCID50) per ml was used as a measure of the infectivity of each deletion mutant. Mutant viruses derived from transfections in HeLa cells were used to infect U87 CD4.CXCR4 cells. Each virus concentration was tested in triplicate at a starting dilution of 1:10, followed by four-fold serial dilutions.

The cells were plated the day before infection at a density of 3 x 10 3 cells / well. On the day of transfection, the culture baths were removed from the plate in cells and 90 μl of diluted virus was added. Days 1, 3 and 6 After infection, the virus was removed from the plate and 200 μl of culture medium was added. On days 6 and 8 after infection, the supernatant was collected for p24 ELISA analysis. The dilution of the virus < which caused 50% of the culture to become infected (TCID50) was determined according to the method of Reed and Muench (Aldovini A. and B. Walter 1990, Dulbecco R. 1988). 3. Results Viruses containing sequential point deletions within the SPI domain of Gag (Figure 12) were characterized for particle production, infectivity, Gag processing and sensitivity to DSB. The results of these experiments were used to identify SPI residues associated with DSB activity. As expected, the effect of point deletions on the production of viral particles varied with a function of the proximity of the change of the proteolytic cleavage site. The results of these experiments are summarized in Figure 13. Viruses with deletions in residues E366, A367 and M368 were the most affected, generating < 25% of the number of particles normally observed in wild type virus infection. In vitro infectivity assays were used to characterize the capacity of the deletion mutants for support virus replication. These experiments indicated that the deletion of individual residues at any of the five positions E365 to Q369 resulted in a virus < which was either non-infectious or significantly impaired for replication (Figure 13). In contrast, starting with the V370 residue and extending away from the CA-SP1 cleavage site, none of the characterized point deletions resulted in a decrease in virus infectivity (Figure 13). With the exception of viruses with deletions at positions 1376 and M377, all mutant viruses exhibited a normal or almost normal phenotype of Gag processing (Figure 13). The results of these three sets of experiments allowed the design and interpretation of experiments to identify the genetic determinants of DSB activity. The sensitivity of DSB was determined in experiments that characterized the effect of DSB in a late step in the processing of Gag, the cleavage of CA-SP1. Specifically, these trials measured the ability of DSB to interrupt CA-SP1 processing. As seen, for example, Example 8, the defect induced by DSB in the Gag processing correlates with the ability of the compound to inhibit viral replication. The results of these experiments indicate that the suppression of an individual residue in any of the six positions E365 to V370 significantly reduces the effect of DSB on the CA-SP1 processing (Figure 14). In contrast, starting with the T372 residue and extending away from the CA-SP1 cleavage site, all the characterized point deletions were completely sensitive to the DSB-induced interruption of the CA-SP1 processing (Figure 14). The SP1 residues associated with DSB activity consist of contiguous E365 to V370 residues. Residues A364 through V370 were inserted in the analogous position of the SPI domain of Gag in the SIV retrovirus resistant to DSB (Mac 239 isolate). Additionally, the N-terminus of the CA protein of this chimeric virus was modified by cumulative substitution of residues found in SIV with specific residues of HIV-1. This approach is summarized in Figure 15. Then, the effect of DSB on the Gag processing phenotype of each of the chimeric viruses was determined. As shown in Figure 15, the SIV.DM virus exhibits a Gag processing phenotype indicative of DSB sensitivity. Thus, the minimum sequence of HIV-1 CA-SP1-specific residues that need to be inserted to rescue DSB activity in SHIVs range from V362 to V370.

Table 1. PCR Mutagenesis primers The resistance and mutagenesis data presented above suggest that the amino acid sequence GHKARVL-AEAMSQV in the region of the Gag CA-SPl cleavage site of HIV-1 serves as a genetic determinant of viral sensitivity to DSB. Extension of the determinants of DSB sensitivity to other lentiviruses: CA-SPl chimeras as animal efficacy models for development of maturation inhibitors The development of anti-HIV-1 therapeutic products has been impeded by the lack of an efficacy model animal This lack of an animal model is mainly due to the inability of most animals HIV-1 strains to replicate and cause disease in non-human primates. In some cases, this problem has been overcome through the use of chimeric viruses "that incorporate the regions of interest of the HIV-1 viral target into a viral SIV structure" that will support replication in a non-human primate. The most notable example of this approach comprises chimeric HIV-1 / SIV (SHIV) viruses in which the proteins that constitute the infectious virus are exclusively of SIV origin with the exception of Env (gp 120 / gp 41) that is derived of HIV-1. These envelope SHIV chimeras have been used extensively in the development of HIV-1 vaccines. Inhibitors of HIV-1 maturation interrupt Gag's CA-SP1 processing, <which results in the formation and release of non-infectious viral particles that exhibit abnormal core morphology. See, for example, Li et al. Proc Nati Acad Sci U S A. 100: 13555-60 (2003). The betulinic acid derivative DSB is an example of this class of inhibitors. The critical viral genetic determinants that are associated with the activity of maturation inhibitors correlate to the amino acid residues that flank the HIV-1 CA-SPl cleavage site. When this The determinant is introduced in the CA-SPl cleavage sites of the non-HIV-1 viruses resistant to DSB, resulting in the chimeras sensitive to the maturation inhibitor. These chimeric CA-SP1 viruses serve as the basis for an animal efficacy model for HIV-1 maturation inhibitors. The CA-SP1 region of HIV-1 needed to introduce sensitivity to the maturation inhibitor in selected lentiviruses. The amino acid residues of the HIV-1 CA-SPl junction that are determinants of DSB sensitivity were used to replace the corresponding CA-SP1 amino acids in the Simian Immunodeficiency (SIV) genome. Similarly, the amino acid residues of the HIV-1 CA-SPl binding that are determinants of DSB were replaced in feline immunodeficiency virus (Fiv), Bovine Immunodeficiency Virus (BIV), Equine Infectious Anemia Virus ( EIAV), Visna-Maedi virus, and Encephalitis and Caprine Arthritis virus (CAEV). Table 2 depicts the Gag polypeptide sequence for HIV-1, SIV, FIV, EIAV and BIV in the region of the CA-SPl cleavage site.

Table 2. Comparison of sequences in the region of the region of HIV-1 CA-SPl cleavage site with SIV, FIV, EIAV and BIV CA SPI NC HIV-1 GHKARVL AEAMSQVTNPATIM IQKG (SEQ ID NO: 76) FIV GYKMQLL AEALTKVQ WQS (SEQ ID NO: 77) EIAV KQKMMLL AKALQ TGLA (SEQ ID NO: 78) BIV KSKMQFL VAAMKEMGIQSPIPAV PHTPEAYA SQTS (SEQ ID NO: 79) The CA-SP1 sequence of HIV-1 used for the Replacement is as follows: CA SPI GHKARVL AEAMSQV (SEQ ID NO: 80) The method described above to generate the chimeric provirus DNA clone of SHIV CA-SPl was used to generate IVF, EIAV and BIV provirus clones that contain selected waste or the extended region of the CA-SPl region of HIV-1 that replaces the sequence type corresponding wild (Figure 16).

The chimeric DNA-provirus clone of SHIV CA-SPl was generated by site-directed mutagenesis using normal molecular biology techniques. Briefly, the Unique sites of restriction enzymes in the SIV Gag that surround the CA-SP1 region were identified, namely, BamHl (in the matrix) and Sbf-I (in NC). Starting from the region of CA-SP1 where mutagenesis is proposed, two overlapping primers were synthesized, a forward primer and a reverse primer incorporating the mutated sequence, ie HIV CA-SPl, at its 5 'ends. Using the wild-type SIV provirus DNA as a template, two separate PCR reactions were established to amplify the SIV-Gag fragments in either the mutagenesis site direction (CA-SP1 region), i.e., produce two amplified fragments that overlap in the mutated CA-SPl region, a Bam HI-CA-SPl fragment, and a CA-SPl-Sbf-I fragment. In a third PCR reaction, Bam HI-CA-SP1 and CA-SPl-Sbf-I were fixed in their common HIV-CA-SPl sequence and amplified with a direct SIV Bam Hl primer and a Sbf primer. -I of reverse SIV to generate a gag fragment of chimeric SHIV CA-SPl. The chimeric SHIV CA-SPl PCR fragment was cloned into the BamHl-Sbf-I window of the SIV provirus clone by replacing the wild-type SIV-Gag sequence to produce the SHIV CA-SPl provirus cDNA clone. .

Similarly, unique restriction enzyme cloning sites circling the CA-SP1 regions in the IVF genome have been identified (Genbank accession number NC_001482), EIAV (GA # AF016316) and BIV (GA # M32690) ( Figure 16). Specific FIV / EIAV / BIV-HIVlCA-SPl chimeric primers are synthesized together with specific primers of the FIV, EIAV or BIV genome that incorporate the specific sequence of the cloning site. These primers together with the corresponding provirus DNA clone as a template (FIV, EIAV or BIAV) in PCR reactions generate the chimeric HIV-1 CA-SP1 fragment. The HIV-1 chimeric CA-SPl fragment is digested with the appropriate restriction enzyme and cloned into the SacI-EcoRI window of the IVF provirus.; or (ii) the KasI-EcoRV window of the EIAV provirus; or (iii) the BsrGI-Apal window of the BIV provirus that replaces the corresponding wild-type sequence (Figure 16). The cIVimeric FIV / EIAV / BIV-HIV-1CA-SP1 provirus DNA clones were sequenced to confirm the presence of the proposed mutations. Based on the results observed, which indicate the transfer of sensitivity of DSB, additional constructions are generated using the previous strategy in order to optimize the results.

In summary, a chimeric virus was generated in which the CA-SP1 determinant of sensitivity to HIV-1 maturation inhibitor has replaced the Gag analog region in the simian immunodeficiency virus (SIV) resistant to the maturation inhibitor. The transfer of this region of HIV-1 in the SIV genome results in phenotype sensitive to maturation inhibitor. The infection of a non-human primate with this HIV-1 / SIV chimeric virus should result in an animal efficacy model for the therapeutic development of maturation inhibitors. Analogous approaches are used to prepare and characterize HIV-1 CA-SP1 chimeras with FIV, BIV and EIAV. These additional chimeric DSB-sensitive viruses should allow the development of additional animal efficacy models for the study of HIV-1 maturation inhibitors. Uses of mutant and chimeric viruses The mutant and chimeric viruses of the present invention, as described above, are useful in a variety of cell-based as well as animal-based assays. By comparing the phenotypes associated with a virus that is resistant to DSB, with a virus that is sensitive to DSB, compounds that act by a mechanism similar to that of DSB can be identified. Thus, the invention includes a method for identifying a compound that inhibits cleavage of p25 to p24 in wild type HIV-1, but does not inhibit CA-SP1 processing in HIV-1 containing a deletion in the CA region. -SPl. The compounds obtained by this method are also included in the present invention. SIV chimeras and other lentiviruses that do not easily infect humans have additional advantages. First of all, these viruses pose a lower security risk to laboratory workers. As a result, cell-based assays can be carried out with less risk to identify new compounds that inhibit CA-SP1 processing, by way of example. The lower risk may allow for trials that can not be performed easily or safely with HIV, and may also decrease the cost of these trials. Additionally, chimeric viruses are useful in animal models. For example, chimeric SIV that is sensitive to DSB can be used to identify new compounds that inhibit CA-SP1 processing, for example, to identify pharmaceutical compositions, routes of administration and dose regimens for the treatment of the disease; and to study the effect of combination therapies, such as DSB with protease inhibitors. Since SIV is generally limited to the infection of monkeys, the generation of additional lentiviral chimeras allows studies of animals to be carried out on animals that are less expensive, easier to handle, have a more rapid progress of the disease or other are more appropriate for a particular aspect of human disease, by way of example. Additionally, animal models can be used to identify pharmaceutical compositions suitable for the treatment of animal diseases, of interest in the treatment of companion animals and other high-value animals, such as breeds of agricultural reproduction and racing horses. Chimeric viruses can be derived from any retrovirus. For example, HIV-1-2 derivative, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus ( FIV), Bovine Immunodeficiency Virus (BIV), encephalitis virus and goat arthritis (caev), Equine infectious anemia virus (EIAV), Visna-maedi virus, or feline leukemia virus (FeLV). These chimeric viruses can be used in the methods of the invention described elsewhere herein. For example, these recombinant non-HIV-1 lentiviruses can be used in a method to identify a compound that inhibits Gag viral p25 protein processing (CA-SP1) to p24 (CA), the method of comparing ability of this compound to inhibit the replication of a non-wild type HIV-1 lentivirus with the recombinant DSB-sensitive variant thereof. This inhibition can occur in a cell; in an animal; or in vitro. Construction and use of viruses or polypeptides with epitope tags The present invention also relates to recombinant retroviruses with epitope tags in the CA-SP1 region of Gag. Epitope tags can be inserted into the CA domain and / or the SPI domain. Suitable labels are well known to those skilled in the art, and include the HA epitope of haemagglutinin (YPYDVPDYA) (SEQ ID NO: 81), the VP7 epitope of bluetongue virus (QYPALT) (SEQ.

ID NO: 82), a-tubulin epitope (EEF), the indicator (DYKDDDDK) (SEQ ID NO: 83), and VSV-G (YTDIEMNRLGK) (SEQ ID NO: 84). Examples of SPI containing epitope tags are illustrated in Figure 17. These epitope tagged viruses and fragments thereof are useful in identifying new compounds < They inhibit the processing of CA-SPl in vi tro, in cell-based assays, and in vivo, including in animal models. Additional uses of these epitope-tagged viruses and fragments thereof are described elsewhere herein. Polynucleotides, polypeptides and antibodies of the invention The invention also includes isolated polypeptides and polynucleotides. In one embodiment, the invention includes polypeptides at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% identical to an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23) (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). In another embodiment, the invention includes polynucleotides that encode the above polypeptides. The polynucleotides of the invention include degenerate variants, such as those which differ in the third base of the codon but which nevertheless code for the same amino acid due to the "degeneracy" of the coding. The term "approximately" as used herein refers to a value that is 10% more or less than the stated value, and preferably 5% more or less. The polypeptides and polynucleotides of the invention are useful in the methods of the invention. In one aspect, they can be used in an in vi tro assay to identify compounds that bind to the Gag CA-SPl region. In another, they can be used in the production of antibodies useful in other methods described elsewhere in this. In another, a polynucleotide can be inserted into a vector and then into a host cell for the production of the polypeptide. The above modalities are examples and it is not proposed < that are limiting. The present invention comprises a polynucleotide (comprising a sequence encoding an amino acid sequence containing a mutation in the p25 protein of HIV-1 Gag (CA-SP1), this mutation resulting in a decrease in the inhibition of processing p25 (CA-SP1) to p24 (CA) by DSB The polynucleotide of the invention includes a mutation that is optionally located at or near the CA-SPl cleavage site or located in the SPI domain of CA-SPl. This mutation may be present in an amino acid sequence that is selected from the group consisting of GHKARVLVEAMSQV (SEQ ID NO: 2) and SHKARILAEVMSQV (SEQ ID NO: 3) The polynucleotide of this invention also refers to sequences designated as SEQ ID NO: 3. NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. The invention also includes a vector comprising the polynucleotide, a host cell comprising the vector and a method for producing the polypeptides that comprises incubating the host cell in a medium and recovering the polypeptide from the medium. The invention further includes a polynucleotide that hybridizes under severe conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. The invention also includes a polynucleotide which hybridizes to SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 10 or 12, which contains a mutation that results in a decrease in the processing inhibition of p25 to p24 by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic, and also wherein the mutation is optionally located at or near the CA-SPl cleavage site or in the SPI domain of CA-SPl. The invention also relates to a vector comprising the polynucleotides, a host cell <; comprising the vector and a method for producing the polypeptides, comprising incubating the host cell in a medium, and recovering the polypeptide from the medium. "Near" or "adjacent", as used in reference to polypeptides, is proposed to include approximately 50, approximately 25, approximately 20, or approximately 15 reference point residues. For example, close encompass approximately 50, approximately 25, approximately 20, or approximately 15 residues on either side of the HIV-1 Gag CA-SPl cleavage site; more preferably about ten residues on either side of the HIV-1 Gag CA-SPl cleavage site; and preferably about seven residues on either side of the HIV-1 Gag CA-SPl cleavage site. With reference to polynucleotides, the terms "near" or "adjacent" refer to about 150, about 75, about 60, about 45, or about 30 nucleotides from the reference point. "Isolated" means altered "by the hand of man" from the natural state. If a composition or substance occurs in nature, it has been changed or removed from its original environment, or both, when it is in its "isolated" form. "Also, the" isolated "nucleic acid molecules of the invention are proposed to are a nucleic acid molecule, DNA or RNA, that has been removed from its native environment, eg, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. isolated include molecules of recombinant DNAs maintained in heterologous host cells or purified DNA molecules (partially or substantially) in solution. Isolated RNA molecules include RNA transcripts in vivo or in vi tro of AD molecules. of the present invention. The isolated nucleic acid molecules according to the present invention further include these molecules produced in a synthetic manner. "Polynucleotide" refers in general to any polyribonucleotide or polydeoxyribonucleotide, which may be RNA or AD? unmodified or RNA or AD? modified. "Polynucleotides" includes, but is not limited to, AD? single strand and double strand, AD? which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-female and double-stranded regions, hybrid molecules comprising AD? and RNA which may be single-stranded or more typically double-stranded or a mixture of double-stranded and single-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or AD ?, or both RNA and AD ?. The term polynucleotide also includes the AD? or RNA that they contain one or more modified bases and the DNA or RNA with structures modified for stability or for other reasons. "Modified" bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications have been made to AD? and to AR ?; thus, "polynucleotide" encompasses biochemical, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of AD? and RNA characteristics of viruses and cells. "Polynucleotide" also encompasses relatively short polynucleotides, often referred to as oligonucleotides. "Polypeptide" refers to any peptide or protein comprising two or more amino acids joined together by peptide bonds or modified peptide bonds, i.e., peptide isosteres. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers and to longer chains, generally referred to as proteins. The polypeptides may contain amino acids different from the 20 amino acids encoded by genes. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-transductional processing, or by chemical modification that are well known in the art. These modifications are well described in basic texts and in more detailed monographs, as well as in voluminous search literature. Modifications can occur in any part of a polypeptide, including the structure of the peptide, the side chains of amino acids and the amino or carboxyl terms. It will be appreciated that the same type of modification may be present in the same or variable degree at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. The polypeptides can be branched as a result of ubiquitination, and they can be cyclic, with or without branching. The cyclic, branched and branched cyclic polypeptides can result from natural post-translational processes or can be made by synthesis methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent bonding of flavin, covalent attachment of a heme moiety, covalent bonding of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent binding of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, mutilation, myristoylation, oxidation, proteolytic processing, phosphorylation, phenylation, racemization, selenoilation, sulfation, amino acid addition t-RNA mediated protein such as arginilation, and ubiquitination. "Mutant" as the term is used herein, is a polynucleotide or polypeptide that differs respectively from a reference polynucleotide or polypeptide. A chemical mutant of a polynucleotide differs in the nucleotide sequence of another reference polynucleotide. Changes in the nucleotide sequence of the mutant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. The nucleotide changes can result in substitutions, additions, deletions, fusions and truncations of amino acids in the polypeptide encoded by the reference sequence, as discussed below. A typical mutant of a polypeptide differs in the amino acid sequence of another reference polypeptide. In general, the differences they are limited so that the sequences of the reference polypeptide and the variant are closely similar together and in many identical regions. A mutant and reference polypeptide may differ in the amino acid sequence by one or more substitutions, additions, deletions in any combination. An inserted or substituted amino acid residue can be encoded or not by the genetic code. A mutant of a polynucleotide or polypeptide can be one that occurs naturally such as an allelic variant, or it can be a mutant that is not known and that occurs naturally. Mutants that do not occur naturally of the polynucleotides and polypeptides can be made by mutagenesis techniques or by direct synthesis. In this manner, the mutant, (or fragments, derivatives or analogs) of a polypeptide encoded by any of the polynucleotides described herein can be (i) one in which at least one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue (a conserved amino acid residue, or at least one but less than ten conserved amino acid residues) and this residue of substituted amino acid may be one or not encoded by the genetic code, (ii) one in which one or more d amino acid residues includes a substituent group, (iii) one in which the mature polypeptide is fused to another compound, such as a compound for increasing the half-life of the polypeptide (eg, polyethylene glycol), or (iv) one in which additional amino acids are fused to the mature polypeptide, such as a peptide from the IgG: Fc fusion region or leader sequence or secretory or an eme sequence is used for the purification of mature polypeptide or a proprotein sequence. These eme mutants are judged to be within the reach of those skilled in the art of the teachings herein. The polynucleotides encoding these mutants are also encompassed by the invention. "Mutant" as used herein is equivalent to the term "variant". Substitutions of amino acids loaded with other charged amino acids and with neutral or negatively charged amino acids are included. Additionally, one or more of the amino acid residues of the polypeptides of the invention (eg, arginine and lysine residues) can be deleted or substituted with another residue to eliminate unwanted processing by protease such as, for example, furins or kexins. The prevention of aggregation is highly desirable The aggregation of proteins not only gives result in a loss of activity, but it is also problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2: 331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)). In this way, the polypeptides of the present invention can include one or more substitutions, deletions or additions of amino acids, and be of natural mutations or human manipulation.

As indicated, the changes are so preferred of minor nature, such as substitutions preservatives of amino acids that do not affect in a significant protein folding or activity (see Table 3).

Table 3. Conservative amino acid substitutions Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine However, in some embodiments, it is desirable to use non-conservative amino acid substitutions. For example, non-conservative amino acid substitution is used to render a DSB-resistant DSB-sensitive virus. The polynucleotides encompassed by this invention can have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of identity with a reference sequence, provided that the reference polynucleotide codes for an amino acid sequence that contains a mutation in the CA-SP1 protein, the mutation that results in the decrease in the processing inhibition of p25 to p24 by a 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. The polynucleotides also encompassed by this invention include those mutations that are "undetectable", in which the different codons code for the same amino acid (wobble). "Identity" is a measure of the identity of the nucleotide sequence or amino acid sequence. The term "identity" is used interchangeably with the word "homology" in the present. In general, the sequences are aligned so that the highest order correspondence is obtained. "Identity" per se has a recognized significance in the art and can be calculated using published techniques. While there are several methods for measuring the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those skilled in the art. The methods commonly used to determine the identity or similarity between two sequences' include, without limitation, those described in Baxevanis and Oullette, Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Second Edition, Wiley-Interscience , New York, (2001). Methods to determine identity and similarity are encoded in computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the packet of GCS program (Devereux, J. et al., Nucleic Acids Research 12 (1): 387, (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J. Molec. Biol. 215: 403, ( 1990)). A polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence, proposes that the nucleotide sequence of polynucleotides be identical to the reference sequence except that the sequence of polynucleotide can include up to five point mutations per 100 nucleotides of the reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or replaced with another nucleotide, or a number of nucleotides up to 5% of the nucleotides Totals in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5 'or 3' terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, interspersed either individually between nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Similarly, for a polypeptide having an amino acid sequence having at least, for example, 95% of "identity" to a reference amino acid sequence, it is proposed that the amino acid sequence of the polypeptide be identical to the reference sequence except that the polypeptide sequence can include up to five amino acid alterations per 100 amino acids of the reference amino acid. To obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence can be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence can be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed either individually between the residues in the reference sequence or in one or more contiguous groups within the reference sequence. The reference sequence (query) may be the complete nucleotide sequence of any of the nucleotide sequences of the invention or any polynucleotide failing (e.g., a polynucleotide that encodes the amino acid sequence of the invention and / or deletion C). -terminal).

If any particular nucleic acid molecule has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% of Identity or perhaps identical to, for example, the nucleotide sequences of the invention can be determined in conventional manner using known computer programs, such as the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wl 53711). BESTFIT uses the local homology algorithm of Smith-Waterman, (Advances in Applied Mathematics 2: 482-489 (1981)), to adjust the best homology segment between two sequences. When BESTFIT or any other program of the sequence invention is used to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are adjusted, such that the percentage identity it is calculated on the full length of the reference nucleotide sequence and that separations in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. In a specific embodiment, the identity between a sequence of the present invention and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Congo, App. Biosci, 6: 237-245 (1990)). The preferred parameters used in a FASTDB alignment of the DNA sequence to calculate the percent identity are: Matrix = unit, k-tuple = 4, Penalty of Mal Correspondence = 1, Union Penalty = 30, Randomization Group Length = 0, Cut Score = 1, Separation Penalty = 5, Separation Size Penalty = 0.05, Window Size = 500 or the length of the subject nucleotide sequence, whichever is shorter. According to this modality, if the subject sequence is shorter than the reference sequence due to 5 'or 3' deletions, not due to internal deletions, a manual correction is made to the results to take into account the fact that the program FASTDB does not account for the 5 'and 3' truncations of the subject sequences when calculating percent identity. For subject sequences truncated at the 5 'or 3' ends, relative to the query sequence, the identity percent is corrected by calculating the number of bases of the query sequence that are 5 'and 3' of the subject sequence , which do not correspond / align, as one percent of the total bases of the query sequence. A determination is made of whether a nucleotide is corresponded / aligned by the results of alignment of the FASTDB sequence. This percentage is then subtracted from the identity percent, calculated by the previous FASTDB program using the specified parameters, to arrive at a final percentage of identity score. This corrected score is what is used for the purposes of this modality. Only bases outside the 5 'and 3' bases of the subject sequence, as exhibited by FASTDB alignment, which are not matched / aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. . For example, a subject sequence of 90 bases is aligned to a query sequence of 100 bases to determine percent identity. Deletions occur at the 5 'end of the subject sequence and therefore, the FASTDB alignment does not show an alignment / alignment of the first 10 bases at the 5 end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5 'and 31 unrequited endpoints / total number of bases in the query sequence) so that 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases corresponded perfectly, the final percent identity would be 90%. In another example, a subject sequence of 90 bases is compared to a query sequence of 100 bases. In At this time, the deletions are identical deletions so there are no bases in 5 'or 3' of the subject sequence, they are not matched / aligned with the query. In this case, the identity percent calculated by FASTDB is not manually corrected. Only the 5 'and 3' bases of the subject sequence that are not matched / aligned with the query sequence are manually corrected. No other manual corrections are made for the purposes of this modality. The present application relates to nucleic acid molecules which have at least about 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity or that are identical to the nucleic acid sequence described herein, or fragments thereof, although they encode a polypeptide < which has the functional activity described. This is because even though a particular nucleic acid molecule does not code for a polypeptide having the functional activity described, one skilled in the art will still know how to use the nucleic acid molecule, for example, as a hybridization probe or a polymerase chain reaction (PCR) primer. The uses of the nucleic acid molecules of the present invention which do not code for a polypeptide having the functional activity described include, inter alia: (1) isolating the variants thereof in a cDNA library; (2) in situ hybridization (eg, "FISH") to determine the cellular location or presence of the described sequences, and (3) Northern blot analysis to detect mRNA expression in specific tissues. As used herein, the term "PCR" refers to the polymerase chain reaction which is the subject of U.S. Patent Nos. 4,683,195 and 4,683,202 to Mullis et al., As well as to improvements not known in the art. technique. In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques can be employed within the skill of the art. These techniques are fully explained in the literature. See, for example, Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. The term "stringent conditions", when used herein to refer to homology in hybridization, is based on the combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions, and well known in the art. the technique (Sambrook, et al., supra). The invention includes a molecule isolated nucleic acid comprising, a polynucleotide that hybridizes under severe conditions of hybridization to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for example, the sequence complementary to the coding and / or non-coding sequence (ie, transcribed, untranslated) of any polynucleotide or a polynucleotide fragment as described herein. For "severe hybridization conditions" incubation is proposed overnight at 42 ° C in a solution comprising, or alternatively consisting of: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Denhardt 5x solution, 10% dextran-sulfate, and 20 μg / ml salmon sperm DNA, sheared, denatured, followed by washing in O.lx SSC at approximately 65 ° C . The polypeptides encoded by these polynucleotides are also encompassed by the invention. The invention also includes a cgue virus comprising the polynucleotides of the invention, and wherein the virus includes a retrovirus comprising the polynucleotides, and wherein the retrovirus can be a member of the group consisting of HIV-1, HIV-1-2, HTLV-I, HTLV-II, SIV, avian leukosis virus (ALV), endogenous avian retrovirus (EAV), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIV), or feline leukemia virus (FeLV). The invention further includes a polypeptide that contains a mutation in the CA-SP1 protein, the mutation that results in a decrease in the inhibition of processing of p25 to p24 by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid , and also wherein the mutation is optionally located at or near the CA-SP1 cleavage site or located in the SPI domain of SEQ ID NO: 5 or SEQ ID NO: 7 (originating polynucleotide sequences) which codes for the CA-SPl protein. This polypeptide may be encoded by a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9, or may comprise a sequence that is selected from the group consisting of of GHKARVLVEAMSQV (SEQ ID NO: 2) and SHKARILAEVMSQV (SEQ ID NO: 3). The polypeptide of this invention can be further encoded by a polynucleotide cge hybridizes to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. The invention also includes a polypeptide encoded by a polynucleotide that hybridizes to SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 10 or 12, which contains a mutation that results in a decrease in the processing inhibition of p25 to p24 by acid 3-0- (3 ', 3'- dimethylsuccinyl) betulinic, and also wherein the mutation is optionally located at or near the cleavage site of CA-SP1 or in the SPI domain of CA-SPl. The polypeptide of this invention further includes polypeptides that are part of a chimeric or fusion protein. These chimeric proteins can be derived from species that include, but are not limited to: primates, including apes and man; rodents, including rat and mouse; feline bovine sheep; including goat and sheep; canine or porcine. The fusion proteins may include sequences of synthetic peptides, bifunctional antibodies, peptides linked to proteins of the above species, or with linker peptides. The polypeptides of the invention can be further linked with detectable labels; metal compounds; cofactors; chromatography separation labels, such as, but not limited to: histidine, protein A, or the like, or linkers; blood stabilization portions such as, but not limited to: transferrin or the like; therapeutic agents and so on. The invention also includes a carbon atom that selectively binds to an amino acid sequence that contains a mutation in the CA-SP1 protein that results in a decrease in the inhibition of p25 (CA-SP1) processing to p24 (CA) by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid and also wherein the mutation is optionally located at or near the CA-SPl cleavage site or in the SPI domain of CA-SPl. The invention also includes an antibody that selectively binds to the polypeptide having the mutation comprising a sequence that is one of GHKARVLVEAMSQV (SEQ ID NO: 2), SHKARILAEVMSQV (SEQ ID NO: 3). This antibody can selectively bind to the polypeptide encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. This antibody can also be bound selective to the polypeptide encoded by a polynucleotide that hybridizes under highly stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. The invention also includes an antibody that binds selectively to SPI, which will make it possible to distinguish SPl from CA-SPl (p25). The invention also includes the antibody that selectively binds to CA (p24), which will distinguish CA from CA-SP1. The invention also includes an antibody that selectively binds to CA-SP1, which will distinguish CA from CA-SP1. The invention further includes an antibody that selectively binds at or near the CA-SPl cleavage site. He The antibody of this invention can be a polyclonal antibody, a monoclonal antibody or the antibody can be chimeric or functional, or part of a fusion protein. The invention further includes a portion of any antibody of this invention, including single chain, light chain, heavy chain, CDR, F (ab ') 2, Fab, Fab', Fv, sFv, or dsFv, or any combination thereof. . As used herein, an antibody "binds selectively" to a target peptide when it binds to the target peptide and does not bind significantly to unrelated proteins. The term "selectively binds" also comprises determining whether the antibody binds selectively to the target mutant sequence relative to a native target sequence. An antibody that "selectively binds" a target peptide is equivalent to an antibody that is "specific" to an objective peptide, as used herein. An antibody is still considered to bind selectively to a peptide even if it also binds to other proteins that are not substantially homologous to the target peptide as long as these proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it will be understood that the antibody that binds to the peptide will still be selective despite of the degree of cross reactivity. In another embodiment, determining whether the antibody selectively binds to the mutant target sequence comprises: (a) determining the binding affinity of the antibody for the mutant target sequence and for the native target sequences; and (b) comparing the binding affinities determined in this way, the presence of a higher binding affinity for the mutant target sequence than for the native one indicating that the antibody binds selectively to the mutant target sequence. The invention further relates to an antibody immobilized in an insoluble carrier comprising any of the antibodies described herein. The immobilized antibody in an insoluble carrier includes multi-well plates, culture plates, culture tubes, test tubes, beads, spheres, filters, electrophoresis material, microscope slides, membranes, or affinity chromatography medium. The invention also includes labeled antibodies, comprising a detectable signal. The labeled antibodies of this invention are labeled with a detectable molecule, which includes an enzyme, a fluorescent substance, a chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidite, electro dense substance, and a radioisotope, or any combination thereof. The invention further includes a method for producing a hybridoma comprising fusing a mammalian myeloma cell with a mammalian B cell that produces a monoclonal antibody that selectively binds to an amino acid sequence containing a mutation in the CA- protein. SP1, this mutation that results in a decrease in the inhibition of p25 to p24 processing by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid and a hybridoma that produces any of the monoclonal antibodies described herein. The invention further includes a method for producing an antibody comprising culturing a hybridoma that produces the monoclonal antibodies described herein in an appropriate medium and isolating the antibodies from the medium, as is well known in the art. The invention also includes the production of polyclonal antibodies comprising the injection, either an injection or multiple injections of any of the polypeptides of the invention into any animal known in the art that is useful for the production of polyclonal antibodies, including, in a manner enunciative and without limitation, mouse, rat, hamster, rabbit, goat, sheep, servant, guinea pig, or primate, and recover the antibodies in sera produced in the same. The invention includes high avidity or high affinity antibodies produced therein. The invention also includes B cells produced from the species listed for further use in cell fusion processes for the preparation of hybridomas producing monoclonal antibodies as described herein. The invention further relates to a kit comprising the antibody or a portion thereof as described herein, a container comprising the antibody and instructions for use, a kit comprising the polypeptides of the invention and instructions for use and a kit comprising the polynucleotide of the invention, a container comprising the polynucleotide and instructions for use, or any combination thereof. These equipment will include, but are not limited to, nucleic acid detection equipment, which may or may not use immunoassay and PCR equipment. These kits are useful for clinical diagnostic use and provide standardized reagents as required in current clinical practice. These teams will provide either information regarding the presence or absence of mutations before treatment or to monitor the progress of the patient during therapy. The teams of the invention can also be used to provide standardized reagents for use in research laboratory studies. Compound of the Invention In one aspect, the invention also relates to a compound, a method for using a compound, a method for identifying a compound and the like. The term "a", "an", or "an", as used in the present invention may refer to either the singular or the plural. For example, "a compound" encompasses one or more compounds. Compounds useful in the methods of the present invention include betulinic acid and betulin derivatives which are depicted in U.S. Patent Nos. 5,679,828 and 6,172,110 respectively, and in U.S. Patent Applications Nos. 60 / 443,180 and 10 / 670,797, which are incorporated herein by reference. Additional useful compounds include oleanolic acid derivatives described by Zhu et al. (Bioorg, Chem Lett 11: 3115-3118 (2001)); oleanolic acid derivative and promolic acid described by Kashiwada et al. (J. Nat. Prod. 61: 1090-1095 (1998)); 3-O-acyl-ursolic acid derivatives described by Kashiwada et al. (J. Nat. Prod. 63: 1619-1622 (2000)); and 3-alkoxylamido-3-deoxy-betulinic acid derivatives, described by Kashiwada et al. (Chem. Pharm. Bull. 48: 1387-1390 (2000)). (All references incorporated as reference). In some embodiments, the compounds useful in the present invention include, without limitation, those betulinic acid derivatives having the general Formula I and dihydrobetulinic acid derivatives of the formula II: or a pharmaceutically acceptable salt thereof, wherein R is a substituted and unsubstituted C2-C20 carboxycyl, R 'is hydrogen, substituted and unsubstituted C2-C10 alkyl, or an aryl group. Preferred compounds are those in which R is one of the substituents in Table 4, below, and R 'is hydrogen. In other embodiments, useful compounds include betulin and dihydrobetulin derivatives of Formula III: or a pharmaceutically acceptable salt thereof, wherein, R1 is a substituted and unsubstituted C_-C20 carboxycyl, or an ester thereof; R2 is hydrogen, C (C6H5) _, or a substituted and unsubstituted C2-C20 carboxycyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or di-alkylamino, or -OR4, wherein R4 is hydrogen, C_4alkanoyl, benzoyl or a C2-C20 substituted and unsubstituted carboxyacyl; where the dashed line represents an optional double bond between C20 and C29. Preferred compounds useful in the present invention are those where R. is one of the substituents in Table 4, R. is hydrogen or one of the substituents in Table 4 and R. is hydrogen. Table 4: Preferred Substituents for R, R ', R1, R2; The most preferred compounds are 3-0- (3 ', 3) acid dimethylsuccinyl) betulinic acid, 3-0- (3 ', 3' -dimethylsuccinyl) dihydrobetulinic acid, 3-0- (3 ', 3'-dimethylsuccinyl) betulin, and 3-0- (3', 3'-dimethylsuccinyl or flutaryl) ) dihydrobetulin. A particularly preferred compound is 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. In some embodiments, the compounds useful in the present invention are described by Formulas IV, V, VI and VII.

Rn = is -0R14 or -NHR--; R.2 = C00R.7, COO A, or CH20R17 R.3 = -H, halogen, amino, mono- or di-alcguilamino optionally substituted, or -0R16; R.4 = -H, substituted or unsubstituted C2-C20 carboxyacyl; R.5 = -H, substituted or unsubstituted C2-C20 carboxyacyl; R.6 = -H, C4-G_alkanoyl, benzyl, or substituted or unsubstituted C2-C20 carboxyacyl; R-7 = -H, C (C6H5) 3, or C2-C20 carboxycyl substituted or unsubstituted where the dashed line represents optional link between C20 and C29, and where A = Na +, K +, or other cation, where The R38 portions other than hydrogen are bound to R33 oxygen by a covalent bond to the carbonyl carbon. Preferred compounds are those where R38 is not hydrogen. In further embodiments, any of R38, R40 and / or R41 are methyl. In some embodiments, the compounds useful in the methods of the invention also include those described in the provisional application U.S. Patent No. 60 / 559,358, which is incorporated by reference in its entirety. In one aspect, these compounds are described by reference to the following compounds VIII to XI: In some embodiments, the compounds useful in the present invention have the general Formula VIII: or a pharmaceutically acceptable salt or ester thereof wherein A is a fused ring of the Formula: (i) (ii) (iii) wherein the ring carbons designated x and y in the Formulas of A are the same as the ring carbons designated x and y in Formula VIII: R 51 is a carboxyalkanoyl, wherein the alkanoyl chain may be optionally substituted by one or more hydroxy or halo, or may be interrupted by a nitrogen, sulfur or oxygen atom, or combinations thereof; R52, R53 and RS4 are independently hydrogen, methyl, halogen or hydroxy, carbonyl or -COOR66, wherein R66 is alkyl or carboxyalkyl, wherein the alkyl chain may be optionally substituted by one or more of hydroxyl or halo, or may be interrupted by nitrogen, sulfur or oxygen atom, or combinations thereof; R55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more of hydroxy or halo, or R55 is a carboxyl or hydroxymethyl; R56 is hydrogen, methyl, hydroxy or halogen; R57 and R58 are independently hydrogen or C.alkyl; R59 is CH2 or CH3; R60 is hydrogen, hydroxy or, methyl; R61 is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more of hydroxy or halo; R62 is hydrogen or methyl; R63 is hydrogen or methyl; R64 is hydrogen or hydroxy; R65 is hydrogen if C12 and C13 form an individual ring, or R65 is absent if C12 and C13 form a double bond; and where the straight dashed line represents an optional double bond between C12 and C13 or C20 and C29. with the condition that when A is then R51 can not be glutaryl or succinyl when there is a double bond between C12 and C13; when A is (ii) and R61 is methyl, then R51 can not be succinyl; when A is (iii) and R52, R53 and R63 are each hydrogen, then R51 can not be succinyl; and with the condition that A (i) can not be when R52 and R "are both methyl and a double bond exists between C12 and C13. In some embodiments, R51 is a carboxy (C2_6) alkoxycarbonyl group or a carboxy (C2_6) alkoxy group (C. 6) alkylcarbonyl. Suitable groups are selected from the group consisting of: According to the invention, in some embodiments, the compounds have Formula IX: where R51, R54, R55, R56, R57, R58 and R64 are as defined previously for Formula VIII. In one embodiment, R56 is β-methyl, R58 is hydrogen, R55 is hydroxymethyl and R51 is 3 ', 3'-dimethylglutaryl, 3', 3'-dimethylsuccinyl, glutaryl or succinyl. In another embodiment, R56 is hydrogen, R57 and R58 are both methyl, R55 is carboxyl and R51 is 3 ', 3' -dimethylglutaryl, 3 ', 3'-dimethylsuccinyl, glutaryl or succinyl. In some embodiments, R55 is carboxyalkoxycarbonyl, alkoxycarbonyl, alkanoyloxymethyl, carboxyalkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more of hydroxy or halo, or R55 is a carboxyl or hydroxymethyl. In some embodiments, R55 is selected from a group consisting of carboxyl, hydroxymethyl, -C02 (CH2) nCOOH, -C02 (CH2) nCH3, -CH2OC (0) (CH2) nCH_, -CH.OC (O) (CH2 ) nCOOH, -CO (CH2) nCH3 and -CO (CH2) nCOOH. In some embodiments, R55 is selected from a group consisting of Y In some embodiments, R-- is hydroxymethyl In some embodiments R55 is carboxyl. In some embodiments n is from 0 to 20, and preferably from 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is from 1 to 6. In some embodiments, n is from 2 to 6. In some embodiments, the compounds useful in the present invention have Formula X: wherein R51, R59, R60 and R61 are as defined above for Formula VIII. In one embodiment, R55 is 3 ', 3'-dimethylglutaryl, 3', 3'-dimethylsuccinyl, glutaryl or succinyl. In some embodiments, R6I is methyl, methoxycarbonyl, carboxyalkoxycarbonyl, alkanoyloxymethyl, alkoxymethyl or carboxyalkoxymethyl, any of which is optionally substituted by one or more of hydroxy or halo. In some embodiments, R61 is selected from the group consisting of methyl, -C02 (CH2) nCOOH, -COC (O) (CH2) nCH3, -CO (CH2) nCH3 and -CO (CH2) nCOOH. In some embodiments, n is from 0 to 20, or preferably from 0 to 6. In some embodiments, n is from 1 to 10. In some embodiments, n is from 2 to 8. In some embodiments, n is 1 a 6. In some modalities, n is from 2 to 6. In some modalities, R61 is methyl. In some embodiments, R61 is methoxycarbonyl. In some embodiments, R61 is selected from the group consisting of methoxymethyl and ethoxymethyl. In some embodiments, the methyl groups found in R61 may be substituted with a halogen or a hydroxy. In some embodiments, the compounds useful in the present invention have Formula XI: wherein R51, R52, R53, R54, are as defined above for Formula VIII. In one modality, R51 is 3 ', 3' -dimethylglutaryl, 3 ', 3'-dimethylsuccinyl, glutaryl or succinyl. In one embodiment, both R52 and R53 are methyl. Any triterpene falling within the scope of Formula VIII can be used. According to the invention, in some embodiments, the compounds of Formula VIII are selected from the group consisting of uvaol derivatives, ursolic acid, erythrodiol, equinoxic acid, oleanolic acid, sumaresinolic acid, lupeol, dihydrolupeol, methyl ester of betulinic acid. and methyl ester of dihydrobetulinic acid, 17-a-methyl-androstenediol, androstenediol, and 4,4-dimethylaminosteanediol. In some embodiments, the compounds of the present invention are defined as Formula VIII, wherein R52 and R53 are both methyl. In some embodiments, the compounds of the present invention are defined as Formula VIII, wherein R51 is 3 ', 3'-dimethylsuccinyl. In some embodiments, the compounds of the present invention are defined as Formula VIII, wherein R51 is succinyl, ie, According to the invention, in some embodiments, the stereochemistry of the substituents of the side chain. In some embodiments, the compounds of the present invention are defined as Formula VIII wherein A is (i) and R55 is in the β-position. In some embodiments, the compounds of the present invention are defined as Formula VIII, wherein A is (i) and R56 is in the β-position.

In some embodiments, the compounds of the present invention are defined as in Formula VIII, where A is (i) and R64 is in position a. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and R57 is a-methyl, and R58 is hydrogen. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i), R58 is a-methyl and R57 is hydrogen. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (i) and both R57 and R58 are methyl. In some embodiments, the compounds of the present invention are defined as in Formula VIII, wherein A is (ii) and R61 is in the β-position. In some embodiments, 3 ', 3' -dimethylsuccinyl is in the C3 position. In some embodiments, the compounds of Formula IX are 3-0- (3 ', 3' -dimethylsuccinyl) uvaol; 3-0- (3 ', 3' -dimethylsuccinyl) erythrodiol; 3-0- (3 ', 3'-dimethylsuccinyl) equinoxical acid or 3-0- (3', 3'-) acid dimethylsuccinyl) sumaresinolic. In some embodiments, the compounds of Formula X are 3-0- (3 ', 3' -dimethylsuccinyl) lupeol; 3-0- (3 ', 3' -dimethylsuccinyl) dihydrolupeol; 3-0- (3 ', 3' -dimethylsuccinyl) 17β-methyl ester-betulinic acid; or 3-0- (3 ', 3' -dimethylsuccinyl) 17β-methylester-dihydrobetulinic acid. In some embodiments, the compounds of Formula XI are 3-0- (3 ', 3' -dimethylsuccinyl) -4,4-dimethylaminosteanediol; 3-0- (3 ', 3' -dimethylsuccinyl) 17a-methylandrostenediol; 3-0- (3 ', 3' -dimethylsuccinyl) androstenediol. In a further embodiment the invention includes compounds and methods using the compounds of Formula XII: where R72 is one of: wherein Z is hydroxy or halogen; and R73 is lower alkyl, such as methyl, ethyl or propyl. In further embodiments, the compounds useful in the present invention are betulin derivative compounds of Formula XIII: or a pharmaceutically acceptable salt or prodrug thereof, wherein R- is C3-C20alkanoyl, carboxyalkanoyl, carboxyalkenoyl, alkoxycarbonylalkanoyl, alqueniloxicarbonilalcanoilo, cianoalcanoilo, hydroxyalkanoyl, aminocarbonilalcanoilo, hidroxiaminocarbonilalcanoilo, monoalquilaminocarbonilalcanoilo, dialquilaminocarbonilalcanoilo, heteroarilalcanoilo, heterocyclylalkanoyl, heterociclilcarbonilalcanoilo, heteroarilaminocarbonilalcanoilo, heterociclilaminocarbonilalcanoilo, cianoaminocarbonilalcanoilo, alquilsulfonilaminocarbonilalcanoilo, arilsulfonilaminocarbonilalcanoilo, sulfoaminocarbonilalcanoilo, fosfonoaminocarbonilalcanoilo, tetrazolilalcanoilo, phosphono, sulfo, fosfonoalcanoilo, sulfoalcanoilo, alquilsulfonilalcanoilo or alquilfosfonoalcanoilo; R8- is formyl, carboxyalkenyl, heterocyclyl, heteroaryl, -CH2SR94, (¡) (¡I) (iii) (iv) (vi) (vü) (vüi) R 83 is hydroxyl, isopropenyl, isopropyl, 1'-hydroxyisopropyl, 1'-haloisopropyl, 1'-thioisopropyl, 1'-trifluoromethylisopropyl, 2'-hydroxyisopropyl, 2'-haloisopropyl, 2'-thioisopropyl, 2'-trifluoromethylisopropyl, 1 ' -hydroxyethyl, 1 '- (alkoxy) ethyl, 1 '- (alkoxyalkoxy) ethyl, 1' - (arylalkoxy) ethyl; 1'- (arylcarbonyloxy) ethyl, 1 '- (oxo) ethyl, 1' - (hydroxyl) -1 '- (hydroxyalkyl) ethyl, 1' (oxo) oxazolidinyl, 1 ', 2'-epoxyisopropyl, 2' -haloisopropenyl , 2 '-hydroxy isopropenyl, 2' -aminoisopropenyl, or wherein Y is -SRU1 or -NR113R114; Rm is methyl; Rll2 is hydrogen or hydroxyl; R113 and R114 are independently hydrogen, alkyl, alkanoyl, arylalkyl, heteroarylalkyl, arylsulfonyl or arylaminocarbonyl; or Rm and Rn. they may be taken together with the nitrogen to which they are attached to form a heterocycle, wherein the heterocycle may optionally include one or more additional nitrogen, sulfur or oxygen atoms; m is zero to three; R84 is hydrogen; or R83 and R84 can be taken together to form oxo, alkylimino, alkoxyimino or benzyloxyimino; R85 is C2-C20al < alkyl, alkenyl, C2-C20carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, cyano, cyanoalkyl, alkylthioalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkanoylaminoalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, heterocyclylcarbonylalkyl, cicloalquilcarbonilalquilo, heteroarilalquilaminocarbonilalquilo, arilalqui1-aminocarbonyl, heterocyclylalkylamino-alkylcarbonyl, carboxialquilaminocarbonilalquilo, arilsulfonilaminocarbonilalquilo, alquilsulfonilaminocarbonilalquilo, arilfosfonoamino-alkylcarbonyl, alquilfosfonoaminocarbonilalquilo or hydroxyimino (amino) alkyl; R86 is hydrogen, phosphono, sulfo, cyano, alkyl, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, cycloalkyl, heterocyclyl, aryl, heteroaryl, carboxyalkyl, alkoxycarbonylalkyl, or cyanoalkyl; R87 or R88 are independently hydrogen, alkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl, alkoxycarbonylaminoalkoxyalkyl, alkoxycarbonylaminoalkyl, aminoalkoxyalkyl, alkylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, or cycloalkyl, or R87 and R8 can together with the nitrogen atom to which they are attached form a heterocyclyl or heteroaryl group, in wherein the heterocyclyl or heteroaryl may optionally include one or more additional nitrogen, sulfur or oxygen atoms; R89 is hydrogen, phosphono, sulfo, cyano, alkyl, alkylsilyl, cycloalkyl, carboxyalkyl, alkoxycarbonyloxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, cyanoalkyl, phosphonoalkyl, sulfoalkyl, alkylsulfonyl, alkylphosphono, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, or dialkoxyalkyl; R9o AND R_? are independently hydrogen, hydroxy, cyano, alkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxy, carboxyalkyl, alkanoyloxyalkyl, alkoxyalkyl, alkoxyalkoxyalkyl, alcoxicarbonilaminoalcoxialquilo, alkoxycarbonylaminoalkyl, aminoalkoxyalkyl, alkylcarbonylaminoalkyl, heterocyclyl, heterocyclylalkyl, aryl, arylalkyl, arylcarbonylaminoalkyl, ariisulfonilo, or cycloalkyl, or alkyl interrupted by one or more oxygen atoms, or R90 or R91 may together with the nitrogen atom to which they are attached form a heterocyclyl group, wherein the heterocyclyl may optionally include one or more additional nitrogen, sulfur or oxygen atoms; R_2 and R-3 are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloalkyloxy, heterocyclylaminoalkyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R__ and R93 may together with the atom of nitrogen to which they are attached form a heterocyclyl group, wherein the heterocyclyl may optionally include one or more additional nitrogen, sulfur or oxygen atoms, or R92 and R93 may together with the nitrogen atom to which they are attached form an alkylazo group and it is one to six; R94 is hydrogen, alkyl, alkenyl, arylalkyl, carboxyalkyl, carboxyalkenyl, alkoxycarbonylalkyl, alkenyloxycarbonylalkyl, cyanoalkyl, hydroxyalkyl, carboxybenzyl, aminocarbonylalkyl; R95 and R96 are independently hydrogen, alkyl, alkoxycarbonyl, alkoxyaminoalkyl, cycloalkyloxy, heterocyclylaminoalkyl, cycloalkyl, cyanoalkyl, cyano, sulfo, phosphono, sulfoalkyl, phosphonoalkyl, alkylsulfonyl, alkylphosphono, alkoxyalkyl, heterocyclylalkyl, or R95 and R96 may together with the nitrogen atom to which they are attached form a heterocyclyl group, wherein the heterocyclyl may optionally include one or more additional nitrogen, sulfur or oxygen atoms, or R95 and R96 may together with the atom of nitrogen to which they join form a group alquilazo; R97 is hydrogen, alkyl, alkenyl, carboxyalkyl, amino, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, alkoxycarbonyl, cyanoalkyl, alkylthioalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, alkanoylaminoalkyl, aminocarbonylalkyl, alkylaminocarbonylalkyl, dialkylaminocarbonylalkyl, heterocyclylcarbonylalkyl, cycloalkylcarbonylalkyl, heteroarylalkylaminocarbonylalkyl, arylalkylaminocarbonylalkyl, heterocyclylalkyl-aminocarbonylalkyl, carboxyalkylaminocarbonylalkyl, arylsulfonylaminocarbonylalkyl, alkylsulfonylamino-carbonylalkyl, or hydroxyimino (amino) alkyl, • R98 and R99 are independently hydrogen, methyl or ethyl, preferably hydrogen or methyl; and d is from one to six. The alkyl groups and the alkyl-containing groups of the compounds of the present invention can be straight and branched chain alkyl groups, preferably having from one to ten carbon atoms. In some embodiments, the alkyl groups or alkyl-containing groups of the present invention may be substituted with a C3.7 cycloalkyl group. In some embodiments, the cycloalkyl group may include, without limitation, a cyclobutyl, cyclopentyl or cyclohexyl group. Also, included within the scope of the present invention are pharmaceutically acceptable salts non-toxic to the compounds of the present invention. These salts can be prepared in itself during the final isolation and purification of the compounds or by reacting separately the purified compound in its free acid form with a suitable inorganic or organic base and by isolating the salt formed in this manner. These may include cations based on alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, N-methyl-glucamine and the like. Also, included within the scope of the present invention are the pharmaceutically esters acceptable non-toxic compounds of the present invention. Ester groups are preferably of the type that hydrolyze relatively easily under physiological conditions. Examples of pharmaceutically acceptable esters of the compounds of the invention include C.sub.6 alkyl esters wherein the alkyl group is straight or branched chain. Acceptable esters also include C5_7 cycloalkyl esters as well as arylalkyl esters, such as, but not limited to, benzyl. C.sub.4 alkyl esters are preferred. In some embodiments, the esters are selected from the group consisting of esters of alkylcarboxylic acid, such as esters of acetic acid, and esters of mono- or di-alkyl phosphate, such as methylphosphate ester or dimethylphosphate ester. The esters of the compounds of the present invention can be prepared according to conventional methods. Certain compounds are the derivatives listed above referred to as "prodrugs". This includes compounds within the scope of formula VIII to XI, by way of example. The term "prodrug" refers to compounds that are rapidly transformed in vivo by an enzymatic or chemical process, to produce the parent compound of the above formulas, for example, by hydrolysis in blood. A complete analysis is provided by Higuchi, T. and V. Stella in Pro-durgs as Novel Delivery Systems. Vol. 14. A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association, Pergamon Press, 1987. Useful prodrugs may be esters, for example, of the compounds of formulas VIII, IX, X and XI . In some prodrug moieties, a lower alkyl group is substituted with one or more hydroxy or halo groups by a suitable acid. Suitable acids include, for example, carboxylic acids, sulfonic acids, phosphoric acid or lower alkyl esters thereof, and phosphonic acid or lower alkyl esters thereof. For example, suitable carboxylic acids include alkylcarboxylic acids, such as acetic acid, arylcarboxylic acids and arylalkylcarboxylic acids. Suitable sulfonic acids include alkylsulfonic acids, arylsulfonic acids and arylalkylsulfonic acids. Suitable phosphoric and phosphonic esters are methyl or ethyl esters. In some embodiments, C3 acyl groups having dimethyl or oxygen groups at the C3 'position may be the most active compounds. This observation suggests that these types in acyl groups may be important to the improved anti-HIV activity. In one embodiment, the invention relates to a method for treating HIV-1 infection in a patient at administering a compound that inhibits the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but does not significantly affect other Gag processing steps, wherein the compound is a compound of formula I until XIII. In one embodiment, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), but does not it significantly affects other Gag processing steps, wherein the compound is a compound of formula I through XIII, with the exception of DSB. In one embodiment, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), but does not it significantly affects other steps of Gag processing, wherein the compound is different from a compound of formula I to VII, or is different from a compound of formula I to XIII. In one embodiment, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of Gag viral p25 protein (CA-SP1) to p24.

(CA), but that does not significantly affect other Gag processing steps, where the compound is different of a compound of the formula I to XI; or in other modalities it is different from I to XIII. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), in a cell but without significantly affecting other Gag processing steps, in wherein the compound is a compound of the groups of formulas I through XIII. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), in a cell but which does not significantly affect other Gag processing steps, wherein the compound is different from a compound of the groups of formulas I to VII; or in other embodiments, is different from a compound of formula I through XIII. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), in one cell but without significantly affecting other Gag processing steps, in wherein the compound is different from a compound of the groups of formulas I through XI; or in other embodiments is different from a compound of formula I through XIII. In another embodiment, the invention relates to a method for treating human blood or human blood products by inhibiting the processing of the viral p25 protein. from Gag (CA-SPl) to p24 (CA), in one cell but without significantly affecting other Gag processing steps, wherein the compound is a compound of formula I through XIII. In another embodiment, the invention relates to a method for treating human blood or human blood products by inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), in a cell but without significantly affecting it other Gag processing steps, wherein the compound is different from a compound of formula I through VII; or in other embodiments is different from a compound of formula I through XIII. Synthesis of DSB and related compounds The reaction of botulinum acid and dihydrobetulinic acid with dimethylsuccinic anhydride produced a mixture of 3-0- (2 ', 2'-dimethylsuccinyl) and 3-0- (3', 3'-dimethylsuccinyl) botulinum acid and dihydrobetulinic acid, respectively. The mixtures were successfully separated by HPLC on a preparative scale producing pure samples. The structures of these isomers were assigned by long-interval H-13C COZY tests. The botulinum acid and dihydrobetulinic acid derivatives of the present invention were all synthesized by refluxing a solution of botulinum acid or dihydrobetulinic acid, dimethylaminopyridine (1). molar equivalent), and an appropriate anhydride (2.5-10 molar equivalents) in anhydrous pridine (5-10 mL). The reaction mixture was then diluted with ice water and extracted with CHC13. The organic layer was washed with water, dried over MgSO4, and concentrated under reduced pressure. The residue was chromatographed using HPLC on a semipreparative scale or on a silica gel column to produce the product. Botulinum 3-0- (3 ', 3'-dimethylsuccinyl) 70% yield preparation (starting with 542 mg of betulinic acid); MeOH crystallization gave colorless needles; p.f. 274 ° -276 ° C.; [a] D19 + 23.5 ° (c = 0.71), CHCl3-MeOH [1: 1]); FABMS positive m / z 585 (M + H) \ - FABMS Negative m / z 583 (M-H) -; HR-FABMS calculated for C36H5706 585.4155, found m / z 585.4161; NMR * H (pyridine-d5): 0.73, 0.92, 0.97, 1.01, 1.05 (each 3H, s; 4- (CH3) 2, 8-CH3, 10-CH3, 14-CH.), 1.55 (6H, s, 3'-CH3 x 2), 1.80 (3H, s, 20-CH3), 2.89, 2.97 (each one H, d, J = 15.5 Hz, H-2 '), 3.53 (H, m , H-19), 4.76 (HH, dd, J = 5.0, 11.5 Hz, H-3), 4.78, 4.95 (each ÍH, br,, H-30). 3-0- (3 ', 3'-dimethylsuccinic acid) dihydrobetulinic acid: yield 24.5% (starting with 155.9 mg dihydrobetulinic acid); crystallization of MeOH-H20 gave colorless needles; p.f. 291 ° -292 ° C; [] D20.13.4 ° (c = ll, CHC13-MeOH [1: 1], NMR "H (pyridine-d5): 0.85, 0.94 (each 3H, d, J = 7.0 Hz, 20- (CH3) 2 ), 0.75, 0.93, 0.97, 1.01, 1.03 (each 3H, s; 4- (CH3) 2, 8-CH3, 10-CH3, 14-CH3), 1.55 (6H, s; 3 '-CH3 x 2), 2.89, 2.97 (each lH, d, J = 15.5 Hz; -2 '), 4.77 (ÍH, dd, J = 5.0, 11.0 Hz, H-3); Analysis calculated for C3-H_80-.5 / 2H2O: C 68.43, H 10.04; found C 68.64, H 9.78. The synthesis of 3-0- (3 ', 3' -dimethylglutaryl) betulinic acid was described in U.S. Patent No. 5,679,828, as compound No. 4. 3-0- (3 ', 3'-dimethylglutaryl) acid ) dihydrobetulinic: yield 93.3% (starting with 100.5 mg of dihydrobetulinic acid); Needle crystallization MeOH-H20 gave colorless needles; p.f. 287 ° -289 ° C; [a] D20-17.9 ° (c = 0.5, CHCl 3 -MeOH [1: 1]); XH NMR (pyridine-d5): 0.86, 0.93 (each 3H, d, J = 6.5 Hz; 20- (CH3) 2), 0.78, 0.92, 0.96, 1.02, 1.05 (each 3H, s; 4- (CH3) 2, 8-CH3, 10-CH3, 14-CH.), 1.38, 1.39 (each 3H, s; 3 '-CH3 x 2), 2.78 (4H, m, H2 -2 'and 4'), 4.76 (lH, dd, J = 4.5, 11.5 Hz, H-3). Analysis calculated for C 37 H 60 O 6: C 73.96, H 10.06; Found C 73.83, H 10.10. The synthesis for 3-O-diglycolyl-betulinic acid was described in U.S. Patent No. 5,679,828, as compound No. 5. 3-O-diglycolyl-dihydrobetulinic acid: yield 79.2% (initiating with 103.5 mg of dihydrobetulinic acid ); amorphous whitish powder; [α] D20-9.8 ° (c = 1, CHC13 MeOH [1: 1]); XH-NMR (pyridine-d5): 0.79, 0.87 (each 3H, d, J = 6.5 Hz, 20- (CH3) 2), 0.87, 0.88, 0.91, 0.98, 1.01 (each 3H, s; 4- (CH3) 2, 8-CH3, 10-CH3, 14-CH3), 4.21, 4.23 (each 2H, s, H2-2 'and 4'), 4.57 (lH, dd, J = 6.5, 10.0 Hz, H-3); Analysis calculated for C34H5407.2H20: C 66.85, H 9.57; found C 67.21, H 9.33. The synthesis of 3-0- (3 ', 3' -dimethylsuccinyl) betulin and 3-0- (3 ', 3' -dimethylglutaryl) betulin was described in U.S. Patent Application No. 10 / 670,797.

Methods for inhibiting HIV with a compound The methods for "inhibiting HIV" or "inhibiting HIV" as used herein means any interference with, inhibition of, and / or prevention of HIV using the methods of the invention. As such, inhibition methods are useful in inhibiting HIV infectivity, inhibiting p25 processing, inhibiting viral maturation, forming virions exhibiting altered phenotypes, and the like. Preferably, the methods of the invention act in the processing of p25 in the cells of an animal, but are not limited by that method of action. A method of inhibiting HIV with a compound may be pertinent to a method of treating HIV infection in a patient. Therefore, a method for inhibiting HIV with a compound is formed in a similar manner to treat a patient.

Methods for inhibiting HIV-1 replication in cells of an animal include contacting infected cells with a compound of formula I through XIII, above. Related embodiments include a method for treating an HIV-1 infection in a patient comprising administering a compound of formula I to XIII; a method for inhibiting the processing of p25 either in a cell, in vivo and / or in vi tro by the administration of a compound that inhibits the processing of p25; and a method for treating human blood or human blood products by administering a compound of formula I to XIII. Also included is a method for identifying a compound that inhibits either p25 processing, HIV maturation, HIV infection, HIV virion phenotypes and the like. In one embodiment, the compound is a derivative of botulinum acid, betulin, or dihydrobetulinic acid or dihydrobetulin and which includes the preferred substituents of Table 4. Preferred compounds include, but are not limited to, 3-0- (3 ') acid. , 3'-dimethylsuccinyl) botulinum, 3-0- (3 ', 3'-dimethylsuccinyl) betulin, 3-0- (3', 3 '-dimethylglutaryl) betulin, (3', 3'-dimethylglutaryl) dihydrobetulinic acid , 3-0- (3 ', 3'-dimethylglutaryl) botulinum acid, acid (3', 3'- dimethylglutaryl) dihydrobetulinic, 3-0-diglycolyl-betulinic acid and 3-0-diglycolyl-dihydrobetulinic acid. In one embodiment, the invention relates to a method for inhibiting the replication of HIV-1 in cells of an animal by contacting infected cells with a compound that inhibits the processing of Gag's viral p25 protein (CA-SP1) to p24 (CA), but which does not significantly affect other steps of Gag processing, wherein the compound is a compound of formulas I through XIII above. In one embodiment, the invention relates to a method for inhibiting the replication of HIV-1 in cells of an animal by contacting infected cells with a compound that inhibits the processing of Gag's viral p25 protein (CA-SP1) to p24 (CA), but which does not significantly affect other Gag processing steps, wherein the compound is a compound of formulas I through XIII, with the exception of DSB. In one embodiment, the invention relates to a method for inhibiting the replication of HIV-1 in cells of an animal by contacting infected cells with a compound that inhibits the processing of Gag's viral p25 protein (CA-SP1) to p24 (CA), but that does not significantly affect other Gag processing steps, where the compound is one that is excluded from formulas I until VI. In one embodiment, the invention relates to a method for treating HIV-1 infection in a patient by administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), but does not it significantly affects other Gag processing steps, wherein the compound is one different from those of formulas I through XIII. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting other steps of Gag processing, in wherein the compound is a compound of formulas I through XIII. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting other steps of Gag processing, in wherein the compound is a compound different from those of formulas I through VI. In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting other steps of Gag processing, in wherein the compound is a compound different from those of formulas I through XIII.

In another embodiment, the invention relates to a method for inhibiting the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting other steps of Gag processing, in wherein the compound is a compound different from those of formulas I through XI. In another embodiment, the invention relates to a method for treating human blood or human blood products by inhibiting the processing of the p25 viral protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting it. other steps of Gag processing, wherein the compound is a compound of formulas i through XIII. In another embodiment, the invention relates to a method for treating human blood or human blood products by inhibiting the processing of the p25 viral protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting it. other steps of Gag processing, wherein the compound is a compound different from those of formulas I through VI. In another embodiment, the invention relates to a method for treating human blood or human blood products by inhibiting the processing of the p25 viral protein from Gag (CA-SP1) to p24 (CA) in a cell, but without significantly affecting it. other steps of Gag processing, where the compound is a compound different from those of Formulas I through XIII. The method described herein, further comprises contacting the cells with one or more drugs selected from the group consisting of antiviral agents, antifungal agents, antibacterial agent, anti-cancer agent, immunostimulatory agents, and combinations thereof. The method may include the treatment of human blood products. The invention can also be used in conjunction with a method of treating cancer comprising administering to an animal one or more anti-neoplastic agents, exposing an animal to an amount of radiation killing cancer cells, or a combination of both. Methods for identifying compounds The invention further includes a method for identifying compounds that inhibit HIV replication in cells of an animal described herein. In one embodiment, the method comprises: (a) contacting a Gag polypeptide comprising an CA-SP1 cleavage site with a test compound; (b) adding a labeled substance that binds selectively at or near the CA-SP1 cleavage site; Y (c) measuring the binding of the test compound at or near the CA-SPl cleavage site.

Substances labeled by molecules include labeled antibodies or labeled DSB and the label includes a enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidite, electro dense substance, such as gold, osmium tetraoxide, lead acetate or uranyl, and radioisotope, antibodies labeled with these molecules substances or a combination thereof. Assays may include, without limitation, ELISA, double and individual intercalation techniques, immunodiffusion techniques or immunoprecipitation, as is known in the art ("Immunoassay Handbook, 2nd ed.," D. ild, Nature Publishing Group , (2001)). These methods for identification may also include, but are not limited to, Western blotting assays, colorimetric assays, electron microscopy and light techniques, confocal microscopy, or other known techniques. A method for identifying egue compounds that inhibit HIV replication in cells of an animal further comprises: (a) contacting a Gag protein comprising a wild-type CA-SPl cleavage site, with a HIV-1 primed at the presence of a test compound; (b) separately, put in contact a Gag protein comprising a CA-SP1 cleavage site a mutant or a protein comprising an alternative protease cleavage site with HIV-1 protease in the presence of the test compound; and (c) comparing the cleavage of native wild type Gag protein to the amount of cleavage of the mutant Gag protein or to the amount of cleavage of the peptide comprising an alternative protease cleavage site. Step (b) above is performed as a control in order to eliminate the compounds that can bind directly to, and therefore inhibit, the protease enzyme. The above method also includes the method wherein the wild-type CA-SP1 cleavage site, mutant CA-SP1 or alternative protease is contained within a peptide or recombinant peptide fragment. The method for identifying HIV-1 inhibiting compounds described herein also includes a method wherein the peptide or protein is labeled with a fluorescent portion and a fluorescence quenching portion, each attached to opposite sides of the excision site of CA-SP1, and wherein the detection comprises measuring the signal of the fluorescent portion, or wherein the peptide or protein is labeled with two fluorescent portions, each linked to opposite sides of the CA-SP1 cleavage site, and wherein the detection involves measuring the transfer of energy fluorescent from one portion to the other in the presence of HIV-1 test compound and protease and compare the transfer of fluorescent energy to acguella observed when the same processing is applied to a peptide comprising a sequence comprising a mutation in the CA-SP1 cleavage site or comprising a sequence containing another cleavage site. Examples of assays based on fluorescence on protease activity are well known in the art. In this example, a protease substrate is labeled with green fluorescent ink molecules, which fluoresce when the substrate is cleaved by the protease enzyme (Molecular Probes, Protease Assay Kit). The method for comparing the excision, above, also includes the use of a labeled antibody that selectively binds to CA or SPl or CA-SPl in order to measure the extent to which the test compound inhibits CA-SPl cleavage. . The antibody can be labeled with a labeled molecule from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avbidin, electrodense substance, and radioisotope, or combinations thereof. The method also includes the use of an antibody to a specific epitope tag sequence to selectively detect CA-SP1 (p25) or SPI, in which the amino acid sequence for that epitope tag has been handled according to methods normal in the technique. Suitable labels are well known to those skilled in the art, and include the HA epitope of haemagglutinin (YPYDVPDYA) (SEQ ID No.: 81), VP7 epitope of bluetongue virus (QYPALT) (SEQ ID NO. .: 82), epitope of -tubulin (EEF), indicator (DIKDDDDK) (SEQ.

ID. No .: 83), and VSV-G (YTDOEMNRLGK) (SEQ ID No .: 84). Examples of SPI containing epitope tags are illustrated in Figure 17. As an example, the epitope tag sequence FLAG (Sigma-Aldrich) is inserted into the p2 region (SPI) of Gag by olicleotide-directed mutagenesis of a Gag expression plasmid. The presence of the spl domain in the protein expressed in the cell is then detected using commercially available anti-FLAG monoclonal antibodies (Sigma-Aldrich). (Hopp, TP Biotechnology 6: 1204-1210 (1988)) The method for identifying compounds that disrupt CA-SP1 cleavage also includes the addition of a compound to HIV-1 infected cells and the detection of cleavage products of CA-SPl to lyse and analyze the cells by the virions released. The method included in the invention can be performed using a Western blot analysis of viral proteins and by detecting p25 using an antibody to p25 or where the mixture is analyzed by gel electrophoresis of viral proteins and image the metabolically labeled proteins, or where the mixture is analyzed using immunoassays using an antibody that binds selectively to p25 or an antibody that binds selectively in order to distinguish p25 from p24. The invention includes the use of an antibody to a specific epitope tag sequence inserted into the C-terminal domain of SPI to selectively detect p25 or SPI. For example, an intercalation ELISA assay can be performed where p25 or p24 in the detergent solubilized virus are captured using an antibody that selectively binds to the Gag CA region, antibody that binds to a multi-cavity plate . After a washing step, the bound p25 is detected using an antibody to an epitope tag inserted in SPI, which is conjugated to an appropriate detection reagent (eg, alkaline phosphatase for an enzyme-linked immunosorbent assay). Viruses related by cells treated by compounds acting by this mechanism will generally have increased p25 levels compared to untreated virions. The method described relates to an antibody that selectively binds to p25, or an antibody that selectively binds to SP1, or an antibody to an epitope tag sequence inserted into SPI, which is labeled with a selected molecule of the group consisting of enzyme, fluorescent substance, emulsifying substance, horseradish peroxidase, alkaline phosphatase, biotin, avidite, electrodense substance, and radioisotope, or combinations thereof. "Infected cells", as used herein, include cells naturally infected by membrane fusion and subsequent insertion of the viral genome into the cells, or transfection of the cells with viral genetic material through artificial means. These methods include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, lipid-mediated transfection, electroporation, or infection. The invention can be practiced by infecting target cells in vitro for an infectious strain of HIV and in the presence of the test compound, under appropriate culture conditions and for varying periods of time. Cells infected by the supernatant fluid can be processed and loaded onto a polyacrylamide gel for the detection of virus levels, by methods that are well known in the art. Uninfected and untreated cells can be used as negative and positive controls of infection, respectively. Alternatively, the invention can be practiced by culturing the target cells in the presence of the test compound before infecting the cells with a strain of HIV. The invention also includes a method for identifying compounds that inhibit replication of HIV-1 in the cells of an animal, comprising: (a) contacting a test compound with wild-type virus isolate and separately with isolates of viruses having reduced sensitivity to botulinum 3-0- (3 ', 3'-dimethylsuccinyl); and (b) selecting test compounds that are more active against wild type virus isolate compared to virus isolates having reduced sensitivity to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. This invention further includes a method for identifying compounds that act by any of the mechanisms mentioned above, which comprises treating cells infected or transfected with HIV-1 with a compound, then analyzing the virus particles released by the cells treated with the compound by sectioning thin and transmission electron microscopy, by normal methods well known in the art. A compound acts by the above mechanism if particles are detected that exhibit spherical condensed nuclei that are acéntricos with respect to the viral particle and / or an electrodensa layer of increasing form just inside the viral membrane. For electronic microscopic studies, the Infected cells or centrifuged viral pellets obtained from the supernatant fluid can be contacted with a fixative, such as a freshly prepared glutaraldehyde or paraformaldehyde, and / or osmium tetraoxide or other fixative compatible with electron microscopy that has been known in the art. The virus from the supernatant fluid or cells is dehydrated and embedded in an electroradiator polymer such as an epoxy or methacrylate resin, thinly cut using an ultramicrotome stained using electrodense dyes such as uranyl acetate, and / or lead citrate , and it is seen in a transmission electron microscope. Uninfected and untreated cells can be used as negative and positive infection controls, respectively. Alternatively, the invention can be practiced by culturing the target cells in the presence of the test compound before infecting the cells with an HIV strain. The maturation defects caused by the compounds of the present invention are determined by the presence of morphologically abnormal viral particles, as compared to the controls, as described herein. For cell culture studies, cells infected with virus can be observed for the formation of syncytia, or the supernatant can be tested for the presence of HIV particles. The virus present in the supernatant can be collected to infect other candid cultures to determine infectivity. Also included in the invention is a method for determining whether an individual is infected with HIV-1, is susceptible to treatment by a compound that inhibits p25 processing, the method comprising taking the patient's blood, genotyping viral RNA and determining if the viral RNA contains mutations in the cleavage site of CA-SPl. The invention also includes a method for identifying compounds that act by the aforementioned mechanisms, which comprises testing by a combination of the methods described herein. The HIV Gag protein and fragments thereof for use in the assays mentioned above can be expressed or synthesized using a variety of methods familiar to those skilled in the art. Gag can be produced in a system of transcription and translation in vi tro using a lysate of rabbit meticulous cells. The Gag expressed in this system has been shown to be processed sequentially in a pattern similar to that observed in infected cells (Pettit, S.C. et al., J. Virol. 76: 10226-10233 (2002)). In addition, the Gag expressed by this method is capable of mounting on immature viral particles when fused to the cytoplasmic, retroviral, type D self-assembly domain, heterologous (Saalian, M. et al., J. Virol. 76: 10811-10820 (2002)). Plasmid pDAB72 available from the NIH AIDS Reagent Program can be used for this purpose (Erickson-Viitanen, S. et al., AIDS Res. Hum. Retroviruses., 5: 577-91 (1989); Sidhu MK et al. , Biotechniques, 18: 20, 22, 24 (1995)). Other in vitro transcription / translation systems based on wheat germ or bacterial lysates can also be used for this purpose. HIV Gag can also be expressed in transfected cells using a variety of commercially available expression vectors. Plasmid p55-GAG / GFP, available from the NIH AIDS Reagent Program, can be used to express the green fluorescent protein-Gag protein fusion protein of HIV in mammalian cells for drug interaction studies (Sandefur, S. et al., Virol. 72: 2723-2732 (1998)). This construction will allow the capture and purification of the Gag fusion protein using GFP-specific monoclonal antibodies. In addition, Gag can be expressed in cells using recombinant viral vectors, such as those used in the vaccinia, adenovirus, or baculovirus virus systems. Gag can also be expressed by infecting HIV cells or by transfecting cells with proviral DNA. Finally, Gag can be expressed in yeast or bacterial cells transformed with the appropriate expression vectors.

In addition to the Gag proteins expressed in cells or in vi tro using cell lysates, the peptides corresponding to the various regions of Gag can be synthesized commercially from the use of normal techniques and peptide synthesis. The invention further encompasses compounds identified by the methods of this invention and / or a compound that inhibits replication of HIV-1 according to the methods of this invention and pharmaceutical compositions comprising one or more compounds as described herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers. Pharmaceutical Compositions The compounds according to the present invention have been found to possess anti-retroviral activity, particularly anti-HIV. The salts and other formulations of the present invention are expected to have improved solubility in water, and improved oral bioavailability. Also, due to the improved solubility in water, it will be easier to formulate the salts of the present invention in pharmaceutical preparations. Additionally, the compounds according to the present invention are expected to have improved biodistribution properties. In one embodiment, the compounds are those of Formula I through XIII, in another, they are different compounds of the compounds of Formula I through XIII. This invention also includes a pharmaceutical composition comprising a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), but does not significantly affect other Gag processing steps, or inhibits the maturation of virus particles released from infected, treated cells, such as the compounds of Formula I through XIII. The invention includes a pharmaceutical composition comprising one or more compounds described herein, or pharmaceutically acceptable salts, esters or prodrugs thereof, and pharmaceutically acceptable carriers, wherein the compound is of Formulas I through XIII above, or Preferred, wherein the compound is selected from the group consisting of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid, 3-0- (3', 3'-dimethylsuccinyl) betulin, 3-0- (3 ', 3' -dimethylglutaryl) betulin, 3-0- (3 ', 3' dimethylsuccinyl) dihydrobetulinic acid, 3-0- (3 ', 3'-dimethylglutaryl) betulinic acid, (3', 3'-dimethylglutaryl) acid dihydrobetulinic, 3-0-diglycolyl-betulinic acid and 3-O-diglycolyl-dihydrobetulinic acid. The pharmaceutical compositions according to the invention further comprise one or more drugs selected from an antiviral agent, antifungal agent, anticancer agent or an agent immunostimulator. The pharmaceutical compositions of the present invention may comprise at least one of the compounds of Formulas I through XIII described herein. The pharmaceutical compositions according to the present invention may also further comprise other antiviral agents, such as, but not limited to, AZT (zidovudine, RETROVIR "", GlaxoSmithKine), 3TC (lamivudine, EPIVIR ™, GlaxoSmithKine), AZT + 3TC , (COMBIVIR "*, GlaxoSmithKIine), AZT + 3TC + abacavir (TRIZIVIR®, GlaxoSmithKine), ddl (didanosine, VIDEX®, Bristol-Myers Squibb), ddC (zalcitabine, HIVID®, Hoffmann-La Roche), D4T (Stavudine, ZERIT®, Bristol-Myers Squibb), abacavir (ZIAGEN®, GlaxoSmithKIine), tenofovir (VIREAD®, Gilead Sciences), nevirapine (VIRAMUNE®, Boehringer Ingelheim), delavirdine (Pfizer), emtricitabine (EMTRIVA®, Gilead Sciences), efavirenz (SUSTIVA®, DuPont Pharmaceuticals), saquinavir ( I? VIRASE®, FORTOVASE®, Hoffimann-LaRoche), ritonavir (? ORVIR®, Abbott Laboratories), indinavir (CRIXIVA? ™, Merck and Company), nelfinavir (VIRACEPT®, Pfizer), amprenavir (AGE? ERASE®, GlaxoSmithKIine ), adefovir (PREVEO®, HEPSERA®, Gilead Sciences), atazanavir (Bristol-Myers Squibb), fosamprenavir (LEXIVA®, GlaxoSmithKine) and hydroxyurea (HYDREA®, Bristol-Meyers Squibb), or any other antiretroviral drug or antibody in combination each other, or associated with a biologically based therapeutic product, such as, for example, peptides derived from gp41, enfurvitide (FUZEON®, Roche and Trimeris) and T-1249, or soluble CD4, antibodies to CD4, and conjugates of CD4 or anti -CD4, or as the present one is presented additionally. Additional suitable antiviral agents for optimal use with one of the compounds of Formulas I through XIII of the present invention may include, without limitation, amphotericin B (FUNGIZONE®); Ampligen (unrequired RNA) developed by Hemispherx Biopharma; BETASERO? ® (ß-interferon, Chiron); butylated hydroxytoluene; Carrosyn (polymannnoacetate); Castanospermina; Contracan (stearic acid derivative); Pharmatex cream (containing benzalkonium chloride); 5-unsubstituted derivative of zidovudine; penciclovir (DE? AVIR® Novartis); famciclovir (FAMVIR® Novartis); acyclovir (ZOVIRAX® GlaxoSmithKine); citofovir (VISTIDE "* Gilead); ganciclovir (CYTOVENE®, Hoffman LaRoche); dextran-sulfate; D-penicillamine (3-mercapto-D-valine); FOSCARNET® (trisodium forsphonioformate; AstraZeneca); fusidic acid; glycyrrhizin (a constituent of licorice root); HPA-23 (ammonium-21-tungsto-9-antimonate); ORNIDYL® (eflornithine, Aventis); nonoxynol; pentamidine isethionate (PE? TAM-300); Peptide T (octapeptide sequence, Peninsula Laboratories); Phenytoin (Pfizer); INH or isoniazid; ribavirin (VIRAZOLE®, Valeant Pharmaceuticals); rifabutin, ansamycin (MYCOBUTIN® Pfizer); CD4-IgG2 (Progenies Pharmaceuticals) or other molecules based on CD4 or containing CD4; Trimetrexate (Medimmune); suramine and analogues thereof (Bayer); and WELLFERON® (a-interferon, GlaxoSmithKIine). The pharmaceutical compositions of the present invention may also further comprise immunomodulators. Immunomodulators suitable for optimal use with a betulinic acid or betulin derivative of the present invention according to the present invention may include, without limitation and without limitation, ABPP (Bropririmine); Ampligen (unrequired RNA) Hemispherx Biopharma; anti-interferon-to-human antibody; ascorbic acid and derivatives thereof interferon-β; Ciamexon; cyclosporin; cimetidine; CL-246,738 colony stimulating factors, including GM-CSF dinitrochlorobenzene; HE2000 (Hollis-Eden Pharmaceuticals) inteferon- ?; glucan; hyperimmune gamma-globulin (Bayer) immuthiol (sodium diethylthiocarbamate); interleukin-1 (Hoffmann-LaRoche; Amgen), interleukin-2 (IL-2) (Chiron) isoprinosine (inosino-pranobex); Krestina; LC-9018 (Yakult) lentinan (Yamanouchi); LF1695; methionine-enkephalin Minho C; muramyl tripeptide, MTP-PE; Naltrexone (Barr Laboratories); RNA immunomodulator; REMUNE ™ (Immune Response Corporation); RETICULOSE® (Advanced Viral Research Corporation); shosaikoto; ginseng; thymic tumor factor; Thymopentin; thymosin factor 5; thymosin 1 (ZADAXIN®, SciClone), thymostimulin, TNF (tumor necrosis factor, Genentech), and vitamin preparations. The pharmaceutical compositions of the present invention may also further comprise anti-cancer therapeutic agents. Suitable anticancer therapeutics for optional use include an anticancer composition effective to inhibit neoplasia comprising a compound, or a pharmaceutically acceptable salt or prodrug of the anticancer agent, which can be used for combination therapy includes, but is not limited to, anticancer agents. alkylation, such as busulfan, cis-platinum, mitomycin C, and carboplatin antifungal agents, such as colchicine, vinblastine, taxol, such as paclitaxel (TAXOL®, Bristol-Meyers Squibb) docetaxel (TAXOTERE®, Aventis), topo inhibitors I , such as camptothecin, irinotecan and topotecan (HYCAMTIN®, GlaxoSmithKIine), topo and II inhibitors, such as doxorubicin, daunorubicin and etoposides such as VP16; RNA / DNA antimetabolites, such as 5-azacytidine, 5-fluorouracil and methotrexate, DNA antimetabolites, such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea, thioguanine, and antibodies such as trastuzumab (HERCEPTIN ®, Genentech), and rituximab (RITUXAN®, Genentech and Biogen-Idec), melphalan, chlorambucil, cyclophosphamide, ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin, bleomycin, mitoxantrone, eliptinium, fludarabine, octreotide, retinoic acid, tamoxifen, alanosine, and combinations thereof. The invention further provides methods for providing antibacterial therapeutic compounds, antiparasitic therapeutic compounds, and antifungal therapeutic compounds for use in combination with the compounds of the invention and pharmaceutically acceptable salts thereof. Examples of antibacterial therapeutics include compounds such as penicillins, ampicillin, amoxicillin, cyclacillin, epicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, cephalexin, cepharadine, cef doxyl, cefaclor, cefoxitin, cefotaxime, ceftizoxime, cefinoxoxine. , ceftriáxona, moxalactam, imipenem, clavulanate, timentina, sulbactam, erythromycin, neomycin, gentamicin, streptomycin, metronidazole, chloramphenicol, clindamycin, lincomycin, quinolones, rifampin, sulfonamides, bacitracin, polymyxin B, vancomycin, doxycycline, metacycline, minocycline, tetracycline, amphotericin B, cycloserine, ciprofloxacin, norfloxacin, isoniazid, ethambutol, and nalidixic acid, as well as the derivatives and altered forms of each of these compounds.

Examples of antiparasitic therapeutics include bithionol, diethylcarbamazine citrate, mebendazole, metrifonate, niclosamine, niridazole, oxamniquine and other derivatives of quinine, piperazine citrate, praziquantel, pyrantel pamoate and thiabendazole, as well as derivatives and altered forms of each of these compounds. Examples of antifungal therapeutic products include amphotericin B, clotrimazole, conazole nitrate, flucytosine, griseofulvin, ketoconazole and miconazole, as well as derivatives and alternating forms of each of these compounds. Antifungal compounds also include aculeacin A and papulocandin B. The preferred animal subject of the present invention is a mammal. By the term "mammal" an individual is proposed that corresponds to the Mammalia class. The invention is particularly useful in the treatment of human patients. The term "treatment" means the administration to subjects of a compound of Formulas I through XIII or a compound identified by one or more assays within the present invention, for purposes that may include prevention, improvement or cure of a pathology related to retrovirus. . These compounds for treating a subject that are identified by one or more assays within the present invention are identified as compounds having the ability to interrupt Gag processing, as described herein. The term "inhibits interaction" as used herein, means that it prevents, or reduces the rate, direct or indirect association of one or more molecules, peptides, proteins, enzymes, or receptors; or that prevents or reduces the normal activity of one or more molecules, peptides, proteins, enzymes or receptors. Medications are considered to be provided "in combination" with another if they are provided to the patient concurrently or if the time between the administration of each medication is such to allow an overlap of the biological activity. In a preferred embodiment, at least one compound of the above Formulas I to XIII comprises an individual pharmaceutical composition. The pharmaceutical compositions for administration according to the present invention may comprise at least one compound of the above Formulas I to Xlll or compounds identified by one or more assays within the present invention. These compounds for treating a subject that are identified by one or more assays within the present invention are identified as compounds that have the ability to interrupt Gag processing, described in the present. The compounds according to the present invention are further included in a pharmaceutically acceptable form, optionally combined with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purposes. The amounts and regimens for the administration of a compound of Formulas I through XIII according to the present invention can be easily determined by those skilled in the clinical art to treat a retroviral pathology. For example, administration can be by parenteral route, such as subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, transmucosal, ocular, rectal, intravaginal or buccal. Alternatively, or concurrently, administration may be by the oral route. The administration may be as a nasal or oral spray, or topically, such as powders, ointments, drops or a patch. The dose administered depends on the age, health and weight of the recipient, type of previous or concurrent treatment, if any, frequency of treatment, and the nature of the desired effect. The compounds and methods of the invention are additionally useful. For example, these compounds can be used prophylactically, to minimize the risk of infection. In another modality, you can use a compound to minimize the extent of the illness of an infected person. The invention also relates to novel compounds for treating HIV in an infected individual. In a modality, the invention is particularly useful in stimulating an immune response in a person infected with HIV. For example, by allowing non-infectious virus to be released from infected cells, these infected cells continue to be exposed to antigens and can be effectively targeted by the immune system or other therapies directed against these antigens. In another example, by continuing to allow the release of non-infectious viruses, an infected individual continues to develop an immune response to the virus without suffering the detrimental effects of this virus. The invention is also useful in expanding the scope of treatment, and offers new means to treat the disease in patients in need thereof. In another embodiment, the invention can be practiced in a patient who does not respond to another therapy for reasons other than viral resistance. For example, conventional methods for treating HIV, as known in the art, are associated with deleterious side effects. In one embodiment, the methods and compositions of the invention are useful in the treatment of a patient without a reduction in one or more harmful side effects. In one embodiment, the invention includes a method for treating a patient with a compound that does not have a particular side effect or has less than a particular side effect. The bioavailability of the drugs is also relevant in the treatment. In one embodiment, the invention can be practiced such that the compounds are more effectively absorbed in infected cells. In one embodiment, the invention encompasses improved methods for distributing a drug to an HIV infected cell. Compositions within the scope of this invention include all compositions comprising at least one compound of Formulas I through XIII above according to the present invention in an amount effective to achieve its intended purpose. As individual needs vary, the determination of optimal ranges of effective amounts of each component is within the skill of the technique. For example, a dose may comprise 0.0001 mg to 10 g / kg of body weight. Typical doses comprise about 0.1 to about 100 mg / kg of body weight. Preferred doses comprise about 1 to about 100 mg / kg of body weight of the active ingredient. The most preferred doses comprise about 5 to about 50 mg / kg of body weight.

Administration of a compound of the present invention may also optionally include prior, concurrent, subsequent therapy. or attached using immune system boosters or immunomodulators. In addition to the pharmacologically active compounds, a pharmaceutical composition of the present invention may also contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate the processing of the active compounds into preparations that can be used in a pharmaceutical form. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees and capsules, and also cgue preparations can be administered rectally, such as suppositories, as well as solutions suitable for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of the active compounds, together with the excipient. The pharmaceutical preparations of the present invention are made in a manner that is self-recognized, for example, by means of conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes. In this way, the preparations Pharmaceuticals for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of the granules, after adding suitable auxiliary products, if desired, or it is necessary, to obtain tablets or cores of dragons. Suitable excipients are, for example, fillers such as saccharide, for example, lactose or sucrose, mannitol or sorbitol; cellulose and / or calcium phosphate preparations, such as tricalcium phosphate or calcium acid phosphate; as well as binders such as starch paste, using, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents such as the aforementioned starches and also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, such as sodium alginate, may be added. The auxiliary products are all of the above, flow regulating and lubricating agents, for example, silica, talc, stearic acid and salts thereof, such as magnesium stearate or calcium stearate, and / or polyethylene glycol. Dragee groups are provided with suitable coatings that, if you want, are resistant to gastric juices. For this purpose, concentrated saccharide solutions may be used which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to reduce the gastric juice resistant coatings, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses. Other pharmaceutical preparations that can be used orally include soft-setting capsules made of gelatin, as well as sealed, soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Soft-fit capsules can contain the active compounds in the form of granules that can be mixed with fillers such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and optionally, stabilizers. In flat capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty acids or liquid paraffin. In addition, they can be add stabilizers. Possible pharmaceutical preparations which can be used rectally include, for example, suppositories which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use rectal gelatin capsules consisting of a combination of the active compounds with a base. Possible base materials include liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons. Formulations suitable for parenteral administration include aqueous solutions of the active compounds in the water soluble form, for example, water soluble salts. In addition, suspensions of the active compounds may be administered as appropriate suspensions of oily injection. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic esters of fatty acids, such as ethyl oleate, triglycerides or glycol 400. Aqueous injection suspensions which may contain substances that increase the viscosity of the suspension they include, for example, sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension may also contain stabilizers. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert eluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils such as cotton, peanut, corn, germ, olive oils, of castor bean and sesame, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and esters of sorbitan fatty acids, and mixtures thereof. The suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, sorbitol and polyoxyethylene sorbitol esters, cellulose, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and combinations thereof. Pharmaceutical compositions for topical administration include formulations suitable for administration to the skin, mucosa, lung or eye surfaces. The The compositions can be prepared as a pressurized or non-pressurized dry powder, liquid or suspension. The active ingredients in non-pressurized powder formulations can be mixed in a finely divided form in an inert pharmaceutically acceptable carrier, including but not limited to, mannitol, fructose, dextrose, sucrose, lactose, saccharin or other sugars or sweeteners. The pressurized composition may contain a compressed gas, such as nitrogen, or a liquefied gas propellant. The propellant may also contain an active ingredient on the surface, which may be a liquid or solid nonionic or anionic agent. The anionic agent may be in the form of a sodium salt. A formulation for use in the eye will comprise a pharmaceutically acceptable ophthalmic carrier, such as an ointment, oils, such as vegetable oils, or an encapsulating material. The regions of the eye to be treated include the corneal region, or internal regions such as the iris, lens, ciliary body, anterior chamber, posterior chamber, aqueous humor, vitreous humor, choroid or retina. The compositions for rectal administration may be in the form of suppositories. The compositions for use in the vagina may be in the form of suppositories, creams, foams or in implanted vaginal inserts.

The compositions can be administered in the form of liposomes. Liposomes can be made from phospholipids, phosphatidylcholines (lecithins) or other lipoidal, natural or synthetic compounds, as is known in the art. Any non-toxic pharmaceutically acceptable lipid capable of forming liposomes can be used. The liposomes can be multilamellar or monolamellar. A pharmaceutical formulation for systemic administration according to the invention can be formulated for enteral, parenteral or topical administration. In fact, all three types of formulation can be used simultaneously to achieve systemic administration of the active ingredient. Formulations suitable for oral administration include hard or soft gelatin capsules, dragees, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. The compounds described above or in the compounds identified by no or more assays within the present invention and which have the ability to interrupt the processing of Gag, can also be administered in the form of an implant when combined with a slow release carrier. , biodegradable. Alternatively, the compounds of the present invention can be formulated as a transdermal patch for continuous release of the active ingredient. The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations may be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is proposed that the present invention cover the modifications and variations of this invention with the condition that they come within the scope of the appended claims and their equivalents. EXAMPLES Example 1 Antiviral activity against primary HIV-1 isolates A strong virus inhibition assay was used to evaluate the anti-viral activity of DSB against primary HIV-1 isolates propagated in PMBC. Briefly, serial dilutions of DSB were made in the medium in 96-well tissue culture plates. 25-250 TCID50 of virus and 5 x 105 PBMC stimulated with PHA were added to each cavity. On days 1, 3 and 5 after infection, the medium was removed from each well and replaced with fresh medium containing DSB at the appropriate concentration. On day 7 after infection, the supernatant was removed from the culture of each cavity for p24 detection of virus replication and 50% inhibitory concentrations (IC50) were calculated by normal methods. Table 5 shows the potent anti-viral activity of DSB against a panel of primary HIV-1 isolates. DSB is displayed at activity levels similar to the approved drugs that were tested in parallel. In an important way, the activity of DSB was not restricted by the use of co-receptor. Table 5: Inhibitory activity (IC50) of DSB and two drugs tested against a panel of primary HIV-1 type B isolate. Isolated HIV-1 clinical denoted by * were isolated in Panacos. All other virus isolates were obtained from the NIH AIDS Reference Repository.

-IC, n (nM) Mean 6.1 4.4 28.8 Note: R5 and R4 refer to the chemokine receptors CCR5 and CXCR4, respectively. The toxicity of DSB was analyzed when incubated with PBMC stimulated with PHA for 7 days at a range of concentrations, then determining cell viability using the XTT method. The cytotoxic concentration at 50% was > 30 μM, which corresponds to a therapeutic index of approximately 5000. Example 2 Anti-viral activity of DSB against drug-resistant HIV-1 isolates: DSB activity was tested against a panel of resistant HIV-1 isolates to approved drugs. These viruses were obtained from the NIH Reference and AIDS Research Reagents Program. Assays were performed using viruses propagated in PBMCs with a p24 endpoint (above), or using cell line targets (MT-2 cells) and a terminal point of cell annihilation. The MT-2 test format was as follows. Serial dilutions of DSB, or with each approved drug, were prepared in 96-well plates. To each sample cavity was added medium containing MT-2 cells at 3 x 10 5 cells / mL and virus inoculum at a concentration necessary to result in 80% annihilation of the cell targets at 5 days after the infection (Pl). On day 5 after infection, virus induced cell annihilation was determined by the XTT method and the compound's inhibitory activity was determined. Table 6 shows the potent anti-viral activity of DSB against a panel of drug-resistant HIV-1 isolates. The results were not significantly different from those obtained with the panel of wild-type isolates (Table 5), demonstrating that DSB retains its activity against virus strains resistant to all major classes of approved drugs. Table 6: Inhibitory activity (IC50 nM) of DSB against a panel of drug-resistant HIV-1 isolates. The assays were performed in fresh PBMC with a p24 terminal point except for the NNRTI-resistant isolates crue were performed in MT-2 cells with a cell viability terminal point (XTT). ICso (nM) * Resistant to crease. Note: R5 and X4 refer to the chemokine receptors CCR5 and CXCR4, respectively. Example 3 DSB inhibits replication of HIV-1 in a later step in the virus life cycle To distinguish the inhibitory activity of DSB against early and late replication targets, a multinuclear activation of a galactosidase indicator (MAGI) was used. In this trial, the targets are HeLa cells that they stably express CD4, CXCR4, CCR5 and an indicator construct consisting of the galactosidase gene (modified to be localized to the nucleus) driven by a truncated HIV-1 LTR. The infection of these cells results in Tat expression that drives the activation of the β-galactosidase reporter gene. The expression of β-galactosidase in infected cells is detected using the chromogenic substrate X-gal. As shown in Table 7, the T-20 entry inhibitor, the NRTI AZT and the NNRTI nevirapine caused significant reductions in the expression of the β-galactosidase gene in MAGI cells infected with HIV-1 due to their ability to interrupt early steps in viral replication that affect the expression of the Tat protein. In contrast, the protease inhibitor indinavir targets a late step in the replication of the virus (after Tat expression) and does not prevent the expression of the β-galactosidase gene in this system. Similar results were obtained with DSB as with indinavir, indicating that DSB blocks the replication of the virus at a point in time after the completion of the integration of the proviral DNA and the synthesis of the viral transactivation protein (Table 7).

Table 7: Effect of DSB and input inhibitors (the peptide T-20 of CGP41), RT (AZT and Nevirapine) and protease (indinavir) in the expression of β-galactosidase in MAGI cells infected with HIV-1. DMSO control did not contain drug.

Kanamoto et al. (Antimicrob Agents Chemother., April; 45 (4): 1225-30, (2002)) have also reported that DSB acts in a late step in HIV replication. However, they reported that the compound inhibits the release of the virus from chronically infected cells. In contrast, those present using a variety of experimental systems indicate that DSB does not have a significant effect on virus release (e.g., Example 6). Example 4 DSB does not inhibit HIV-1 protease activity It was previously determined that DSB has no effect on the HIV-1 protease function using a cell-free fluorometric assay that characterized the activity of the enzyme by following excision of a protein. synthetic peptide substrate. The results of these experiments indicated that at concentrations up to 50 μg / mL that the DSB does not have effect on protease function. As a result of the observation that DSB blocks virus replication at a later stage, studies were also conducted using a recombinant form of the Gag protein, which is a more relevant system than the synthetic peptide substrate used in the initial trials. The use of the recombinant Gag protein as a substrate resulted in a similar experimental result. In these experiments, DSB does not interrupt the processing of the Gag protein mediated by protease at concentrations as high as 50 μg / mL. In contrast, as expected, the protease inhibitor indinavir blocked the processing of the Gag protein at 5 μg / mL (Figure 1). Example 5 DSB causes a defect in the final step of Gag processing (cleavage of CA-SPl) that has been associated with viral maturation defects. In order to better define the mechanism of action of DSB, a detailed examination of the viruses produced was undertaken. of cell lines infected with HIV-1 treated with DSB. Briefly, H9 cells chronically infected with HIV-1IIIB isolate were treated with DSB at 1 μg / mL for a period of 48 hours. Indinavir was used as a control. At the time point of 48 hours, the spent medium was removed and fresh medium containing the compound was added. A 24, 48 and 72 hours after the addition of the fresh compound, both the cells and the supernatant were recovered for analysis. The level of virus in the culture supernatant was determined and Western blots were used to characterize viral protein production in both cell-associated and cell-free viruses. As observed in the previous experiments, DSB did not cause a significant reduction in the amount of virus produced by chronically infected H9 cells, however, there was a defect in the processing of Gag in both cell-associated and cell-free viruses. This defect takes the form of an additional band in Western blots cgue corresponding to p25 (Figure 2). This p25 band results from incomplete processing of the capsid CA-SP1 precursor. Treatment with DSB of cell lines chronically infected with HIV-2 and SIV exhibited normal Gag processing consistent with the observed lack of antiviral activity against these viruses. The Gag processing defect seen in the presence of DSB is completely different from that observed with the protease inhibitor indinavir (Figure 2). As discussed above, mutations at the cleavage site of p25 to p24 that prevents processing are associated with defects in viral maturation and infectivity (Wiegers K. et al., J. Virol. 72: 2846-54 (1998) ).

As discussed above, (C.T. Wild et al., XIV Int. AIDS Conf. Barcelona, Spain, Abstract MoPeA3030, (July 2002)), abnormal processing of p25 to p24 is also seen in other incipient maturation defects. These include mutations in the late Gag domain (PTAP) or defects in the viral assembly mediated by TSG-101 cgue interrupts germination (Garrus, J.E et al., Cell, 107: 55-65, (2001); De irov, D. G. et al. , J. Virology 76: 105-117, (2002)). However, these mutations cause inhibition of virus release, while treatment with DSB does not have a significant effect on the release of the virus. The morphology of these mutant-maturing / germinating mutants is also quite different from that of the DSB treatment (see Example 6). In addition, mutations that interfere with viral RNA dimerization and lead to the production of immature virus with defective core structures give a similar Gag processing phenotype (Liang, C. et al., J. Virology, 73: 6147-6151 , (1999)). However, in these cases, the incorporation of RNA is inhibited and the morphology of the released particles is different from those following the treatment with DSB (see Example 6).

Example 6 Treatment with DSB affects the maturation of HIV-1 as determined by electron microscopy (EM) It has been shown that Gag mutations of HIV-1 that interrupt the processing of p25 to p24 cause non-infectious viral particles characterized by an internal morphology distinct from the normal virus (Wiegers K. et al., J Virol 72: 2846-54 (1998)). To determine if the virus generated in the presence of DSB exhibited this distinct morphology, the following experiment was carried out. HeLa cells were transfected with the infectious molecular clone pNL4-3 of HIV-1 and treated as described above with DSB. After treatment, the infected cells treated with DSB were fixed in glutaraldehyde and analyzed by MS. The results of this analysis are shown in Figure 3. These results are consistent with a compound that interrupts the processing of p25 to p24 that generates morphologically normal, non-infectious viral particles. 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB) is an example of a compound that interrupts the processing of p25 to p24 and potently inhibits replication of HIV-1. However, this compound does not inhibit the activity of PR, and its action is specific for the processing step of p25 to p24, not other steps in Gag processing. Additionally, treatment with DSB results in the abnormal morphology of the particle of HIV, described above. Example 7 Selection in vi tro for HIV-1 isolates resistant to compounds that interrupt the processing of the Gag viral capsid protein (CA) of the protein precursor CA-peptide separator 1. A series of experiments were carried out to select virus resistant to inhibition by 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid (DSB), an inhibitor of HIV-1 maturation. For each experiment, either the NL4-3 or RF virus isolate was used to infect two cell cultures. After infection, one culture was maintained in growth medium containing DSB, while the other culture was maintained in parallel in growth medium lacking DSB. In one experiment, H9 cells that have been infected with RF viruses were maintained in the presence or absence of increasing concentrations of DSB (0.05-16 μg / ml). The cells were passed every 2-3 days with the addition of fresh drug. Virus replication was monitored by p24 ELISA every 7 days. At this time, cultures treated with DSB with high levels of p24 were co-cultured with fresh, uninfected H9 cells at a 1: 1 ratio of cells in the presence of lx or 2x the original concentration of DSB. After 8 weeks of co-culture, the virus free of Cells were harvested from the culture containing DSB at a concentration of 1.6 μg / ml and used to infect fresh H9 cells. Every 7 days, the virus from cultures containing high levels of p24 was passed through cell-free infection in the presence of lx or 2x the original concentration of DSB. After 5 weeks of cell-free passage, the culture virus containing 3.2 Ug / ml of DSB was harvested and used to infect MT-2 cells. Virus replication in MT-2 cells was monitored by observing the formation of syncytia microscopically. Every 1-3 days, the cells were washed to remove the incoming viruses, and fresh drug was added to the culture under selection. Every 3-4 days, after the emergence of extensive syncytia in the culture under selection, the supernatant of each culture was collected and passed through a 0.45 μm filter to remove cell debris. This filtered virus supernatant was then used to infect fresh MT-2 cells in the presence or absence of fresh drug. After 4 rounds of cell-free infection (approximately 2 weeks in culture), with the drug concentration at 3.2 μg / ml, concentrated solutions of virus were collected and frozen for further analysis. In a second experiment, a concentrated virus solution derived from the molecular clone pNL4-3 (5.7 x 104 TCID50) it was used to infect MT-2 cells (6 x 10 6 cells) and the cultures were maintained in the presence or absence of DSB at a concentration of 1.6 μg / ml. Every 1-3 days, the cells were washed to remove the virus from the entrance, fresh drug was added to the culture under selection. The replication of the virus was monitored by microscopically observing the formation of syncytia. Every 3-7 days, after the emergence of extensive syncytia in the culture under selection, the supernatant of each culture was collected and passed through a 0.45 μm filter to remove cell debris. This filtered virus supernatant was then used to infect fresh MT-2 cells in the presence or absence of fresh drug. After 5 rounds of cell-free infection, and each subsequent round, the drug concentration doubled. After 10 rounds of cell-free infection (approximately 7 weeks in culture), then the drug concentration reached 12.8 μg / ml, concentrated solutions of virus were collected and frozen for further analysis. Example 8 Characterization of HIV-1 isolates selected for resistance to compounds that interrupt the processing of the Gag viral capsid protein (CA) of the protein precursor CA-peptide separator 1.

Concentrated solutions of virus derivatives as described above were further analyzed in both phenotypic and genotypic ways to characterize the nature of their drug resistance. The resistance of the viruses to 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid (DSB) was determined in virus replication assays. Briefly, concentrated virus solutions were first titrated to H9 cells by quantifying p24 levels (by ELISA) in cultures 8 days after infection with 4-fold serial dilutions of virus. The virus entry was then normalized for a second assay in which each virus was cultured for 8 days in the presence of 4-fold serial dilutions of the drug. The IC50 for each virus was determined as the dilution of the drug that reduced the p24 endpoint level by 50% compared to the control without drug. In these tests, the two concentrated solutions of independently derived viruses resulted in IC 50 values greater than 2 A for DSB, compared to an IC 50 of 0.02 μ M for viruses that have been grown in parallel in the absence of the drug. In a subsequent series of experiments, the A364V mutation was managed in the AD? proviral of? L4-3 of HIV-1, which was subsequently transfected into HeLa cells. The resulting virus was collected and used to test DSB activity in a test of viral replication, as described above. In these assays, the DSB-resistant virus resulted in an IC50 value of 0.1 μM whereas wild-type NL4-3 gave an IC50 value of 0.01 μM to determine if the resistant viruses were able to escape the excision defect of CA-SPl elicited by DSB in the wild-type virus, concentrated solutions of each virus grown in either the presence or absence of the drug were analyzed by Western blotting. The viruses were pelleted through a 20% sucrose cushion of filtered culture supernatants that were harvested 60 hours after infection and 18 hours after the cells had been washed and fresh drug added. The viruses were lysed, and the amount of each virus was normalized by quantifying p24 levels in each sample. Western blot analysis of the viral proteins in each sample demonstrated that drug-resistant viruses do not contain the CA-SP1 product in the presence of DSB, confirming that these viruses were resistant to the effects of the drug in this cleavage event. Finally, to identify the genetic determinants of DSB resistance, the complete PR and Gag coding regions of the viral genomes were amplified by high fidelity RT-PCR for sequencing.

Viral RNA was purified from each virus lysate prepared as described above and digested with D? Asa to remove any AD? pollutant. The RT-PCR products were then gel purified to remove any non-specific PCR product. Finally, both strands of the fragments of AD? The resulting sequences were sequenced using overlap of a series of primers. Two amino acid mutations were identified that are independently capable of conferring resistance to DSB, a substitution of alanine to valine in the Gag polyprotein at residue 364 in isolate? L4-3 and at residue 366 in isolate RF. These are the first and third residues, respectively, the 3 'address of the CA-SP1 cleavage site (the? -term of SPI). Alanine is highly conserved in each of these positions throughout the types of HIV-1 in the database. Example 9 Determinants of activity of the HIV-1 maturation inhibitor, DSB, correlate to the CA-SP1 domain of the Gag protein To further define the molecular determinants of DSB activity, a series of chimeric viruses were prepared of which They inserted residues near the cleavage site CA-SP1 of the DSB-sensitive virus, HIV-1 in the analogue region of the Gag protein.

DSB-resistant retrovirus, SIV. The characterization of these SIV / HIV chimeras (SHIV) with respect to DSB activity allowed further identification of minimal HIV-1 Gag sequences both necessary and sufficient for DSB activity. Materials and methods Construction of SHIV DNA clones Three panels of SHIV were generated using SIVmac239 resistant to DSB as the residue binding structure of the Gag CA-SPl region of DSB resistant pNL4-3 of HIV-1. The CA-SPl sequences for these SHIV constructs are shown in Figure 20. All DNA mutagenesis was carried out using the PCR-overlap-PCR strategy (Ho, SN, HD Hunt, RM Horton, JK Pullen, and LR Pease (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77: 51-59) and other normal molecular cloning approaches (Sambrook, J., EF Fritsch, and T. Maniatis (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Cell culture and DNA transfection HeLa cells were maintained in DMEM (Invitrogen) (supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin) and passed to confluence. All plasmid DNAs were prepared using the midiprep kit (Qiagen). HeLa cells were transfected with wild type SIVmac239, HIV-1 pNL4-3 or SHIV proviral DNAs using the FuGENE 6 transfection reagent (Roche). Briefly, the cells were seeded in a 6-well plate (Corning) at a concentration of 1.5 x 10 5 cells per well the day before use and allowed to reach 60 to 80% confluence on the day of transfection. For each transfection, 3 μl of FuGE? E 6 was diluted in 100 μl of serum-free DMEM followed by the addition of 1 μg of AD ?. After gentle mixing, the mixture of ADβ-lipid complexes was gently added dropwise to the cells in 1.5 ml of complete DMEM medium. Twenty-four hours after transfection, the medium containing the AD? -FuGE? E 6 complexes was removed and 1.5 ml of fresh DMEM was added to the transfected cells. At 48 hours after transfection, both the cells and the culture supernatant were harvested for further analysis. SDS-PAGE and Western Blotting To characterize the effect of incorporation of residues from the CA-SP1 domain of HIV-1 in SIV into viral particle production and processing of the Gag polyprotein, Western blotting was performed. Briefly, 48 hours after transfection, culture medium containing viral particles was collected and it was clarified by centrifugation at 2,000 rpm and 4 ° C for 20 minutes in a Sorvall RT 6000B centrifuge. The supernatants containing the particles were then concentrated through a pad of 20% sucrose in a microcentrifuge at 13,000 rpm at 4 ° C for 120 minutes and the pellets were resuspended in lysis buffer (150 mM Tris-HCl, Triton X-100 5%, deoxycholate 1%, 0.1% sodium dodecyl sulfate [SDS], pH 8.0). For cell lysates, at 48 hours post-transfection, the cells were washed once with PBS and lysed) 150 mM Tris-HCl, 5% Triton X-100, 1% deoxycholate, pH 8.0) followed by centrifugation at 13,000 rom at 4 ° C for 5 minutes to remove nuclear fractions. Viral pellets and cell lysates were separated on a Bis-Tris gel of 12% NuPAGE (Invitrogen) and transfected to a nitrocellulose membrane (Invitrogen) followed by blocking in PBS buffer containing 5% Tween and 5% dry milk powder. The membrane was incubated with anti-p27 McAb from SIVmac251 (NIH AIDS Research and Reference Reagent Program) and hybridized with goat anti-mouse horseradish peroxidase (Sigma). For the membrane containing HIV-1 proteins the membrane was incubated with immunoglobulin from patients infected with HIV-1 (HIV-Ig) (NIH AIDS Reseca and Reference Reagent Program) and hybridized with goat anti-human horseradish peroxidase (Sigma). The immune complex was visualized with a system ECL (Amersham Pharmacia Biotech) according to the instructions manufactured by the manufacturer. Effect of DBS on Gacr processing of SHIV To address the effect of Gag substitutions on the ability of DBS to inhibit CA-SP1 processing, HeLa cells were transfected with wild-type SIVmac239 DNA from pNL4-3 of HIV-1 or SHIV provirales when using the procedure described above. The DSB at a concentration of 1 μg / ml or DMSO (control without drug) was maintained throughout the entire culture period and was performed as described previously SDS-PAGE / Western Blot to analyze viral proteins derived from these cultures. Only viruses that were completely sensitive to the effects of DSB were classified as sensitive, whereas those with residual resistance were classified as resistant. As such, the classification in experiment 9 differs from the previous classification, since viruses that were not completely resistant were classified as sensitive. For this reason, SHIV.DM was classified as resistant to DSB in experiment 9, but was previously classified as DSB-sensitive.

Results Characterization of SHIV from Gag Three panels of HIV-1 / SIV Gag chimeras were prepared (Figure 20). Panel 1 consisted of virus containing a structure in which residues of the SPI domain of HIV-1 have been inserted. The HIV-1 inserts in these chimeras varied in size from individual point substitution (SHV DA) to complete replacement of the SPI domain of SIV with the SPI sequence of HIV-1 (SHIV DN). Panel 2 consisted of viruses containing the same SP-1 substitutions plus the inclusion of the two C-terminal CA residues of HIV-1 (LM to VL). The SHIVs in panel 3 were identical to those in panel 2 except that, in addition to the substitutions in the SPI domain and the two C-terminal HIV-1 CA residues (LM to VL), each of these chimeras also incorporated a change from Q (SIV) to H (HIV-1) in the 6th? m position in the 5 'direction (P6) from the CA-SPl cleavage site. The level of viral particle release of transfected cells and the Gag processing profile for each of the SHIVs was determined (Figure 21). All of the SHIVs in panel 1 behaved similarly with respect to particle production and Gag processing; these chimeric viruses exhibited almost wild-type levels of particle production and a normal profile of Gag processing compared to the SIV of origin. The majority of the cumes in panel 2 characterized by normal Gag processing profiles, while the FC cellular expression of SHIV and 11 was somewhat reduced (Figure 21A). For all SHIVs in panel 2, the amount of virus production in relation to cell-associated expression was comparable to the SIV of origin. In contrast to the SHIVs of panels 1 and 2, most of the chimeras in panel 3 exhibited defects in Gag processing. Of these, GH, Gl and 23 exhibited a normal Gag processing profile. It is clear from these results that the six change residues from Q to H in the 5 'direction from the CA-SPl cleavage site affect the ability of SIV-PR to process chimeric Gag proteins. The results of panel 2 of SHIV, comprised in them that have HIV-1 residues in both the SPI and CA domains, are shown in Figure 21. As described above, this SHIV panel is identical to panel 1 except that In addition to substitutions in the SPI domain, each of these chimeras also incorporates the 2 C-terminal residues of HIV-1 Gag (VL of HIV-1 replaces LM of SIV). As with the viruses in panel 1, the chimeras in panel 2 were characterized by normal Gag processing profiles (Figure 21) and in the meantime something was reduced (Figure 21A), the cell expression of FC and 11 of the SHIVs, the The proportion of viruses found in the supernatant of cells transfected with cgue DNA encoded for these SHIV was proportional to the level of viral release observed for all viruses in panel 2. The results of panel 3 of SHIV with viruses containing HIV-1 residues both in the SPI domains as Gag CA are shown in Figure 21. This SHIV panel is identical to those in panel 1 except that in addition to the substitutions in the domain is SPI, each of these chimeras also incorporates the two C residues -terminals of HIV-1 (LM from SIV to VI for HIV-1) plus a change from Q (SIV) to H (HIV-1) in the 6th position at the 5 'direction of the CA-SPl cleavage site. Unlike the first two panels, most of the chimeras in panel 3 exhibited defects in Gag processing (Figure 21). Of these SHIV, only GH, Gl and 23 exhibited a normal Gag processing profile. It is clear from these results that the six change residues from Q to H in the 5 'direction from the CA-SPl cleavage site have a significant effect on the ability of the SIV protease to process the resulting chimeric Gag protein. Sensitivity to SHIV from Gag to DSB Each of the SHIVs in panels 1, 2 and 3 were characterized for their sensitivity to DSB. As described above, DSB interrupts the cleavage of HIV-1 CA-SP1 This leads to the release of non-infectious viral particles that exhibit normal core morphology (Li, F., R. Goila-Gaur, K. Salzwedel, NR Kilgore, M. Reddick, C. Matallana, A. Castillo, D. Zoumplis , DE Martin, JM Orenstein, GP Allaway, E. 0. Freed, and CT Wild. (2003) PA-457: a potent HIV inhibitor that disrupts core condensation by targeting a step in Gag processing Proc. Nati. Acad. Sci. USA 100: 13555-13560.). In the current study, cells expressing SHIV were cultured in the presence of DSB at a concentration of 1 μg / ml for a period of 48 hours. At the end of this time, the virus was harvested from the culture supernatant and the Gag processing profile was analyzed for each chimeric virus and compared to the processing of the Gag protein in the absence of the compound (Figure 22). As can be seen in Figure 22, none of the SPI of SPI (panel 1) exhibited sensitivity to DSB. Even SHIV DN, which contains the SPI domain of HIV-1 complete, exhibited a normal profile of Gag processing in the presence of the compound or concentrations in excess of the IC50 determined in tissue culture (Figure 22) (Li et al.) . The results of the panel 1 chimeras demonstrate that the determinants of DSB activity include Gag regions of HIV-1 outside the SPI domain. The results of the SHIVs of panel 2 are identical to those observed for panel 1.

In a specific manner, none of these CA-SPl cassettes exhibited sensitivity to DSB (Figure 22). Since the panel 2 viruses contained the C-terminal VL amino acid residues, in addition to the SP1 sequence of HIV-1, these results show that HIV-1 Gag residues different from those immediately flanking the HIV-1 site. Excision of CA-SPl play a role in the sensitivity of DSB. As shown in Figure 22A, DSB does not interrupt the processing of CA-SP1 for a subset of the SHIVs in panel 3. Of these chimeric viruses, SHIV 23 and Gl exhibited a level of DSB sensitivity comparable to isolated NL4-3 of prototypic HIV-1 (Figure 22A). In repeated experiments, the magnitude of the effect of the compound on each of these viruses, as determined by the relative ratios of CA-SP1 to CA, was almost identical. Also in panel 3, SHIV GH exhibited some level of sensitivity to DSB, however, at a qualitative level, the activity observed against this virus was reduced compared to that observed with SHIV 23 and Gl. For SHIV GH, the effect of DSB on Gag processing was reduced to the point where the very weak CA-SPl band observed in the immunoblot is not apparent in Figure 22A. A 5X increase in the amount of viral proteins loaded in the gel improved the sensitivity of the transfer assay Western at a level that allowed the DSB-mediated processing defect to be observed for the SHIV GH (Figure 22B). For the rest of the viruses in panel 3, the effect of the compound can not be terminated due to defects related to the sequence in the Gag processing (Figure 21B). The results of the SHIVs in panel 3 indicate that the His residue in the 6th? P, at position in the 5 'direction from the CA-SPl cleavage site plays an important role in the sensitivity of DSB and what significant portions of both CA as of SPI beyond the immediate vicinity of the cleavage site are necessary for DSB activity. Analysis We prepared (Figure 20) and characterized three panels of SIV / HIV-1 chimeras. All SHIV of panels 1 and 2 behaved similarly with respect to the effect of SPI or CA / SP1 substitutions on Gag protein processing, viral particle release and DSB sensitivity (Figures 21 and 22). Although some of the viruses in panel 2 (ie FD and 11) were characterized by a reduction in the level of viral particle production, this effect was most likely due to a complete reduction in the amount of virus generated by the cells transfected (Figure 21A). The fact that none of the SHIV of panels 1 and 2 was sensitive to DSB as determined by the effect of the compound on the CA-SP1 processing indicates that the Gag sequence outside the immediate vicinity of the HIV-1 CA-SPl cleavage site plays a critical role in the activity of DSB. In contrast to the viruses in panels 1 and 2, three members of the SHIVs of panel 3 exhibited some degree of sensitivity to DSB. Among these viruses, SHIV 23 and Gl exhibited sensitivity type NL4-3 to DSB while SHIV GH exhibited a somewhat reduced level of sensitivity to the compound. Of the additional viruses in panel 3, the effect of CA-SP1 substitutions on Gag processing makes it impossible to determine the effect of the compound on the remaining chimeras. The effect of DSB on the Gag processing profile of these three panels of the SHIV of CA-SPl suggests that the determinants of compound activity include a relatively large region of Gag flanking the CA-SPl cleavage site. Comparison of the DSB activity against SHIV 11 of panel 2 (insensitive) with SHIV 23 of panel 3 (sensitive) indicates that the His residue located at 6 o'clock position CA in the 5 'direction of the cleavage site it is critical to the activity of DSB (Figure 22B). Consistent with this observation, in vitro resistance selection studies have identified a mutation in this position that confers the same level of insensitivity to DSB.

The results here provide the existence of both high and low affinity binding sites in the CA-SP1 region. A low affinity interaction will result in partial DSB activity (ie, SIVm3) whereas a high affinity binding will give complete sensitivity to the compound (i.e., SHIV 23). EXAMPLE 10 Genotyping of viral isolates As shown above, sequence polymorphisms in HIV have been shown to correspond to the ability of a virus to replicate in the presence of DSB. The majority of sequence polymorphisms are grouped in Gag, especially in the region coding for CA-SP1. Accordingly, the genotyping of a viral isolate can be used to easily determine whether replication of this virus by DSB, or any other compound that interferes with p25 processing, is likely to be inhibited. The results of this genotyping are useful in, for example, the determination of whether a viral infection can be treated in a patient with DSB, or any other compound that interferes with p25 processing in a similar manner, or in determining the emergence of resistant variants during a course of treatment with DSB. Genotyping can be performed by several methods. In some modalities, genotyping is performed by sequencing. Methods An individual frozen aliquot (approximate volume 1.2 ml) of plasma is obtained from each patient. The plasma sample is stored at -70 ° C until it is ready for processing. Each sample is identified using the three-digit patient ID number. On the day of processing, each plasma sample is rapidly thawed in a 37 ° C water bath and then placed on ice. A 140 μl aliquot of plasma is removed to a separate tube for nucleic acid purification using the QIAamp Mini Viral RNA Purification Purification Kit (Qiagen). The rest of the plasma sample is transferred to a separate tube for brief centrifugation at low speed (3 minutes at 8,000 rpm) to clarify the plasma. One ml of the clarified supernatant is then transferred to a fresh tube and centrifuged for 2 hours at full speed (13,000 rpm) to pellet the virus. The supernatant is carefully removed using a pipette and transferred to a separate tube for storage at -70 ° C as a precaution against possible disruption of the viral pellet. The viral pellet is resuspended in 140 μl of PBS and stored at -70 ° C as a backup sample in the event that sufficient product is not obtained of RT-PCR of the initial aliquot of non-sedimented plasma.

Table 8 Gag HIV-1 primers CA-SP1 primers of HIV-1 conserved in type B: After purification, the viral RNA is eluted in a final volume of approximately 60 μl. Are used initially only 7 μl of this concentrated solution as a template for reverse transcription using the StrataScrip First Strand Synthesis System (Stratagene). The rest of the concentrated RNA solution is stored at -70 ° C as a backup. The primer for reverse transcription (R + 625) binds approximately 625 bp in the 6 'direction of the CA-SPl cleavage site. All primers to be used for RT-PCR and sequencing in this project have been designated to bind to Gag regions that are highly conserved among HIV-1 type B isolates and have been validated using plasma samples from different patients. The reverse transcription reaction is performed in a total volume of 50 μl. Only 5 μl of this reaction is initially used as a template for PCR amplification of the CA-SPl region using the PicoMaxx High Fidelity PCR Master Mix (Stratagene). The rest of the reaction is stored at -20 ° C as a backup. A "nested" PCR strategy is used in two steps that has been found to It provides a high performance of very clean DNA product. The forward and reverse primers for the first round PCR amplification (F-625 and R + 525) bind approximately 625 bp of the 5 'and 525 bp direction in the 3' direction of the CA-SPl cleavage site, respectively. It is not typically visible to the product by agarose gel analysis after this first PCR reaction. The initial PCR reaction is performed in a total volume of 50 μl. After cyclization, 5 μl of this reaction is removed and used as a template in a second-round "nested" PCR reaction using primers F-575 and R-450, which bind to the gag regions that they are internal to the regions to which the initial pair of primers is fixed. The rest of the first round PCR reaction is stored at -20 ° C as a backup. The forward and reverse primers for the second round PCR reaction bind approximately 575 bp in the 5 'and 450 bp direction in the 3' direction of the CA-SPl cleavage site, respectively. Five μl of the final "nested" PCR reaction is removed by analysis of DNA products by agarose gel electrophoresis. If, as expected, the reaction contains only a prominent band of the size provided for the desired product (approximately 1.1 kb), and the yield is estimated to be sufficient to allow sequencing (ie, approximately 200 ng total), then 40 μl of the reaction is removed for purification of the DNA product using the MinElute PCR Purification Kit (Qiagen).

The remaining approximately 5 μl of the reaction is stored at -20 ° C as a backup. After elution of the purified DNA product, an appropriate volume (corresponding to at least 40 ng of DNA) is transferred to two tubes, each containing a different sequencing primer (one for each DNA strand). The strand sequencing primer "+" (F-300) binds approximately 300 bp in the 5 'direction of the CA-SPl cleavage site. The strand sequencing primer "-" (R + 275) sets approximately 275 bp in the 3 'direction of the CA-SPl cleavage site. The template / primer mixture is sent for sequencing and analysis. The rest of the purified DNA product is stored at -20 ° C as a backup. The resulting sequence analysis provides overlapping readings for each strand of DNA to help resolve any ambiguity in any individual sequencing reaction. If ambiguities are found in both sequencing reactions, additional sequencing reactions are analyzed using alternate validated sequencing primers in the case where the problem is in the homogeneity of the Gag region to which the original sequencing primers are fixed. These will include two "+" strand primers (F-375 and F-125) that fix approximately 375 and 125 bp in the 5 'direction and two strand primers "-" (R + 100 and R + 400) that bind approximately 100 and 400 bp in the 3 'direction of the CA-SP1 cleavage site, respectively. If the final "nested" PCR reaction contains significant background bands when analyzed on an agarose gel (ie, greater than about 10% of the total yield), or if the sequencing fails to produce clear sequence, then the product of The desired DNA is purified by running the complete PCR reaction (re-amplified from a back-up sample if necessary) on an agarose gel and cleaving the desired band. The DNA is then purified from the agarose using the QIAEX II Gel Extraction Kit (Qiagen) and the eluate is prepared for sequencing as described above. If the PCR reaction fails to produce sufficient product for sequencing, then additional RT-PCR or PCR reactions may be run, if necessary, using any of the backup samples summarized above and additional sets of validated primers, including four forward primers which fix approximately 550, 375, 300 and 125 bp in the 5 'direction (F- 550, F-375, F-300 and F-125) and three inverse primers that fix approximately 400, 275, and 100 bp in the 3 'direction (R + 400, R + 275 and R + 100) of the excision site of CA-SPl. For example, excellent results have been obtained using the R + 525 primer for reverse transcription and primers F-575 and R + 450 for individual round PCR amplification. The F-550 and R + 400 primers work well for PCR amplification. The resulting genotype is then mapped to the genotype of the viruses identified elsewhere herein to determine whether the virus is inhibited or not inhibited by DSB. Further confirmation of the genotyping results can be obtained by monitoring experiments of the viral isolates. Example 11 Genetic load during treatment with DSB To determine the change in the HIV-1 phenotype during a course of treatment with DSB, the genotype of the virus population in each patient before dosing and at the end of the study (day 28) it is obtained to determine if a mutation has occurred during the course of the treatment. If mutations are identified at the end of the study, samples that were not present before dosing, then intermediate samples taken on days 7 and 10 after dosing they are also genotyped to determine when the mutation occurred. Using this method, mutations are determined which may occur in the total population of the virus during the course of the study. A mutation will be identified as a variation greater than 25% in the designation of amino acids for a given codon. Once a mutation has been identified, a chromatogram of the wild-type data is reviewed to determine the amino acid identities at that position in the minor virus populations. If none of the resistance mutations listed above are identified using these criteria, then the chromatograms of each reaction are reviewed to determine if some minor species (less than 25% of the total population) are present in each of the relevant positions. Having now fully described this invention, it will be understood by those skilled in the art that it may be performed within a broad and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any modality thereof. All patents, applications and publications cited herein are fully incorporated by reference in their entirety.

It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention.

Claims (2)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. Method for treating HIV-1 infection in a patient, characterized in that it comprises administering a compound that inhibits the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but without significantly affecting other processing steps of GAg. 2. Method according to claim 1, characterized in that the method does not significantly reduce the amount of virions released from infected cells in the patient. Method according to claim 1, characterized in that the method has no significant effect on the amount of RNA incorporation in virions released from the patient's cells. Method according to claim 1, characterized in that the compound inhibits the maturation of virions released in cells of the patient. Method according to claim 1, characterized in that a majority of the virions released from the patient's cells exhibit an altered phenotype with relation to the wild-type virions, wherein the phenotype is selected from the group consisting of: (a) virions having electrodense, spherical nuclei that are acentric with respect to the viral particle; (b) virions that have increasingly dense electrodense layers that are just inside the viral membrane; (c) virions that have reduced infectivity or do not have infectivity; and (d) the combination of any of (a) through (c). 6. Method for treating HIV-1 infection in a patient, characterized in that it comprises administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), wherein the compound binds to a polypeptide encoded by a polynucleotide having a sequence at least about 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) approximately nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) about nucleotides 1858-1920 of SEQ ID NO: 19. 7. Method according to claim 6, characterized in that the polynucleotide has a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) approximately nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) approximately nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 8. • Method for treating HIV-1 infection in a patient, characterized in that it comprises administering a compound that inhibits the processing of Gag viral p25 protein ( CA-SP1) to p24 (CA), and wherein the compound binds to a polypeptide having a sequence at least about 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 9. Method according to claim 8, characterized in that the polypeptide has a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? A? PDCK ILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (c) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118); and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). 10. Method for treating HIV-1 infection in a patient, characterized in that it comprises administering a compound that binds to the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), and wherein the compound is joins a cgue polypeptide having a sequence selected from any of SEQ ID NOS: 121 to 443. 11. Method according to any of claims 1-10, characterized in that HIV-1 infection in a patient comprises infection with an HIV -1 that does not respond to other HIV-1 therapies. Method according to any of claims 1-10, characterized in that the patient with HIV-1 infection does not respond otherwise to other HIV-1 therapies. 13. Method according to any of claims 1-10, characterized in that it further comprises administering to the patient a further compound which is an immunomodulatory agent, an anticancer agent, an antibacterial agent, an antifungal agent, an antiviral agent or a combination thereof. same. 14. Method according to any of claims 1-10, characterized in that the patient is administered the compound in combination with at least one other antiviral agent. 15. Method according to claim 14, characterized in that the other antiviral agent is selected from the group consisting of: zidovudine; lamivudine; didanosine; zalcitabine; stavudine; abacavir; nevirapine; delavirdine; emtricitabine; efavirenz; sacguinavir; ritonavir; indinavir; nelfinavir; amprenavir; tenofovir; adefovir; atazanavir; fosamprenavir; hydroxyurea; AL-721; ampligene, butylated hydroxytoluene; polimannoacetate, castanospermine; contracan; pharmatex cream, CS-87, penciclovir, famciclovir, acyclovir, citofovir, ganciclovir, dextran-sulfate, D-penicillamine phosphono trisodium phosphonoformate, fusidic acid, HPA-23, eflornithine, nonoxynol, pentamidine isethionate, T peptide, phenytoin, isoniazid, ribavirin , rifabutin, ansamycin, trimetrexate, SK-818; suramin; UAOOl; enfuvirtide; gp41-peptides derived from gp41; antibodies to CD4; Soluble CD4; molecules that contain CD4; CD4-IgG2; and combinations thereof. 16. Method for inhibiting the processing of the p25 viral protein from Gag (CA-SP1) to p24 (CA), characterized in that it comprises administering a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 ( CA), but does not significantly affect other Gag processing steps. 17. Method for inhibiting the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), characterized in that it comprises administering a compound that binds to a polypeptide encoded by a polynucleotide having a sequence of at least about 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) approximately nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 18. Method according to claim 17, characterized in that the polynucleotide has a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) approximately nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) about nucleotides 1858-1920 of SEQ ID NO: 19. 19. Method for inhibiting the processing of Gag viral p25 protein (CA-SP1) to p24 (CA), characterized in that it comprises administering a compound that binds to a polypeptide having a sequence at least about 70% identical to a sequence selected from group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 20. Method according to claim 19, characterized in that the polypeptide has a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 2. 3); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 21. Method for inhibiting the processing of Gag viral p25 protein (CA-SP1) to p24 (CA), characterized in that it comprises administering a compound that binds to a polypeptide having a sequence selected from any of SEQ ID NO: 121 to 443. 22. Method for treating human blood products, characterized in that it comprises contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), but does not affect Significantly other Gag processing steps. 23. Method according to claim 22, characterized in that the inhibition does not significantly reduce the amount of virions released from infected cells treated with the compound. Method according to claim 22, characterized in that the inhibition has no significant effect on the amount of RNA incorporated in the virions released from infected cells treated with the compound. 25. Method according to claim 22, characterized in that the compound inhibits the maturation of released virions and infected cells treated with the compound. 26. Method according to claim 22, characterized in that a majority of the virions released from treated infected cells exhibit an altered phenotype in relation to the wild-type virions wherein the phenotype is selected from the group consisting of: (a) virions that have electrodense, spherical nuclei that are acentric with respect to the viral particle; (b) virions that have increasingly dense electrodense layers that are just inside the viral membrane; (c) virions that have reduced infectivity or do not have infectivity; and (d) the combination of any of (a) through (c). 27. Method for treating human blood products, characterized in that it comprises contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SP1) to p24 (CA), where the compound binds to a polypeptide encoded by a polynucleotide having a sequence at least about 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) approximately nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) approximately nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 28. Method according to claim 27, characterized in that the polynucleotide has a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) approximately nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 29. Method for treating human blood products, characterized in that it comprises contacting the blood products with a compound that inhibits the processing of the viral p25 protein from Gag (CA-SPl) to p24 (CA), in wherein the compound binds to a polypeptide having a sequence at least about 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 30. Method according to claim 29, characterized in that the polypeptide has a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 31. Method for treating human blood products, characterized in that it comprises contacting the blood products with a compound that inhibits the processing of the p25 viral protein from Gag (CA-SP1) to p24 (CA), where the compound binds to a polypeptide having a sequence selected from any of SEQ ID NO: 121 to 443. 32. Method according to any of claims 1 to 10, 16 to 31, characterized in that the compound inhibits the interaction of HIV protease with CA -SPl. 33. Method according to claim 32, characterized in that the compound directly inhibits the interaction of the HIV protease with CA-SP1. 34. Method according to claim 32, characterized in that the compound indirectly inhibits the interaction of the HIV protease with CA-SP1. 35. Method according to any of claims 1 to 10, 16 to 31, characterized in that the compound binds to the viral Gag protein such that the interaction of the HIV protease with CA-SP1 is inhibited. 36. Method according to any of claims 1 to 10, 16 to 31, characterized in that the The compound binds at or near the cleavage site of the Gag p25 viral protein (CA-SP1) to p24 (CA). 37. Method according to any of claims 1 to 10, 16 to 31, characterized in that the compound is a dimethylsuccinyl-betulinic acid derivative or a dimethylsuccinyl-betulin derivative. 38. Method according to claim 37, characterized in that the compound is selected from the group consisting of: 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid; 3-O- (3 ', 3' -dimethylsuccinyl) betulin; 3-0- (3 ', 3'-dimethylglutaryl) betulin; 3-0- (3 ', 3'-dimethylsuccinyl) dihydrobetulinic acid; 3-0- (3 ', 3' -dimethylglutaryl) betulinic acid; (3 ', 3'-dimethylglutaryl) dihydrobetulinic acid; 3-0-diglycolyl-betulinic acid; 3-0-diglycolyl-dihydrobetulinic acid; and combinations thereof. 39. Method according to any of claims 1 to 10, 16 to 31, characterized in that the compound is not a derivative of betulin or dihydrobetulin of Formulas I to III: or a pharmaceutically acceptable salt thereof, wherein, R is a substituted and unsubstituted C2-C20 carboxycyl, R 'is hydrogen, substituted and unsubstituted C2-C10 alkyl, or an aryl group; R1 is a substituted and unsubstituted C2-C20 carboxycyl, R2 is hydrogen, C (C6H_) 3, or a substituted and unsubstituted C-C20 carboxycyl; and R3 is hydrogen, halogen, amino, optionally substituted mono- or dialkylamino, or -OR4, where R4 is hydrogen, C1.4alkanoyl, benzoyl or a C2-C20 substituted and unsubstituted carboxylic acid; where the hyphenated line represents an optional double link between C20 and C29. 40. Mutant Lentivirus, characterized in that it comprises a mutation in the nucleic acid sequence coding for Gag that returns to the replication of the mutant lentivirus less sensitive to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB). 41. Mutant Lentivirus according to claim 40, characterized in that the virus Gag polypeptide does not bind to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. 42. Mutant Lentivirus according to claim 40, characterized in that the mutation further makes the mutant lentivirus less sensitive to the inhibition of p25 (CA-SP1) to p24 (CA) processing by DSB. 43. Mutant Lentivirus according to claim 40, characterized in that it comprises a mutation in the nucleotide sequence coding for Gag p25 protein (CA-SP1). 44. Mutant Lentivirus according to claim 43, characterized in that it encodes a Gag polypeptide wherein one or more are deleted or replaced. more amino acids in the amino acid sequence in the CA-SP1 region. 45. Mutant Lentivirus according to claim 40, characterized in that the mutation occurs in the nucleotide sequence coding for an amino acid sequence at least about 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? ANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (C) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118), - and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). 46. Mutant Lentivirus according to claim 40, characterized in that the mutation occurs in the nucleotide sequence coding for the amino acid sequence selected from the group consisting of from: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 47. Mutant Lentivirus according to claim 40, characterized in that the mutation occurs in the nucleotide sequence at least about 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) approximately nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 48. Mutant Lentivirus according to claim 40, characterized in that the mutation occurs in the nucleotide sequence selected from the group coting of: (a) nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) approximately nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) approximately nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 49. Mutant Lentivirus according to claim 40, characterized in that the CA-SP1 mutation occurs in the codon coding for the amino acid selected from the group that cots of: (a) Amino Acid 1 (Glycine) in GHKARVLAEAMSQVTN (SEQ ID NO: 117); (b) Amino Acid 2 (Histidine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) OR GHKARVLAEAMSQVTN (SEQ ID NO: 117); (c) Amino Acid 6 (Valine or Isoleucine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (d) Amino Acid 7 (Leucine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) OR GHKARVLAEAMSQVTN (SEQ ID NO: 117); (e) Amino Acid 8 (Alanine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (f) Amino Acid 10 (Alanine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (g) Amino Acid 11 (Methionine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (h) Amino Acid 12 (Serine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) Amino Acid 13 (Glutamine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) OR GHKARVLAEAMSQVTN (SEQ ID NO: 117); (j) Amino Acid 14 (Valine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (k) Amino Acid 15 (Threonine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (1) Amino Acid 16 (Asparagine) in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); and (m) Amino Acid 45 (Alanine) in HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIA (SEQ ID NO: 120). 50. Mutant Lentivirus according to claim 40, characterized in that the mutation in the CA-SP1 sequence results in a change in the encoded amino acid selected from the group coting of: (a) Glycine in GHKARVLAEAMSQVTN (SEQ ID NO: 117 ); (b) Histidine to Glutamine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (c) Tyrosine histidine at amino acid 2 in SHKARILAEAMSQVTN (SEQ ID NO: 116) OR GHKARVLAEAMSQVTN (SEQ ID NO: 117); (d) Valine or Isoleucine to Leucine at amino acid 6 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (e) Leucine to Methionine at amino acid 7 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (f) Alanine to Valine at amino acid 8 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (g) Alanine to Valine at amino acid 10 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (h) Methionine to Leucine at amino acid 11 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) Serine to Lysine at amino acid 12 in SHKARILAEAMSQVTN (SEQ ID NO: 116) OR GHKARVLAEAMSQVTN (SEQ ID NO: 117); (j) Glutamine to glutamic acid at amino acid 13 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (k) Valine alanine at amino acid 14 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (1) Threonine to Leucine at amino acid 15 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (m) Asparagine to Alanine at amino acid 16 in SHKARILAEAMSQVTN (SEQ ID NO: 116) or GHKARVLAEAMSQVTN (SEQ ID NO: 117); (n) Alanine to Threonine at amino acid 45 in HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIA (SEQ ID NO: 525). 51. Mutant Lentivirus according to claim 40, characterized in that the mutation occurs in the polynucleotide coding for the amino acid sequence selected from any of SEQ ID NO: 121 to 443. 52. Mutant Lentivirus according to any of the claims 40 to 51, characterized in that it is HIV-1 mutant. 53. Recombinant non-HIV-1 retroviruses, characterized in that the replication of which is inhibited by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB). 54. Non-recombinant HIV-1 retroviruses according to claim 53, characterized in that DSB inhibits the processing of Gag viral p25 protein (CA-SP1) to p24 (CA) but does not significantly inhibit other steps of Gag processing. 55. Recombinant HIV-1 non-retrovirus according to claim 53, characterized in that the Gag polypeptide of the virus binds to 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. 56. Recombinant HIV-1 non-retrovirus according to claim 53, characterized in that it contains a p25 protein (CA-SP1) comprising an amino acid sequence at least about 70% identical to the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 57. Recombinant HIV-1 non-retrovirus according to claim 56, characterized in that it contains a p25 protein (CA-SP1) comprising an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 58. Recombinant HIV-1 non-retrovirus according to claim 53, characterized in that it comprises a polynucleotide encoding CA-SP1, which is at least about 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) approximately nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) approximately nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 59. Recombinant HIV-1 non-retroviruses according to claim 53, characterized in that it comprises a polynucleotide encoding CA-SP1, having a selected sequence of the group consisting of: (a) approximately nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (f) approximately nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 60. Recombinant HIV-1 non-retroviruses according to claim 53, characterized in that it contains a p25 protein (CA-SP1) comprising an amino acid sequence. selected from any of SEQ ID NO. 121 to 443. 61. Retroviruses of recombinant HIV-1 from according to claim 53, characterized in that it has a nucleotide sequence at least about 70% identical to the sequence selected from the group consisting of: (a) SEQ ID NO: 90, (b) SEQ ID NO: 92, (C) SEQ ID NO: 94, (d) SEQ ID NO: 96, (e) SEQ ID NO: 98. 62. Retroviruses non-recombinant HIV-1 according to claim 53, characterized in that it has a nucleotide sequence selected from the group which consists of: (a) SEQ ID NO: 90; (b) SEQ ID NO: 92; (C) SEQ ID NO: 94; (d) SEQ ID NO: 96; and (e) SEQ ID NO: 98. 63. Recombinant HIV-1 non-retrovirus according to claim 53, characterized in that it is derived from a virus selected from the group consisting of HIV-2, HTLV-1, HTLV-II. , SIV, avian leukosis virus (ALV), endogenous avian retrovirus (VAS), mouse mammary tumor virus (MMTV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), encephalitis virus and Capina arthritis (CAEV), Visna-maedi virus, and feline leukemia virus (FeLV). 64. Recombinant HIV-1 non-retrovirus according to claim 63, characterized in that it is derived from a virus selected from the group consisting of: (a) SIV; (b) FIV; (C) EAIV; (d) BIV; (e) CAEV; and (f) Visna-Maedi virus. 65. Animal model of lentivirus infection, characterized in that it comprises a non-human animal host infected with the recombinant HIV-1 retrovirus of claim 53. 66. Animal model according to claim 65, characterized in that the animal host Non-human is selected from the group consisting of: (a) a cat; (b) a primate; (c) a monkey; (d) a horse; (e) a sheep; (f) a goat; (g) a cow; (h) a mouse (i) a rat; and (j) a bird. 67. Method for making a lentivirus non-recombinant HIV-1 sensitive to inhibition by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB), characterized in that it comprises replacing the polynucleotide sequence coding for CA-SPl of a non-HIV-1 lentivirus that is not sensitive to DSB with the sequence encoding CA-SP1 of a DSB-sensitive HIV-1 virus. 68. Method according to claim 67, characterized in that the polynucleotide sequence encoding CA-SP1 in the non-HIV-1 recombinant lentivirus, comprises a sequence at least about 70% identical with a sequence selected from the group consisting of : (a) approximately nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) approximately nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 69. Method according to claim 68, characterized in that the polynucleotide sequence coding for CA-SP1 in the non-recombinant HIV-1 lentivirus, comprises a sequence selected from the group consisting of: (a) ) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) approximately nucleotides 1370-1413 of SEQ ID NO: 18; (f) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 70. Method according to claim 67, characterized in that the CA-SP1 protein of the lentivirus does not Recombinant HIV-1 sensitive to DSB comprises at least about 70% amino acid sequence identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 71. Method according to claim 67, characterized in that the CA-SP1 protein from the recombinant HIV-1 non-HIV-1 lentivirus comprises an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? ANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (C) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118); and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). 72. Method according to claim 67, characterized in that the CA-SP1 protein of the lentivirus does not Recombinant HIV-1 sensitive to DSB has an amino acid sequence selected from any of SEQ ID? O: 121 a 443. 73. Method according to claim 67, characterized in that it comprises: (a) deleting from the genome of lentivirus, the nucleotides corresponding to nucleotides 1370-1413 of SEQ ID NO: 18; and (b) inserting nucleotides 1370-1413 of SEQ ID NO: 18 or nucleotides 1857-1899 of SEQ ID NO: 19 in the non-HIV-1 lentivirus region. 74. Recombinant HIV-1 non-Lentivirus characterized in that it is produced by the method of any of claims 67 to 73. 75. Antibody, characterized in that it binds selectively at or near the Gag CA-SPl cleavage site. 76. Antibody according to claim 75, characterized in that it binds to an amino acid sequence at least about 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 77. Antibody according to claim 75, characterized in that it is linked to an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCK ILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 2. 3); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 78. Antibody according to claim 75, characterized in that it binds to a sequence of amino acids selected from any of SEQ ID NOS: 121 to 443. 79. Antibody according to claim 75, characterized in that it selectively binds to a polypeptide comprising a mutation in the HIV CA-SPl polypeptide which results in a decrease in the inhibition of the processing of p25 (CA-SP1) to p24 (CA) by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid. 80. Antibody according to claim 79, characterized in that the mutation is located at or near the cleavage site of CA-SP1 or in the SP domain of CA-SP1. 81. Antibody according to claim 79, characterized in that the polypeptide is selected from the group consisting of: (a) GHKARVLVEAMSQV (SEQ ID NO: 2); (b) SHKARILAEVMSQV (SEQ ID NO: 3); (C) SQKARILAEAMSQVTN; (d) GQKARVLAEAMSQVTN (e) SYKARILAEAMSQVTN (f) GYKARVLAEAMSQVTN (g) SHKARLLAEAMSQVTN (h) GHKARLLAEAMSQVTN (i) SHKARIMAEAMSQVTN (j) GHKARVMAEAMSQVTN (k) SHKARILAEVMSQVTN 1) GHKARVLAEVMSQVTN; m) SHKARILVEAMSQVTN n) GHKARVLVEAMSQVTN O) SHKARILAEALSQVTN p) GHKARVLAEALSQVTN q) SHKARILAEAMLQVTN r) GHKARVLAEAMLQVTN s) SHKARILAEAMSEVTN t) GHKARVLAEAMSEVTN (u) SHKARILAEAMSQATN v) GHKARVLAEAMSQATN w) SHKARILAEAMSQVLN x) GHKARVLAEAMSQVLN y) SHKARILAEAMSQVTA (z) GHKARVLAEAMSQVTA (a) HKARILAEAMSQVTNPATIMIQKGNFR QRKTVKCFNCGKEGHIT. 82. Antibody according to claim 79, characterized in that it binds selectively to an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. 83. Antibody according to claim 75, characterized in that it is selected from the group consisting of: (a) an antibody that selectively binds to SPI but not to CA-SP1; (b) an antibody that selectively binds CA-SP1 but not CA; and (c) an antibody that selectively binds to CA but not to CA-SPl. 84. Antibody in accordance with the claim 75, characterized in that it inhibits the binding of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid to Gag. 85. Isolated CA-SP1 polypeptide, characterized in that it is at least 70% identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (c) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 2. 3); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118), - and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). 86. CA-SP1 polypeptide isolated according to claim 85, characterized in that it is identical to a sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (C) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 87. Isolated CA-SP1 polypeptide, characterized in that it is encoded by a polynucleotide sequence at least 70% identical to a sequence selected from the group consisting of: (a) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) about nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) approximately nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) approximately nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 88. CA-SP1 polypeptide isolated according to claim 87, characterized in that it is encoded by a polynucleotide sequence selected from the group consisting of: (a) ) about nucleotides 1243-1435 of SEQ ID NO: 18; (b) approximately nucleotides 1729-1920 of SEQ ID NO: 19; (c) about nucleotides 1344-1435 of SEQ ID NO: 18; (d) about nucleotides 1828-1920 of SEQ ID NO: 19; (e) about nucleotides 1370-1413 of SEQ ID NO: 18; (1) about nucleotides 1857-1899 of SEQ ID NO: 19 (g) about nucleotides 1372-1419 of SEQ ID NO: 18; (h) about nucleotides 1858-1905 of SEQ ID NO: 19; (i) about nucleotides 1372-1434 of SEQ ID NO: 18; and (j) approximately nucleotides 1858-1920 of SEQ ID NO: 19. 89. Polypeptide isolated from the CA-SP1 region of HIV-1, characterized in that it consists of an amino acid sequence selected from any of SEQ ID NO: 121 to 443. 90. Polypeptide isolated from HIV CA-SP1 polypeptide, characterized in that it contains a mutation that results in a decrease in the inhibition of p25 processing by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic 91. Isolated polypeptide according to claim 90, characterized in that the mutation is located at the cleavage site of CA-SP1 or in the SPI domain. 92. Polypeptide isolated in accordance with claim 90, characterized in that it is encoded by a polynucleotide selected from the group cgue consists of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 9. 93. Polypeptide isolated according to claim 90, characterized in that it contains a mutation in which it results in a decrease in the inhibition of p25 processing by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic acid; wherein the mutation occurs in the nucleic acid sequence encoding a polypeptide selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? A? PDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (c) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 2. 3); (d) TACQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 24); (e) SHKARILAEAMSQV (SEQ ID? O: 25); (f) GHKARVLAEAMSQV (SEQ ID? O: 26) (g) SHKARILAEAMSQVT? (SEQ ID? O: 116); (h) GHKARVLAEAMSQVT? (SEQ ID? O: 117); (i) SHKARILAEAMSQVT? SATIM (SEQ ID? O: 118); and (j) GHKARVLAEAMSQVT? PATIM (SEQ ID? O: 119). 94. Isolated polypeptide according to claim 90, characterized in that it is selected from group consisting of: GHKARVLVEAMSQV (SEQ ID NO: 2); SHKARILAEVMSQV (SEQ ID NO: 3); SQKARILAEAMSQVTN; GQKARVLAEAMSQVTN; SYKARILAEAMSQVTN; GYKARVLAEAMSQVTN; SHKARLLAEAMSQVTN; GHKARLLAEAMSQVTN; SHKARIMAEAMSQVTN; GHKARVMAEAMSQVTN; SHKARILAEVMSQVTN; GHKARVLAEVMSQVTN; SHKARILVEAMSQVTN; GHKARVLVEAMSQVTN; SHKARILAEALSQVTN; GHKARVLAEALSQVTN; SHKARILAEAMLQVT; GHKARVLAEAMLQVTN; SHKARILAEAMSEVTN; GHKARVLAEAMSEVTN; SHKARILAEAMSQATN; GHKARVLAEAMSQATN; SHKARILAEAMSQVLN; (x) GHKARVLAEAMSQVLN; (y) SHKARILAEAMSQVTA; (z) GHKARVLAEAMSQVTA; and (aa) HKARILAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHI. 95. Isolated polypeptide according to claim 90, characterized in that it is encoded by an isolated polynucleotide that hybridizes under severe conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7 and 10. 96. Polypeptide isolated from according to claim 90, characterized in that it is part of a chimeric or fusion protein. 97. Isolated polynucleotide, characterized in that it encodes an amino acid sequence containing a mutation in a p25 protein of HIV Gag (CA-SP1), the mutation that results in a decrease in the inhibition of p25 processing (CA-SP1) ) to P24 (CA) by 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid. 98. Isolated polynucleotide according to claim 97, characterized in that the decrease in the inhibition of p25 processing is due to a decrease in the inhibition of the HIV-1 protease interaction with Gag. 99. Polynucleotide isolated in accordance with Claim 97, characterized by the decrease in the inhibition of p25 processing is due to a decrease in the binding of 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid to Gag. 100. Isolated polynucleotide according to claim 97, characterized in that the decrease in p25 processing inhibition is due to a decrease in DSB binding at or near the Gag CA-SPl cleavage site. 101. Isolated polynucleotide according to claim 97, characterized in that the mutation is located at or near the cleavage site of CA-SP1 or in the SPI domain of CA-SP1. 102. Isolated polynucleotide according to claim 97, characterized in that the mutation is located on or near the polynucleotide that codes for the amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVT? SATIM (SEQ ID? O: 21); (b) K? WMTETLLVQ? A? PDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVT? PATIM (SEQ ID? O: 22); (C) TACQGVGGPSHKARILAEAMSQVT? SATIM; (SEQ ID? O: 23); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118); and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 103. Isolated polynucleotide according to claim 97, characterized in that it is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 9. 104. Polynucleotide isolated according to claim 97, characterized in that it has at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6. 105. Isolated polynucleotide according to claim 97, characterized in that it has at least about 80% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 9. 106. Isolated polynucleotide according to claim 97, characterized in that it has at least about 95% identity to a polynucleotide selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7. 107. Isolated polynucleotide according to claim 97, characterized in that it has at least about 80% identity to a polynucleotide of SEQ ID NO: 10. Vector, characterized in that it comprises the isolated polynucleotide according to claim 97. 109. Hosting cell, characterized in that it comprises the vector of claim 108. 110. Method for producing a polypeptide, characterized in that it comprises incubating the host cell of claim 190. in a medium and recover the polypeptide from the medium. 111. Method for identifying a compound that inhibits replication of HIV-1 in cells of an animal, characterized in that it comprises: (a) contacting a polypeptide comprising an CA-SPl cleavage site with a test compound; (b) adding a labeled substance that binds selectively at or near the CA-SP1 cleavage site; Y (c) measuring the binding of the marking substance to the polypeptide. 112. Method of compliance with the claim 111, characterized by the compound being bound at or near the cleavage site of CA-SPl. 113. Method according to claim 111, characterized in that the compound inhibits the interaction of HIV-1 protease with the cleavage site of CA-SP1. 114. Method according to claim 111, characterized in that the polypeptide comprising a CA-SP1 cleavage site is a fragment of the recombinant polypeptide or peptide. 115. Method of compliance with the claim 111, characterized in that the labeled substance is a labeled antibody specific for CA-SP1; and wherein the measurement comprises measuring the change in the amount of the labeled antibody bound to the protein in the presence of the test compound compared to a control. 116. Method of compliance with claim 111, characterized in that the labeled substance is 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid, and wherein the method comprises measuring the change in the amount of 3-0- (3 ', 3'-dimethylsuccinyl) labeled betulinic bound to the protein in the presence of the test compound, compared to a control. 117. Method according to claim 111, characterized in that the mark on the marked substance is an enzyme, a fluorescent substance, a substance chemiluminescent, horseradish peroxidase, alkaline phosphatase, biotin, avidin, an electrodense substance, radioisotope, or a combination thereof. 118. Method for identifying a test compound that inhibits replication of HIV-1 in the cells of an animal, characterized in that it comprises: (a) contacting a polypeptide comprising a CA-SPl cleavage site with a protease in the presence of a test compound; and (b) detecting the interaction of the protease with the polypeptide. 119. Method according to claim 118, characterized in that the protease consists essentially of HIV-1 protease. 120. Method of compliance with the claim 118, characterized in that the polypeptide is labeled with two fluorescent portions, each linked to opposite sides of the CA-SPl cleavage site, and wherein the detection comprises measuring the transfer of fluorescent energy from one portion to the other in the presence of the test compound 121. Method according to claim 118, characterized in that the effect of the test compound on cleavage of the polypeptide is detected by measuring the binding of an antibody to at least one of SPI, p24 (CA), or CA-SPl (p25) 122. Method according to claim 121, characterized in that the labeled antibody is labeled with a molecule selected from the group consisting of enzyme, fluorescent substance, chemiluminescent substance, horseradish peroxidase, alkaline phosphatase, biotin, avidin, electrodense substance, radioisotope, and combinations thereof. 123. Method according to claim 118, characterized in that it comprises: (a) contacting a first polypeptide, comprising an HIV-1 wild type CA-SPl cleavage site, with HIV-1 protease in the presence of the test compound; (b) contacting a second polypeptide, comprising a mutant CA-SPl cleavage site or an alternative protease cleavage site with HIV-1 protease in the presence of the test compound, wherein the cleavage of the mutant cleavage or alternative protease cleavage site is not substantially inhibited by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid; (c) detecting the interaction of HIV-1 protease with the first and second peptides; and (d) comparing the cleavage of the first polypeptide to cleavage of the second polypeptide. 124. Method according to claim 123, characterized in that the region of the wild-type CA-SP1, mutant CA-SPl or alternative protease cleavage site region is contained within a polypeptide or recombinant peptide fragment. 125. Method according to claim 123, characterized in that the polypeptide is labeled with a fluorescent portion and a fluorescence quenching portion, each linked to opposite sides of the CA-SP1 cleavage site, and wherein the detection comprises measuring the signal of the fluorescent portion. 126. Method of compliance with the claim 123, characterized in that at least one of the first or the second polypeptide further comprises an epitope tag. 127. Method of compliance with the claim 123, characterized in that (a) and (b) are presented in a cell. 128. Method according to claim 123, characterized in that the detection of the interaction of the compound with the first and second polypeptides comprises gel electrophoresis. 129. Method for identifying compounds that inhibit the replication of HIV-1 in the cells of an animal, characterized in that it comprises: (a) contacting a test compound with cells infected with a first virus, replication of which is sensitive to inhibition by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB); (b) contacting a test compound with infected cells with a second virus having significantly reduced sensitivity to (DSB); (c) determine the phenotype of treated cells infected with the first virus and the second virus; and (d) selecting a test compound that is more active against the first virus compared to the second virus. 130. Method according to claim 129, characterized in that the phenotype is selected from the group consisting of: (a) viral replication; (b) excision of CA-SPl; and (c) production of an altered virion phenotype. 131. Method for identifying a compound that inhibits replication of HIV-1 in cells of an animal, characterized in that it comprises: (a) applying a test compound to cells infected with a lentivirus, the replication of which is sensitive to inhibition by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB); and (b) analyzing a lysate derived from cells infected to determine if cleavage of the CA-SP1 protein has occurred. 132. Method according to claim 131, characterized in that the lysate is obtained by lysing at least one of (a) infected cells; or (b) the viral particles released. 133. Method according to claim 131, characterized in that the analysis to determine if the excision of the CA-SP1 protein has been present comprises measuring the presence or absence of p25. 134. Method according to claim 131, characterized in that the analysis to determine if cleavage of the CA-SP1 protein has occurred comprises performing a western blot of viral proteins and detecting p25 using an antibody to p25. 135. Method according to claim 131, characterized in that the analysis to determine if cleavage of the CA-SP1 protein has been carried out comprises performing gel electrophoresis of viral proteins and imaging the metabolically labeled proteins. 136. Method according to claim 131, characterized in that the analysis to determine if the cleavage of the CA-SP1 protein has occurred comprises performing an immunoassay. 137. Method according to claim 133, characterized in that the immunoassay comprises: (a) capturing p25 and p24 on a substrate using an antibody that binds selectively to p24; (b) detecting the presence or absence of p25 in the substrate with an antibody that binds selectively to p25. 138. Method according to claim 131, characterized in that an epitope tag sequence is inserted into SPI, and wherein the assay comprises selective detection of p25 using an antibody to the epitope tag. 139. Method for identifying a compound that inhibits replication of HIV-1 in the cells of an animal, characterized in that it comprises: applying a test compound to cells infected with a lentivirus, the replication of which is sensitive to inhibition by acid -0- (3 ', 3'-dimethylsuccinyl) etulinic (DSB), and subsequently analyze the viral particles released by the cells for an altered virion phenotype. 140. Method of compliance with the claim 139, characterized in that the analysis comprises transmission electron microscopy. 141. Method according to claim 139, characterized in that the altered phenotype is selected from the group consisting of: (a) virions having spherical electrodense nuclei that are acentric with respect to the viral particle; (b) virions having increasingly dense electrodense layers that are just inside the viral membrane; (c) virions that have reduced infectivity or do not have infectivity; and (d) the combination of any of (a) through (c). 142. Method according to any of claims 129, 131 or 139, characterized in that the lentivirus, the replication of which is sensitive to inhibition by 3-0- (3 ', 3' -dimethylsuccinyl) betulinic acid (DSB), is HIV-1 143. Method according to any of claims 129, 131 or 139, characterized in that the lentivirus, the replication of which is sensitive to inhibition by 3-0- (3 ', 3' -dimethylsuccinyl) etulinic acid (DSB), is a non-HIV-1 recombinant lentivirus comprising the nucleotide coding for HIV-1 CA-SP1, and wherein the lentivirus is derived from a lentivirus selected from the group consisting of: (a) SIV; (b) FIV; (c) EAIV; (d) BIV; (e) CAEV; (f) Visna-Maedi virus; and (g) HIV-2. 144. Method according to claim 131, characterized in that the virus having significantly reduced sensitivity to DSB is selected from the group consisting of: (a) SIV; (b) FIV; (c) EAIV; (d) BIV; (e) CAEV; (f) Visna-Maedi virus; (g) HIV-2; and (h) HIV-1 mutant. 145. Method for identifying a compound, characterized in that it comprises: (a) measuring the progress of the disease in treated animals, infected with a lentivirus, where the lentivirus is sensitive to 3-O- (3 ', 3'-dimethylsuccinyl) ) betulinic (DSB); (b) measure the progress of the disease in untreated animals infected with the lentivirus; (c) compare the response of treated and untreated animals. 146. Method for identifying a compound, characterized in that it comprises: (a) administering the compound to animals infected with a first lentivirus, wherein the replication of the first lentivirus is inhibited by 3-O- (3 ', 3'-dimethylsuccinyl) betulinic (DSB); (b) administering the compound to animals infected with a second lentivirus, wherein the replication of the second lentivirus is not substantially inhibited by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB); (c) measure and compare the progress of the disease in animals infected with the first lentivirus, in comparison with animals infected with the second lentivirus. 147. Method for identifying a compound, characterized in that it comprises measuring the interaction of the compound with an amino acid sequence selected from the group consisting of: (a) KNWMTETFLVQNANPDCKTILKALGPAATLEEMMTA CQGVGGPSHKARILAEAMSQVTNSATIM (SEQ ID NO: 21); (b) KNWMTETLLVQNANPDCKTILKALGPGATLEEMMTA CQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 22); (c) TACQGVGGPSHKARILAEAMSQVTNSATIM; (SEQ ID NO:

2. 3); (d) TACQGVGGPGHKARVLAEAMSQVTNPATIM (SEQ ID NO: 24); (e) SHKARILAEAMSQV (SEQ ID NO: 25); (f) GHKARVLAEAMSQV (SEQ ID NO: 26) (g) SHKARILAEAMSQVTN (SEQ ID NO: 116); (h) GHKARVLAEAMSQVTN (SEQ ID NO: 117); (i) SHKARILAEAMSQVTNSATIM (SEQ ID NO: 118), - and (j) GHKARVLAEAMSQVTNPATIM (SEQ ID NO: 119). 148. Method for determining if a patient is infected with HIV-1 that is susceptible to treatment by a compound that inhibits p25 processing, characterized in that it comprises: (a) obtaining a patient specimen; (b) obtaining nucleic acid encoding CA-SP1 from the specimen; (c) genotyping the viral nucleic acid; (d) determining whether the nucleic acid codes for a variant of CA-SP1, the processing of which is sensitive to inhibition by 3-0- (3 ', 3'-dimethylsuccinyl) betulinic acid (DSB). 149. Method for treating a disease in a patient in need thereof, characterized in that: (a) identifying a compound that inhibits the processing of Gag p25 viral protein (CA-SP1) to p24 (CA), but has no effect significant in other steps of Gag processing; (b) obtain regulatory approval for the sale and use of the compound; packaging the compound for the sale and treatment of a disease in a patient in need thereof.