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WO1991019817A1 - VECTEURS D'EXPRESSION CONTENANT LE GENE nef DU VIH AUGMENTANT L'EFFICACITE DE LA REPLICATION - Google Patents

VECTEURS D'EXPRESSION CONTENANT LE GENE nef DU VIH AUGMENTANT L'EFFICACITE DE LA REPLICATION Download PDF

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WO1991019817A1
WO1991019817A1 PCT/US1991/004333 US9104333W WO9119817A1 WO 1991019817 A1 WO1991019817 A1 WO 1991019817A1 US 9104333 W US9104333 W US 9104333W WO 9119817 A1 WO9119817 A1 WO 9119817A1
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hiv
vector
eli
gene
nef
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William A. Haseltine
Ernest Terwilliger
Erik Langhof
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is directed to a vector comprising a replication-competent HIV-1 provirus or HIV-2 provirus and a sequence which enhances the replication efficiency of said provirus.
  • the human immunodeficiency virus (HIV-I, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders [Barre-Sinoussi, et al. , Science 220:868-871 (1983); Gallo et al, Science 224:500-503 (1984); Levy et al., Science 225:840-842 (1984); Popovic et al., Science 224:497-500 (1984); Sarngadharan et al., Science 224:506-508 (1984); Siegal et al., N. Enel. J. Med.
  • AIDS acquired immune deficiency syndrome
  • the disease is characterized by a long asymptomatic period followed by progressive degeneration of the immune system and the central nervous system.
  • Studies of the virus indicate that replication is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture [Zagury et al., Science 231:850-853 (1986)].
  • the expression of the virus in infected patients also appears to be regulated to enable evasion of the immune response.
  • HIV-I Molecular studies of the regulation and genomic organization of HIV-I show that it encodes a number of genes [Ratner et al., Nature 313:277-284 (1985); Sanchez-Pescador et al., Science 227:484-492 (1985);
  • Retroviruses are typically classified as belonging to one of three subfamilies, namely oncoviruses, spumaviruses and lentiviruses. Infection by oncoviruses is typically associated with malignant disorders. These viruses typically contain a single-stranded, plus-strand RNA genome of approximately 8,000 to 10,000 nucleotides encompassing the gag, pol and env. genes, as well as long terminal repeat (LTR) sequences. Oncoviruses typically contain an oncogene. It is generally believed that spumaviruses are not pathogenic in vivo, although they induce foamy cytopathic changes in tissue culture. Infection by lentiviruses is generally slow and causes chronic debilitating diseases after a long latency period. Lentiviruses, in addition to the gag, pol. and env genes possess a number of additional genes with regulatory functions.
  • HIV human immunodeficiency viruses
  • HIV-1 shares the gag, pol and env genes, respectively with the other retroviruses [Haseltine, W.A. , Journal of Acquired Immune Deficiency Syndrome, 1:217-240 (1988)]. HIV-1 also possesses additional genes modulating viral replication.
  • the HIV-1 genome encodes vif. vpr. tat, rev. vpu and nef proteins [Haseltine, W.A. , Journal of Acquired Immune Deficiency Syndrome, supra1.
  • the long terminal repeats (LTRs) of HIV contain cis-acting sequences that are important for integration, transcription and polyadenylation. Additional cis-acting signals allow regulation of HIV sequences by some of the novel HIV gene products [Haseltine, W.A. , Journal of Acquired Immune
  • Nucleotide sequences from HIV-2 and simian immunodeficiency virus (SIV) also contain the structural genes, gag, pol. and env as well as regulatory sequences such as tat, rev, and nef [Guyader, M. , et al. , Nature. 326:662-669 (1987); Chakrabarti, L. , et al., Nature 328:543-547 (1987), which are incorporated herein by reference] .
  • the ungulate lentiviruses such as the Visna virus, caprine arthritis, and encephalitis virus (CAEV) and the equine infectious anemia virus (EIAV) , which are the retroviruses most closely related to these primate immunodeficiency viruses, lack the region which specifies nef. namely the long open-reading frame that begins near the 3' end of the envelope (env) and extends into the 3' LTR.
  • the nef protein specified by the HIV and SIV viruses share common features, including post-translational modification by addition of the fatty acid, myristic acid at the amino terminus and regions of conserved amino acid sequences.
  • the protein encoded by the HIV-1 nef gene is typically 206 amino acid long, although some variations occur depending upon the strain.
  • antibodies to nef protein are found in infected individuals and in infected monkeys. This would suggest that nef plays an important role in natural infection. However, this is contradicted by other data. For example, the nef protein is prematurely truncated in many replication-competent HIV-1 isolates.
  • nucleotides corresponding to a replication-competent HIV provirus and a sequence which makes it easier to cause infection in a wider range of cells and a wider range of animals with the provirus in order to be able to develop animal models for treating the disease.
  • HIV segment and also containing a sequence of nucleotides corresponding to the nef gene of the ELI strain of HIV (nef gene segment) .
  • the HIV segment of this vector would also contain a functional vpu gene segment or a functional vpr gene segment. Still more preferably, the HIV segment of this vector would contain both a functional vpu and a functional vpr gene segment.
  • a vector comprising an ELI gene segment.
  • this vector also contains a heterologous gene.
  • the vectors can be used in vivo and in vitro to transfect a wide variety of cell lines.
  • Figure 1 is a schematic diagram of an HIV genome and a series of vectors prepared.
  • Figure 2 is a schematic diagram of a portion of the HXB ELI 1-fs vectors.
  • Figure 3 is a schematic diagram of a portion of the HXB ELI 2 and HXB ELI 2-fs vectors.
  • Figure 4 is a schematic diagram of a vector containing an HIV segment, a functional nef gene, a funcitonal vpu gene, and a functional vpr gene.
  • Figure 5 is a schematic diagram of a portion of the
  • Figure 6 is a chart of p24 production in peripheral blood lymphocytes (PBL) over time by a variety of vectors.
  • Figure 7 is a chart of p24 production in monocyte/macrophage cultures transfected by various vectors over time.
  • Figure 8 is a chart of p24 production in PBL transfected by various vectors over time.
  • HIV segment a vector containing a sufficient number of nucleotides corresponding to an HIV genome to express HIV gene products necessary for viral replication and infectivity
  • ELI nef gene segment a sequence of nucleotides corresponding to a nef gene of the ELI strain to enhance viral replication
  • the HIV segment corresponds to nucleotides of the HIV-1 or HIV-2 genomes. More preferably, the HIV segment corresponds to nucleotides of the HIV-1 genome. Preferably, the HIV segment does not correspond to the entire ELI strain of HIV-1.
  • the nef gene of ELI has its initiation codon located at position 8822, which is immediately after the end of the env reading frame and extends through most of the U3 region of the 3' LTR. This gene encodes a protein with an apparent molecular weight of about 25-27 kd.
  • LAV ELI a French clone from a Zairian isolate was isolated in 1983 from a 24 year old woman with AIDS by co-cultivation of patient peripheral blood lymphocytes with fresh PHA-stimulated PBMC. There are 47 differences in amino acid sequence (out of a total of 207) between the nef product of LAV ELI and that of the IIIB-derived Hxorf clone used in our previous studies of nef [Terwilliger, E.F. , et al., J. Virol. 60:754 (1986)].
  • a clone e.g., LAV ELI
  • LAV ELI which contains a functional nef gene.
  • This ELI nef segment can preferably be inserted in the presently described vector containing the HIV segment in the location where the nef segment from the other HIV-1 or HIV-2 strains used would be. If one used an HIV segment with a nef gene, one can excise that nef gene and insert the ELI nef segment.
  • the HIV segment will contain a sufficient number of nucleotides corresponding to all HIV genes necessary for replication and infection.
  • this segment will also contain nucleotides corresponding to a functional vpu gene or a functional vpr gene. More preferably, it will contain nucleotides corresponding to both functional vpu and vpr genes .
  • the VPU sequence in the HXB2 provirus does not contain an initiation codon for vpu and therefore does not express a functional vpu gene.
  • the HIV segment has nucleotides corresponding to HXB2 strain one would insert an initiation codon immediately upstream and in frame with the vpr sequence.
  • the clones BH8, BH10, LAV ELI, etc. contain a nucleotide sequence which results in a functional vpu gene.
  • the HXB2 strain has a premature termination codon and does not express a functional vpr protein. If one is using an HIV segment that corresponds to such a vor gene, which is not considered functional, one can insert a functional vpu gene from a strain that expresses a functional vpr gene product such as LAV BRU, LAV ELI, etc.
  • vectors can be synthesized by a variety of methods known to the person of ordinary skill in the art. A variety of different vectors which can be prepared are shown in Figures 1-4.
  • ELI nef sequences can be constructed and inserted within the 3' end of an HIV sequence, such as one having nucleotides corresponding to the replication competent HXB2 (also referred to as HXBc2) provirus.
  • Compatible restriction sites can be generated by standard techniques where needed such as by oligonucleotide-directed mutagenesis. For example, a
  • HXB-ELI 1 consists entirely of HXB2 sequences as far 3' as the native
  • HXB-ELI-2 contains only HXB2 sequences as far 3' as this Eco
  • the LAV ELI sequence extends from this position 3' to the Sac 1 site at 9607, as in the case of HXB ELI-1.
  • sequence from the vector corresponding to a sequence between the Sal 1 site 5820 and a kpn 1 site at position 6382 can be excised and replaced with the corresponding segment from BH10, which includes a functional vpu gene.
  • sequence from the vector corresponding to a sequence between the Sal 1 site 5820 and a kpn 1 site at position 6382 can be excised and replaced with the corresponding segment from BH10, which includes a functional vpu gene.
  • Proviral plasmids containing the ELI nef gene and the HIV segment vector described above can readily be made by inserting this sequence into a plasmid backbone by known means such as use of restriction enzyme sites.
  • a sufficient number of nucleotides permits additions, deletions and substitutions as long as the claimed functional ability is not lost. For example, if one is referring to the functional ability of the nef gene in terms of viral replication, then replication of the resultant virus must be greater than by an isogenic nef virus.
  • the vector is used to transfect a desired cell by standard techniques. For example, one can transfect a cell in vitro using, for example, the calcium phosphate coprecipitation technique or DEAE dextran technique. Alternatively, this vector can be used to transfect living cells in vivo. Such techniques are well known to the skilled artisan.
  • nef gene segment of the present invention By use of the nef gene segment of the present invention, one is able to take an HIV strain, for example, the HXB2 strain, which does not replicate well in peripheral blood mononuclear cells (PBMC) as well as purified primary human monocytese or monocytoid cell lines and obtain enhanced infectivity in such cell lines.
  • PBMC peripheral blood mononuclear cells
  • the virus can establish a stable infection via integration of the virus into the host DNA.
  • the native virus is initially carefully regulated so that it does not appear too "aggressive".
  • the provirus formed by the vectors will be more aggressive.
  • the IIIB precursor of HXB2 spontaneously mutated to a nef"phenotype.
  • This nef " precursor of HXB2 replicated poorly in PBMC.
  • a compensatory mutation arose in 3' env sequences of the precursor to HXB2 to produce a virus that could replicate well in PBMC.
  • the HXB2 virus contains the original nef mutation as well as the compensatory mutation in
  • the 3' env compensatory mutation has a negative affect on growth in the presence of closely related nef sequences such as the BH8 and BH10 nef genes. This negative interaction does not extend to divergent nef sequences such as the ELI nef gene.
  • BH8 and BH10 nef is a negative regulator with HXB2 3' env sequences.
  • BH8 and BH10 nef should be positive regulators with ELI 3' env sequences.
  • ELI nef is a positive regulator with either HXB2 or ELI 3' env sequences.
  • ELI nef is an essential gene for growth in primary monocytes and macrophages. Accordingly, various tropisms exhibited by HIV strains will not be exhibited by vectors of the present invention. Thus, such vectors can be used to transform a wider range of cells than the strains typically can.
  • this vector to transfect cells in vitro or in vivo can permit an improved system for screening drugs and understanding the pathogenesis of the virus. Since the range of the cells transformed by this vector is wider than that with other laboratory strains such as the IIIB strain, one is able to explore the affects of a drug on the spread of the virus in a wider range of infected cells than is typical.
  • the parameters observed include syncytia formation, single-cell killing, levels of supernatant of HIV p24 core protein such as determined by RIA, immunoprecipitation of metabolically labelled viral proteins with AIDS patient serum, etc.
  • the vector is preferably used n vitro with primary monocytes/macrophages.
  • the vector can also be used in vivo to "infect" cells.
  • the present vectors result in proviral clones which replicate much more aggressively in primary lymphocytes than the typical lab strains and should replicate much more effectively in the SCID-HU mice.
  • Other preferred animal models include mammals other than humans. Preferred examples of such mammals would include rabbits, mice, rats, cats and primates. Preferred primates include chimpanzees and monkeys.
  • Administration can be any of a variety of routes including parenteral administration such as intramuscular, intraperitoneal, intravenous or subcutaneous.
  • Knowing the amount and the conditions necessary to establish "infection” (transfection) permits the development of a model assay to establish effectiveness of a preselected compound in vivo.
  • one can prepare a vector comprising a sufficient number of nucleotides corresponding to a nef gene of the ELI strain of HIV-1 to enhance viral replication (ELI nef gene segment) , a promoter sequence and a polyadenylation sequence, but not containing all the ELI encoding nucleotide sequences.
  • the vector contains an LTR sequence, more preferably an HIV LTR sequence.
  • the promoter can be the LTR or a separate promoter such as another viral promoter, e.g., the SL3 promoter.
  • This vector would preferably contain a heterologous gene. In one embodiment, the heterologous gene would be a marker gene.
  • Marker genes are well known in the art, for example, the chloramphenicol acetyltransferase (CAT) gene or a growth hormone such as human growth hormone (hGH) .
  • the vector can be cotransfected into a cell with a vector comprising a replication competent HIV provirus, or alternatively into a cell line transfected by a replication competent HIV, e.g., HXB2 or HXB-BH8. This would permit further anlaysis of the interaction between the ELI nef segment and other viral genes.
  • the use of this vector should also increase the range of host that can be transfected by such virus.
  • Vectors HXB2 is a full-length infectious provirus derived from the IIIB strain of HIV-1 [Fisher, A.G. , et al. , Nature 316:262-265 (1985)] its nef, yor and VPU genes are defective as a consequence of single point mutations in each gene. There is a BamHI site at nucleotide 8506, whose location was used in preparing a variety of plasmids. See Figure 1.
  • HXB-ELI 1 consists entirely of HXB2 sequences as far 3' as the native BamHI site at position 8506, within the env and rev gene sequences upstream of nef. and LAV ELI sequences between that position and a Sad site at position 9607. See Figures 1 and 2. The remainder of the 3' LTR sequence is derived from HXB2.
  • HXB-ELI-2 the 5' junction between the HXB2 and LAV ELI sequence is shifted downstream to a new Eco RI site at position 8792, only 30 bp upstream from the nef initiation codon. See Figures 1 and 3.
  • HXB-ELI-2 contains only HXB2 sequences as far 3' as this Eco RI site.
  • the LAV ELI sequence extends from this position 3' to the Sacl site at 9607, as in the case of HXB-ELI-1.
  • HXB-BH8 the 3' terminus at the native Xhol site contained sequences derived from the BH8 provirus.
  • This strain is a IIIB strain, which contains a complete nef open reading frame and was originally referred to as HXB-orf. See Figure 5.
  • the vpr + . VPU " * ' HXB-BH8-R provirus was derived from the vpr " .
  • the proviral sequence of HXB-BH8 between an Apa 1 site at position 2008 and a Sal 1 site at position 5820 was replaced with the corresponding sequence from clone LAV BRU. This segment includes a functional vpr reading frame.
  • HXB-BH8 sequence between 5820 and a kpn 1 site at position 6382 was then excised and replaced with the corresponding segment from BH10, a closely related IIIB clone. This segment includes a functional VPU gene. The same replacements were made in HXB-ELI 1 to generate HXB-ELI 1-R. See Figure 4.
  • the HXB-ELI 1-R sequence was inserted into the known plasmid pBR322 between the EcoRV and PvuII sites to generate the plasmid DFCI-HT, which contains the HXB-ELI-1-R sequence with a pBR322 backbone. A sample of this plasmid has been deposited with the
  • PBMC Peripheral blood mononuclear cells
  • Infectious supernatant equivalent to 20 ng of p24 was added to each well of primary cells. The infected cell cultures were incubated overnight and the growth medium was then carefully decanted from each well and replaced with fresh medium. After the last medium change samples for p24 assays were collected every third day before the cultures were fed.
  • PBMC peripheral blood mononuclear cells
  • Monocytes were obtained from the PBMC by fractionation on Percoll gradients. Contaminating T cells were from the low density cells removed by rosetting with neuraminidase treated sheep red blood cells.
  • the purity of the monocyte population was verified by immunoperoxidase staining of cytopin spin cell preparations of the isolated monocytes. More than 95% of the cells stained with Leu3-M and ⁇ 5% were positive for Leu 18 (CD20) , Leu 3 (CD4) , or Leu 2 (CD8) . 4 x 10° monocytes were cultured as described for PBMC cultures to maintain a comparable growth environment. Viral stocks were used as described for PBMC cultures and samples for p24 assays were harvested at the same points.
  • HXB-BH8 IIIB-derived nef-positive (HXB-BH8) and negative (HXB2) viruses were also transfected as controls.
  • HXB2 contains a point mutation within nef resulting in a premature termination codon being inserted into the reading frame after amino acid 123. This truncated nef protein is not functional.
  • HXB-BH8 previously called Hx-orf
  • Hx-orf does not contain this mutation, and produces a full-length nef product [Terwilliger, E.F., et al. , J. Virol. 60:754 (1986)].
  • HXB-BH8 A lag in levels of supernatant p24 was detectable in the culture transfected with HXB-BH8 as compared to the one containing HXB2 at early times post-transfection. A marked difference in the pattern of syncytia formation was also apparent. Replication of virus derived from HXB-ELI 1 following transfection was not substantially different from that of HXB2. In different experiments the kinetics of virus infection exhibited by HXB-ELI 1 were either equivalent to or modestly enhanced over those displayed by HXB2.
  • HXB-ELI-nef virus upon the infected cells, whether evaluated by syncytia formation or by decreases in total cell numbers over time. Similar results were obtained in repeat experiments, sometimes with even larger differences in kinetics between the nef-positive and nef-negative HXB-ELI 1 clones.
  • Immunoprecipitation of metabolically labelled infected cell lysates with a nef-specific polyclonal rabbit serum revealed the presence of a 27kD nef protein in lysates from cells infected with either the HXB-BH8 or HXB-ELI 1 viruses. This band was not visible in lysates from cells transfected with either HXB2 or HXB-ELI 1-fs.
  • HXB-BH8 Transfection by HXB-BH8 confirmed the earlier report that introduction of a functional nef gene into the HXB2 provirus results in a virus which replicates more slowly on CD4 + T cells than does the nef " counterpart.
  • the LAV ELI nef gene appeared to display a marked enhancing effect upon the replication of the virus in the Jurkat cells. This positive effect was larger and in the opposite direction from the smaller negative effect of IIIB nef observed in the context of the HXB-BH8/HXB2 pair of viruses.
  • a repeat experiment using independently derived clones of the HXB-ELI 1 pair of proviruses yielded comparable results, eliminating the possibility that a random secondary change at some distal site within one of the constructs was responsible for the results.
  • HXB-ELI 2 contains a smaller insertion of LAV ELI sequences than HXB-ELI 1 (See Figure 1 and 3).
  • the 3' end of the 800 base pair insert remains the same but the 5' end is shifted downstream to just 30 base pairs 5' to the nef initiation codon.
  • HXB2 is defective in two other regulatory genes in addition to nef. the vpu [Cohen, E.A. , et al., J. AIDS 3:11 (1990)] and VET [Alizon, M. , et al., Cell 46:83 (1986)] genes.
  • HXB-BH8-R for restored provirus, which was derived from HXB-BH8, intact vpr and vpu frames have been restored by recombination with sequences from other IIIB clones and LAV ELI as described earlier (See Figure 1) .
  • HXB-BH8-R therefore represents a provirus which would be predicted to be fully functional for all the HIV genes.
  • HXB-ELI 1 Identical recombinations were made in HXB-ELI 1 to generate HXB-ELI 1-R. See Figure 4.
  • HXB-BH8-R and HXB-ELI 1-R are therefore isogenic except for the 1.1 kb of LAV ELI sequence in HXB-ELI 1-R which replaces the corresponding IIIB sequence in HXB-BH10-R.
  • IIIB replicates very well in T-cell lines but poorly in PBMC, and is particularly weak in either primary monocytes or onocytoid cell lines [Koyanagi, Y. et al., Science. 236:819 (1987)]. Barely detectable levels of p24 antigen production were observed within the supernatants of PBMC infected with the HXB2 virus. See Figure 6. Similarly, the HXB-BH8 and HXB-BH8-R viruses also did not exhibit significantly increased activity in the PBMC over HXB2 ( Figure 6) . No detectable replication by any of these three viruses was observed in the monocyte macrophage cultures ( Figure 7) .
  • Figures 6 and 7 both show p24 production over time for cells transfected with HXB-ELI 1 (ELI-1 nef + , open circle), HXB-ELI 2 (ELI-2 nef + ; solid square) HXB-BH8 (BH8 nef + , open square); HXB-ELI 1-fs (ELI-lfs nef " , solid circle); HXB-ELI 2-fs (ELI-2fs nef " , solid triangle) and HXB2 (HXBc2 nef " , open triangle).
  • HXB-ELI 1 ELI-1 nef + , open circle
  • HXB-ELI 2 ELI-2 nef + ; solid square
  • HXB-BH8 BH8 nef + , open square
  • HXB-ELI 1-fs ELI-lfs nef " , solid circle
  • HXB-ELI 2-fs ELI-2fs nef " , solid triangle
  • HXB-ELI 1 and HXB-ELI 2 viruses exhibited strong replication within the PBMC, attaining maximum levels of p24 antigen production, of ca. 26 ng/ml and ca. 33 ng/ml respectively, on Day 17 and 20 following infection ( Figure 6) .
  • the HXB-ELI 1-R virus was even more aggressive, achieving a peak virus production level 3-5-fold higher than HXB-ELI 1 or ELI 2. This was more than 50-fold higher than the maximum virus titer attained by any of the viruses containing IIIB nef sequences.
  • supernatant p24 levels were again either barely within or below the limit of detection.
  • HXB-ELI 1-R again attained a several-fold higher maximum titer than the other two viruses. No p24 could be detected in the supernatants of monocytes infected with either of the HXB-ELI-nef-defective isogenic viruses. See, for example, Figure 8, where HXB-ELI 1-R is represented by open squares, HXB-ELI by open triangles and HXB2 by open circles, which shows the results of another typical experiment.
  • HXB-ELI 1-R While coding changes in other reading frames have also been introduced into HXB-ELI 1-R as compared to the original HXB-ELI 1, the magnitude of the effects we have characterized for the vpu and vpr during virus infection of Jurkat cells is entirely sufficient to account for the boost in virus titers seen in both the PBMC and monocyte cultures infected with HXB-ELI 1-R.
  • nef can modestly down regulate expression from the HIV LTR in trans. This was accomplished by co-transfection into C0S-7 cells of an HIV LTR-CAT construct together with either a nef expression vector or an isogenic vector containing a frameshift in the nef seqeunce. Each plasmid included an SV40 origin of replication. However, similar results were obtained using HIV LTR elements cloned from either HXB2 or LAV ELI, and regardless of which strain of nef was being expressed. Together with the replication studies, this suggests that the down regulatory effects may be secondary phenomena.

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Abstract

On décrit des vecteurs contenant un nombre suffisant de nucléotides pour exprimer les produits génétiques du VIH nécessaires à la réplication efficace, et contenant également un segment du gène nef de la souche ELI. Ces vecteurs peuvent servir à transfecter une grande diversité de cellules et d'hôtes destinés à l'usage dans les systèmes de dosage.
PCT/US1991/004333 1990-06-20 1991-06-18 VECTEURS D'EXPRESSION CONTENANT LE GENE nef DU VIH AUGMENTANT L'EFFICACITE DE LA REPLICATION Ceased WO1991019817A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996011696A1 (fr) * 1994-10-13 1996-04-25 The Regents Of The University Of California Methode de criblage de medicaments anti-vih
EP0754223A4 (fr) * 1994-02-14 1997-09-17 Macfarlane Burnet Ctre Med Res Souches non pathogenes de vih-1
CN113913460A (zh) * 2021-10-08 2022-01-11 武汉波睿达生物科技有限公司 一种检测可复制慢病毒的方法及其应用

Citations (1)

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EP0754223A4 (fr) * 1994-02-14 1997-09-17 Macfarlane Burnet Ctre Med Res Souches non pathogenes de vih-1
WO1996011696A1 (fr) * 1994-10-13 1996-04-25 The Regents Of The University Of California Methode de criblage de medicaments anti-vih
US5639619A (en) * 1994-10-13 1997-06-17 Regents Of The University Of California Screening assay for anti-HIV drugs using the Vpr gene
US5721104A (en) * 1994-10-13 1998-02-24 Regents Of The University Of California Screening assay for anti-HIV drugs
CN113913460A (zh) * 2021-10-08 2022-01-11 武汉波睿达生物科技有限公司 一种检测可复制慢病毒的方法及其应用

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JPH05509232A (ja) 1993-12-22
EP0535161A1 (fr) 1993-04-07
EP0535161A4 (en) 1993-12-01
CA2085880A1 (fr) 1991-12-21

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