JP2002517261A - Herpes simplex virus origin binding protein-N-terminal truncation (OBP-NT) - Google Patents

Abstract

  (57) [Summary] The present invention relates to herpesvirus OBP-NT polypeptides, polynucleotides encoding OBP-NT polypeptides, and methods of producing such polypeptides by recombinant techniques. Also provided are methods of utilizing OBP-NT polypeptides to screen antiviral compounds.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to polynucleotides encoding newly identified polypeptides, their production and use, and variants, agonists and antagonists thereof, and their uses. In particular, the present invention relates to polynucleotides encoding polypeptides, hereinafter referred to as herpes simplex virus N-terminal truncation or "OBP-NT".

BACKGROUND OF THE INVENTION Herpesviruses form a medically important family of viruses, including three subfamilies, alphaherpesvirus, betaherpesvirus and gammaherpesvirus. It is known that these viruses can produce infectious virus in susceptible cells as well as re-activate their response to stimuli while remaining resident in the host. Herpes simplex virus types 1 and 2 (HSV
-1, HSV-2) is, for example, keratitis leading to blindness from HSV-1 infection,
It is an alpha herpes virus known to induce several diseases in humans, including encephalitis and cold sores, neonatal diseases derived from HSV-2 infection, and genital herpes. HSV-1 and HSV-2 are very homologous viruses, which are indistinguishable until 1962, when two serotypes were identified. Since over 70 years have passed since their isolation, such viruses are the most intensively studied herpesviruses and are the archetypal alphaherpesvirus subfamily. The sequencing of the HSV-1 genome was completed in 1988, when 72 genes encoding 70 unique proteins were identified. From this time, four more genes were finally identified, and another six genes were assumed. H
SV-2 was recently sequenced and 74 similar unique proteins were identified. Further unknown proteins may be encoded in the HSV-2 genome by HSV-1, analogy. Despite the vast amount of research using HSV-1, many issues remain regarding the ecology of this virus and HSV-2. HSV-1 and HSV as targets for the development of antiviral agents
It is particularly preferred to utilize the V-2 gene.

[0003] HSV-1 infects 40-80% of the general public, while HSV-2 infects 20-65% of the general public. HSV-2 infection is particularly pronounced in prostitute and homosexual groups, reaching as high as 90%. Current antiviral agents reduce pain, lesion intensity and hair loss and are only partially effective. Accordingly, there is a need for antiviral agents that have reduced time to healing and improved efficacy overall. In addition, the ability to shock the virus to reactivate from the incubation period is also very advantageous and has implications for disease modification. Clearly, there is a need for agents such as the OBP-NT polypeptides of the present invention that have the advantage of being useful for screening compounds for antiviral activity. Such factors are also useful in determining their role in the pathogenesis of infection, dysfunction and disease. There is also a need to identify and characterize such factors and their antagonists and agonists in order to find ways to prevent, ameliorate or cure infections, dysfunctions and diseases.

[0004] Origin binding protein encoded in the UL9 gene of HSV-1
rotein: OBP) was described when its binding activity to two homologous regions within the viral DNA replication origin (Sites I and II, also referred to separately as BoxI and BoxII) was described, ie, in 1986. It was first identified and further characterized in 1988 (Elias, P. et al., 1986. Proc. Nat'l. Acad. Sci. USA 83: 6322-6.
326; Elias, P. and IR Lehman. 1988. Proc. Nat'l. Acad. Sci. USA 85: 2.
959-2963). The three viral origins of DNA replication, two copies of oriS and one copy of oriL are contained within the HSV-1 and HSV-2 genomes (Spaete, RR and Frenkel, N. 1985. Proc. Nat '). l. Acad. Sci. USA 82: 694
-698; Stow, ND and EC McMonagle. 1983. Virology 130: 427-438; Dola.
n, A. et al., 1998. J. Virol. 72: 2010-2021). oriS has one copy of each of OBP binding sites I and II, and a third homologous sequence site III and AT
And all these regions are required for the origin to function effectively (see FIG. 1: Elias, P. et al., 1990. J. Biol. Chem. 265: 17167-). 17173)
. oriL contains two copies of each of sites I and III and an AT-rich strand and is required for the origin to function effectively (Lockshon, D. and Gallowa
y, DA1988. Mol. Cell. Biol. 8: 4018-4027). Despite much research in the art, the ability of OBP to interact with site III is controversial; on the other hand, purified OBP does not interact with oligonucleotides containing site III. OBP from infected cell extracts was identified as capable of binding site III oligonucleotides (Olivo, PD et al., 1988. Proc. Nat'l.
Acad. Sci. USA 85: 5414-5418; Bruckner, RC et al., 1991. J. Biol. Chem. 266.
: 2669-2674; Hazuda, DJ et al., 1991, J. Biol. Chem. 266: 24621-24626; Dabrowsk
i, CE and PASchaffer, 1991, J. Virol. 65: 3140-3150). Furthermore, it has been suggested that purified OBP interacts with site III within the context of the complete viral origin oriS (Elias, P. et al., 1990, J. Biol. Chem. 265: 17167-17173; K.
off, A. et al., 1991, J. Virol. 65: 3284-3292; Elias, P. et al., 1992, J. Biol. Chem. 2
67: 17424-17429). The availability of viral mutants has revealed that OBP is essential for virus growth and DNA replication (Malik, AK et al., 1992).
, Virology 190: 702-715). OBP also measures the requirements for helicase activity and specific dimerization sequences encoded in the N-terminus and the ability to bind the origin sequence encoded in the C-terminal half of the protein for viral DNA replication. Tested in a transient replication assay with cells (Hazuda, DJ et al., 1992, J. Biol. Chem.
267: 14309-14315; Malik, AK and SKWeller, 1996, J. Virol. 70: 7859-.
7866; Lee, SS-K. And IRLehman, 1997, Proc. Nat'l. Acad. Sci. USA. 94:28.
38-2842). Subsequent studies characterized nucleotides important for origin binding activity and revealed the relationship between OBP origin binding and origin-directed DNA replication (Challberg. MD, 1991, Seminars in Virology 2). : 247-256; Hernandez, T
R. et al., 1991, J. Virol. 65: 1649-1652; Dabrowski, CE et al., 1994, Mol. Cell. Bi.
ol. 14: 2545-2555).

[0005] The UL9 (OBP) reading frame is encoded in a 5.0-5.2 kilobase RNA in HSV-1. In the electrophoretic mobility shift assay (EMSA) following incubation of the HSV-1 infected cell extract with the radiolabeled HSV-1 origin sequence,
BP consists of the protein component of a protein: DNA complex called "complex B" (Barada
ran, K. et al., 1996, J. Virol. 70: 5673-5679). Furthermore, the UL of the HSV-1 genome
Novel RNAs 6.3-6.4 and 4.0-4.1 kilobases in length transcribed from 9 regions have been described (Deb, SP et al., 1993, Biochem and Biophys Res. Comm.
193: 617-623; Baradaran, K. et al., 1994, J. Virol. 68: 4251-4261). UL8.
The 4.0-4.1 kilobase RNA, designated No. 5, was found to encode a novel origin binding protein, OBP, and was subjected to in-frame translation at the C-terminal (DNA-bound) half of OBP. (See FIG. 2). Although the function of this protein has not yet been elucidated, DNA replication has been postulated to have a role in switching the packaging of the viral genome into the capsid. OBPC is HSV
-1 infected cell extract and the radiolabeled HSV-1 source sequence, after incubation, contains the protein component of the protein: DNA complex designated "complex A" by EMSA (Baradaran, K. et al., 1996, J. Virol. 70: 5673-5679). Complex A is origin site I
And II, and was unable to bind to site III due to a single nucleotide difference between sites I and III in the OBP binding region (Dabrowski, CE and PA Schaffer, 1991, J.Virol. 65: 3
140-3150). An RNA of 6.3-6.4 kilobases, termed UL 9.5, has not yet been mapped, but was hypothesized to encode a protein that overlaps at the N-terminus of OBP (
Baradaran, K. et al., 1994, J. Virol. 68: 4251-4261).

DISCLOSURE OF THE INVENTION HSV-1 OBP-NT, HSV-2 OBP-NT and VZV OBP-NT
It is an object of the present invention to provide a polypeptide identified as an OBP-NT polypeptide from a herpes virus, including NT. Polynucleotides encoding OBP-NT polypeptides, particularly HSV-1 OBP-NT, HSV-2 OBP-NT and VZV OBP-
It is a further object of the present invention to provide a polynucleotide encoding a polypeptide referred to as NT. In a particularly preferred embodiment of the invention, the polynucleotide comprises an HSV comprising the sequence shown in Table 1 (SEQ ID NO: 1) or a variant thereof, including the full length gene.
-1 consists of the region encoding the OBP-NT polypeptide. Another particularly preferred embodiment of the present invention is an OBP-NT protein from HSV-1 comprising the amino acid sequence of Table 1 (SEQ ID NO: 2) or a variant thereof. Another aspect of the present invention is an isolated nucleic acid molecule encoding OBP-NT, particularly HSV-1 OBP-NT, including mRNA, cDNA, genomic DNA. Further embodiments of the present invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising them.

[0007] According to another aspect of the present invention there is provided the use of a polypeptide of the present invention for therapeutic or prophylactic purposes, particularly for genetic immunization. Among the particularly preferred embodiments of the present invention are naturally occurring allelic variants of OBP-NT and the polypeptides encoded thereby. In another aspect of the present invention, biologically, diagnostically, prophylactically, clinically or prophylactically useful variants of the polypeptide of HSV-1 OBP-NT and compositions comprising them Is provided. Among them, a particularly preferred embodiment of the present invention is a variant of the HSV-1 OBP-NT polypeptide encoded by a naturally occurring allele of the OBP-NT gene. In a preferred embodiment of the present invention, there is provided a method for producing the OBP-NT polypeptide described above. According to another aspect of the present invention, including as an antiviral agent, including, for example, antibodies or inhibitors of the interaction between polynucleotides and polypeptides,
Inhibitors for the polypeptides of the invention are provided.

[0008] According to certain preferred embodiments of the present invention, the products, compositions, and OBP-N
Method for evaluating T expression, method for treating disease, method for testing genetic variants, OBP-NT
The polypeptide or polynucleotide is administered to an organism and the virus, especially HSV-
Provided are methods for raising an immunological response to C.1. According to certain preferred embodiments of the present invention and other embodiments of the present invention, there are provided polynucleotides that hybridize to an OBP-NT polynucleotide sequence, particularly under stringent conditions. In certain preferred embodiments of the present invention, there are provided antibodies against the OBP-NT polypeptide.

In another embodiment of the invention, a method of identifying a compound that binds or otherwise interacts with a polypeptide or polynucleotide of the invention and inhibits or activates its activity, which is to be screened. Under conditions that bind the compound and the polypeptide or polynucleotide of the invention or cause another interaction therebetween,
A polynucleotide or polypeptide is contacted with a compound to assess binding or other interaction with the compound, such binding or interaction producing a detectable signal in response to the binding interaction with the polypeptide or polynucleotide. And then detecting the presence or absence of a signal resulting from the binding or interaction of the compound with the polypeptide or polynucleotide, thereby allowing the compound to be a polypeptide or polynucleotide. Provided is a method comprising determining whether to bind to or otherwise interact with and activate or inhibit its activity. According to yet another aspect of the present invention, there are provided OBP-NT agonists and antagonists, preferably virucidal or virostatic agonists and antagonists. According to a further aspect of the present invention there is provided a composition comprising an OBP-NT polynucleotide or OBP-NT polypeptide for administration to a unicellular or multinuclear cell organism. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art upon reading the following description and reading other portions of the present disclosure.

BEST MODE FOR CARRYING OUT THE INVENTION As described in more detail below, the present invention relates to OBP-NT polypeptides and polynucleotides. Specifically, the present invention relates to, for example, HSV-2OB
OBP- of other herpesviruses, including P-NT and VZV OBP-NT
OB of HSV-1, related by amino acid sequence homology to NT protein
The present invention relates to P-NT polypeptides and polynucleotides. In particular, the present invention relates to HSV-1 OBP-NT having the nucleotide and amino acid sequences shown in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Table 1 OBP-NT polynucleotide and polypeptide sequences

[0012] (A) HSV-1 of OBP-NT polynucleotide sequence [SEQ ID NO: 1] 5'-ATGAACGACCGCCCCTTCCACCGACTTATCGTCCAGGTGGAAAGCCTTCATCGCGTGGGC CCCAACCTTCTGAACAACTACGACGTCCTCGTTCTGGACGAGGTTATGTCGACGCTGGGC CAGCTCTATTCGCCAACGATGCAGCAACTGGGCCGCGTGGATGCGTTAATGCTACGCCTG CTGCGCATCTGTCCTCGGATCATCGCCATGGACGCAACCGCCAACGCGCAGTTGGTGGAC TTCCTGTGCGGTCTCCGGGGCGAAAAAAACGTGCATGTGGTGGTCGGCGAGTACGCCATG CCCGGGTTTTCGGCGCGCCGGTGCCTGTTTCTCCCGCGTCTGGGGACCGAGCTCCTGCAG GCTGCCCTGCGCCCGCCCGGGCCGCCGAGCGGCCCGTCTCCGGACGCCTCTCCGGAGGCC CGGGGGGCCACGTTCTTTGGGGAGCTGGAAGCGCGCCTTGGCGGGGGCGATAACATCTGC ATTTTTTCGTCGACGGTCTCCTTCGCGGAGATCGTGGCCCGGTTCTGCCGTCAGTTTACG GACCGCGTGCTGTTGCTTCACTCGCTCACCCCCCTCGGGGACGTGACCACGTGGGGCCAA TACCGCGTGGTTATATACACGACGGTCGTAACCGTGGGCCTCAGCTTCGATCCCCTGCAC TTTGATGGCATGTTCGCCTACGTGAAACCCATGAACTACGGACCGGACATGGTGTCCGTG TACCAGTCCCTGGGACGGGTGCGCACCCTCCGCAAGGGGGAGCTACTGATTTACATGGAC GGCTCCGGGGCGCGCTCGGAGCCCGTCTTTACGCCCATGCTCCTTAATCACGTGGTCAGT TCCTGCGGCCAGTGGCCCGCGCAGTTC TCCCAGGTCACAAACCTGCTGTGTCGCCGGTTC AAGGGGCGCTGTGACGCGTCGGCATGCGACACGTCGCTGGGGCGGGGGTCGCGCATCTAC AACAAATTCCGTTACAAACACTACTTTGAGAGATGCACGCTGGCGTGTCTCTCGGACAGC CTTAACATCCTTCACATGCTGCTGACCCTAAACTGCATACGCGTGCGCTTCTGGGGACAC GACGATACCCTGACCCCAAAGGACTTCTGTCTGTTTTTGCGGGGCGTACATTTCGACGCC CTCAGGGCCCAGCGCGATCTACGGGAGCTGCGGTGCCGGGATCCCGAGGCGTCGCTGCCG GCCCAGGCCGCCGAGACGGAGGAGGTGGGTCTTTTCGTCGAAAAATACCTCCGGTCCGAT GTCGCGCCGGCGGAAATTGTCGCGCTCATGCGCAACCTCAACAGCCTGATGGGACGCACG CGGTTTATTTACCTGGCGTTGCTGGAGGCCTGTCTCCGCGTTCCCATGGCCACCCGCAGC AGCGCCATATTTCGGCGGATCTATGACCACTACGCCACGGGCGTCATCCCCACGATCAAC GTCACCGGAGAGCTGGAGCTCGTGGCCCTGCCCCCCACCCTGAACGTAACCCCCGTCTGG GAGCTGTTGTGCCTGTGCAGCACCATGGCCGCGCGCCTGCATTGGGACTCGGCGGCCGGG GGATCTGGGAGGACCTTCGGCCCCGATGACGTGCTGGACCTACTGACCCCCCACTACGAC CGCTACATGCAGCTGGTGTTCGAACTGGGCCACTGTAACGTAACCGACGGACTTCTGCTC TCGGAGGAAGCCGTCAAGCGCGTCGCCGACGCCCTAAGCGGCTGTCCCCCGCGCGGGTCC GTTAGCGAGACGGACCACGCGGTGGCGCTGTTCAAGATAATCTGGGGCGAACTGTTTGGC GTGCAGATGGCCAAAAGCACGCAGACGTTTCCCGGGGCGGGGCGCGTTAAA AACCTCACC AAACAGACAATCGTGGGGTTGTTGGACGCCCACCACATCGACCACAGCGCCTGCCGGACC CACAGGCAGCTGTACGCCCTGCTTATGGCCCACAAGCGGGAGTTTGCGGGCGCGCGCTTC AAGCTACGCGTGCCCGCGTGGGGGCGCTGTTTGCGCACGCATCATCCGCGCGATCGATCCCAGCCGCGCCATCATGCGCGCGACGCCCATCAGCGCGACGCCCATCAGCGCGAGCCCCATCAGCGCGACGCCCATCAGCGCGACGCCCATCAGCGCGCGCACCCATCATACCTGCGTCGCGCGCTCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCCC at

[0013] (B) OBP-NT polypeptide sequences in this table polynucleotide herpes simplex virus type 1 deduced from the sequence (HSV-1) [SEQ ID NO: _{2] NH 2 -MNDRPFHRLIVQVESLHRVGPNLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRL LRICPRIIAMDATANAQLVDFLCGLRGEKNVHVVVGEYAMPGFSARRCLFLPRLGTELLQ AALRPPGPPSGPSPDASPEARGATFFGELEARLGGGDNICIFSSTVSFAEIVARFCRQFT DRVLLLHSLTPLGDVTTWGQYRVVIYTTVVTVGLSFDPLHFDGMFAYVKPMNYGPDMVSV} YQSLGRVRTLRKGELLIYMDGSGARSEPVFTPMLLNHVVSSCGQWPAQFSQVTNLLCRRF KGRCDASACDTSLGRGSRIYNKFRYKHYFERCTLACLSDSLNILHMLLTLNCIRVRFWGH DDTLTPKDFCLFLRGVHFDALRAQRDLRELRCRDPEASLPAQAAETEEVGLFVEKYLRSD VAPAEIVALMRNLNSLMGRTRFIYLALLEACLRVPMATRSSAIFRRIYDHYATGVIPTIN VTGELELVALPPTLNVTPVWELLCLCSTMAARLHWDSAAGGSGRTFGPDDVLDLLTPHYD RYMQLVFELGHCNVTDGLLLSEEAVKRVADALSGCPPRGSVSETDHAVALFKIIWGELFG VQMAKSTQTFPGAGRVKNLTKQTIVGLLDAHHIDHSACRTHRQLYALLMAHKREFAGARF KLRVPAWGRCLRTHSSSANPNADIILEAALSELPTEAWPMMQGAVNFSTL * -OH

[0014] (C) herpes simplex virus type 2 (HSV-2) a polynucleotide sequence [SEQ ID NO: 3] containing OBP-NT ORF sequence of 5'- ATGAACGACCGCCCCTTCCACCGTCTCATCGTGCAGGTGGAAAGTCTTCATCGCGTGGGC CCGAACCTGTTGAACAACTACGACGTGCTCGTCACGAAGTCACTCATGTCGACGTTGGGC CAACTGTACTCGCCGACGATGCAGCAGCTGGGCCGCGTCGACGCGTTGATGCTGCGCCTG CTGCGCACGTGCCCGCGGATCATCGCCATGGACGCCACCGCCAACGCGCAGCTGGTGGAC TTTCTGTGCAGCCTCCGGGGCGAAAAGAACGTTCACGTGGTCATCGGGGAGTACGCCATG CCCGGATTTTCGGCGCGCCGTTGTCTGTTTCTCCCGCGCCTGGGGCCCGAGGTCCTGCAG GCGGCCCTGCGCCGCCGGGGGCCGGCGGGCGGGGCGCCCCCCCCGGACGCCCCCCCGGAC GCCACCTTCTTCGGGGAGCTGGAGGCGCGCCTGGCCGGCGGAGATAACGTCTGCATCTTC TCGTCGACGGTCTCCTTCGCGGAGGTCGTTGCCAGGTTCTGCCGGCAGTTTACGGACCGC GTGCTGCTGCTCCACTCGCTCACCCCGCCCGGCGACGTGACCACATGGGGCCGGTACCGG GTGGTCATCTACACAACGGTCGTGACGGTGGGCCTTAGCTTCGATCCGCCGCACTTTGAC AGCATGTTCGCCTACGTGAAACCCATGAACTACGGGCCGGACATGGTGTCCGTGTACCAG TCGCTGGGGCGGGTACGGACTCTCCGCAAGGGGGAGCTGCTGATCTACATGGACGGGTCC GGGGCGCGCTCGGAGCCCGT CTTTACGCCCATGCTGCTCAACCACGTGGTGAGCGCCAGC GGGCAGTGGCCGGCACAGTTTTCCCAGGTGACGAACCTGTTGTGCCGCCGGTTCAAAGGG CGCTGCGACGCGTCGCACGCCGACGCGGCGCAGGCGCGGGGGTCGCGCATCTACAGCAAA TTCCGGTACAAGCACTACTTCGAGAGGTGCACGCTGGCGTGCCTCGCGGACAGTCTTAAC ATCCTCCACATGCTTCTGACCCTCAACTGCATGCACGTGCGGTTCTGGGGCCACGACGCC GCGCTGACCCCGAGGAACTTTTGTCTGTTTTTGCGGGGGATACATTTTGATGCCCTGAGG GCCCAGCGGGATCTGCGGGAGCTGCGCTGCCAGGACCCCGACACGTCCCTGTCGGCCCAG GCCGCCGAGACGGAGGAGGTGGGCCTTTTCGTCGAAAAGTACCTCCGGCCGGACGTCGCG CCGGCCGAGGTGGTCGCGCTCATGCGCGGCCTCAACAGCCTGGTCGGCCGCACGCGGTTC ATCTACCTGGTGCTGCTGGAGGCCTGTCTTCGCGTCCCCATGGCCGCCCATAGCAGCGCC ATCTTCCGGCGGCTTTACGACCACTACGCCACGGGCGTCATCCCCACGATCAACGCCGCC GGAGAGCTGGAGCTTGTGGCCCTACACCCCACCCTAAACGTCGCCCCCGTCTGGGAGCTG TTCCGTCTGTGCAGCACCATGGCCGCGTGCCTGCAGTGGGACTCGATGGCCGGGGGGTCG GGGCGAACCTTTAGCCCCGAGGACGTGCTGGAGCTGCTGAACCCCCACTACGACCGCTAC ATGCAGCTGGTGTTCGAACTGGGCCACTGTAACGTGACCGACGGCCCCTTGCTGTCGGAG GACGCGGTTAAGCGCGTGGCCGACGCCCTGAGCGGCTGCCCCCCGCGCGGGTCCGTGAGC GAGACGGAGCACGCGCTGTCGCTGTTCAAGATCATCTGGGGCGA ACTGTTCGGGGTGCAG CTGGCCAAGAGCACGCAGACGTTTCCCGGGGCGGGGCGCGTTAAAAACCTCACCAAGCGA GCCATCGTGGAGCTGCTGGACGCCCACCGCATCGACCACAGCGCCTGCCGGACGCACAGA CAGCTGTACGCGCTGCTGATGGCCCATAAGCGGGAGTTTGCGGGCGCGCGCTTCAAGCTG CGCGCGCCCGCGTGGGGGCGCTGCTTGCGCACGCACGCCTCCGGCGCCCAGCCCAACACT GACATCATTCTCGAGGCGGCTCTGTCGGAGCTTCCCACCGAGGCCTGGCCCATGATGCAG GGGGCGGTGAACTTTAGCACCCTATAA-3 '

[0015] (D) herpes simplex virus type 2 deduced from ORF sequence of a polynucleotide (HSV-2) OBP-NT polypeptide sequence [SEQ ID NO: 4] NH2-MNDRPFHRLIVQVESLHRVGPNLLNNYDVLVTKSLMSTLGQLYSPTMQQLGRVDALMLRL LRTCPRIIAMDATANAQLVDFLCSLRGEKNVHVVIGEYAMPGFSARRCLFLPRLGPEVLQ AALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEVVARFCRQFTDR VLLLHSLTPPGDVTTWGRYRVVIYTTVVTVGLSFDPPHFDSMFAYVKPMNYGPDMVSVYQ SLGRVRTLRKGELLIYMDGSGARSEPVFTPMLLNHVVSASGQWPAQFSQVTNLLCRRFKG RCDASHADAAQARGSRIYSKFRYKHYFERCTLACLADSLNILHMLLTLNCMHVRFWGHDA ALTPRNFCLFLRGIHFDALRAQRDLRELRCQDPDTSLSAQAAETEEVGLFVEKYLRPDVA PAEVVALMRGLNSLVGRTRFIYLVLLEACLRVPMAAHSSAIFRRLYDHYATGVIPTINAA GELELVALHPTLNVAPVWELFRLCSTMAACLQWDSMAGGSGRTFSPEDVLELLNPHYDRY MQLVFELGHCNVTDGPLLSEDAVKRVADALSGCPPRGSVSETEHALSLFKIIWGELFGVQ LAKSTQTFPGAGRVKNLTKRAIVELLDAHRIDHSACRTHRQLYALLMAHKREFAGARFKL RAPAWGRCLRTHASGAQPNTDIILEAALSELPTEAWPMMQGAVNFSTL * -COOH

[0016] (E) herpes virus VZV OBP-NT polynucleotide sequences [SEQ ID NO: 2] 5'-ATGGGTTTTAAACGTTTGATTGTGCAACTTGAAAGCCTACACCGCGTATCCAGCGAAGCT ATCGACAGCTACGACGTATTAATACTGGATGAGGTAATGTCAGTGATTGGACAATTATAC TCCCCCACAATGAGACGTCTTTCCGCGGTTGATAGCCTATTATATCGTCTTTTAAATCGC TGTTCTCAAATTATCGCGATGGATGCTACAGTAAACTCGCAGTTTATTGATTTAATCTCC GGATTGCGTGGAGATGAAAACATACACACAATTGTGTGTACATACGCGGGAGTTGGGTTC TCCGGAAGAACTTGCACGATCCTGCGTGATATGGGCATCGACACGCTTGTGCGAGTCATT AAACGATCTCCTGAACACGAGGATGTACGTACCATACACCAACTACGTGGAACATTTTTT GACGAACTAGCACTACGATTACAATGTGGGCATAACATCTGTATATTTTCATCAACTTTA TCGTTTTCGGAGCTAGTTGCTCAGTTTTGTGCAATATTTACAGACTCTATTCTTATTTTA AACTCAACTCGGCCCCTATGTAATGTAAACGAATGGAAACATTTTCGCGTGTTGGTGTAC ACTACCGTCGTGACCGTTGGATTGAGTTTTGACATGGCTCATTTTCATAGCATGTTTGCT TACATAAAGCCAATGTCATATGGGCCGGATATGGTATCGGTCTACCAGTCATTAGGGCGT GTACGTTTATTGCTACTTAATGAAGTTTTGATGTACGTCGATGGCTCAAGGACCAGATGC GGACCCCTGTTCTCGCCAATGTTACTAAACTTTACCATCGCAAATAAATTTCAATGGTTT CCTACACACAC CCAAATAACTAACAAACTGTGCTGTGCATTTAGGCAACGATGTGCAAAT GCATTTACACGCTCGAACACCCATCTCTTCTCAAGATTTAAATACAAACACCTTTTCGAG AGATGCTCTCTTTGGAGTTTAGCCGATAGCATTAATATCTTACAAACTCTTTTGGCCTCT AACCAAATTTTGGTTGTATTGGATGGCATGGGTCCAATAACGGACGTTTCCCCAGTTCAA TTTTGTGCATTTATACACGATCTCAGACATAGCGCTAACGCCGTAGCTTCCTGTATGCGT TCTCTTAGACAGGACAATGACAGCTGCTTGACCGATTTTGGCCCTTCCGGATTTATGGCC GATAACATTACCGCGTTTATGGAAAAGTATCTTATGGAGTCAATTAATACCGAAGAACAA ATTAAAGTATTTAAAGCCCTTGCATGTCCAATAGAACAGCCTAGACTAGTCAATACGGCA ATATTGGGGGCGTGTATACGAATACCTGAAGCGTTGGAAGCATTTGACGTATTTCAAAAA ATATACACGCACTACGCTTCCGGTTGGTTTCCCGTCCTGGACAAAACCGGGGAATTTAGC ATCGCGACTATAACTACCGCCCCAAATTTAACCACACATTGGGAGCTGTTTCGCCGTTGT GCCTATATTGCAAAAACACTCAAGTGGAATCCGTCCACCGAAGGCTGTGTAACACAAGTT TTGGATACGGACATTAATACACTTTTCAATCAACACGGGGATTCGCTGGCTCAACTAATA TTTGAGGTTATGCGCTGTAACGTTACTGACGCTAAGATTATATTAAACCGCCCGGTTTGG CGAACAACCGGATTCTTAGATGGATGCCATAATCAATGCTTCCGTCCAATCCCTACAAAA CACGAATATAACATTGCTCTATTTCGTTTAATTTGGGAACAATTATTTGGCGCCCGCGTA ACTAAAAGTACCCAGACCTTTCCGGGAAGTACTCG TGTGAAAAACCTAAAAAAAAAAGAT CTAGAAACTTTACTTGATTCAATTAACGTGGATCGTTCTGCATGTCGTACCTACCGCCAG TTGTATAACCTGCTTATGAGCCAGCGCCATTCGTTCTCTCAACAGCGTTACAAAATTACT GCCCCCGCTTGGGGGCACGCCACGTGCGGTCATTGCACTTGGCGATCATTGCACTTGGCGACGATTGCACTTGGCGAGG

[0017] (F) VZV OBP-NT polypeptide sequence deduced from the polynucleotide sequence [SEQ ID NO: 6] NH2-MGFKRLIVQLESLHRVSSEAIDSYDVLILDEVMSVIGQLYSPTMRRLSAVDSLLYRLLNR CSQIIAMDATVNSQFIDLISGLRGDENIHTIVCTYAGVGFSGRTCTILRDMGIDTLVRVI KRSPEHEDVRTIHQLRGTFFDELALRLQCGHNICIFSSTLSFSELVAQFCAIFTDSILIL NSTRPLCNVNEWKHFRVLVYTTVVTVGLSFDMAHFHSMFAYIKPMSYGPDMVSVYQSLGR VRLLLLNEVLMYVDGSRTRCGPLFSPMLLNFTIANKFQWFPTHTQITNKLCCAFRQRCAN AFTRSNTHLFSRFKYKHLFERCSLWSLADSINILQTLLASNQILVVLDGMGPITDVSPVQ FCAFIHDLRHSANAVASCMRSLRQDNDSCLTDFGPSGFMADNITAFMEKYLMESINTEEQ IKVFKALACPIEQPRLVNTAILGACIRIPEALEAFDVFQKIYTHYASGWFPVLDKTGEFS IATITTAPNLTTHWELFRRCAYIAKTLKWNPSTEGCVTQVLDTDINTLFNQHGDSLAQLI FEVMRCNVTDAKIILNRPVWRTTGFLDGCHNQCFRPIPTKHEYNIALFRLIWEQLFGARV TKSTQTFPGSTRVKNLKKKDLETLLDSINVDRSACRTYRQLYNLLMSQRHSFSQQRYKIT APAWARHVYFQAHQMHLAPHAEAMLQLALSELSPGSWPRINGAVNFESL * -COOH

Polypeptide The polypeptide of the present invention is a polypeptide of Table 1 [SEQ ID NO: 2, 4 or 6] (
Especially mature polypeptides) and polypeptides and fragments, in particular having the biological activity of OBP-NT, having at least 70% identity to the polypeptides of Table 1 or the relevant parts thereof, preferably to the polypeptides of Table 1 At least 80%
Polypeptides and fragments having at least 90% identity to the polypeptides of Table 1, even more preferably at least 95% identity to the polypeptides of Table 1, further comprising such polypeptides And generally such a portion of the polypeptide comprises at least 30 amino acids, more preferably at least 50 amino acids.

The present invention also provides a compound of the formula: X— (R ₁ ) _m — (R ₂ ) — (R ₃ ) _n —Y wherein X at the amino terminus is hydrogen or metal and Y at the carboxyl terminus Hydrogen or metal, R ₁ and R ₃ are any amino acid residues, and m is 1 to 1
000 is an integer of 0 or 0, n is an integer of 1 to 1000 or 0, and R ₂ represents the amino acid sequence of the present invention, in particular, the amino acid sequence of Table 1]. In the above formulas, R ₂ is bonded to R ₁ amino terminal residues at its left, its carboxy terminal residue is directed to bind to R ₃ in the right. When m and / or n is greater than 1, the chain of amino acid residues represented by any of the R groups may be either a heteropolymer or a homopolymer, with a heteropolymer being preferred.

A fragment is a variant polypeptide having an amino acid sequence that entirely, but not entirely, the amino acid sequence of a polypeptide described above. OB
Like the P-NT polypeptide, the fragment is “free-standing
) "Or they may be comprised in one part or region, most preferably a single contiguous region, a larger polypeptide forming a single larger polypeptide. Preferred fragments include truncated polypeptides having some of the variants, such as, for example, the amino acid sequence of Table 1 or a contiguous series of residues including the amino or carboxyl-terminus. Also preferred are degraded forms of the polypeptides of the present invention in a host, particularly HSV-1. In addition, alpha helices and alpha helix forming regions, beta sheets and beta sheet forming regions, turns and turn forming regions, coils and coil forming regions, hydrophilic regions, hydrophobic regions, alpha amphiphilic regions, beta amphiphilic regions, flexible regions Also preferred are fragments characterized by structural or functional attributes, such as fragments that comprise a surface-forming region, a substrate binding region, and a high antigen index region.

[0021] Biologically active fragments are also preferred fragments. Biologically active fragments are those that mediate the activity of OBP-NT, including those that have a similar or improved activity, or that have a reduced undesirable activity. Also included are fragments that are antigenic or immunogenic in an animal, particularly a human. Particularly preferred are fragments consisting of receptors or domains of enzymes that confer a function essential for the survival of HSV-1 or the ability to initiate or maintain disease in an individual, particularly a human.

Variants that are fragments of the polypeptides of the present invention can be used for the production of the corresponding full-length polypeptides by peptide synthesis methods. Thus, these variants can be used as intermediates for producing the full-length polypeptides of the invention. In addition to the standard one and three letter designations for amino acids, "X" or "X
The term "aa" is also used to describe certain polypeptides of the present invention. "
"X" and "Xaa" mean that any of the twenty naturally occurring amino acids can be at such designated positions in the polypeptide sequence.

Polynucleotides Another aspect of the present invention relates to isolated polynucleotides, including full-length genes, encoding OBP-NT polypeptides having the deduced amino acid sequences of Table 1, including closely related polynucleotides and polynucleotides thereof. About the variant. Using the information herein, such as the polynucleotide sequence shown in Table 1 [SEQ ID NO: 1, 3 or 5], using HSV-1 KOS as a starting material, cloning a chromosomal DNA fragment from the virus, Using standard cloning and screening methods to determine and subsequently obtain full-length clones, OBP-N
A polynucleotide of the invention encoding a T polypeptide can be obtained. For example, to obtain a polynucleotide sequence of the present invention such as the sequence shown in Table 1, a library of clones of chromosomal DNA of HSV-1 KOS in Escherichia coli or other suitable host is derived from the partial sequence. With a radiolabeled oligonucleotide, preferably a 17-mer or longer oligonucleotide. The clones having the same DNA as the probe can then be distinguished using stringent conditions. Alternatively, novel messenger RNAs (mRNAs) encoding the polypeptides were identified by Northern blot analysis and radiolabeled oligonucleotides or complementary RNA fragments derived from subsequences, preferably 17-mers or longer. Can be probed. Then
The 5 'and 3' ends of the new gene can be mapped and the gene cloned. By sequencing individual clones identified with sequencing primers designed from the original sequence, it is possible to extend the sequence in both directions and determine the full-length gene sequence. Conveniently, such sequencing is performed using denatured double-stranded DNA prepared from a plasmid clone. A suitable technique is Ma
niatis, T., Fritsch, EF and Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbo
r, New York (1989) (see, inter alia, screening by hybridization 1.90 and sequencing of denatured double-stranded DNA template 13.70). For example, the polynucleotide shown in Table 1 [SEQ ID NO: 1] is H
MRNA harvested after infection with SV-1 was found by subjecting to Northern blot analysis.

The DNA sequence in Table 1 has approximately the same number of amino acid residues as shown in Table 1 [SEQ ID NO: 2] and a protein having an estimated molecular weight that can be calculated from the molecular weight of amino acid residues known in the art. Has an open reading frame. The polynucleotide of SEQ ID NO: 1 between nucleotide number 1 of SEQ ID NO: 1 and the stop codon starting at nucleotide number 2131 encodes the polypeptide of SEQ ID NO: 2. The OBP-NTs of the present invention are structurally associated with other proteins of the herpesvirus family, particularly, for example, UL9 and UL8.5 of HSV-1.

The present invention provides a polynucleotide sequence that is identical over its entire length to the nucleotide coding sequence in Table 1. The present invention also relates to the coding sequence for the mature polypeptide or fragment thereof as well as the reading sequence in the reading frame having other coding sequences, such as coding sequences for leader or secretory sequences, pre, pro or prepro protein sequences. A coding sequence for a polypeptide or fragment is provided. Polynucleotides also include non-coding 5 'and nucleotides, such as, for example, transcribed but not translated sequences, termination signals, mRNA stabilizing sequences, introns, polyadenylation signals, and additional coding sequences encoding additional amino acids. Non-coding sequences, including but not limited to 3 'sequences. For example, it can encode a marker sequence that facilitates purification of the fusion polypeptide. In certain embodiments of the invention, the marker sequence is a pQE vector (Qiagen
Inc.), Gentz et al., Proc. Natl. Acad. Sci. USA (1989) 86.
: A hexa-histidine peptide as described in 821-824;
Or an HA tag (Wilson et al., Cell, 37: 767 (1984)). Polynucleotides of the present invention include, but are not limited to, polynucleotides consisting of structural genes and naturally associated sequences that regulate gene expression.

A preferred embodiment of the present invention is a polynucleotide having from nucleotide 1 shown in SEQ ID NO: 1 of Table 1 to a nucleotide immediately upstream of nucleotide 2131 or a nucleotide containing the nucleotide, wherein HSV-1 O
Encodes a BP-NT polypeptide.

The present invention also provides a compound of the formula: X- (R₁)_m− (R_Two)-(R_Three)_n-Y wherein X at the 5 'end of the molecule is hydrogen or a metal, or
To form a covalent bond, and Y at the 3 'end of the molecule is hydrogen or metal, or
Together with X forms a covalent bond, and R₁And R_ThreeAre each independently
M is an integer of 1 to 3000 or 0, and n is 1 to 300
An integer of 0 or 0;_TwoIs the nucleic acid sequence of the present invention, particularly a nucleus selected from Table 1.
Acid sequence]. In the polynucleotide of the above formula, R _Two Means that the 5 'terminal residue is R₁And the 3 'terminal residue has R_Three
Orientated to join. If m and / or n is greater than 1, R
The chain of nucleic acid residues represented by any of the groups
Any one may be used, and a heteropolymer is preferred. In a preferred embodiment,
When X and Y together form a covalent bond, the polynucleotide of the above formula
A tide is a closed circular polynucleotide whose formula is such that the second strand is complementary.
The polynucleotide may be a double-stranded polynucleotide exhibiting a first strand. Another specific example
Wherein m and / or n is an integer between 1 and 1000.

Most preferably, the polynucleotide of the present invention is derived from HSV-1, but is also preferably obtained from other organisms of the same family taxonomically. As used herein, the term "polynucleotide encoding a polypeptide" refers to a polypeptide of the present invention, in particular, a viral polypeptide, more specifically, a polypeptide of OBP-NT having the amino acid sequence shown in Table 1. Includes polynucleotides containing sequences encoding the peptides. The term includes a single contiguous or non-contiguous region encoding the polypeptide (e.g., interrupted by an inserted sequence or editing), as well as additional regions that may include coding and / or non-coding sequences. And polynucleotides. The present invention further relates to variants of the polynucleotides described herein that encode variants of the polypeptide having the deduced amino acid sequence of Table 1. Variants that are fragments of the polynucleotides of the invention can be used for the synthesis of full-length polynucleotides of the invention.

Further preferred embodiments are those wherein several, a few, 5-10, 1-5, 1-3, 2 or 1 or 0 amino acid residues are substituted, deleted or deleted in any combination. An OB having the amino acid sequence of the OBP-NT polypeptide of Table 1 added.
Polynucleotide encoding a P-NT variant. Particularly preferably, OBP-
Silent substitutions, additions and deletions that do not alter the properties or activity of NT.

A further preferred embodiment of the present invention relates to an OBP- having the amino acid sequence shown in Table 1.
At least 7 over the entire length of the polynucleotide encoding the NT polypeptide.
Polynucleotides having 0% identity and polynucleotides complementary to such polynucleotides. Also most preferred are polynucleotides consisting of a region having at least 80% identity over its entire length to a polynucleotide encoding an OBP-NT polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides having at least 90% identity over their entire length to the same are particularly preferred, with those having at least 95% identity being particularly preferred. Furthermore, among those having at least 95% identity, those having at least 97% identity are more preferred, and those with at least 98% and at least 99% identity are particularly preferred. Those with at least 99% identity are more preferred.

A preferred embodiment is a polynucleotide that encodes a polypeptide that retains substantially the same biological function or activity as the mature polypeptide encoded by the DNA in Table 1. The invention further relates to polynucleotides that hybridize to the sequences described herein. In this regard, the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein above. As used herein, the terms "stringent conditions" and "stringent hybridization conditions" refer to hybridization occurring only when there is at least 95%, preferably at least 97%, identity between the sequences. Means As an example of stringent hybridization conditions, 50% formamide, 5 × SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured cut salmon sperm DNA overnight at 42 ° C. Incubate and then hybridize the support to 0.1 × SS
C (about 65 ° C.). Hybridization and washing conditions are known and described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL,
Second Edition, Cold Spring Harbor, NY (1989), especially in Chapter 11.

The present invention provides for the use of a suitable library containing the complete gene of the polynucleotide sequence shown in Table 1 under stringent hybridization conditions with a probe having the sequence of the polynucleotide sequence shown in Table 1 or a fragment thereof. Screening,
There is provided a polynucleotide essentially comprising the polynucleotide sequence obtainable by isolating the DNA sequence. Fragments useful for obtaining such polynucleotides include, for example, probes and primers described elsewhere herein.

As further described herein for the analysis of the polynucleotides of the invention, for example, the polynucleotides of the invention as described
And used as a hybridization probe for genomic DNA,
The full-length cDNA and genomic clone encoding BP-NT were isolated and
CDNA and genomic clones of other genes with high identity to the NT gene can be isolated. Such probes generally have at least 15
Bases. Preferably, such a probe consists of at least 30 bases and may have at least 50 bases. Particularly preferred probes have at least 30 bases and no more than 50 bases. For example, the coding region of the OBP-NT gene can be isolated by screening using the DNA sequence in Table 1 to synthesize an oligonucleotide probe. A cDNA, genomic DNA or mRNA library is then screened using a labeled oligonucleotide having a sequence complementary to the sequence of the gene of the invention to determine which members of the library will hybridize to the probe.

The polynucleotides and polypeptides of the present invention, as further described herein with respect to polynucleotide analysis, for example, as research reagents and materials for the discovery of treatment and diagnosis for diseases, especially human diseases. Can be used. Polynucleotides of the present invention that are oligonucleotides derived from the sequences in Table 1 can be used in the methods described herein, but are preferably used in PCR, and the polynucleotides identified herein as a whole or It is determined whether it is transcribed by the virus in partially infected tissues. Such sequences are also useful in diagnosing the stage of infection reached by the pathogen. The present invention also relates to polypeptides wherein additional amino or carboxy terminal amino acids are added to the mature protein or amino acids are added inside the mature polypeptide (eg, where the mature form has one or more polypeptide chains). An encoding polynucleotide is provided. Such sequences may, among other things, play a role in the processing of the protein from the precursor to the mature form, carry the protein, increase or decrease the half-life of the protein, or manipulate the protein for analysis or production. Can be facilitated. As is common in vivo, the added amino acids are processed away from the mature protein by cellular enzymes.

A precursor protein having a mature form of a polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When the prosequence is removed from such an inactive precursor, it is generally activated. Some or all of the prosequence can be removed prior to activation. Generally, such precursors are called proproteins. In addition to the standard symbols A, G, C, T / U for nucleotides, the term "N" can also be used to describe a particular polynucleotide of the invention. "
N "is a nucleic acid having the effect of forming a premature stop codon in such an open reading frame when acting together with adjacent nucleotide positions and reading in the correct reading frame. Unless otherwise preferred, it means that any of the four DNA or RNA bases is at that designated position in the DNA or RNA sequence.

In short, the polynucleotide of the present invention may be a mature protein, a mature protein to which a leader sequence is added (also referred to as a pro? Protein), or a mature protein having one or more pro sequences that are not the leader sequence of a preprotein. It encodes a pre-proprotein which is a precursor, or a precursor sequence and a precursor of a proprotein, which generally has one or more prosequences which are removed during a processing step that produces an active mature form of the polypeptide.

Vectors, Host Cells, Expression Systems The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered with the vectors of the present invention, and the production of polypeptides of the present invention by recombinant techniques. Further, such a protein can be produced using a cell-free translation system and RNA derived from the DNA construct of the present invention. For recombinant production, host cells can be genetically engineered to incorporate an expression system or a portion thereof, or a polynucleotide of the present invention. Introduction of a polynucleotide into a host cell can be performed by calcium phosphate transfection, DEAE-
Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambroo, such as dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
k et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989), can be performed by methods described in several standard laboratory manuals.

Representative examples of suitable hosts include streptococcus, staphyl
ococcus), bacterial cells such as enterococcus, E. coli, streptomyces and Bacillus subtilis; fungal cells such as yeast cells and Aspergillus cells; Drosophila ) Insect cells such as S2 and Spodoptera Sf9 cells; CHO, COS, HeLa, C127, 3T3, BHK, 293, Vero (V
ero) and Bowes melanoma cells; and plant cells.

A wide variety of expression systems can be used to produce the polypeptides of the present invention. Such vectors are, inter alia, chromosomal, episomal and viral derived vectors, such as bacterial plasmid derived, bacteriophage derived, transposon derived, yeast episomal derived, insertion element derived, yeast chromosomal element derived, herpes virus, baculovirus, such as SV40. Derived from combinations thereof, such as vectors derived from viruses such as papovavirus, vaccinia virus, adenovirus, chicken poxvirus, pseudorabies virus and retrovirus, and vectors derived from plasmids such as cosmids and phagemids and genetic factors of bacteriophage. Vector to be used. Expression system constructs may have regulatory regions that control as well as cause expression. Generally, systems or vectors suitable for maintaining, amplifying or expressing the polynucleotide in the host and / or expressing the polypeptide may be used for expression in this regard. Suitable DNA sequences can be obtained, for example, from Sambrook et al., MOLECULAR CLONING.
, A LABORATORY MANUAL (supra), may be inserted into the expression system by any of a variety of well-known conventional techniques.

In order to secrete the translated protein into the endoplasmic reticulum lumen, periplasmic space or extracellular environment, an appropriate secretion signal may be inserted into the expressed polypeptide. These signals may be unique to the polypeptide or may be heterologous signals. The polypeptide of the present invention may be prepared by ammonium sulfate or ethanol precipitation, acid extraction,
Can be recovered and purified from recombinant cell culture by well-known methods, including anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. . Most preferably, high quality liquid chromatography is used for purification. If the polypeptide denatures during isolation and / or purification, it can be reconstituted into its active conformation using well known methods for regenerating proteins.

Diagnostic, Prognostic, Serotyping and Mutation Assays The present invention also relates to the use of the OBP-NT polynucleotides of the present invention for use as diagnostic reagents. O in eukaryotes, especially mammals, especially humans
Detection of BP-NT will provide a diagnostic method for diagnosis of disease. OBP
-Eukaryotes that can infect or possibly infect organisms containing the NT gene (hereinafter, "
Individuals), especially mammals, especially humans, can be detected at the nucleic acid level by various methods.

Nucleic acids for diagnosis are obtained from cells and tissues of infected individuals, such as neuronal organs, blood, mucous membranes, epithelium and skin. Genomic DNA may be used directly for detection or may be enzymatically amplified by using PCR or other amplification methods before subjecting it to analysis. RNA, cDNA and genomic DNA can be used in a similar manner. With amplification, the species and strain of virus present in an individual can be characterized by genotyping the viral genes. Deletions and insertions can be detected by a change in the size of the amplified product relative to the genotype of the control sequence. Point mutations can be identified by hybridizing the amplified DNA to a labeled OBP-NT polynucleotide sequence. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences can also be detected by altering the electrophoretic mobility of the DNA fragment in the gel, with or without denaturing agents, or by direct DNA sequencing. See, for example, Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S1 protection or chemical cleavage methods. For example, Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
)checking.

Cells carrying mutations or polymorphisms (allelic mutations) in the gene of the invention
DNA or RNA by various techniques, such as to allow for serotyping
Can be detected at the level. For example, RT-PCR can be used to detect mutations in RNA. It is particularly preferred to use RT-PCR in combination with an automatic detection system, such as GeneScan. RNA, cDNA or genomic DNA can also be used for PCR or RT-PCR for the same purpose. As an example, OBP-
Mutations can be identified and analyzed using PCR primers complementary to the nucleic acid encoding the NT polypeptide. Examples of typical primers are shown in Table 2 below.

Table 2 Primers for Amplifying OBP-NT Polynucleotide SEQ ID NO: Primer Sequence 5 5′-CCTGTCGTCGGAGTTTTACCCAG-3 ′ 65′-GAGGTGCGGTCCCGAGACTTATAG-3 ′

The present invention also provides a compound of the formula: X- (R₁)_m− (R_Two)-(R_Three)_n-Y wherein X at the 5 'end of the molecule is hydrogen or metal, and Y at the 3' end of the molecule is water.
Element or metal;₁And R_ThreeIs a nucleic acid residue, m is an integer of 1 to 20 or
Is 0; n is an integer of 1 to 20 or 0;_TwoIs the primer sequence of the present invention
And especially a primer sequence selected from Table 2]. In the polynucleotide of the above formula, R _Two Means that the 5 'terminal residue is R₁And the 3 'terminal residue has R_Three
Orientated to join. if m and / or n is greater than 1,
The chain of the nucleic acid residue represented by any of the R groups is a heteropolymer or a homopolymer.
Any of them may be used, and preferably a region complementary to the polynucleotide region in Table 1 is used.
It is a telomer. In a preferred embodiment, m and / or n are 1 to
It is an integer between 10.

The present invention further provides a primer in which 1, 2, 3 or 4 nucleotides have been removed from the 5 ′ and / or 3 ′ end. In particular, these primers
It can be used for amplification of OBP-NT DNA isolated from a sample derived from an individual. Primers may be used to amplify a gene isolated from an infected individual and subject the gene to various methods for DNA sequence studies. In this way, mutations in the DNA sequence may be detected, the mutation may be used to diagnose infection, and the serotype and / or classification of the infectious agent may be performed. The invention further relates to diseases, preferably viral infections, more preferably HSV-
1. A method of diagnosing an infection according to 1., wherein the increase in the expression level of a polynucleotide having the sequence of Table 1 is determined from a sample derived from the individual. Increasing or decreasing the expression of the OBP-NT polynucleotide can be performed by any method known in the art for quantifying a polynucleotide, for example, amplification, PC
It can be measured using R, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

In addition, a diagnostic assay according to the invention for detecting overexpression of the OBP-NT protein compared to a normal control tissue sample can be used, for example, to detect the presence of an infection. Assay techniques that can be used to determine levels of OBP-NT protein, in a sample derived from a host are well-known to those of skill in the art. Such assays include radioimmunoassays, competitive binding assays, Western blot analysis and ELISA assays. Differences in RNA or genomic sequence between viruses of different phenotypes can also be measured. Sequence variation is observed in some or all viruses of a particular phenotype, but if neither virus has lost the phenotype, the sequence variation may be responsible for the phenotype. In this way, chromosomal regions that confer pathogenicity, growth characteristics, survival characteristics and / or ecological niche characteristics can be identified.

Differential expression The polynucleotides and polypeptides of the present invention may be used as reagents in a differential screening method. Many differential screening and differential display methods exist in the art and can use the polynucleotides and polypeptides of the present invention. For example, the differential display method is described in Chuang et al., J. Bacteriol. 175: 2026-2036.
(1993). This method uses a randomly primed RT-P
A gene expressed in an organism is identified by identifying an mRNA present using CR. By comparing pre-infection and post-infection characteristics, genes that are up- and down-regulated during infection can be identified, RT-PCR products can be sequenced and matched to an "unknown" ORF .

Gene expression patterns may be analyzed using RT-PCR. For RT-PCR using the polynucleotides of the present invention, messenger RNA is isolated from virus-infected tissues, followed by reverse transcription of RNA samples primed with random hexanucleotides, followed by PCR using gene-specific primer pairs. To evaluate the amount of each mRNA. Determination of the presence and amount of a particular mRNA species by quantification of the resulting PCR product provides information about the viral genes transcribed in the infected tissue. Analyze gene transcription at different times of infection to gain in-depth knowledge of gene regulation in viral pathogenesis and gain a clear understanding of which gene products are targets for antiviral drug screening Can be. Due to the gene-specific properties of the PCR primers used,
It will be appreciated that viral mRNA preparations need not always include mammalian RNA. Optimally, in a very short time, TRIzole (GIBC
O-BRL) mechanically crushed in the presence, then TRIzole reagent and DNAa
Viral mRNA is prepared from infected murine lung tissue by performing se treatment according to the manufacturer's instructions to remove contaminating DNA. Preferably, the process is optimized by finding conditions that maximize the amount of HSV-1 RNA detected by probing Northern with an appropriately labeled sequence-specific oligonucleotide or RNA probe. Each of these methods may have advantages depending on the particular application.
One skilled in the art will select the solution that is most relevant to the particular application.

Antibodies The polypeptides of the invention or variants thereof, or cells expressing them, can be used as an immunogen to obtain antibodies immunospecific for such polypeptides. "Antibodies" as used herein include monoclonal and polyclonal antibodies, chimeric antibodies, single-chain antibodies, monkey and humanized antibodies, and Fab fragments, including the products of a Fab immunoglobulin library. Antibodies obtained against the polypeptides of the present invention can be obtained by administering the polypeptides or fragments, analogs or cells bearing the epitope, preferably to non-human animals, using conventional protocols. For preparing monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. For example, Kohler, G. and Mils
tein, C., Nature, 256: 495-497 (1975); Kozbor et al., Immunolo.
gy Today, 4:72 (1983); Cole et al., MONOCLONAL ANTIBODIES AND CANCER.
Various techniques such as those described in THERAPY, Alan R Liss, Inc., pp. 77-96 (1985).

Using techniques for producing single-chain antibodies (US Pat. No. 4,946,778),
Single chain antibodies against the polypeptides of the present invention can be produced. In addition, transgenic mice or other organisms, such as other mammals, can be used to express humanized antibodies. Alternatively, anti-OBP-
Antibody genes that have binding activity for the polypeptide may be selected from a repertoire of PCR-amplified v genes of human lymphocytes screened for retention of NT or from an intact library (McCafferty, J. et al. Et al., Natu
re 348: 552-554 (1990); Marks, J. et al., Biotechnology 10:
779-783 (1992)). The affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al., Nature 3).
52: 624-628 (1991)).

When two antigen binding domains are present, each domain may be directed to a different epitope, referred to as a “bispecific” antibody. Clones expressing the polypeptide of the present invention can be isolated or identified using the above-mentioned antibodies, and the polypeptide can be purified by affinity chromatography. Thus, inter alia, infection using an antibody against the OBP-NT polypeptide,
In particular, it can treat viral infections. Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of the invention. "Antigenically equivalent derivative" encompasses a polypeptide that is specifically recognized by a particular antibody or equivalent thereof, wherein the antibody, when raised against a protein or polypeptide of the invention, It interferes with the physical interaction between the pathogen and the mammalian host. "Immunologically equivalent derivative" encompasses a polypeptide or equivalent thereof, which, when used in a suitable formulation to raise antibodies in vertebrates, results in the binding of the antibody to the pathogen and mammalian host. Acts to prevent immediate physical interaction between

The polypeptide, eg, an antigenically or immunologically equivalent derivative, or a fusion protein thereof, is used as an antigen to immunize a mouse or other animal, such as a rat or chicken. Fusion proteins can confer stability on a polypeptide. The antigen is
For example, by conjugation, an immunogenic carrier protein, such as bovine serum albumin (BSA) or keyhole limpet hemocyanin
mocyanin: KLH). Alternatively, a multi-antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof, has sufficient antigenicity to improve immunogenicity. No need to use a carrier. Preferably, the antibody or variant thereof is modified to reduce immunogenicity in the individual. For example, if the individual is a human, the antibody is most preferably "humanized"; for example, Jones, P. et al., Nature 321: 522-525 (1986) or Tem.
As described in pest et al., Biotechnology 9: 266-273 (1991), the complementarity determining regions of antibodies from hybridomas have been grafted onto human monoclonal antibodies.

For use of the polynucleotides of the present invention in genetic immunization, preferably direct injection of plasmid DNA into muscle (Wolff et al., Hum. Mol. Genet 1: 363 (1
992); Manthorpe et al., Hum. Gene Ther. 4: 419 (1963)), Delivery of DNA complexed with specific protein carriers (Wu et al., J. Biol Chem. 264: 1698).
5 (1989)), DNA co-precipitation using calcium phosphate (Benvenisty & Reshe
f, PNAS USA 83: 9551 (1986)), D into various forms of liposomes.
NA enclosure (Kaneda et al., Science 243: 375 (1989)), particle bombardment (Ta
ng et al., Nature 356: 152 (1992); Eisenbraun et al., DNA Cell Biol.
12: 791 (1993)) and in vivo infection with cloned retroviral vectors (Seeger et al., PNAS USA 81: 5849 (1984)).

Antagonists and Agonists-Assays and Molecules In addition, polypeptides and polynucleotides of the invention can be used to interact with small molecule substrates, for example, in cells, cell-free preparations, chemical libraries, and natural product mixtures. Binding to the ligand can also be evaluated. These substrates and ligands may be natural substrates and ligands, or may be structural or functional mimetics. For example, Coligan et al., Current Protocols in Immunology 1 (2): Fifth.
See Chapter (1991).

The present invention is directed to identifying compounds that enhance (agonist) or block (antagonist) the action of OBP-NT polypeptides or polynucleotides, especially those compounds that are virological and / or virucidal. Methods for screening compounds are provided. Screening methods include high throughput techniques. For example, to screen for agonists or antagonists,
A synthetic reaction mixture comprising OBP-NT polypeptide and a labeled substrate or ligand of such polypeptide, a cellular compartment such as the nucleus, cytoplasm or membrane, or any preparation thereof, may be an OBP-NT agonist or antagonist. Incubate in the presence or absence of no candidate molecule. The ability of a candidate molecule to agonize or antagonize an OBP-NT polypeptide is reflected in reduced binding of a labeled ligand or reduced production of a product from such a substrate. Molecules that bind and have no effect, ie, that do not elicit the effects of the OBP-NT polypeptide, will most likely be good antagonists. Molecules that bind well and increase the rate of product formation from the substrate are agonists. Detection of the rate and level of product formation from the substrate can be enhanced by using a reporter system. Reporter systems useful in this regard include colorimetrically labeled substrates that are converted to products, reporter genes that respond to changes in OBP-NT polynucleotide or polypeptide activity, and binding assays known in the art, It is not limited to these.

Another example of an assay for an OBP-NT antagonist is to convert OBP-NT and a potential antagonist to OBP-N under conditions suitable for a competitive inhibition assay.
A competition assay that mixes with a T-binding molecule, a recombinant OBP-NT binding molecule, a natural substrate or ligand, or a substrate or ligand mimetic. The OBP-NT molecule can be labeled, for example, with a radioactive or colorimetric compound, and the number of OBP-NT molecules bound to the binding molecule or converted to product can be accurately determined to assess the effects of potential antagonists. . Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polynucleotides and / or polypeptides of the present invention, thereby inhibiting and eliminating their activity. Potential antagonists also include OB
A closely related protein or antibody that binds to the same site on a binding molecule, such as a binding molecule that does not induce the activity induced by P-NT, and blocks the action of OBP-NT by excluding OBP-NT from binding And small organic molecules, peptides and polypeptides.

[0058] Potential antagonists bind to and occupy the binding site of the polypeptide, thereby preventing binding to viral or cellular binding molecules, thereby disrupting normal biological activity. Includes small molecules. Examples of small molecules are small organic molecules,
Includes, but is not limited to, peptides, peptide-like molecules. Other potential antagonists include antisense molecules (for a description of these molecules, see Okano, J., Neurochem. 56: 560 (1991); OLIGODEOXYNUC
LEOTIDES AS ANTISENSE INHIBTORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988)). Preferred potential antagonists include compounds related to OBP-NT and variants thereof. Each of the DNA sequences obtained herein can be used for the discovery and development of antiviral compounds. The protein encoded by the expression can be used as a target for screening antiviral drugs. In addition, antisense sequences that regulate the expression of the coding sequence of interest can be constructed using DNA sequences encoding the amino-terminal region of the encoded protein or sequences that facilitate translation of individual mRNAs. Using the antagonists and agonists of the present invention, for example, to inhibit disease,
Can be treated.

Vaccine Another aspect of the present invention is a method of eliciting an immunological response in an individual, especially a mammal, wherein the OBP- suitable to generate an antibody and / or T cell immune response.
A method comprising inoculating an individual with NT, or a fragment or variant thereof, and protecting the individual against infection, especially viral infection, especially HSV-1 infection. Also provided are methods of delaying virus replication due to such an immunological response. Yet another aspect of the present invention is a method of eliciting an immunological response in an individual, the method comprising expressing an OBP-NT or a fragment or variant thereof in vivo, wherein the OBP-NT or a fragment or variant thereof is expressed in vivo. Delivering a nucleic acid vector directing the expression of the variant to the individual and eliciting an immunological response to produce an antibody and / or T cell immune response, including, for example, cytokine producing or cytotoxic T cells; A method comprising protecting the individual from the disease, whether or not the disease is already established in the individual. One example of administering a gene is
By administering the gene to the desired cells as a coating on the particles. Such nucleic acid vectors can be DNA, RNA, modified nucleic acids or DNA / RNA.
A hybrid may be included.

[0060] A further aspect of the invention is directed to OBP-NT or a protein encoded thereby in an individual capable of eliciting or eliciting an immunological response therein when introduced into the individual. An immunological composition for eliciting an immunological response, comprising recombinant OBP-NT or a protein encoded thereby, wherein said OBP-N
The present invention relates to a composition comprising DNA encoding and expressing T or an antigen of a protein encoded thereby. Immunological responses can be used therapeutically and prophylactically, and can be derived from antibody immunization or from cell-mediated immunity, such as that arising from CTLs or CD4 + T cells.

The OBP-NT polypeptide or fragment thereof may or may not itself produce antibodies, but may comprise a fusion protein that will have the ability to stabilize the first protein and have immunogenic and protective properties. It can be fused with a co-protein capable of producing. Such a fusion recombinant protein preferably further solubilizes the antigenic coprotein, such as glutathione-S-transferase (GST) or beta-galactosidase, or facilitates its production and purification. Consists of relatively large coproteins. In addition, the coprotein may act as an adjuvant in providing systemic stimulation of the immune system of the organism receiving the protein. The coprotein may be linked to either the amino or carboxy terminus of the first protein. The present invention relates to compositions, especially vaccine compositions and methods, comprising a polypeptide or polynucleotide of the invention and an immunostimulatory DNA sequence as described in Sato, Y. et al., Science, 273: 352 (1996). provide.

The use of polypeptides as antigens to immunize a host can produce specific antibodies that confer protection against viral infection, for example, by blocking viral replication or reactivation of the virus from its latent period. it can. The present invention also includes a vaccine formulation comprising the immunogenic recombinant protein of the present invention and a suitable carrier. Parenteral administration, including, for example, subcutaneous, intramuscular, intravenous or intradermal administration, is desirable because proteins can be destroyed in the stomach. Formulations suitable for parenteral administration are:
Aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, antibacterial agents and solutes that render the formulation isotonic with the body fluids of the individual, preferably blood; suspending agents or thickening agents And sterile aqueous and non-aqueous suspensions. The prescription is
It can be provided in unit-dose or multi-dose containers, for example, sealed ampules and vials, and stored in a lyophilized condition which requires only the addition of a sterile liquid carrier immediately before use. Vaccine formulations may also have adjuvant systems to enhance the immunogenicity of the formulation, such as an oil-in-water system, and other systems known in the art. The dosage depends on the specific activity of the vaccine and can be readily determined by routine experimentation. Although the invention has been described with reference to certain OBP-NT proteins, the description also includes fragments of the native protein as well as similar proteins having additions, deletions or substitutions that do not substantially alter the immunogenicity of the recombinant protein. It will be understood to include.

Compositions, Kits and Administration The present invention also relates to compositions comprising the above-described polynucleotides or polypeptides or agonists or antagonists thereof. The polypeptides of the present invention can be used in combination with non-sterile or sterile carriers for cells, tissues or organs, such as pharmaceutical carriers suitable for administration to a subject. Such compositions comprise, for example, a vehicle addition or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline,
Includes dextrose, water, glycerol, ethanol and combinations thereof. The formulation must suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more components of the composition of the invention as described above.

[0064] The polypeptides and other compounds of the present invention may be used alone or in combination with other compounds, such as therapeutic compounds. The pharmaceutical composition can be of any effective, convenient, or convenient, including, for example, topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, nasal, transdermal routes, among others. It is administered in a good way. In treatment and prophylaxis, the active ingredient is administered to the individual as an injectable composition, for example, a sterile, preferably isotonic, aqueous dispersion.

The composition can also be a topical formulation in the form of, for example, ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, impregnated bandages and sutures and aerosols, Suitable conventional additives such as preservatives, solvents to aid drug penetration,
It may include emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, such as cream or ointment bases, ethanol or oleyl alcohol for lotions. Such carriers may comprise from about 1 to 98% by weight of the formulation, and more usually will comprise up to about 80% by weight of the formulation.

For administration to mammals, especially humans, the daily dose of active agent is 0.01 mg
/ Kg to 10 mg / kg, typically about 1 mg / kg. In any case,
The actual dose most suitable for an individual will be determined by the consulting physician and will vary with the age, weight and response of the individual. The above doses are exemplary of the average case. Of course, for some individuals higher or lower dose ranges are appropriate and they are also within the scope of the present invention. Conveniently, the vaccine composition is in the form of an injection. Conventional immune adjuvants can be used to enhance the immune response. A suitable unit dose for the vaccine is 0.5 antibody.
This dose is administered 1 to 3 times, preferably at intervals of 1 to 3 weeks. At the indicated dose range, no detrimental toxic effects of the compounds of the present invention are observed which would prevent its administration to appropriate individuals. Each of the references disclosed herein is incorporated herein by reference in its entirety. The contents of any patent application for which this application claims priority is hereby incorporated by reference.

Terminology Certain terms frequently used herein are defined below to facilitate their understanding. “Disease (s)” means diseases caused by and associated with infection by viruses, including herpes labialis, herpes encephalitis, keratitis. A "host cell" is a cell that has been transformed, transfected or infected, or is capable of being transformed, transfected or infected by an exogenous polynucleotide sequence or virus.

“Identity” is known in the art and is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. Also, in the art, "identity" may
It refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by pairing between the chains of the sequences. "Identity" means Computational Mo
lecular Biology, Lesk, AM, Oxford University Press, New York, 198.
8; Biocomputing: Informatics and Genome Projects, Smith, DW, Academ
ic Press, New York, 1993; Computer Analysis of Sequence Data, Part I
, Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994.
Sequence Analysis in Molecular Biology, Von Heinje, G .; Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.
Ed., M Stockton Press, New York, 1991; and Carlio, H and Lipman, D.
, SIAM J. Applied Math., 48: 1073 (1988) (
The present invention is not limited to these, but can be easily determined by known methods. The preferred method of determining identity is designed to give the largest match between the sequences tested. Moreover, methods for determining identity are codified in publicly available computer programs. Preferred computer programming methods for determining the identity between two sequences are described, for example, in the GCG Program Package (Devere
ux, J. et al., Nucleic Acids Research (1984) 12 (1): 387), BLASTP
BLASTN and FASTA (Atschul, SF et al., J. Molec. Biol. (1990) 215:
403-410), but is not limited thereto. The BLAST X program is available from NCBI and other sources (BLAST Manual, Altshul, S. et al., NCBI NLM NIH Bethesda, MD20).
894; Altschul, S .; J. et al. Mol. Biol., 215: 403-410 (1990).
Publicly available from)). In addition, well-known Smith Waterman (Smith Waterm
an) The identity can also be determined using an algorithm.

The parameters for polypeptide sequence comparison include the following: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-45.
3 (1970) Comparative matrix: Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89
: 10915-10919 (1992) BLOSSUM62 Gap penalty: 12 Gap length penalty: 4 A useful program for these parameters is the Genetics Computer Group.
Publicly available as a "gap" program from p. Madison WI. The above parameters are default parameters for peptide comparison (without penalty for end gaps). Parameters for polynucleotide comparisons include: 1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-45.
3 (1970) Comparison matrix: match = + 10, mismatch = 0 Gap penalty: 50 Gap length penalty: 3 A useful program for these parameters is Genetics Computer Group
Publicly available as a "gap" program from p. Madison WI. The above parameters are default parameters for nucleic acid comparison.

Preferred identities for “identity” for polynucleotides and polypeptides
The taste is shown in (1) and (2) below. (1) Further, specific examples of the polynucleotide may be less than the control sequence of SEQ ID NO: 1.
At least 50, 60, 70, 80, 85, 90, 95, 97 or 100% identical
Isolated polynucleotide sequences, including polynucleotide sequences having potential
May be identical to the control sequence of SEQ ID NO: 1, or
May have up to a certain number of nucleotide changes compared to a control sequence.
No. Such changes can be due to deletions, substitutions (transitions) of at least one nucleotide.
Or transversions) or insertions.
Is the position of the 5 'or 3' end of the control nucleotide sequence or their terminal positions.
At positions between the positions, individually or interspersed at nucleotides in the control sequence.
Or as one or more contiguous groups in a control sequence.
The total number of nucleotides in SEQ ID NO: 1 and individual percent identity values (divided by 100
) And subtract the product from the total number of nucleotides in SEQ ID NO: 1.
Determine the number of nucleotide changes. This is explained by the following equation: n_n≤x_n− (X_nY) where n_nIs the number of nucleotide changes, x_nIs all nucleotides in SEQ ID NO: 1
Y is, for example, 0.5 for 50%, 0.6 for 60%,
70% is 0.70, 80% is 0.80, 85% is 0.8
5, 90% is 0.90, 95% is 0.95, 97%
Is 0.97, 100% is 1.00, and is the product operator, x _n Non-integer product of y and y is rounded down to the nearest integer and x_nDeducted from
Good. The change in the polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 is
Nonsense, missense or frame shift in the coding sequence
Can cause mutations, and consequently,
The polypeptide encoded by the otide changes.

For example, the polynucleotide sequence of the invention may be identical to the control sequence of SEQ ID NO: 2, ie, may be 100% identical, or may have up to a certain number of amino acids as compared to the control sequence. (In that case, the identity%
Is less than 100%). Such changes can be due to deletion, substitution (at least one nucleic acid).
Transitions, including transitions and transversions) or insertions, wherein the alteration is at a position 5 'or 3' of the control polynucleotide sequence or at a position between those terminal positions, and in the nucleic acid in the control sequence. Or interspersed, or as one or more contiguous groups in a control sequence. The total number of amino acids in SEQ ID NO: 2 is multiplied by a value obtained by dividing an integer indicating individual identity by 100, and the product is subtracted from the total number of amino acids in SEQ ID NO: 2 to calculate the number of nucleic acid changes for the% identity value. To determine. Alternatively This is illustrated by the following _{equation: n n ≦ x n - (} x n · y) wherein, n _n is the number of amino acid alterations, x _n is the total number of amino acids in SEQ ID NO: 2, y is, for example, 0.70 for 70%, 0.80 for 80%, and 85
% Is 0.85, etc. is a product operator, and the non-integer product of _xn and y is rounded down to the nearest integer and then subtracted from _xn .

(2) Further specific examples of the polypeptide are at least 50, 60, 70, 80, 85, 90, 95, 97 or 100 relative to the polypeptide control sequence of SEQ ID NO: 2.
% Polypeptides, which polypeptides may be identical to the control sequence of SEQ ID NO: 2, or up to a certain number of amino acids as compared to the control sequence. May be changed. Such changes are
Selected from the group consisting of deletion, substitution (including conservative and non-conservative substitutions) or insertion of at least one amino acid, wherein the alteration is at the amino or carboxy terminal position of the control polypeptide sequence or at their terminal position. Intervening positions may occur individually or interspersed with amino acids in the control sequence, or as one or more contiguous groups in the control sequence. The number of amino acid changes is determined by multiplying the total number of amino acids in SEQ ID NO: 2 by a value obtained by dividing an integer indicating the identity by 100, and subtracting the product from the total number of amino acids in SEQ ID NO: 2. This is described by the following equation: n _a ≦ x _a - in (x _a · y) formula, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2,
For example, y is 0.5 for 50%, 0.6 for 60%, 0.70 for 70%, 0.80 for 80%, and 0.5 for 85%. 85,
90% is 0.90, 95% is 0.95, 97% is 0.9
Is 7, 100% if 1.00, • is the symbol for the multiplication operator, and wherein any non-integer product of x _a and y is the rounded down to the nearest integer prior to subtracting it from x _a.

For example, the polypeptide sequence of the invention may be identical to the control sequence of SEQ ID NO: 2, ie, may be 100% identical, or may have up to a certain number of amino acids as compared to the control sequence. (In which case the% identity is 1
Less than 00%). Such changes can be at least one amino acid deletion, substitution (
Conservative or non-conservative substitutions) or insertions, wherein the alteration is at an amino or carboxy terminal position of the control polypeptide sequence or at a position between those terminal positions, at an amino acid in the control sequence. It may occur individually or interspersed, or as one or more contiguous groups in a control sequence. Number of amino acid changes for percent identity value by multiplying the total number of amino acids in SEQ ID NO: 2 by the individual percent identity value (divided by 100) and subtracting the product from the total number of amino acids in SEQ ID NO: 2 To determine. This is described by the following _{equation: n a ≦ x a - (} x a · y) wherein, n _a is the number of amino acid alterations, x _a is the total number of amino acids in SEQ ID NO: 2, y is, For example, 70% is 0.70, 80% is 0.80, 85
A% if 0.85 etc., • is the symbol for the multiplication operator, and wherein any non-integer product of x _a and y is the rounded down to the nearest integer prior to subtracting it from x _a.

“Isolated” means changed “by the hand of man” from its natural state, ie, in the case of natural products, altered or removed from its original environment or both. means. For example, a polynucleotide or polypeptide naturally occurring in an organism is not `` isolated, '' but the same polynucleotide or polypeptide separated from its coexisting material in its natural state is the term used herein. As "isolated". Furthermore, polynucleotides or polypeptides that have been introduced into an organism by transformation, genetic manipulation, or by any other method, are still in the organism, even if the organism is alive or dead. , "Isolated".

“Polynucleotide (s)” generally refers to either polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" refers to single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, single-stranded and Hybrid molecules comprising RNA, which is a mixture of double-stranded regions, and DNA and RNA which may be single-stranded or more typically a mixture of double- or triple-stranded regions or single- and double-stranded regions But not limited thereto. Further, as used herein, "polynucleotide" refers to a triple-stranded region consisting of RNA or DNA, or both RNA and DNA. The chains in these regions may be from the same molecule or from different molecules. The regions may include all one or more of these molecules, but more typically include only some regions of the molecule. One of the molecules of the triple helix region is often an oligonucleotide. As used herein, "polynucleotide (s)"
The term also includes the above DNAs or RNAs containing one or more modified bases. That is, DNA or RNA having a backbone that has been modified for stability or for other reasons is also "polynucleotide (s)" as that term is intended herein. In addition, DNA or RNA (only two examples are shown) that include unusual bases such as inosine or modified bases such as tritylated bases are also polynucleotides as that term is used herein. It will be appreciated that a variety of modifications have been made to DNA and RNA and have been used for many useful purposes, as will be known to those skilled in the art. As used herein, the term "polynucleotide" refers to such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as viruses and cells, such as, for example, simple and complex cells. Includes characteristic DNA and RNA chemical forms. "Polynucleotide (s)" also encompasses relatively short polynucleotides, often referred to as oligonucleotide (s).

“Polypeptide (s)” refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide (s)" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 amino acids encoded by the gene. "Polypeptide (s)" have been modified by either natural processes, such as processing and other post-translational modifications, or by chemical modification techniques. Such modifications are described in detail in the basic text and in more detailed research articles, as well as in the extensive research literature, which are well known to those skilled in the art. It will be apparent that the same type of modification may be present at the same or varying degrees at several sites in a given polypeptide. Also, a given peptide may have many types of modifications. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme moieties, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, phosphotidylinositol Bond, crosslink, cyclization, disulfide bond formation, demethylation, covalent crosslink formation, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, sugar chain formation, GPI anchor formation, hydroxylation Iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipidation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosyl , Selenoylation, sulfation, arginylation Addition of amino acids to proteins mediated transfer RNA, and encompasses ubiquitination. For example, PROTEINS
−STRUCTURE AND MOLECULAR PROPERTIES, 2nd edition, TE Creighton, WHF
reeman and Company, New York (1993) and Wold, F., POSTTRANSLATIO
NAL COVALENT MODIFICATION OF PROTEINS, Posttranslational Protein Modific
ations: Perspectives and Prospects, pp. 1-12, edited by BC Johnson, Academic P
ress, New York (1983); Seifter et al., Meth Enzymol. (1990) 182:
626-646, and Rattan et al., Protein Synthesis: Posttranslational Mod
ifications and Aging, Ann NY Acad Sci (1992) 663: 48-62. Polypeptides may be branched or cyclic with or without branching. Cyclic, branched and branched cyclic polypeptides are the result of natural post-translational processing and can likewise be synthesized in entirely synthetic ways.

As used herein, the term “variant (s)” is each polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. Is a peptide. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not be different from the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. The variant and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis methods or by direct synthesis or other recombinant methods known to those skilled in the art.

EXAMPLES The following examples are performed using standard techniques well known and routine to those of skill in the art, except as otherwise described in detail. The examples are illustrative and do not limit the invention.

Characterization of Gene Expression After Infection with HSV-1 Tissue culture cells are infected with HSV-1 strain KOS and harvested after infection for RNA isolates. HSV-1 infected tissue culture cells are effectively disrupted and treated in the presence of chaotropic agents and RNase inhibitors to obtain a mixture of cellular and viral RNA. The RNA may be a poly A + selected for use in a Northern blot that hybridizes an RNA probe specific for a certain region in the HSV-1 genome. RNase-free of total or poly A + selected RNA, D
Nase-free, DNA- and protein-free preparations are, for example, 5'RACE (cDNA
It is suitable for mapping the 5 'end of the gene of interest using a technique such as a method of rapidly growing the end; Gibco-BRL). The unique primer pair for reverse transcription PCR (RT-PCR) used in the 5'RACE technique is HSV-1 strain KOS
From the published sequence of HSV-1 strain 17, which is homologous to the sequence of

Example 1 RNA Isolation, Northern Blotting and Identification of the 5 'End Complementary radiolabeling of a polynucleotide having the DNA sequence shown in Table 1 (SEQ ID NO: 1), for example, by conventional means Using the obtained RNA fragment as a probe, poly A + RNA harvested from host cells infected with HSV-1 strain KOS was identified by Northern blotting. a) Tissue culture cells such as Vero (monkey kidney cells) were placed in a tissue culture medium [10% heat-inactivated fetal bovine serum (FBS; HyClon) in a 850 cm ² tissue culture roller bottle.
e Laboratories, Inc.), 1% penicillin-streptomycin (Gibco BRL, Li
fe Technologies), 5 ml / L of 200 mM L-glutamine (Gibco BRL, Lif
e Technologies), 10 ml / L 7.5% (w / v) sodium bicarbonate (Gibco
Dulbecco's modified Eagle's medium supplemented with BRL, Life Technologies (DMEM)
Gibco BRL, Life Technologies)] and incubated at 37 ° C. H
SV-1 (strain KOS) is added to a small amount of tissue culture medium at a multiplicity of infection of 10 plaque forming units per cell to infect the cells. After 1 hour of infection at 37 ° C., additional DMEM is added and the infected cells are harvested 17 hours after infection.

According to the manufacturer's instructions, TRI Reagent® (Molecular Research Center)
ter, Inc.). In a biologically safe cabinet,
The tissue culture medium was removed and 6.7 ml of TRI reagent (75 cells
Harvest the infected tissue culture cells by adding 1 ml per mg). Then
Stir until the mixture is homogeneous. Typically, for isolation, two 850 cm ^Two Use a roller bottle. Each contains about 500 mg of cells. So
Centrifuged at 12,000 xg, 4 ° C for 10 minutes to remove insoluble material
Then, the supernatant is transferred to a new test tube and left at room temperature for 5 minutes. To this mixture: 1.
Add 34 ml of BCP phase separation reagent (Molecular Research Center, Inc .; TRI)
0.2 ml per 1 ml of reagent), vigorously shake for 15 seconds, and leave at room temperature for 5 minutes
. The mixture is then centrifuged at 12000 × g at 4 ° C. for 20-30 minutes.
. Transfer about 75% of the colorless upper aqueous phase (containing RNA) to a new tube and transfer to 3.35 m
of isopropanol (0.5 ml of isopropanol per ml of TRI reagent)
Panor). The sample is agitated and left at room temperature for 5 minutes. Allow the sample to dry until solids form.
Freeze in a lyeice / ethanol bath and centrifuge at 12000 xg at 4 ° C for 30 minutes
Apply to separation. The supernatant was removed and the RNA pellet was air-dried at room temperature for 10 minutes.
Resuspend in 3 μl TNM buffer (75 μl T / 100 mg starting material)
NM buffer; TNM: 40 mM Tris-HCl (pH 8.0), 10 mM NaCl
, 6 mM MgCl_Two). 75 units of RNase-free DNase per 100 μl of RNA
1 (Gibco BRL, Life Technologies) was added to remove the DNA.
Incubate for minutes. To this is added 10% of the sample volume of 4M LiCl.
Transfer this sample to an Eppendorf centrifuge tube and add an equal volume of phenol: chloroform (
1: 1). The sample is centrifuged at 13000 xg for 3 minutes in an Eppendorf centrifuge tube.
After centrifugation, the aqueous phase is transferred to a new tube and extracted with an equal volume of chloroform.
The aqueous phase is again transferred to a new tube and twice the sample volume of ethanol is added. sample
Are frozen in a dry ice / ethanol bath until solids form, and
Centrifuge at 13000 × g, 4 ° C. for 30 minutes in a centrifuge tube. Remove ethanol
Wash the RNA with 75% ethanol (minimum 1 ml), agitate and eppendle
Centrifuge at 13000 rpm for 5 minutes at 4 ° C in a centrifuge tube to pellet the RNA.
To the shape. The RNA pellet was air dried for 10 minutes and placed in 200 μl of TNM buffer.
Resuspend again. Determine RNA concentration by measuring absorbance at 260 nm
. 50 μg of RNA was combined with 10 units of DNase I (RNase and protease free;
(Worthington Biochemical Corp.) again and incubate at 37 ° C for 30 minutes.
Cuvette, followed by addition of 4M LiCl, extraction and sedimentation as described above.
And quantify. RNA characteristics were determined by electrophoresis of 1 μg in 1% agarose gel.
And visualized by staining with ethidium bromide [Sambrook et al., Mo.
lecular Cloning, A Laboratory Manual (supra)].

The poly (A) purified mRNA isolation kit (Ambion) was prepared according to the manufacturer's instructions.
Used for poly A + selected RNA. The tissue culture medium is removed in a biologically safe cabinet and the infected tissue culture cells are harvested by adding 10 ml of lysis solution per 2 × 10 ⁸ cells. Typically, three 850 cm ² roller bottles are used for isolation, each containing about 4.6 × 10 ⁷ cells. Combine the lysates, transfer to a 50 ml test tube and stir until homogeneous. Two departure capacities (20m
Add dilution buffer of 1) to this lysate and mix by inverting. The lysate was centrifuged at 12000 × g at 4 ° C. for 15 minutes to remove particulate cell debris,
Transfer the supernatant to a clean 50 ml conical tube. Add one pre-filled vial of oligo dT cellulose to the lysate and mix by inversion. The oligo dT resin is pelleted by gently rocking the tube at room temperature for 60 minutes and centrifuging at 2000 × g for 3 minutes at room temperature. The supernatant is removed and the binding buffer 1
Wash the resin by adding 0 ml, invert and mix well, pelletizing the resin as described above. This wash is repeated twice more with 10 ml of binding buffer. The resin is then washed by resuspension in 10 ml of wash buffer, inverted and mixed well to pellet the resin as described above.
This wash is repeated two more times using 10 ml of wash buffer. After the third wash, the oligo dT resin is resuspended in 1 ml of wash buffer and transferred to a spin column placed in a sterile microfuge tube. Centrifuge the spin column at 5000 xg for 10 seconds at room temperature and measure the absorbance of the flow-through at 260 nm to ensure it is less than 0.05. Place the spin column in a new microfuge tube and elute the poly A + RNA by adding 200 μl of pre-warmed (65 ° C.) elution buffer and centrifuge at 5000 × g for 30 seconds at room temperature.
This elution procedure is repeated with 200 μl of additional elution buffer, and polyA + RNA is precipitated by adding 40 μl of 5 M sodium acetate, 2 μl of glycogen and 2.5 volumes of 100% ethanol. The RNA is stored at -80 ° C for 30 minutes and recovered by centrifugation at 12,000 xg, 4 ° C for 20 minutes. The supernatant is removed and the RNA pellet is air dried at room temperature for 10 minutes and resuspended in 40 μl of DEPC-treated water / 0.1 mM EDTA. Determine the concentration of RNA by measuring the absorbance at 260 nm; store the RNA preparation at -80 ° C until use [Sambrook et al., Molecular Cloning, A Laboratory Manual, supra].

B) Northern blotting of HSV-1 poly A + selected RNA to identify new transcripts Vero of HSV-1 (KOS) infection 17 hours after infection
Poly A + selected RNA (5 μg) isolated from cells or isolated from uninfected (mock) Vero cells is run on a 1% agarose gel prepared using 10 × denaturing gel buffer (Ambion). The RNA samples are each suspended in 10 μl of DEPC-treated water and 3 volumes (30 μl) of sample loading dye (Ambion).
The sample is heated to 65 ° C. before loading on the gel to denature the secondary structure of the RNA. Gels were run at 65 volts for 3-4 hours in 1x gel running buffer (Ambi
Run on). After electrophoresis, vacuum transport was performed at 5 inches Hg for 90 minutes, and the RNA was transferred to a 5xSSC, 10 mM NaOH positively charged nylon membrane (B
rightStar-Plus; Ambion). After transfer, the membrane is lightly rinsed with 1x gel running buffer and UV cross-linked (Stratagene). The crosslinked RNA membrane can be stored at -20 ° C in a container that protects the membrane from physical damage.

Using the riboprobe in vitro transcription system (Promega), the UL8 /
A riboprobe is generated from a plasmid specific to the UL9 region (riboprobe 8
R-3 '; K. Baradaran, CE Dabrowski and PA Schaffer, 1994, J. Virol.
68: 4251-4261). The p8R-3 ′ plasmid DNA is linearized with XbaI and 2 μg is transcribed and radiolabeled according to the manufacturer's instructions. Probe Quant (
(ProbeQuant) G-50 microcolumn (Pharmacia), followed by centrifugation in an Eppendorf microcentrifuge tube at 3000 rpm for 2 minutes, and the operation is repeated to remove free nucleotides and purify the riboprobe. The membrane that binds RNA
Prehybridization / hybridization solution of 10 ml (Ambion) per membrane 100 cm ^2, 1 hour at 75 ° C., is prehybridized. The prehybridization solution was removed and 1 × 10 ⁶ c per 1 ml of 8R-3 ′
Replace with 10 ml of a hybridization solution consisting of a prehybridization / hybridization solution preheated to the hybridization temperature (75 ° C.) plus pm. The membranes were hybridized overnight at 75 ° C., and then each was washed twice at room temperature for 5 minutes twice with a low stringency wash (Low Stringency).
Wash Solution: Ambion), and each was washed with a high stringency wash solution (High Stringency Wash Solution: Ambion) three times at 75 ° C. for 20 minutes.
Attach to autoradiogram.

C) Rapid Amplification of the 5 ′ cDNA Termination of RNA Samples from Infected Cells To synthesize single stranded cDNA with high GC content transcripts, total RNA (1 μg) was prepared according to the manufacturer's instructions. Reverse transcription using a 5 'RACE system (Gibco BRL, Life Technologies) that rapidly amplifies cDNA ends. Each reaction is primed using gene-specific primer 1 (100 nM GSP1; 5'-GCA GAT GTT ATC GCC-3 '). Controls without Super Script II reverse transcriptase added are also included. All samples are then treated with the RNase mixture and the cDNA is purified using a glass mix DNA isolation system, including the 5 'RACE kit. After purification, terminal deoxynucleotidyl transferase (TdT
) Was used to attach dCTP to the end of the cDNA sample, and the reduced anchor primer (5) was used.
'RACE kit) and a second gene-specific primer (GSP
2; 5′-GAT GCA GAC GTT ATC TC-3 ′). PCR
Reactions are allowed to associate on ice in 0.2 ml membrane tubes according to the instructions for PCR of dC-terminal cDNA. The reaction tube was placed directly on ice at the initial denaturation temperature (94 ° C).
Transfer to a pre-equilibrated thermal cycler. The PCR reaction was performed as follows:
Performed on a Kin Elmer Gene Amp PCR System 9600; 9
The reaction is carried out at 4 ° C. for 2 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 1 minute and 30 seconds, followed by 72 ° C. for 5 minutes, and maintained at a temperature of 5 ° C.
An aliquot (10 μl) is electrophoresed on a 6% acrylamide gel in 1 × TBE (Novex) and stained with ethidium bromide. The size of the PCR product is determined by comparing it with a 100 bp DNA ladder (Gibco BRL, Life Technologies). The nested PCR amplification was then performed using a universal amplification primer (UAP) and a third gene-specific primer (GSP3: 5′-CAU CAU CAU CAU CCG ACC ACC ACA TG-3 ′).
) In accordance with the instructions of the nested amplification procedure. PCR is performed as described above.
Primers used for nested amplification were Clone Amp pAMP1 System
(Gibco BRL, Life Technologies) to sequence specific PCR products. The PCR product is purified from a 6% acrylamide gel according to the manufacturer's instructions and cloned into the pAMP1 vector.

[0086] The PCR product was annealed to the pAMP1 vector prior to standard protocol (Sa
The reaction mixture was transformed into DH5α cells (Gibco BRL, Life Technologies) according to mbrook et al., Molecular Cloning, A Laboratory Manual (supra). Bacteria are placed on LB plates containing 100 μg / ml ampicillin (Digene Diagnostics Inc.), grown at 37 ° C. overnight, and colonies for DNA minipreps are picked. 5 ml using RPM kit (Bio101) according to manufacturer's instructions
The plasmid DNA is isolated from a bacterial culture of Plasmid DNA is analyzed by restriction digestion with BamHI and EcoRI, subjected to electrophoresis on a 6% acrylamide gel in 1 × TBE (Novex), and the insert size is determined by comparison to the 100 bp DNA ladder described above. . The clones were then sequenced with the SP6 promoter primer (Gibco BRL, Life Technologies) (T7 sequencease 7-deaza-dGTP sequencing kit; Amersham Life Science).
To determine the 5 'end of the cDNA. DNA from two or more clones containing the amplified PCR product is sequenced and the 5 'end is defined and used to identify the DNA sequence flanking SEQ ID NO: 1.

Example 2 Characterization of OBP-NT A new protein, a new messenger RNA encoding OBP-NT (mRN
A) was identified by Northern blotting using an RNA probe complementary to the UL8 / 9 region of the HSV-1 genome (see Example 1). Attempts to map the RNA encoded within this region have been described previously (De
b, SP et al., 1993, Biochem and Biophys Res. Comm. 193: 617-623; Baradaran, K
Et al., 1994, J. Virol. 68: 4251-4261), current studies utilized mRNA isolated at a very late time point (17 hours after infection). As described above, mRNA 5
'Ends identified and previous mapping experiments reveal 3' ends (
Baradaran, K. et al., 1994, J. Virol. 68: 4251-4261), allowing the estimation of the OBP-NT open reading frame according to well-established guidance in eukaryotes (Kozak). , M., 1986, Cell 44: 283-292).

Putative OBP-NT Characteristics From this analysis, it was determined that OBP-NT is a well-characterized viral origin binding protein, a protein within OBP that corresponds to amino acid 142 of OBP-NT.
It is presumed to start in methionine and encode in-frame. Thus, OBP-NT has a dimerization domain with OBP (leucine zipper at amino acids 150-171; Hazuda, DJ et al., 1992, J. Biol. Chem. 267: 14309-14315),
Helicase motifs II to VI (Malik, AK and SKWeller, 1996, J. Vi.
rol. 70: 7859-7866) and an origin-specific DNA binding domain at the C-terminus (De.
b, S. and SWDeb., 1991, J. Virol. 65: 2829-2838). Based on these sequences shared between proteins, OBP-NT contains amino acids 150-1
It is believed that through the conserved leucine zipper region of 71 (OBP numbering), it can form a homodimer by itself and a heterodimer with OBP. These proteins may also form dimers with viral or cellular proteins that have not yet been identified. OBP-NT contains conserved helicase motifs II to V
The motifs I and Ia, which retain I but are lost for nucleotide binding and helicase activity, are critical and are not expected to function as helicases, such as monomers or homodimers. However, OBP-NT may, for example, function as a helicase of a heterodimer with OBP, and the OBP itself may have similar or different properties as compared to the OBP homodimer. OBP-NT is also presumed to bind to sequences within the viral origin of replication. An experiment illustrating this capability is described below.

B) OBP-NT Binds to the Origin Sequence of the Virus Evidence showing the ability of OBP-NT to bind to sequences within the viral origin of replication (oriS and oriL) has been published in many publications or elsewhere. Can be found in results from experiments performed to characterize origin binding by members of the OBP family, OBP and OBPC. For example, published electrophoretic mobility shift assay (EMSA) experiments in which extracts from HSV-1 infected cells were incubated with radiolabeled oriS DNA (sites I, II or III; see FIG. 1) Thing). In these experiments, two or three virus-specific complexes, complex A (containing OBPC and site I or site II DNA), appearing as a single band or doublet band (Baradaran, K. et al., 1994, J. .Virol. 68:
4251-4261) and complex B (including OBP and site I, II or III DNA) (Dabrowski, CE and PA Schaffer, 1991, J. Biol.
Virol. 65: 3140-3150; Weir, HM et al., 1989, Nucl. Acids Res. 17: 1409-1425;
Hardwicke, MA and PASchaffer, 1995, J. Virol. 69: 1377-1388). In the presence of an antibody facing the C-terminal amino acid of OBP (common to OBP-NT and OBPC), complex A shifts to a higher molecular weight complex (
Super shift). Protein identified as complex B formed at oriS DNA (sites I, II, III): The DNA complex also shifts or has a significant intensity in the presence of the antibody facing the C-terminus of OBP. Decrease (Fig. 8, Da
browski, CE and PASchaffer, 1991, J. Virol. 65: 3140-3150). However, as shown in FIG. 10 herein, complex B formed with extracts from HSV-infected cells was present as a single band in these experiments and was partially purified recombinant OBP. And shows a different electrophoretic mobility from the complex formed by the above (referred to as 9-1). Differences in electrophoretic mobilities obtained between the “complex B” and “complex 9-1” proteins, and these proteins were super-shifted in an EMSA experiment with an antibody facing the C-terminus of OBP. The signal indicates that OBP-related proteins with slightly different molecular weights are present. The result is a full-length OBP
Is present in the complex 9-1 formed with the partially purified OBP, and the OBP in-frame translated slightly smaller protein, ie, O
Consistent with BP-NT being present in complex B from infected cell extracts. Thus, by EMSA, complex B was observed to be one or two bands in the complex formed from the HSV-infected cell extract and the oriS site I or II DNA, and conversely, complex B was not A single band with III; no doublet band of complex B was observed (Dabrow
ski, CE and PASchaffer, 1991, J. Virol. 65: 3140-3150; Weir, HM et al., 1
989, Nucl. Acids Res. 17: 1409-1425; Hardwicke, MA and PASchaffer, 19
95, J. Virol. 69: 1377-1388); the band below the doublet is now OBP
-NT and the upper band is believed to contain OBP. The presence of one or two bands was shown to be dependent on the extract preparation, the time of harvest after infection, and the length of irradiation on the film which obscure two separate bands. OBP-NT
Can bind to sequences within the viral origin of replication by radiolabeled or
Extracts from HSV-1 infected cells incubated with iS DNA were analyzed by EMSA and shown in FIGS. In these experiments, Vero cells were harvested at 850c
m ² tissue culture roller plated 10% FBS-containing DMEM in the bottle, incubated overnight at 37 ° C.. Cells are mock-infected or infected with KSV-1 (KOS) at an MOI of 10 and harvested 3, 6, 9, 12, 15, or 18 hours post-infection (hpi). The cells are pelleted in a desktop centrifuge at 2000 rpm, 4 ° C. for 5 minutes and the pellet is phosphate-buffered saline (PBS; Sambrook et al., Molecu
LAR Cloning, A Laboratory Manual) twice and 50 μl of NET buffer (
A whole cell extract is prepared by resuspension in 50 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA). The pellet is frozen at -80 ° C and
And set to 4 using a Virsonic 550 Ultrasonic Cell Disrupter (VirTis), and sonicated twice for 30 seconds each. Cell debris is pelleted by centrifugation in an Eppendorf microcentrifuge tube at 14000 rpm at 4 ° C for 10 minutes, aliquoted supernatant and stored at -80 ° C. Protein quantification is performed on whole cell extracts using the Bio-Rad Protein Assay (Bio-Rad) according to the manufacturer's instructions for the microassay procedure. Radiolabeled HSV-1 oriS sites I, II, II
I and III (I) probes are prepared as previously described (Dabrow
ski, CE and PASchaffer, 1991, J. Virol. 65: 3140-3150). Site III (
I) The probe has a central branched nucleotide (T) as seen at site I.
Is replaced with G and contains the same sequence as site III. In EMSA, pseudo-infection (1
0 μg protein) or HSV infected (2 μg protein) cell extract in a final volume of 10 μl.
1 of DNA binding buffer (10% glycerol, 50 mM HEPES, pH7.
5 [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Life
Technologies], 0.1 mM EDTA, 0.5 mM DTT, 75 mM NaCl
) At room temperature for 30 minutes, 2 ng of probe and 1.5 μg of poly (dI-dC
Incubate with poly (dI-dC) (Pharmacia). An equal amount of radioactive material was loaded on each gel (35000 cpm) to prepare a 6% polyacrylamide gel, which was subjected to electrophoresis at 0.5 V in 200 × V TBE (Life Technologies) to obtain protein: DNA complex. Separate the body. Sites I, II, II
The results of EMSA performed with whole cell extracts harvested 12 hours after incubation with the I and III (I) probes are shown in FIG. OBPC (Barada
Complex A containing ran, K. et al., 1994, J. Virol. 68: 4251-4261)
I and III (I) probes, but as described previously (Dabrow
ski, CE and PASchaffer, 1991, J. Virol. 65: 3140-3150), not including the site III probe. Complex B, OBP (band above doublet) and OB
The doublet of P-NT (the band below the doublet) has sites I, II and I
Although containing the II (I) probe, only the OBP-NT band below the complex B doublet contains the site III probe. This experiment was performed for OBP, OBP-NT and OBP.
This shows that, despite the common C-terminal DNA binding region of BPC, only OBP-NT can bind to site III within the viral core replication origin. Furthermore, the inability of OBP and OBPC to bind to site III depends on a single nucleotide difference between sites I and III in the core OBP binding domain (T at site III, G at site I). This time, an appropriately sized band was identified from the HSV-infected cell extract, but the purified OBP was
It reveals the difference in results as described above, which did not reveal binding to I (see background).

EMSA was also performed with radiolabeled oriS probes using mock or HSV infected cell extracts harvested long after infection. Site I
As shown in FIGS. 4A and B of the DNAs of and II, the complex B doublet appears by 6 hours post-infection and the intensity increases over 18 hours post-infection. Protein: DNA complexes (C and D), with reduced electrophoretic mobility and at the same time disappearance of mock infected bands, are also seen long after infection. Complex B
The doublet band is of approximately equal intensity for the site I probe, while the smaller OBP-NT complex B band and complex D band are always dominant after infection for the site II probe. In EMSA performed with a site III probe (FIG. 4C), complex B appeared as a separate band, 9 hours post infection, and 18 hours post infection.
The intensity increases until time. Together with this probe, complex D, but not complex C, is also formed; from these experiments, complex C is associated with OBP,
In contrast, complex D appears to be related to OBP-NT. In addition, a low light intensity band is evident, with only a significant increase in intensity at 18 hours post-infection; this band appears to migrate slightly faster than complex A formed with OBPC. Using mock-infected bands for reference, E at sites I, II and III
Sequencing MSA reveals that the single band of complex B formed with the site III probe has the same electrophoretic mobility as the band below the complex B doublet seen with the site I probe. become. From these experiments, it is concluded that OBP and OBPC bind to sites I and II at the viral origin of replication, as previously reported. OBP-NT can also bind to sites I and II, which are the only known OBP family viral proteins that can bind to site III.

C) OBP-NT expression in HSV-1 infected cells 10 amino acid peptide from the N-terminus of OBP-NT (MNDRPFHR
L) using a polyclonal anti-peptide antibody raised against and a protein extract from uninfected (mock) Vero cells, Western blots were used to identify O.
Identify the BP-NT protein. Vero cells were harvested 6 hours after infection (6 hpi) or 18 hours after infection of Vero cells with HSV-1. 10% FB
Vero cells are plated at 2.5 × 10 ⁶ cells in a 100 mm culture dish in DMEM containing S and incubated at 37 ° C. overnight. Cells are mock infected or HSV-1 (KOS) infected at an MOI of 10. At 6 or 18 hours post-infection, cells are scraped into the medium and pelleted by centrifugation at 1500 rpm for 10 minutes using a desktop centrifuge. Each pellet was mixed with 400 μl of phosphate buffered saline (PBS; Sa).
mbrook et al., Molecular Cloning, A Laboratories Manual); 200
μl of lysis buffer (30 mM Tris, pH 8.0, 15 mM EDTA, 3% SD
S) is added and the lysate is incubated on ice for 10 minutes. The lysate is sonicated twice for 30 seconds and 5 volumes of acetone are added. The precipitate is stored at −20 ° C. for 30 minutes and pelleted in an Eppendorf microfuge at 14000 rpm at 4 ° C. for 15 minutes. The pellet was air dried and 400 μl of SDS-PAGE loading buffer containing 0.05% mercaptoethanol (N
OVEX), 4 times each of mock-infected, 6 h and 18 h extracts after infection.
0 μl together with 10 μl (13 μg) of crude extract containing recombinant OBP (rOBP; available from S, Lee and R. Lehman, Stanford University) together with 8% S
Load on DS-PAGE gel. The proteins are separated by electrophoresis at 9 mA / gel overnight and transferred to a PVDF membrane (PolyScreen; NEN Research Products) for 1 hour at 24 V using a GENIE electrophoresis transporter (Idea Scientific Company). For Western blot analysis, block solution [TBS (10
mM Tris, pH 8.0, 150 mM NaCl)] in 5% nonfat dry milk. After rapid washing in TBS, the blot is incubated with the primary antibody diluted 1: 250 in TBS containing 0.05% Tween 20 (TBST). The blot was then digested with TBST for 5 minutes each.
Washed twice, incubated with a secondary antibody (goat anti-rabbit alkaline phosphatase conjugated antibody, IgG; Promega) diluted 1: 4000 in TBST for 30 minutes at room temperature and washed 3 times for 5 minutes each with TBS. I do. NBT (Promega)
66 μl was added to 10 ml of alkaline phosphate substrate buffer (100 mM Tris, pH 8.
The substrate is prepared by adding 0,100 mM NaCl, 5 mM MgCl ₂ ) substrate, followed by 33 μl of BCIP (Promega). Substrate is added to the blot, followed by color development. The blot is washed twice with deionized water to stop the reaction. Western blot results (FIG. 5) show the infected cell extract lane (6 hours post infection).
H) shows a light protein band of the appropriate size (78 kDa by calculation); 18 h after infection, a clearly similar heavy protein band in the infected cell extract lane (18 h); Protein is absent in mock infected cell extract (M). The anti-peptide antibody also identified OBP from the recombinant baculovirus extract, but did not identify OBP from the infected cell extract. This suggests that there may be differences in protein arrangement or modification between these sources.

Example 3 Herpes Virus In Vitro Replication A transient replication assay shows that, as necessary and sufficient to replicate plasmid DNA containing a viral origin of replication in relation to mammalian or insect cells, 7
Species HSV-1 proteins were identified (Stow, ND, 1992, J. Gen. Virol. 73: 313-321).
Boehmer, PE and Lehman, IR, 1997, Annu. Rev. Biochem. 66: 347-384).
However, none of these seven purified proteins or extracts of these proteins from recombinant baculovirus infected cells are sufficient for in vitro directed replication and additional proteins are required. (Skaliter, R. and Lehman, IR, 1994, Proc. Nat'l. Acad. Sci. USA 91).
: 10665-10669; Boehmer, PE and Lehman, IR, 1997, Annu. Rev. Biochem. 66
: 347-384). This time, the OBP-NT protein is located at site I at the origin of replication of the virus.
, II and III have been identified as viral origin binding proteins; these binding sites are contained within the core origin region necessary for efficient replication. Furthermore, OBP-NT is only a viral origin binding protein that binds to the oligonucleotide containing site III. Thus, OBP-NT may provide additional viral functions required for origin-directed replication in vitro in the presence of seven previously identified replication proteins.
In addition to OBP-NT, cellular proteins found to be required for non-origin directional replication may be required for origin directional replication (Skaliter, R. and Lehman, I. et al.
R., 1994, Proc. Nat'l. Acad. Sci. USA 91: 10665-10669).

[0093]

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the core OBP binding domain identified within site I and the homologous DNA sequences of sites II and III for oriS and oriL.

FIG. 2. Characterized motifs of the protein encoded by UL9 mRNA, and UL8.75 and UL8.5mR associated with the UL9 encoded protein.
2 shows a putative protein encoded by NA.

FIG. 3: Harvested and radiolabeled sites I, II, III 12 hours after infection
Or EMSA of HSV-1 infected cell extracts incubated with III (I) DNA.

FIG. 4. Harvested long after infection, a) Radiolabeled site 1DN
A, b) EMSA of uninfected (pseudo) or HSV-1 infected cell extracts incubated with radiolabeled site II DNA and c) radiolabeled site III DNA.

FIG. 5: Uninfected (lane M) cell extract, HSV-1 infected cells harvested 6 or 18 hours post-infection, incubated with polyclonal antibody raised against the N-terminus of OBP-NT Extracts (lanes 6 h, 18 h) or extracts from cells infected with recombinant baculovirus expressing OBP (lane O)
3 shows a western blot of BP).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/19 C12P 21/02 C 4H045 1/21 G01N 33/15 Z 5/10 33/50 Z C12P 21 / 02 33/68 G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 33/68 C12N 5/00 A (72) Inventor Javier J. Garcia United States 19301 South Valley Valley, Paoli, PA 19301 Road No. 29 (72) Inventor Michelle M. Gautica United States 19446 Trambauer Road, Lansdale, PA 2041 F-Term (reference) BA32 BA80 CA01 EA02 GA11 GA19 4B064 AG01 AG32 CA19 CC24 DA01 4B065 AA95 Y AB01 AC14 BA02 CA24 CA44 CA45 4C084 AA02 AA06 BA01 BA02 BA08 BA20 BA22 CA01 CA18 NA14 ZB331 ZB332 4H045 AA10 AA30 BA10 CA03 EA29 EA31 EA52 FA74

Claims (16)

[Claims] 1. An isolated polynucleotide comprising a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. 2. OBP contained in herpes simplex virus type I (HSV-1)
An isolated polynucleotide comprising a polynucleotide that is at least 70% identical to a polynucleotide encoding the same polypeptide expressed by the NT gene. 3. An isolated polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 4. The polynucleotide according to claim 1, comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5. 5. The polynucleotide according to claim 1, which encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. 6. A vector comprising the polynucleotide according to claim 1. 7. A host cell comprising the vector according to claim 6. 8. A method for producing an OBP-NT polypeptide or fragment, comprising culturing the host cell of claim 7 under conditions sufficient to produce an OBP-NT polypeptide or fragment. 9. A polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO: 2. 10. An OBP-NT polypeptide comprising an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. An antagonist which inhibits the activity, binding or expression of the polypeptide according to claim 10. 12. A method of treating an individual in need of anti-herpes virus treatment, comprising administering a therapeutically effective amount of the polypeptide of claim 11. 13. A method for diagnosing a disease associated with the expression or activity of the polypeptide according to claim 9 in an individual, comprising: (a) using the polypeptide according to claim 9 in a sample derived from the individual. A method comprising analyzing the presence or abundance. 14. A method for identifying a compound that interacts with the polypeptide according to claim 9 and inhibits or activates the activity of the polypeptide, wherein the compound and the polypeptide are evaluated in order to evaluate the interaction of the compound. Contacting a composition comprising the polypeptide with the compound to be screened under conditions that allow for interaction between the polypeptides (such interactions can produce a detectable signal in response to the interaction of the compound with the polypeptide). Associated with the second component); then, by detecting the presence or absence of a signal resulting from the interaction of the compound with the polypeptide, the compound interacts with the polypeptide and activates its activity or A method comprising determining whether to inhibit. 15. A method for eliciting an immunological response in a mammal, comprising:
10. The method according to claim 9, which is suitable for generating an antibody and / or T cell immune response.
A method comprising inoculating a mammal with a BP-NT polypeptide or a fragment or variant thereof and protecting the animal from disease. 16. A method of eliciting an immunological response in a mammal, comprising:
Delivering a nucleic acid vector that directs the expression of the OBP-NT polypeptide or a fragment or variant thereof according to claim 9 to elicit an immunological response, and expressing the OBP-NT polypeptide or a fragment or variant thereof. A method that includes causing