CA2330230A1 - Herpes simplex virus origin binding protein n-terminal truncation (obp-nt) - Google Patents

Herpes simplex virus origin binding protein n-terminal truncation (obp-nt) Download PDF

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CA2330230A1
CA2330230A1 CA002330230A CA2330230A CA2330230A1 CA 2330230 A1 CA2330230 A1 CA 2330230A1 CA 002330230 A CA002330230 A CA 002330230A CA 2330230 A CA2330230 A CA 2330230A CA 2330230 A1 CA2330230 A1 CA 2330230A1
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polypeptide
obp
leu
ala
arg
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Christine E. Dabrowski-Amaral
Javier J. Garcia
Michele M. Gorczyca
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SmithKline Beecham Corp
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Abstract

The invention provides herpes virus OBP-NT polypeptides and polynucleotides encoding OBP-NT polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing OBP-NT
polypeptides to screen for antiviral compounds.

Description

Herpes simplex virus Origin binding protein - N-terminal truncation (OBP-NT) FIELD OF THE INVENTION
This invention relates to polynucleotides encoding a newly identified polypeptide, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, the invention relates to polynucleotides encoding the polypeptide hereinafter referred to as herpes simplex virus origin binding protein N-terminal truncation, or "OBP-NT".
BACKGROUND OF THE INVENTION
The herpesviruses make up a medically important family of viruses comprised of three subfamilies, the alphaherpesviruses, betaherpesviruses and gammaherpesviruses.
These viruses are known to be able to produce infectious virus in a susceptible cell, as well as to remain latent in the host, reactivating in response to stimuli. Herpes simplex virus types 1 and 2 {HSV-I, HSV-2) are alphaherpesviruses known to cause several types of disease in humans, including, for example, keratitis leading to blindness, encephalitisand herpes labialis from HSV-1 infection, neonatal disease and genital herpes from infection. HSV-l and HSV-2 are highly homologous viruses which were not distinguished until 1962 when the 2 serotypes were identified. Since its isolation more than 70 years ago, HSV-1 has been the most intensively studied herpesvirus and is the prototype of the alphaherpesvirus subfamily. Sequencing of the HSV-1 genome was completed in 1988, at which time 72 genes encoding 70 unique proteins were identified. Since this time an additional 4 genes have been conclusively identified, with another 6 genes postulated.
HSV-2 has recently been sequenced, with identification of the analogous 74 unique proteins. It is likely that additional as yet unknown proteins are encoded within the HSV-1 and, by analogy, HSV-2 genomes. Despite the vast amount of research with HSV-1, many questions concerning the biology of this virus and of HSV-2 remain. It is particularly preferred to employ HSV-1 and HSV-2 genes and gene products as targets for the development of antiviral reagents.
HSV-1 infects 40 to 80% of the general population, while HSV-2 infects 20 to 65% of the population. HSV-2 infection is particularly significant in the prostitute and homosexual populations, where the frequency may approach 90%. Current antivirals show only partial efficacy with reductions in pain, lesion severity and shedding. A
medical need exists, therefore, for antivirals demonstrating improvement in overall efficacy, with decreased time to healing. In addition, the ability to impact viral reactivation from latency would be a significant advantage and may have disease-modifying implications.
Clearly, there exists a need for factors, such as the OBP-NT embodiment of the invention, that have a present benefit of being useful to screen compounds for antiviral activity. Such factors are also useful to determine their role in pathogenesis of infection.
dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists to find ways to prevent, ameliorate or correct such infection, dysfunction and disease.
The origin binding protein (OBP), encoded within the UL9 gene of the HSV-1 genome, was originally identified in 1986 and further characterized in 1988, when binding activity to 2 homologous domains (site I and site II, also called Box 1 and Box II) within viral origins of DNA replication was described (Elias, P. et al.. 1986. Proc.
Nat'l. Acad. Sci.
USA 83:6322-6326; Elias, P. and I. R. Lehman. 1988. Proc. Nat'1. Acad. Sci.
USA 85:2959 2963). Three viral origins of DNA replication are contained within the HSV-1 and HSV-2 genomes. 2 copies of oriS and 1 copy of oriL (Spaete. R. R, and Frenkel, N.
1985. Proc.
Nat'l. Acad. Sci. USA 82:694-698; Stow, N. D. and E. C. McMonagle. 1983.
Virology 130:427-438; Dolan, A. et al. 1998. J. Virol. 72:2010-2021 ). OriS contains 1 copy each of OBP binding sites I and II, as well as a third homologous sequence site III
and an AT rich stretch; all of these domains are required for efficient origin function (see figure 1; Elias, P.
et al. 1990. J. Biol. Chem. 265:17167-17173). OriL contains 2 copies each of sites I and III
and an AT rich stretch, which are required for efficient origin function (Lockshon, D. and Galloway, D. A. 1988. Mol. Cell. Biol. 8:4018-4027) Despite considerable work in the field, the ability of the OBP to interact with site III remains controversial;
while purified OBP does not appear to interact with oligonucleotides comprising site III, OBP
from infected cell extracts has been identified as being able to bind site III
oligonucleotides (Olivo, P. D. et al. 1988. Proc. Nat7. Acad. Sci. USA 85:5414-5418; Bruckner, R. C. et al.
1991. J. Biol. Chem. 266:2669-2674; Hazuda, D. J. et al. 1991. J. Biol. Chem.
266:24621-24626; Dabrowski, C. E. and P. A. Schaffer. 1991. J. Virol. 65:3140-3150).
Further, purified OBP has been suggested to interact with site III within the context of the complete viral origin oriS (Elias, P. et al, 1990. J. Biol. Chem. 265:17167-17173;
Koff, A. et al.
1991. J. Virol. 65:3284-329; Elias, P. et al. 1992. J. Biol. Chem. 267:17424-17429). The OBP has been shown to be essential for viral growth and DNA replication by the generation of viral mutants (Malik, .A. K. et al. 1992. Virology 190:702-715).
OBP has also been tested in cell-based transient replication assays, which demonstrated the requirement for helicase activity and specific dimerization sequences encoded within the N-terminus, and the ability to bind origin sequences encoded in the C-terminal half of the protein, for viral DNA replication (Hazuda. D. J. et al. 1992. J. Biol. Chem. 267:14309-14315; Malik, A. K. and S. K. Welter. 1996. J. Virol. 70:7859-7866; Lee, S. S.-K. and I. R.
Lehman. 1997.
Proc. Nat'l. Acad. Sci. USA. 94:2838-2842). Subsequent work resulted in the characterization of nucleotides important for origin binding activity, and demonstrated the relationship between the origin binding ability of OBP and origin-directed DNA
replication (Challberg. M. D. 1991. Seminars in Virology 2:247-256; Hernandez, T. R. et al. 1991. J.
Virol. 65:1649-1652; Dabrowski, C. E. et al. 1994. Mol. Cell. Biol. 14:2545-2555).
The UL9 (OBP) open reading frame is encoded within a 5.0-5.2 kilobase RNA in HSV-1. OBP comprises the protein component of the protein:DNA complex designated 'complex B' in electrophoretic mobility shift assays (EMSA) following incubation of HSV-1 infected cell extracts and radiolabeled HSV-I origin sequences (Baradaran, K. et al. 1996.
J. Virol. 70:5673-5679) Novel RNAs 6.3-6.4 and 4.0-4.I kilobases in length have been described, which are also transcribed from the UL9 region of the HSV-1 genome (Deb, S.
P. et al. 1993. Biochem and Biophys Res. Comm. 193:617-623; Baradaran, K. et al. 1994. J.
Virol. 68:4251-4261). The 4.0-4.1 kiIobase RNA, designated UL8.5, was shown to encode a novel origin binding protein, OBPC, which is translated in-frame with the C-terminal (DNA binding] half of OBP ('see figure 2). The function of this protein is as yet unknown, although a role in the switch from DNA replication to packaging of the viral genome into the capsid has been postulated. OBPC comprises the protein component of the protein:DNA complex designated 'complex A' by EMSA following incubation of HSV-infected cell extracts and radiolabeled HSV-1 origin sequences (Baradaran, K.
et al. 1996.
J. V irol. 70:5673-5679) Complex A has been shown to bind to origin sites I
and II; the inability to bind to site III was shown to be due to a single nucleotide difference between sites I and III in the OBP binding domain (Dabrowski, C. E. and P. A.
Schaffer. 1991. J.
Virol. 65:3140-3150). The 6.3-6.4 kilobase RNA, designated UL9.5, has not yet been mapped but has been postulated to encode a protein which overlaps the N-terminus of the OBP (Baradaran, K. et al. 1994. J. Virol. 68:4251-4261).

SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as OBP-NT polypeptides from herpes viruses, including from HSV-1 OBP-NT, HSV-2 OBP-NT, and VZV OBP-NT.
It is a further object of the invention to provide polynucleotides that encode OBP-NT
polypeptides, particularly polynucleotides that encode the polypeptide herein designated HSV-I OBP-NT, HSV-2 OBP-NT and VZV OBP-NT.
In a particularly preferred embodiment of the invention the polynucleotide comprises a region encoding HSV-1 OBP-NT polypeptide comprising a sequence set out in Table 1 [SEQ ID NO: I ] which includes a full length gene, or a variant thereof.
In another particularly preferred embodiment of the invention there is a OBP-NT
protein from HSV-1 comprising the amino acid sequence of Table I [SEQ ID
N0:2], or a variant thereof.
As a further aspect of the invention there are provided isolated nucleic acid molecules IS encoding OBP-NT, particularly HSV-1 OBP-NT, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of OBP-NT and polypeptides encoded thereby.
In another aspect of the invention there are provided biologically, diagnostically, prophylactically, clinically or therapeutically useful variants of polypeptides of HSV-1 OBP-NT and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of OBP-NT polypeptide encoded by naturally occurring alleles of the OBP-NT gene.
In a preferred embodiment of the invention there are provided methods for producing the aforementioned OBP-NT polypeptides.
In accordance with yet another aspect of the invention, there are provided inhibitors to poiypeptides of the inventions, useful as antiviral agents, including, for example, antibodies or inhibitors of interactions between polynucleotides and polypeptides.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing OBP-NT expression, treating disease,._ assaying genetic variation, and administering a OBP-NT polypeptide or polynucleotide to an organism to raise an immunological response against a virus, especially HSV-1.
In accordance with certain preferred embodiments of this and other aspects of the invention there are provided polynucleotides that hybridize to OBP-NT
polynucleotide sequences, particularly under stringent conditions.
In certain preferred embodiments of the invention there are provided antibodies against OBP-NT polypeptides.
In other embodiments of the invention there are provided methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypeptide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction of the compound with the polypeptide or polynucleotide.
In accordance with yet another aspect of the invention, there are provided OBP-NT
agonists and antagonists, preferably virostatic or virocidal agonists and antagonists.
In a further aspect of the invention there are provided compositions comprising a OBP-NT polynucleotide or a OBP-NT polypeptide for administration to a cell or to a multicellular organism.
25. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 represents the nucleotide sequence of the core OBP binding domain defined within site I, and the homologous DNAs of sites II and III, for oriS
and oriL.
Figure 2 represents characterized motifs of the protein encoded by the UL9 mRNA, as well as the predicted proteins encoded by the UL8.75 and UL8.5 mRNAs in relation to the UL9 encoded protein.
Figure 3 represents an EMSA of HSV-1 infected cell extracts harvested 12 hours post-infection, incubated with radiolabelled site I. II, III or III(I) DNAs.
Figure 4 represents an EMSA of uninfected (Mock) cell extracts, or HSV-1 infected cell extracts harvested at increasing time post-infection, incubated with a) S radiolabelled site I DNA, b) radiolabelled site II DNA, and c) radiolabelled site III DNA.
Figure S represents a Western blot of uninfected (lane M) cell extracts, HSV-1 infected cell extracts harvested at 6 or 18 hours post-infection (lanes 6h, 18h), or extracts from cells infected with recombinant baculovirus expressing OBP (lane OBP), incubated with polyclonal antibody generated against the N-terminus of OBP-NT.
I O DESCRIPTION OF THE INVENTION
The invention relates to OBP-NT polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of OBP-NT of HSV-1, which is related by amino acid sequence homology to other herpes virus OBP-NT proteins including, for example, HSV-2 OBP-NT and VZV OBP-NT. The IS invention relates especially to HSV-1 OBP-NT having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

OBP-NT Polynucleotide and Polypeptide Sequences 20 (A)Sequences from HSV-1 OBP-NT polynucleotide sequence [SEQ ID NO:1].
S'-ATGAACGACCGCCCCTTCCACCGACTTATCGTCCAGGTGGAAAGCCTTCATCGCGTGGGC
CCCAACCTTCTGAACAACTACGACGTCCTCGTTCTGGACGAGGTTATGTCGACGCTGGGC
CAGCTCTATTCGCCAACGATGCAGCAACTGGGCCGCGTGGATGCGTTAATGCTACGCCTG
CTGCGCATCTGTCCTCGGATCATCGCCATGGACGCAACCGCCAACGCGCAGTTGGTGGAC

CCCGGGTTTTCGGCGCGCCGGTGCCTGTTTCTCCCGCGTCTGGGGACCGAGCTCCTGCAG
GCTGCCCTGCGCCCGCCCGGGCCGCCGAGCGGCCCGTCTCCGGACGCCTCTCCGGAGGCC
CGGGGGGCCACGTTCTTTGGGGAGCTGGAAGCGCGCCTTGGCGGGGGCGATAACATCTGC
ATTTTTTCGTCGACGGTCTCCTTCGCGGAGATCGTGGCCCGGTTCTGCCGTCAGTTTACG

TACCGCGTGGTTATATACACGACGGTCGTAACCGTGGGCCTCAGCTTCGATCCCCTGCAC
TTTGATGGCATGTTCGCCTACGTGAAACCCATGAACTACGGACCGGACATGGTGTCCGTG
TACCAGTCCCTGGGACGGGTGCGCACCCTCCGCAAGGGGGAGCTACTGATTTACATGGAC
GGCTCCGGGGCGCGCTCGGAGCCCGTCTTTACGCCCATGCTCCTTAATCACGTGGTCAGT

AAGGGGCGCTGTGACGCGTCGGCATGCGACACGTCGCTGGGGCGGGGGTCGCGCATCTAC
AACAAATTCCGTTACAAACACTACTTTGAGAGATGCACGCTGGCGTGTCTCTCGGACAGC
CTTAACATCCTTCACATGCTGCTGACCCTAAACTGCATACGCGTGCGCTTCTGGGGACAC
GACGATACCCTGACCCCAAAGGACTTCTGTCTGTTTTTGCGGGGCGTACATTTCGACGCC
CTCAGGGCCCAGCGCGATCTACGGGAGCTGCGGTGCCGGGATCCCGAGGCGTCGCTGCCG
GCCCAGGCCGCCGAGACGGAGGAGGTGGGTCTTTTCGTCGAAAAATACCTCCGGTCCGAT
GTCGCGCCGGCGGAAATTGTCGCGCTCATGCGCAACCTCAACAGCCTGATGGGACGCACG
S CGGTTTATTTACCTGGCGTTGCTGGAGGCCTGTCTCCGCGTTCCCATGGCCACCCGCAGC
AGCGCCATATTTCGGCGGATCTATGACCACTACGCCACGGGCGTCATCCCCACGATCAAC
GTCACCGGAGAGCTGGAGCTCGTGGCCCTGCCCCCCACCCTGAACGTAACCCCCGTCTGG
GAGCTGTTGTGCCTGTGCAGCACCATGGCCGCGCGCCTGCATTGGGACTCGGCGGCCGGG
GGATCTGGGAGGACCTTCGGCCCCGATGACGTGCTGGACCTACTGACCCCCCACTACGAC
IO CGCTACATGCAGCTGGTGTTCGAACTGGGCCACTGTAACGTAACCGACGGACTTCTGCTC
TCGGAGGAAGCCGTCAAGCGCGTCGCCGACGCCCTAAGCGGCTGTCCCCCGCGCGGGTCC
GTTAGCGAGACGGACCACGCGGTGGCGCTGTTCAAGATAATCTGGGGCGAACTGTTTGGC
GTGCAGATGGCCAAAAGCACGCAGACGTTTCCCGGGGCGGGGCGCGTTAAAAACCTCACC
AAACAGACAATCGTGGGGTTGTTGGACGCCCACCACATCGACCACAGCGCCTGCCGGACC
IS CACAGGCAGCTGTACGCCCTGCTTATGGCCCACAAGCGGGAGTTTGCGGGCGCGCGCTTC
AAGCTACGCGTGCCCGCGTGGGGGCGCTGTTTGCGCACGCACTCATCCAGCGCCAACCCC
AACGCTGACATCATCCTGGAGGCGGCGCTGTCGGAGCTCCCCACCGAGGCCTGGCCCATG
ATGCAGGGGGCGGTGAACTTTAGCACCCTATAA-3' 20 (B)Herpes simplex virus type 1 (HSV-1) OBP-NT polypeptide sequence deduced from the polynucieotide sequence in this table [SEQ ID N0:2J.
NH:-MNDRPFHRLIVQVESLHRVGPNLLNNYDVLVLDEVMSTLGQLYSPTMQQLGRVDALMLRL
LRICPRIIAMDATANAQLVDFLCGLRGEKNVHVWGEYAMPGFSARRCLFLPRLGTELLQ
AALRPPGPPSGPSPDASPEARGATFFGELEARLGGGDNICIFSSTVSFAEIVARFCRQFT

YQSLGRVRTLRKGELLIYMDGSGARSEPVFTPMLLNHWSSCGQWPAQFSQVTNLLCRRF
KGRCDASACDTSLGRGSRIYNKFRYKHYFERCTLACLSDSLNILHMLLTLNCIRVRFWGH
DDTLTPKDFCLFLRGVHFDALRAQRDLRELRCRDPEASLPAQAAETEEVGLFVEKYLRSD
VAPAEIVALMRNLNSLMGRTRFIYLALLEACLRVPMATRSSATFRRIYDHYATGVIPTIN

RYMQLVFELGHCNVTDGLLLSEEAVKRVADALSGCPPRGSVSETDHAVALFKIIWGELFG
VQMAKSTQTFPGAGRVKNLTKQTIVGLLDAHHIDHSACRTHRQLYALLMAHKREFAGARF
KLRVPAWGRCLRTHSSSANPNADIILEAALSELPTEAWPMMQGAVNFSTL*-COOH
35 (C)Polynucleotide sequences comprising Herpes simplex virus type 2 (HSV-2) OBP-NT
ORF sequence [SEQ ID N0:3].
5'-ATGAACGACCGCCCCTTCCACCGTCTCATCGTGCAGGTGGAAAGTCTTCATCGCGTGGGC
CCGAACCTGTTGAACAACTACGACGTGCTCGTCACGAAGTCACTCATGTCGACGTTGGGC

CTGCGCACGTGCCCGCGGATCATCGCCATGGACGCCACCGCCAACGCGCAGCTGGTGGAC
TTTCTGTGCAGCCTCCGGGGCGAAAAGAACGTTCACGTGGTCATCGGGGAGTACGCCATG
CCCGGATTTTCGGCGCGCCGTTGTCTGTTTCTCCCGCGCCTGGGGCCCGAGGTCCTGCAG
GCGGCCCTGCGCCGCCGGGGGCCGGCGGGCGGGGCGCCCCCCCCGGACGCCCCCCCGGAC

TCGTCGACGGTCTCCTTCGCGGAGGTCGTTGCCAGGTTCTGCCGGCAGTTTACGGACCGC
GTGCTGCTGCTCCACTCGCTCACCCCGCCCGGCGACGTGACCACATGGGGCCGGTACCGG
GTGGTCATCTACACAACGGTCGTGACGGTGGGCCTTAGCTTCGATCCGCCGCACTTTGAC
AGCATGTTCGCCTACGTGAAACCCATGAACTACGGGCCGGACATGGTGTCCGTGTACCAG
S TCGCTGGGGCGGGTACGGACTCTCCGCAAGGGGGAGCTGCTGATCTACATGGACGGGTCC
GGGGCGCGCTCGGAGCCCGTCTTTACGCCCATGCTGCTCAACCACGTGGTGAGCGCCAGC
GGGCAGTGGCCGGCACAGTTTTCCCAGGTGACGAACCTGTTGTGCCGCCGGTTCAAAGGG
CGCTGCGACGCGTCGCACGCCGACGCGGCGCAGGCGCGGGGGTCGCGCATCTACAGCAAA
TTCCGGTACAAGCACTACTTCGAGAGGTGCACGCTGGCGTGCCTCGCGGACAGTCTTAAC
IO ATCCTCCACATGCTTCTGACCCTCAACTGCATGCACGTGCGGTTCTGGGGCCACGACGCC
GCGCTGACCCCGAGGAACTTTTGTCTGTTTTTGCGGGGGATACATTTTGATGCCCTGAGG
GCCCAGCGGGATCTGCGGGAGCTGCGCTGCCAGGACCCCGACACGTCCCTGTCGGCCCAG
GCCGCCGAGACGGAGGAGGTGGGCCTTTTCGTCGAAAAGTACCTCCGGCCGGACGTCGCG
CCGGCCGAGGTGGTCGCGCTCATGCGCGGCCTCAACAGCCTGGTCGGCCGCACGCGGTTC
IS ATCTACCTGGTGCTGCTGGAGGCCTGTCTTCGCGTCCCCATGGCCGCCCATAGCAGCGCC
ATCTTCCGGCGGCTTTACGACCACTACGCCACGGGCGTCATCCCCACGATCAACGCCGCC
GGAGAGCTGGAGCTTGTGGCCCTACACCCCACCCTAAACGTCGCCCCCGTCTGGGAGCTG
TTCCGTCTGTGCAGCACCATGGCCGCGTGCCTGCAGTGGGACTCGATGGCCGGGGGGTCG
GGGCGAACCTTTAGCCCCGAGGACGTGCTGGAGCTGCTGAACCCCCACTACGACCGCTAC
O ATGCAGCTGGTGTTCGAACTGGGCCACTGTAACGTGACCGACGGCCCCTTGCTGTCGGAG
GACGCGGTTAAGCGCGTGGCCGACGCCCTGAGCGGCTGCCCCCCGCGCGGGTCCGTGAGC
GAGACGGAGCACGCGCTGTCGCTGTTCAAGATCATCTGGGGCGAACTGTTCGGGGTGCAG
CTGGCCAAGAGCACGCAGACGTTTCCCGGGGCGGGGCGCGTTAAAAACCTCACCAAGCGA
GCCATCGTGGAGCTGCTGGACGCCCACCGCATCGACCACAGCGCCTGCCGGACGCACAGA
ZS CAGCTGTACGCGCTGCTGATGGCCCATAAGCGGGAGTTTGCGGGCGCGCGCTTCAAGCTG
CGCGCGCCCGCGTGGGGGCGCTGCTTGCGCACGCACGCCTCCGGCGCCCAGCCCAACACT
GACATCATTCTCGAGGCGGCTCTGTCGGAGCTTCCCACCGAGGCCTGGCCCATGATGCAG
GGGGCGGTGAACTTTAGCACCCTATAA-3' 30 (D)Herpes simplex virus type 2 (HSV-2) OBP-NT polypeptide sequence deduced from the polynucleotide ORF sequence [SEQ ID N0:4].

LRTCPRIIAMDATANAQLVDFLCSLRGEKNVHWIGEYAMPGFSARRCLFLPRLGPEVLQ
AALRRRGPAGGAPPPDAPPDATFFGELEARLAGGDNVCIFSSTVSFAEWARFCRQFTDR

SLGRVRTLRKGELLIYMDGSGARSEPVFTPMLLNHWSASGQWPAQFSQVTNLLCRRFKG
RCDASHADAAQARGSRIYSKFRYKHYFERCTLACLADSLNILHMLLTLNCMHVRFWGHDA
ALTPRNFCLFLRGIHFDALRAQRDLRELRCQDPDTSLSAQAAETEEVGLFVEKYLRPDVA
PAEWALMRGLNSLVGRTRFIYLVLLEACLRVPMAAHSSAIFRRLYDHYATGVIPTINAA
4O GELELVALHPTLNVAPVWELFRLCSTMAACLQWpSMAGGSGRTFSPEDVLELLNPHYDRY
MQLVFELGHCNVTDGPLLSEDAVKRVADALSGCPPRGSVSETEHALSLFKIIWGELFGVQ
LAKSTQTFPGAGRVKNLTKRAIVELLDAHRIDHSACRTHRQLYALLMAHKREFAGARFKL
RAPAWGRCLRTHASGAQPNTDIILEAALSELPTEAWPMMQGAVNFSTL*-COOH
4S (E) Herpes virus VZV OBP-NT polynucleotide sequence [SEQ ID NO:S].
5'-ATGGGTTTTAAACGTTTGATTGTGCAACTTGAAAGCCTACACCGCGTATCCAGCGAAGCT
ATCGACAGCTACGACGTATTAATACTGGATGAGGTAATGTCAGTGATTGGACAATTATAC
TCCCCCACAATGAGACGTCTTTCCGCGGTTGATAGCCTATTATATCGTCTTTTAAATCGC
TGTTCTCAAATTATCGCGATGGATGCTACAGTAAACTCGCAGTTTATTGATTTAATCTCC
SO GGATTGCGTGGAGATGAAAACA'I'ACACACAATTGTGTGTACATACGCGGGAGTTGGGTTC
TCCGGAAGAACTTGCACGATCCTGCGTGATATGGGCATCGACACGCTTGTGCGAGTCATT

AAACGATCTCCTGAACACGAGGATGTACGTACCATACACCAACTACGTGGAACATTTTTT
GACGAACTAGCACTACGATTACAATGTGGGCATAACATCTGTATATTTTCATCAACTTTA
TCGTTTTCGGAGCTAGTTGCTCAGTTTTGTGCAATATTTACAGACTCTATTCTTATTTTA
AACTCAACTCGGCCCCTATGTAATGTAAACGAATGGAAACATTTTCGCGTGTTGGTGTAC
S ACTACCGTCGTGACCGTTGGATTGAGTTTTGACATGGCTCATTTTCATAGCATGTTTGCT
TACATAAAGCCAATGTCATATGGGCCGGATATGGTATCGGTCTACCAGTCATTAGGGCGT
GTACGTTTATTGCTACTTAATGAAGTTTTGATGTACGTCGATGGCTCAAGGACCAGATGC
GGACCCCTGTTCTCGCCAATGTTACTAAACTTTACCATCGCAAATAAATTTCAATGGTTT
CCTACACACACCCAAATAACTAACAAACTGTGCTGTGCATTTAGGCAACGATGTGCAAAT
IO GCATTTACACGCTCGAACACCCATCTCTTCTCAAGATTTAAATACAAACACCTTTTCGAG
AGATGCTCTCTTTGGAGTTTAGCCGATAGCATTAATATCTTACAAACTCTTTTGGCCTCT
AACCAAATTTTGGTTGTATTGGATGGCATGGGTCCAATAACGGACGTTTCCCCAGTTCAA
TTTTGTGCATTTATACACGATCTCAGACATAGCGCTAACGCCGTAGCTTCCTGTATGCGT
TCTCTTAGACAGGACAATGACAGCTGCTTGACCGATTTTGGCCCTTCCGGATTTATGGCC
IS GATAACATTACCGCGTTTATGGAAAAGTATCTTATGGAGTCAATTAATACCGAAGAACAA
ATTAAAGTATTTAAAGCCCTTGCATGTCCAATAGAACAGCCTAGACTAGTCAATACGGCA
ATATTGGGGGCGTGTATACGAATACCTGAAGCGTTGGAAGCATTTGACGTATTTCAAAAA
ATATACACGCACTACGCTTCCGGTTGGTTTCCCGTCCTGGACAAAACCGGGGAATTTAGC
ATCGCGACTATAACTACCGCCCCAAATTTAACCACACATTGGGAGCTGTTTCGCCGTTGT
ZO GCCTATATTGCAAAAACACTCAAGTGGAATCCGTCCACCGAAGGCTGTGTAACACAAGTT
TTGGATACGGACATTAATACACTTTTCAATCAACACGGGGATTCGCTGGCTCAACTAATA
TTTGAGGTTATGCGCTGTAACGTTACTGACGCTAAGATTATATTAAACCGCCCGGTTTGG
CGAACAACCGGATTCTTAGATGGATGCCATAATCAATGCTTCCGTCCAATCCCTACAAAA
CACGAATATAACATTGCTCTATTTCGTTTAATTTGGGAACAATTATTTGGCGCCCGCGTA
ZS ACTAAAAGTACCCAGACCTTTCCGGGAAGTACTCGTGTGAAAAACCTAAi3AAA.AAAAGAT
CTAGAAACTTTACTTGATTCAATTAACGTGGATCGTTCTGCATGTCGTACCTACCGCCAG
TTGTATAACCTGCTTATGAGCCAGCGCCATTCGTTCTCTCAACAGCGTTACAAAATTACT
GCCCCCGCTTGGGCACGCCACGTGTATTTTCAAGCACATCAAATGCACTTGGCCCCGCAT
GCCGAAGCCATGCTACAATTAGCGCTATCGGAACTGTCCCCGGGATCGTGGCCGCGGATA
3O AACGGGGCGGTAAATTTTGAAAGTTTATAA-3' (F)VZV OBP-NT polypeptide sequence deduced from polynucleotide sequence [SEQ ID N0:6]

KRSPEHEDVRTIHQLRGTFFDELALRLQCGHNICIFSSTLSFSELVAQFCAIFTDSILIL
NSTRPLCNVNEWKHFRVLWTTWTVGLSFDMAHFHSMFAYIKPMSYGPDMVSWQSLGR
VRLLLLNEVLMYVDGSRTRCGPLFSPMLLNFTIANKFQWFPTHTQITNKLCCAFRQRCAN
AFTRSNTHLFSRFKYKHLFERCSLWSLADSINILQTLLASNQILWLDGMGPITDVSPVQ

IKVFKALACPIEQPRLVNTAILGACIRIPEALEAFDVFQKIYTHYASGWFPVLDKTGEFS
IATITTAPNLTTHWELFRRCAYIAKTLKWNPSTEGCVTQVLDTDINTLFNQHGDSLAQLI
FEVMRCNVTDAKIILNRPVWRTTGFLDGCHNQCFRPIPTKHEYNIALFRLIWEQLFGARV
TKSTQTFPGSTRVKNLKKKDLETLLDSINVDRSACRTYRQLYNLLMSQRHSFSQQRYKIT
4S APAWARHWFQAHQMHLAPHAEAMLQLALSELSPGSWPRINGAVNFESL*-COOH

WO 99/64b33 PCT/US99/13140 Polypeptides The polypeptides of the invention include polypeptides of Table 1 [SEQ ID NOS:
2, 4 and 6) (in particular the mature polypeptides) as well as polypeptides and fragments, particularly those which have the biological activity of OBP-NT, and also those which have at least 70% identity to a polypeptide of Table 1 or a relevant portion, preferably at least 80%
identity to a polypeptide of Table 1 and more preferably at least 90% identity to a polypeptide of Table 1 and still more preferably at least 95% identity to a polypeptide of Table 1 and also including portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
The invention also includes polypeptides of the formula:
X-(R 1 )m-(R?)-~R3)n-Y
wherein, at the amino terminus, X is hydrogen or a metal, and at the carboxyl terminus, Y is hydrogen or a metal, R 1 and R3 are any amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R~ is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1. In the formula above R2 is oriented so that its amino terminal residue is at the left, bound to Rl, and its carboxy terminal residue is at the right, bound to R3. Any stretch of amino acid residues denoted by either R group, where m and/or n is greater than l, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
A fragment is a variant polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides. As with OBP-NT polypeptides fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1, or of variants thereof, such as a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus. Degradation forms of the polypeptides of the invention in a host, particularly HSV-1, are also preferred. Further preferred are fragments characterized by structural or functional attributes such as fragrrtents that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.

Also preferred are biologically active fragments which are those fragments that mediate activities of OBP-NT, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of HSV-1 or the ability to initiate, or maintain cause disease in an individual, particularly a human.
Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis;
therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the mventton.
In addition to the standard single and triple letter representations for amino acids, the term "X" or "Xaa" may also be used in describing certain polypeptides of the invention.
"X" and "Xaa" mean that any of the twenty naturally occurring amino acids may appear at such a designated position in the polypeptide sequence.
Polynucleotides Another aspect of the invention relates to isolated polynucleotides, including the full length gene. that encode the OBP-NT polypeptide having a deduced amino acid sequence of Table 1 and polynucleotides closely related thereto and variants thereof.
Using the information provided herein, such as a polynucleotide sequence set out in Table I [SEQ ID NOS:I, 3 and 5], a polynucleotide of the invention encoding OBP-NT
polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from virus using HSV-1 KOS as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a sequence given in Table l, a library of clones of chromosomal DNA of HSV-1 KOS in ~.coli or some other suitable host may be probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent conditions. Alternatively, a novel messenger RNA
(mRNA) encoding a polypeptide can be identified by Northern blot analysis, probed with a radiolabeled oligonucleotide or complementary RNA fragment, preferably a 17-mer or longer, derived from a partial sequence. The 5' and 3' ends of the novel gene can then be mapped and the gene cloned. By sequencing the individual clones thus identified with sequencing primers designed from the original sequence it is then possible to extend the -_ sequence in both directions to determine the full gene sequence. Conveniently, such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( 1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70).
Illustrative of the invention, a polynucleotide set out in Table 1 [SEQ ID
NO:1 ] was discovered following Northern blotting of mRNA harvested following infection with HSV-1.
The DNA sequences set out in Table 1 contain open reading frames encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID N0:2]
with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art. The polynucleotide of SEQ ID NO: I, between nucleotide number 1 and the stop codon which begins at nucleotide number 2131 of SEQ ID
NO: l, encodes the polypeptide of SEQ ID N0:2.
OBP-NT of the invention is structurally related to other proteins of the herpesvirus family, particularly for example LJL9 and LTL8.5 of HSV-1.
The invention provides a polynucleotide sequence identical over its entire length to a nucleotide coding sequence in Table 1. Also provided by the invention is the coding sequence for the mature polypeptide or a fragment thereof, by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro-protein sequence.
The polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences, termination signals, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.
Sci., USA 86:
821-824 ( 1989), or an HA tag (Wilson et al., Cell 37: 767 ( 1984)).
Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
A preferred embodiment of the invention is a polynucleotide comprising nucleotide I
to the nucleotide immediately upstream of or including nucleotide 2131 set forth in SEQ ID
NO:1 of Table I, which encodes the HSV-1 OBP-NT polypeptide.

The invention also includes polynucleotides of the formula:
X-(R 1 )m-(R?)-(R3)n-Y
wherein, at the 5' end of the molecule, X is hydrogen or a metal or together with Y defines a covalent bond, and at the 3' end of the molecule, Y is hydrogen or a metal or together with X defines the covalent bond, each occurrence of R I and R3 is independently any nucleic acid residue, m is an integer between 1 and 3000 or zero , n is an integer between 1 and 3000 or zero, and R2 is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1. In the polynucleotide formula above R~ is oriented so that its 5'end residue is at the left, bound to R1, and its 3'end residue is at the right, bound to R3. Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than l, may be either a heteropolymer or a homopolymer, preferably a heteropolymer. Where, in a preferred embodiment, X and Y together define a covalent bond, the polynucleotide of the above formula is a closed, circular polynucleotide. which can be a double-stranded polynucleotide wherein the formula shows a first strand to which the second strand is complementary. In another preferred embodiment m and/or n is an integer between 1 and 1000.
It is most preferred that the polynucleotides of the inventions are derived from HSV-1, however, they may preferably be obtained from organisms of the same taxonomic family.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a viral polypeptide and more particularly a polypeptide of OBP-NT having an amino acid sequence set out in Table 1. The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having a deduced amino acid sequences of Table 1.
Variants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding OBP-NT
variants, that have the amino acid sequence of OBP-NT polypeptides of Table 1 in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of OBP-NT.

Further preferred embodiments of the invention are polynucleotides that are at least 70% identical over their entire length to a poiynucleotide encoding OBP-NT
polypeptide having an amino acid sequence set out in Table l , and polynueleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over its entire length to a polynucleotide encoding OBP-NT polypeptide and polynucleotides complementary thereto.
In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97oIc are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1.
The invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the invention especially relates to polynucieotides that hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, Sx SSC
(150mM NaCI, lSmM trisodium citrate), ~0 mM sodium phosphate (pH7.6), Sx Denhardt's solution, 10%
dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at about 65°C.
Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., ( 1989), particularly Chapter 1 1 therein.
The invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in Table I under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in Table I or a fragment thereof; and isolating said DNA sequence.
Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.

As discussed additionally herein regarding poiynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding OBP-NT and to isolate cDNA and genomic clones of other genes that have a high S sequence identity to the OBP-NT gene. Such probes generally will comprise at least 15 bases.
Preferably, such probes will have at least 30 bases and may have at least 50 bases.
Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
For example, the coding region of the OBP-NT gene may be isolated by screening using a DNA sequence provided in Table 1 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for I S disease, particularly human disease, as further discussed herein relating to polynucleotide assays.
Polynucleotides of the invention that are oligonucleotides derived from the sequences of Table I may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in pan are transcribed by virus in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection the pathogen has attained.
The invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production. among other things. As generally is the case irt vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
IS

In addition to the standard A, G. C, T/LJ representations for nucleic acid bases, the term "N" may also be used in describing certain polynucleotides of the invention. "N"
means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except it is preferred that N is not a base that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame, would have the effect of generating a premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a pro?protein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
Vectors, host cells, expression The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention. host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
For recombinant production, host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention.
Introduction of a polynucieotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis et al., BASIC METHODS !N MOLECULAR BIOLOGY, ( 1986) and Sambrook et al.. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. ( 1989), such as, calcium phosphate transfection, DEAF-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, enterococci E. coli, streptomyces and Bacillus subtilis cells;
fungal cells, such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, Vero and Bowes melanoma cells: and plant cells.
A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include. among others, chromosomal, episomal and virus-derived-WO 99/64b33 PCT/US99/13140 vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as herpesviruses, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived J from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR CLONING, A
LABORATORY MANUAL, (supra).
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, high perfornrance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
Diagnostic, Prognostic, Serotyping and Mutation Assays This invention is also related to the use of the OBP-NT polynucleotides of the invention for use as diagnostic reagents. Detection of OBP-NT in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease.
Eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the HSV
OBP-NT gene may be detected at the nucleic acid level by a variety of techniques.
Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as cells of neuronal origin, blood, mucosa, epithelium, and skin. Genomic DNA
may be used directly for detection or may be amplified enzymatically by using PCR or other_ amplification technique prior to analysis. RNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of virus present in an individual, may be made by an analysis of the genotype of the viral gene.
Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence. Point mutations can be identified by hybridizing amplified DNA to labeled OBP-NT polynucleotide sequences.
Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230. 1242 ( 1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S 1 protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci.. USA. 8~: 4397-4401 (1985).
Cells carrying mutations or polymorphisms (allelic variations) in the gene of the invention may also be detected at the DNA or RNA level by a variety of techniques, to allow for serotyping, for example. For example, RT-PCR can be used to detect mutations in the RNA. It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as, for example, GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose. PCR or RT-PCR. As an example. PCR primers complementary to a nucleic acid encoding OBP-NT can be used to identify and analyze mutations.
Examples of representative primers are shown below in Table 2.
Table 2 Primers for amplification of OBP-NT polynucleotides SEO ID NO PRIMER SEQUENCE
5 5'-CCTGTCGTCGGAGTTTTACCCAG-3' 6 5'-GAGGTGCGGTCCCGAGACTTATAG-3' The invention also includes primers of the formula:
X-(RI)m-(R2)-(R3)n-Y
wherein, at the ~' end of the molecule, X is hydrogen or a metal, and at the 3' end of the molecule. Y is hydrogen or a metal, RI and R3 is any nucleic acid residue, m is an integer between 1 and 20 or zero , n is an integer between 1 and 20 or zero, and R2 is a primer-sequence of the invention, particularly a primer sequence selected from Table 2. In the polynucleotide formula above R~ is oriented so that its ~' end residue is at the left, bound to R l , and its 3' end residue is at the right, bound to R~. Any stretch of nucleic acid residues denoted by either R group, where m and/or n is greater than 1, may be either a heteropolymer J or a homopolymer, preferably a heteropolymer being complementary to a region of a polynucleotide of Table 1. In a preferred embodiment m and/or n is an integer between I and 10.
The invention further provides these primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. These primers may be used for, among other things, amplifying OBP-NT DNA isolated from a sample derived from an individual. The primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing disease, preferably viral infections, more preferably infections by HSV-l, comprising determining from a sample derived from an individual an increased level of expression of polynucleotide having a sequence of Table 1. Increased or decreased expression of OBP-NT
polynucleotide can be measured using any one of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR. RNase protection, Northern blotting and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over-expression of OBP-NT protein compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a OBP-NT protein, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays.
The differences in the RNA or genomic sequence between viruses of differing phenotypes can also be determined. If a mutation or sequence is observed in some or all of the viruses of a certain phenotype, but not in any viruses lacking that phenotype, then the mutation or sequence is likely to be the causative agent of the phenotype. In this way, chromosomal regions may be identified that confer viral pathogenicity, growth characteristics, survival characteristics and/or ecolo~icat niche characteristics.

Differential Expression The polynucleotides and polynucleotides of the invention may be used as reagents for differential screening methods. There are many differential screening and differential display methods known in the art in which the polynucleotides and polypeptides of the invention may be used. For example, the differential display technique is described by Chuang er al., J.
Bacteriol. 175: 2026-2036 ( 1993). This method identifies those genes which are expressed in an organism by identifying mRNA present using randomly-primed RT-PCR. By comparing pre-infection and post infection profiles, genes up and down regulated during infection can be identified and the RT-PCR product sequenced and matched to ORF
'unknowns'.
RT-PCR may also be used to analyze gene expression patterns. For RT-PCR
using the polynucleotides of the invention, messenger RNA is isolated from virally infected tissue, and the amount of each mRNA species assessed by reverse transcription of the RNA
sample primed with random hexanucleotides followed by PCR with gene specific primer pairs. The determination of the presence and amount of a particular mRNA
species by quantification of the resultant PCR product provides information on the viral genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in viral pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for antivirals. Because of the gene specific nature of the PCR
primers employed it should be understood that the viral mRNA preparation need not be free of mammalian RNA. Optimally the viral mRNA is prepared from infected tissue by mechanical disruption in the presence of TRIzoI (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzoI reagent and DNAase treatment to remove contaminating DNA. Preferably the process is optimised by finding those conditions which give a maximum amount of HSV-1 RNA as detected by probing Northerns with a suitably labelled sequence specific oligonucleotide or RNA
probe.
Each of these techniques may have advantages or disadvantages depending on the particular application. The skilled artisan would choose the approach that is the most relevant with the particular end use in mind.

Antibodies The polypeptides of the invention or variants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such poiypeptides.
"Antibodies" as used herein includes monoclonal and polyclonal antibodies, chimeric, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobulin expression library.
Antibodies generated against the polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues or cells to an animal, preferably a nonhuman, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495-497 ( 1975); Kozbor et al., Immunology Todav 4:
72 ( 1983);
Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY. Alan R.
Liss, Inc. ( 1985).
IS Techniques for the production of single chain antibodies (U.S. Patent No.
4,946,778) can be adapted to produce single chain antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies.
Alternatively phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR
amplified v-genes of lymphocytes from humans screened for possessing anti-OBP-NT or from naive libraries (McCafferty, J. et al., ( 1990), Nature 348, 552-554; Marks, J. et al., ( 1992) Biotechnology l0, 779-783). The affinity of these antibodies can also be improved by chain shuffling (Clackson. T. et al., ( 1991 ) Nature 352, 624-628).
If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
Thus, among others, antibodies against OBP-NT-polypeptide may be employed to treat infections, particularly viral infections.
Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention. The term "antigenically equivalent derivative" as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or-poiypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host. The term "immunoiogically equivalent derivative"
as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
The polypeptide, such as 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. The fusion protein may provide stability to the polypeptide. The antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
Alternatively a multiple antigenic peptide comprising multiple copies of the protein or poiypeptide, or an antigenically or immunologically equivalent poiypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
Preferably, the antibody or variant thereof is modified to make it less immunogenic IS in the individual. For examole_ if the inrliv;rl"~1 ;W,....",.. .~.~
~_.:~_~_. _.
preferably be "humanized"; where the complimentarity determining regions) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest et al., ( 1991 ) Biotechnology 9, 266-273.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1:363. Manthorpe et ai., Hum. Gene Ther.
1963:4, d 19), delivery of DNA complexed with specific protein carriers (Wu et ai., J Biol Chem. 1989: 264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &
Reshef, PNAS USA, 1986:83,9551 ), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science 1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152. Eisenbraun et al.. DNA Cell Biol 1993, 12:791 ) and in vivo infection using cloned retroviral vectors (Seeger et al.. PNAS USA 1984:81,5849).
Antagonists and agonists - assays and molecules Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations. chemical libraries, and natural product mixtures. These substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See, e.g., Coiigan et al.. Current Protocols in Immtrnologv l(2): Chapter ~ ( 1991 ). _ The invention provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of OBP-NT polypeptides or polynucleotides, particularly those compounds that are virostatic and/or virocidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a nucleus, cytoplasm or membrane, or a preparation of any thereof, comprising OBP-NT
polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a OBP-NT agonist or antagonist.
The ability of the candidate molecule to agonize or antagonize the OBP-NT polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of OBP-NT
polypeptide are most likely to be good antagonists. Molecules that bind well and increase the rate of product production from substrate are agonists. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in OBP-NT
polynucleotide or poiypeptide activity, and binding assays known in the art.
Another example of an assay for OBP-NT antagonists is a competitive assay that combines OBP-NT and a potential antagonist with OBP-NT-binding molecules, recombinant OBP-NT binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. OBP-NT can be labeled, such as by radioactivity or a colorimetric compound, such that the number of OBP-NT
molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonists.
Potential antagonists include small organic molecules, peptides, poIypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a poiypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing OBP-NT-induced activities, thereby preventing the action of OBP-NT by excluding OBP-NT from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to viral or cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 ( 1991 );
OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL ( 1988), for a description of these molecules).
Preferred potential antagonists include compounds related to and variants of OBP-NT.
Each of the DNA sequences provided herein may be used in the discovery and development of antiviral compounds. The encoded protein, upon expression, can be used as a target for the screening of antiviral drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The antagonists and agonists of the invention may be employed, for instance, to inhibit and treat diseases.
Vaccines Another aspect of the invention relates to a method for inducing an immunoiogical response in an individual, particularly a mammal which comprises inoculating the individual with OBP-NT, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly viral infection and most particularly HSV-1 infection. Also provided are methods whereby such immunological response slows viral replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of OBP-NT, or a fragment or a variant thereof, for expressing OBP-NT, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T
cell immune response, including, for example, cytokine-producing T cells or cytotoxic T
cells, to protect said individual from disease, whether that disease is already established within the individual or not. One way of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNA hybrid.
A further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunolooical response in such individual to a OBP-NT or protein coded therefrom, wherein the composition comprises a recombinant OBP-NT or protein coded therefrom comprising DNA which codes for and expresses an antigen of said OBP
NT or protein coded therefrom. The immunological response may be used therapeutically-or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells.
A OBP-NT polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties. This fused recombinant protein, preferably further comprises an antigenic co-protein, such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively large co-proteins which solubilize the protein and facilitate production and purification thereof.
Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system. The co-protein may be attached to either the amino or carboxy terminus of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al.
Science 273:
352 ( 1996).
The polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against viral infection, for example by blocking viral replication or viral reactivation from latency.
The invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier. Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation insotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

While the invention has been described with reference to certain OBP-NT
protein, it is to be understood that this covers fragments of the naturally occurring protein and similar proteins with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant protein.
Compositions, kits and administration The invention also relates to compositions comprising the polynucleotide or the polypeptides discussed above or their agonists or antagonists. The polypeptides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline.
buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
The formulation should suit the mode of administration. The invention farther rPlatPC tn rlincmnctir ~nra pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
Alternatively the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams. Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions. Such carriers may constitute from about 1 % to about 98% by weight of the formulation; more usually they will constitute up to about 80%
by weight of the formulation.

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in any evem will determine the actual dosage which will be most suitable for an individual and will vary with the a~~e, weight and response of the particular individual. The above dosages are exemplary of the average case.
There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response. A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
Each reference disclosed herein is incorporated by reference herein in its entirety.
Any patent application to which this application claims priority is also incorporated by reference herein in its entirety.
GLOSSARY
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
"Disease(s)" means and disease caused by or related to infection by a virus, including herpes Iabialis, herpes encephalitis, keratitis.
"Host cell" is a cell which has been transformed, transfected or infected, or is capable of transformation, transfection or infection by an exogenous polynucleotide sequence or virus.
"Identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputin g: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, yon -Heinje, G., Academic Press, 1987: and Sequence Analysis Primer, Gribskov, M.
and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 ( 1988). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG
program package (Devereux, J., et al., Nucleic Acids Research l2(I ): 387 ( 1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403-410 (1990).
The BLAST X program is publicly available from NCBI and other sources (BLAST
Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J.
Mol. Biol. 215: 403-410 ( 1990). The well known Smith Waterman algorithm may also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
1 ) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 ( 1970) Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad.
Sci.
USA. 89:10915-10919 ( 1992) Gap Penalty: 12 Gap Length Penalty: 4 A program useful with these parameters is publicly available as the "gap"
program from Genetics Computer Group, Madison WI. The aforementioned parameters are the default parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following:
I) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970) Comparison matrix: matches = + I 0, mismatch = 0 Gap Penalty: 50 Gap Length Penalty: 3 Available as: The "gap" program from Genetics Computer Group, Madison WI.
These are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be, are provided in ( 1 ) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the reference sequence of SEQ ID NO:1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID NO: I or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO:1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID NO:1, or:
nn ~ xn ' ~xn ' Y) wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90°~0, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn. Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID N0:2, that is it may be 100%
identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
Such alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the S' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of amino acids in SEQ ID N0:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID N0:2, or:

nn ~ xn ' ~xn ~ Y)~
wherein nn is the number of amino acid alterations, xn is the total number of amino acids in SEQ ID NO:?, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., ~ is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn.
(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID N0:2, wherein said palypeptide sequence may be identical to the reference sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence. wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID N0:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID N0:2, or:
na ~ xa ' ~xa ~ Y) wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID N0:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and ~ is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID N0:2, that is it may be 100%
identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity.
Such alterations are selected from the group consisting of at least one amino acid deletion.
substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given 9o identity is determined by multiplying the total number of amino acids in SEQ ID
N0:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID N0:2, or:
na ~ xa ' ~xa ~ Y) wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID N0:2, y is, for instance 0.70 for 70010, 0.80 for 809c, 0.85 for 85°lc etc., and ~ is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from xa.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated". as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation. genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
"Polynucleotide(s)" include, without limitation, 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, and RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term "polynucleotide(s)" also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide(s)" as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells. "Polynucleotide(s)"
also embraces 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 and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids. "Polypeptide(s)" include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, fotmylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation _ and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. See, for instance, PROTEINS -STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York ( 1993) and Wold, F., Posttranslational Protein Modifications:
Perspectives and Prospects, pgs. I-12 in POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York ( 1983);
Seifter et al., Meth. Enrymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:
Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 ( 1992).
Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
"Variant(s)" as the term is used herein, is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a poiynucieotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a 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 discussed 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. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A 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 a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
EXAMPLES
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.

The identification of gene expression following infection with HSV-1 Tissue culture cells are infected with HSV-1 strain KOS and harvested post-infection for RNA isolation. HSV-1 infected tissue culture cells are efficiently disrupted and processed in the presence of chaotropic agents and RNase inhibitor to provide a mixture of cellular and viral RNAs RNA may then be polyA+ selected for use in Northern blotting, with hybridization of a RNA probe specific to a region within the HSV-1 genome.
RNase free, DNase free, DNA and protein free preparations of total or polyA+
selected RNA are suitable for 5' end mapping of the gene of interest, using for example techniques such as 5' RACE (rapid amplification of cDNA ends; Gibco-BRL). Unique primer pairs for i0 Reverse Transcription PCR (RT-PCR), used in the 5' RACE procedure, are designed from the published sequence of HSV-1 strain i7, which is homologous to that of HSV-1 strain KOS.
Example 1 RNA isolation, Northern blotting and 5' end identification The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1 ] was identified from a Northern blot of polyA+ RNA harvested from host cells infected with HSV-1 strain KOS, using a complementary radiolabelled fragment of RNA as probe prepared by routine methods, for example:
a) Viral infection and RNA isolation Tissue culture cells such as Vero (monkey kidney cells) are seeded into 850 cm2 tissue culture roller bottles in tissue culture medium [Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Life Technologies) supplemented with 10% heat inactivated fetal bovine serum (FBS; HyClone Laboratories, Inc.), 1 % penicillin-streptomycin (Gibco BRL, Life Technologies), 5 ml per liter of 200 mM L-glutamine (Gibco BRL, Life Technologies), 10 ml per liter of 7.5% (w/v) sodium bicarbonate solution (Gibco BRL, Life Technologies)] and incubated at 37°C. Cells are infected by addition of HSV-1 (strain KOS) in a low volume of tissue culture medium at a multiplicity of infection of 10 plaque forming units per cell. After a I hour infection at 37°C, additional DMEM is added and infected cells are harvested at 17 hours post-infection.
For total RNA isolation TRI Reagent0 (Molecular Research Center, Inc) is used according to the manufacturer's instructions. In a biological safety cabinet infected tissue culture cells are harvested by removal of tissue culture medium and the addition of 6.7 ml of TRI Reagent~ per roller bottle ( I ml per 75 mg of cells). The mixture is then vortexed until homogenous. Typically, two 850 cm2 roller bottles are used per isolation, each containing approximately 500 mg of cells per roller bottle. The homogenate is centrifuged at 12,000 x g for 10 min at 4°C to remove the insoluble material and the supernatant is transferred to a fresh tube and allowed to stand for S min at room temperature. To this mixture 1.34 ml of BCP Phase Separation Reagent is added (Molecular Research Center, Inc; 0.2 ml per ml of TRI ReagentOO ), vortexed vigorously for IS
seconds and allow to stand for 5 min at room temperature. This mixture is then centrifuged at 12000 x g for 20- 30 min at 4°C. Approximately 75% of the colorless upper aqueous phase (containing RNA) is transferred to fresh tubes and 3.35m1 of isopropanol is added (O.Srril of isopropanol per ml of TRI Reagent). The samples are vortexed and allowed to stand for 5 min at room temperature. Samples are frozen in a dry ice / ethanol bath until solid and centrifuged at 12,000 x g for 30 min at 4°C. The supernatant is removed and the RNA
pellet is air dried for 10 minutes at room temperature and resuspended in 503 ul of TNM buffer (75 ul of TNM buffer per 100mg of starting material; TNM:40 mM Tris-HCl pH 8.0, 10 mM NaCI, 6 mM
MgCl2). DNA is removed by the addition of 75 units of RNase-free DNase 1 (Gibco BRL, Life Technologies) per 100 ul of RNA and incubated at 37°C for 20 min. To this is added 10% of sample volume of 4M
LiCI. The samples are transferred to eppendorf tubes and extracted with an equal volume of phenol:chloroform ( 1:1 v:v). The samples are centrifuged for 3 min at 13,000 rpm in an eppendorf centrifuge, the aqueous phase is transferred to a clean tube and extracted with an equal volume chloroform. Again the aqueous phase is transferred to a new tube and double the sample volume of ethanol is added. The samples are frozen until solid in dry ice / ethanol bath and centrifuged at 13,000 rpm in an eppendorf centrifuge for 30 min at 4°C. The ethanol is removed and the RNA is washed with 75% ethanol (minimum 1 ml), vortexed and the RNA pelleted by centrifugation in an eppendorf centrifuge at 13,000 rpm for 5 min at 4°C. The RNA pellet is air dried for 10 min and resuspended in 200 ul TNM buffer. The concentration of RNA is determined by measuring the absorbance at 260 nm. Fifty micrograms of RNA is digested a second time using 10 units of DNase I (RNase and protease free; Worthington Biochemical Corp.) and incubated at 37°C for 30 min, followed by addition of 4M LiCI, extraction, precipitation and quantitation as described above. The quality of the RNA is assessed by electrophoresis of 1 ug in a 1 % agarose gel and visualized following staining with ethidium bromide [Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra)].
For polyA+ selected RNA the Poly(A)Pure mRNA isolation kit (Ambion) is used according to the manufacturer's instructions. In a biological safety cabinet infected tissue culture cells are harvested by removal of tissue culture medium and addition of 10 ml of Lysis solution per 2 x 108 cells. Typically, three 850 cm2 roller bottles are used per isolation, each containing approximately 4.6 x 107 cells. The lysates are combined, transferred to a 50 ml tube and vortexed until homogenous. Two starting volumes (20 ml) of Dilution Buffer are added to the lysate and mixed by inversion. The lysate is centrifuged at 12,000 x g at 4°C for 15 minutes to remove particulate cellular debris and the supernatant is transferred to a clean 50 ml conical tube. One pre-packaged vial of oligo dT cellulose is added to the lysate and mixed by inversion. The tube is rocked gently for 60 minutes at room temperature and the oligo dT resin is pelleted by centrifugation at 2,000 x g for 3 minutes at room temperature. The supernatant is removed and the resin washed by addition of 10 ml of Binding Buffer, mixed well by inversion, and pelleting the resin as before. This wash is repeated twice more using 10 ml of Binding Buffer. The resin is then washed by resuspension in 10 ml of Wash Buffer, mixed well by inversion, and pelleting the resin as before. This wash is repeated twice more 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. The spin column is centrifuged at 5,000 x g at room temperature for 10 seconds, and the absorbance of the flowthrough is measured at 260 nm to ensure that it is less than 0.05. The spin column is placed into a new microfuge tube and the polyA+ RNA is eluted by addition of 200 pl of prewarmed (65°C) Elution Buffer and centrifugation at 5,000 x g at room temperature for 30 seconds. The elution is repeated with an additional 200 ~tl of Elution Buffer and the polyA+ RNA is precipitated by addition of 40 pl of SM sodium acetate. 2 Itl 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 x g for 20 minutes at 4°C. The supernatant is removed and the RNA pellet is air dried for 10 minutes at room temperature and resuspended in 40 X11 of DEPC-treated water/0.1 mM
EDTA. The concentration of RNA is determined by measuring the absorbance at 260 nm; the RNA
preparations are stored at -80°C until use [Sambrook et al., MOLECULAR
CLONING, A
LABORATORYMANUAL, (supra)].
b) Northern blotting of HSV-1 polyA+ selected RNA to identify novel transcripts PolyA+ selected RNA (S ug) isolated from HSV-1 (KOS) infected Vero cells at 17 hours post-infection, or from uninfected (mock) Vero cells, are run on a 1 %
agarose gel prepared using lOX Denaturing Gel Buffer (Ambion). Each RNA sample is suspended in 10 ~tl of DEPC-treated water and three volumes (30 ~tl) of Sample Loading Dye (Ambion).
Samples are heated to 65°C prior to loading onto the gel to denature RNA secondary structure. Gels are run in IX Gel Running Buffer (Ambion) at 65 volts for 3 to 4 hours.
After electrophoresis the RNA is transferred to a positively charged nylon membrane (BrightStar-Plus; Ambion) by vacuum transfer at 5 inches Hg for 90 minutes in SX SSC, 10 mM NaOH. After the transfer, the membrane is rinsed briefly in 1X Gel Running Buffer and is UV crosslinked (Stratagene). Crosslinked RNA membranes can be stored at -20°C
in a vessel that will protect them from physical damage.
Riboprobes are generated from plasmid DNA specific for the UL8/UL9 region of the HSV genome (riboprobe 8R-3': K. Baradaran, C.E. Dabrowski, and P.A.
Schaffer, 1994.
J. Virol. 68:4251-4261) using the Riboprobe In Vitro Transcription System (Promega).
p8R-3' plasmid DNA is linearized with XbaI and 2 u~ is transcribed and radiolabelled according to the manufacturer's instructions. Riboprobes are purified by application to ProbeQuant G-50 Micro Columns (Pharmacia) followed by centrifugation at 3000 rpm for two minutes in the Eppendorf microcentrifuge, and repeated to remove free nucleotides.
The membrane to which the RNA is bound is prehybridized for 1 hour in 10 ml Prehybridization/Hybridization Solution (Ambion) per 100 cm2 of membrane at 75°C. The prehybridization solution is removed and replaced with 10 ml Hybridization solution consisting of Prehybridization/Hybridization Solution preheated to the hybridization temperature (75°C ) plus 1 x 106 cpm per ml of the 8R-3' riboprobe. The membrane is hybridized overnight at 75°C and then washed twice with Low Stringency Wash Solution (Ambion) for 5 minutes each at room temperature, three times with High Stringency Wash Solution (Ambion) for 20 minutes each at 75°C and autoradiographed.
c) The rapid amplification of 5' cDNA ends from RNA samples derived from infected cells Total RNA ( 1 ug) is reverse transcribed using the 5' RACE System for Rapid Amplification of cDNA Ends (Gibco BRL, Life Technologies) according to the manufacturer's instructions for first strand cDNA synthesis of transcripts with high GC
content. Gene specific primer f ( 100 nM GSP1; 5'-GCA GAT GTT ATC GCC-3') is used to prime each reaction. Controls without the addition of Superscript II
reverse transcriptase are included. All samples are then treated with RNase mix and the cDNA is purified using the GlassMax DNA Isolation System included with the 5' RACE
kit.
Following purification, cDNA samples are tailed with dCTP using terminal deoxynucleotidyl transferase (TdT) and used in a PCR reaction using an Abridged Anchor Primer (supplied with the ~' RACE kit) and a second gene specific primer (GSP2; 5'-GAT
GCA GAC GTT ATC TC-3'). PCR reactions are assembled on ice in 0.2 ml thin-wall tubes, according to the instructions for PCR of dC-tailed cDNA. Reaction tubes are transferred directly from ice to the thermal cycler preequilibrated to the initial denaturation temperature (94°C). PCR reactions are run on a Perkin Elmer GeneAmp PCR
System 9600 ._ as follows: 2 minutes at 94 "C, then 35 cycles of 30 seconds at 94 "C, 30 seconds at 55 "C
and 1 minute, 30 seconds at 72 "C followed by ~ minutes at 72 "C and then a hold temperature of 5 "C. Aliquots ( 10 ul) are then electrophoresed on bolo acrylamide gels in 1 X
TBE (Novex) and stained with ethidium bromide. The size of the PCR product is estimated by comparison to a 100 by DNA Ladder (Gibco BRL, Life Technologies). A nested PCR
amplification is then performed following the instructions for Nested Amplification using the Universal Amplification Primer (UAP) and a third gene specific primer (GSP3; 5' CAU
CAU CAU CAU CCG ACC ACC ACA TG-3~. PCR is performed as described above.
The primers used in the nested amplification contain sequences that are specific for cloning the PCR product using the CloneAmp pAMPl System (Gibco BRL, Life Technologies).
The PCR products are purified from a 6~1o acrylamide gel and cloned into the pAMPI
vector following the manufacturer's instnactions.
After the PCR product is annealed to the pAMPI vector the reaction mixture is transformed into DHSa cells (Gibco BRL, Life Technologies) following standard protocols I S [Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (sc~pra)).
Bacteria are plated on LB plates containing 100 ug/ml ampicillin (Digene Diagnostics Inc.), grown overnight at 37°C and colonies are picked for DNA minipreps. Plasmid DNA is isolated from 5 ml bacterial cultures using the RPM Kit (Bio 101 ) following the manufacturer's instructions. Plasmid DNA is analyzed by restriction digest with BamHI and EcoRI and electrophoresed on 6010 acrylamide gels in IX TBE (Novex) to determine the size of the insert compared to a 100 by DNA ladder as above. Clones are then used in sequencing reactions (T7 Sequenase 7-deaza-dGTP Sequencing Kit; Amersham Life Science) with the SP6 promoter primer (Gibco BRL, Life Technologies) to determine the 5' end of the cDNA.
The 5' end is determined following sequencing of DNA from two or more clones containing amplified PCR products and used to identify the contiguous DNA sequence in SEQ
ID
NO:1.
Example 2 OBP-NT Characterization A novel messenger RNA (mRNA) encoding the newly described protein, OBP-NT, was identified by Northern blotting utilizing an RNA probe complementary to the UL8/9 region of the HSV-1 genome (see example 1). While previous efforts to map RNAs encoded within this region have been described (Deb, S. P. et al. 1993.
Biochem and Biophys Res. Comm. 193:617-623; Baradaran, K. et al. 1994. J. Virol. 68:4251-4261), the current study utilized mRNA isolated at a very late time-point (17 hours post-infectionj.
Identification of the 5' end of the mRNA, as described above, together with knowledge of--the 3' end from previous mappine studies (Baradaran, K. et al. 1994. J. Virol.
68:4251-4261 ), allowed for the prediction of the OBP-NT open reading frame according to well-established guidelines in eukaryotes (Kozak, M. 1986. Cell 44:283-292).
a) Predicted OBP-NT features From this analysis OBP-NT is predicted to encode a protein in-frame within the well-characterized viral origin binding protein, OBP, with the inititiating methionine of OBP-NT
corresponding to amino acid 142 of OBP. Thus, OBP-NT shares with OBP a dimerization domain (leucine zipper at amino acids 150-171; Hazuda,.D.J. et al. 1992. J.
Biol. Chem.
267:14309-14315), helicase motifs II through VI (Malik, A. K. and S. K.
Welter. 1996. J.
ViroI. 70:7859-7866), and the origin specific DNA binding domain at the C-tetnimus (Deb, S. and S. W. Deb. 1991. J. Virol. 65:2829-2838). Based on these shared sequences between the proteins, OBP-NT is expected to be able to form homodimers with itself and heterodimers with OBP, through the conserved leucine zipper region at amino acids 150-171 (OBP numbering). These proteins may also be able to dimerize with as yet undefined viral or cellular proteins. OBP-NT is not expected to function as a helicase as a monomer or homodimer although it retains conserved helicase motifs II through VI, as the missing motifs I and Ia are critical for nucleotide binding and thus helicase activity. However, OBP-NT may function as a helicase as a heterodimer with, for example, OBP, and as such may have similar or different properties as compared to the OBP homodimer. OBP-NT is also predicted to bind sequences within the viral origins of replication;
experiments demonstrating this ability are described below.
b) OBP-NT binding to viral origin of replication sequences Evidence for the ability of OBP-NT to bind to sequences within the viral origins of replication (oriS and oriL) can be found in a number of published sources, in results from experiments performed to characterize origin binding by the other members of the OBP family, OBP and OBPC. For example, electrophoretic mobility shift assays (EMSA) experiments have been published in which extracts from HSV-1 infected cells were incubated with radiolabeled oriS
DNAs (sites I, II or III; see figure I ). In these experiments two to three viral specific complexes can be identified - complex A, comprised of OBPC and site I or site II DNAs (Baradaran, K. et al.
1994. J. Virol. 68:4251-4261), and complex B, comprised of OBP and site I, II
or III DNAs, which appears as a single band or as a doublet (see Dabrowski, C. E, and P. A.
Schaffer. 1991. J. Virol.
65:3140-3150; Weir, H. M. et al. 1989. Nucl. Acids Res. 17:1409-1425;
Hardwicke, M. A. and P.
A. Schaffer. 1995. J. Virol. 69:1377-1388). In the presence of antibody directed against the C-terminal amino acids of OBP (which are also in common with OBP-NT and OBPC), complex A.is shifted to a higher molecular weight complex (supershifted). The protein:DNA
complexes identified as complex B formed with oriS DNAs (sites I, II, III) also shifted or significantly decreased in intensity in the presence of antibody directed against the C-terminus of OBP (Figure 8, Dabrowski, C. E. and P. A. Schaffer. 1991. J. Virol. 65:3140-3150). However, as shown in Figure 10 in this paper, complex B formed with extracts from HSV infected cells appears as a single band in these experiments, and demonstrates a different electrophoretic mobility than the complex (designated 9-I) formed with partially purified recombinent OBP. The resulting difference in electrophoretic mobilities between the 'complex B' and 'complex 9-1' proteins, and the ability of these proteins to be supershifted in EMSA experiments by antibody directed against the C-terminus of OBP, is indicative of the presence of OBP-related proteins of slightly different molecular weights. This result is consistent with the presence of full-length OBP within complex 9-1, formed with partially purified OBP, and a slightly smaller molecular weight protein translated in-frame with OBP, i.e. OBP-NT, within complex B from infected cell extracts. Thus, by EMSA, complex B
is observed to be one or two bands in complexes formed from HSV infected cell extracts and oriS
site I or II DNAs, while in contrast, complex B appears to be a single band with site III as probe; no complex B doublet has been observed (Dabrowski, C. E. and P. A. Schaffer.
1991. J. Virol.
65:3140-3150; Weir, H. M. et al. 1989. Nucl. Acids Res. 17:1409-1425;
Hardwicke, M. A. and P.
A. Schaffer. 1995. J. Virol. 69:1377-1388); the lower band of the doublet is now believed to be comprised of OBP-NT and the upper band comprised of OBP. The presence of one or both bands appears to be dependent on the extract preparation, time of harvest post-infection, and length of exposure to film which may blur the presence of 2 discrete bands. Further evidence for the ability of OBP-NT to bind sequences within the viral origins of replication are shown in figures 3 and 4, following EMSA analysis of extracts from HSV-1 infected cells incubated with radiolabeled oriS
DNAs. For these experiments Vero cells are seeded into 850 cm2 tissue culture roller bottles in DMEM containing 10% FBS and incubated overnight at 37oC. Cells are either mock infected or infected with HSV-1 (KOS) at an MOI of 10 and harvested at 3, 6, 9, 12, 15, or 18 hours post infection (hpi). Total cell extracts are prepared by pelleting the cells at 2000 rpm at 4oC for 5 minutes in a tabletop centrifuge, washing the pellet twice with phosphate buffered saline (PBS;
Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL) and resuspending in 50 ul NET buffer (50 mM Tris, pH 8, 100 mM NaCI, 1 mM EDTA). Pellets are frozen at -80oC, thawed at 37oC and sonicated twice for 30 seconds each on a setting of 4 using a Virsonic 550 Ultrasonic Cell Disrupter {VirTis). The cell debris is pelleted at 14000 rpm in an Eppendorf microcentrifuge for 10 minutes at 4oC, and the supernatant is aliquoted and stored at -80oC.
Protein quantitation is performed on the total cell extracts using the Bio-Rad Protein Assay (Bio-Rad) following the._ manufacturer's instructions for the Microassay Procedure. Radiolabeled HSV-1 oriS sites I, II, III
and III(I) probes are prepared as previously described (Dabrowski, C.E. and P.A. Schaffer. 1991. J.
Virol. 65:3140-3150). The site III(I) probe contains sequences identical to site III with a central divergent nucleotide (T) changed to a G, as found in site I. For the EMSAs, mock infected (10 ug protein) or HSV infected (2 ug protein) cell extracts are incubated with 2 ng of probe and 1.5 ug of poly(dI-dC) ~ poly(dI-dC) (Pharmacia) in DNA binding buffer ( 10% glycerol, 50 mM HEPES, pH
7.5 [N-2-hydroxyelthylpiperazine-N'-2-ethanesulfonic acid; Life Technologies], 0.1 mM EDTA, 0.5 mM DTT, 75 mM NaCI) in a final volume of 10 ul for 30 minutes at room temperature. Equal amounts of radioactivity are loaded onto each gel (35,000 epm) and the protein:DNA complexes are separated by electrophoresis in 6% polyacrylamide gels prepared and run in O.SX TBE (Life Technologies) at 200V. Shown in figure 3 are the results of an EMSA performed with total cell extract harvested 12 hpi incubated with site I, II, III and III(I) probes.
Complex A containing OBPC (Baradaran, K. et al. 1994. J. Virol. 68:4251-4261) is present with site I, II, and III(I) probes but not with the site III probe, as previously described (Dabrowski, C.E. and P.A. Schaffer. 1991. J.
Virol. 65:3140-3150). Complex B, a doublet of OBP (upper band of doublet) and OBP-NT (lower band of doublet), is present with site I, II, and III (I) probes, but only the lower OBP-NT band of the complex B doublet is present with the site III probe. This experiment demonstrates that, despite the common C-terminal DNA binding region present in OBP, OBP-NT and OBPC, only OBP-NT is able to bind to site III within the core viral origin of replication. Further, the inability of OBP and OBPC to bind site III is dependent on the single nucleotide difference between sites I and III within the core OBP binding domain (T in site III, G in site I), and now explains the difference in results described above (see background) whereby purified OBP was shown not to bind site III, although a band of the approximate correct size was identified from HSV infected cell extracts.
EMSAs were also performed with radiolabeled oriS probes using mock or HSV
infected cell extracts harvested at increasing times post infection. As shown in figure 4A and B, with site I
and II DNAs the complex B doublet appears by 6 hpi and increases in intensity through 18 hpi.
Additional protein:DNA complexes (C and D) with decreased electrophoretic mobility are also seen at increasing times post infection, with the concomitant disappearance of the mock infected band.
While the complex B doublet bands are of approximately equal intensity with the site I probe, the lower OBP-NT complex B band and the complex D band predominate at all times post-infection with the site II probe. In EMSAs performed with the site III probe (figure 4C) complex B appears as a single discrete band, appearing at 9 hpi and increasing in intensity up to 18 hpi. With this probe, complex D but not complex C is also formed; from these experiments complex C appears to be OBP-related, whereas complex D appears to be OBP-NT related. In addition, a lower light intensity band is apparent, which only increases significantly in intensity at 18 hpi; this band appears to be of slightly faster mobility than complex A formed with OBPC.
Aligning the site I, II
and III EMSAs, using the mock infected bands for reference, it becomes evident that the single band of complex B formed with the site III probe has an electrophoretic mobility identical to the lower band of the complex B doublet seen with the site I probe. From these experiments it we conclude that OBP and OBPC binds to sites i and II in the viral origins of replication, as previously reported. OBP-NT is also able to bind to sites I and II, and further, is the only viral protein of the known OBP family able to bind to site III.
c) OBP-NT Expression in HSV-1 Infected Cells OBP-NT protein is identified in HSV infected cells by Western blot, using a polyclonal anti-peptide antibody raised against a 10 amino acid peptide from the N-terminus of OBP-NT
(MNDRPFHRL), and protein extracts from uninfected (mock) Vero cells, Vero cells harvested at 6 hours post infection (hpi), or Vero cells harvested at 18 hpi with HSV-1. Vero cells are seeded at 2.5 x 106 cells per 100 mm dish in DMEM containing 10% FBS and incubated overnight at 37oC.
Cells are either mock-infected or infected with HSV-I (KOS) at an MOI of 10.
At 6 or 18 hpi cells are scraped into the medium and pelleted at 1500 rpm for 10 minutes in a tabletop centrifuge. Each pellet is resuspended in 400 ul phosphate buffered saline (PBS; Sambrook et al., MOLECULAR
CLONING, A LABORATORY MANUAL); 200 ul of lysis buffer (30 mM Tris, pH 8.0, 15 mM EDTA, 3% SDS) is added and the lysates are incubated on ice for 10 minutes. The lysates are sonicated twice for 30 seconds, and 5 volumes of acetone are added. The precipitates are stored at -20oC for minutes and pelleted for 15 minutes at 4oC in an Eppendorf microcentrifuge at 14000 rpm. The pellets are allowed to air dry, resuspended in 400 ul of SDS-PAGE loading buffer (NOVEX) containing 0.05% ~-mercapotoethanol, and 40 ul each of the mock, 6 hpi and 18 hpi extracts is 25 loaded onto an 8% SDS-PAGE gel, along with 10 ul ( 13 ug) of a crude extract containing recombinant OBP (rOBP; gift of S. Lee and R. Lehman, Stanford University).
Proteins are separated by electrophoresis overnight at 9 mA/gel and transferred to a PVDF
membrane (PolyScreen; NEN Research Products) for 1 hr at 24 V using a GENIE
electrophoretic transfer apparatus (Idea Scientific Company). For Western blot analysis, the membrane is incubated in 30 blocking solution [5% skim milk in TBS ( 10 mM Tris, pH 8.0, 150 mM NaCI)]
for I hr at room temperature. After a quick wash in TBS, the blot is incubated with the primary antibody diluted 1:250 in TBS containing 0.05% Tween 20 (TBST) for 1 hr at room temperature.
The blot is then washed three times for 5 min each with TBST, incubated with the secondary antibody (goat anti-rabbit alkaline phosphatase conjugated antibody, IgG; Promega) diluted I :4000 in TBST for 30 min at room temperature, and washed three times for 5 min each with TBS. The substrate is prepared by adding 66 ul of NBT (Promega) to 10 ml of alkaline phosphatase substrate buffer (100 mM Tris, pH 8.0, 100 mM NaCI, 5 mM MgCl2), followed by the addition of 33 ul of BCIP
(Promega). The substrate is added to the blot, and color development is followed. The blot is washed twice with deionized water to stop the reaction. The results of the Western blot (Figure 5) show a faint protein band of the approximate expected size (78 kDa by calculation) in the 6 hpi infected cell extract lane (6h), and a heavy protein band of the same size is evident in the 18 hpi infected cell extract lane (18h); this protein is not present in mock infected cell extracts (M). This anti-peptide antibody also identified OBP from the recombinant baculovirus extract, but did not identify OBP from infected cell extracts, suggesting the possibility of a difference in protein configuration or modification between these sources Example 3 Herpes Viral in vitro replication Seven HSV-I proteins have been identified as nesessary and sufficient for replication of plasmid DNA containing a viral origin of replication in the context of a mammalian or insect cell, by transient replication assays (Stow, N. D. 1992.
J.
Gen. Virol. 73:313-321; Boehmer, P. E. and Lehman, I. R. 1997. Annu. Rev.
Biochem. 66:347-384). However, neither these seven purified proteins, nor extracts of these proteins from recombinant baculovirus infected cells, are sufficient for origin-directed replication in vitro, suggesting that additional proteins are required (Skaliter, R. and Lehman, I. R. 1994. Proc. Natl. Acad. Sci. USA 91:10665-10669;
Boehmer, P. E. and Lehman, I. R. 1997. Annu. Rev. Biochem. 66:347-384). The OBP-NT protein has now been identified as a viral origin binding protein capable of binding to the sequences defined as sites I, II and III in the viral origins of replication; these binding sites are contained within the core origin regions required for efficient replication. Further, OBP-NT is the only viral origin binding protein which binds to an oligonucleotide containing site III. Thus, OBP-NT is likely to suppiy the additional viral functionality required, in the presence of the seven previously identified replication proteins, for origin-directed replication in vitro. In addition to OBP-NT, it is possible that cellular proteins such as found to be required for non-origin directed replication, may be necessary for origin-directed replication (Skaliter, R. and Lehman, I. R. 1994. Proc. Nat'I. Acad. Sci. USA 91:10665-10669).

SEQUENCE LISTING
<110> Dabrowski-Amaral, Christine E.
Gorczyca, Michele M.
Garcia, Javier J.
<120> Herpes Simplex Virus <130> P50775 <140> Unknown <141>
<150> 60/088,770 <151> 1999-06-10 <160> 6 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 2133 <212> DNA
<213> Unknown <400> 1 atgaacgaccgccccttccaccgacttatcgtccaggtggaaagccttcatcgcgtgggc60 cccaaccttctgaacaactacgacgtcctcgttctggacgaggttatgtcgacgctgggc120 cagctctattcgccaacgatgcagcaactgggccgcgtggatgcgttaatgctacgcctg180 ctgcgcatctgtcctcggatcatcgccatggacgcaaccgccaacgcgcagttggtggac240 ttcctgtgcggtctccggggcgaaaaaaacgtgcatgtggtggtcggcgagtacgccatg300 cccgggttttcggcgcgccggtgcctgtttctcccgcgtctggggaccgagctcctgcag360 gctgccctgcgcccgcccgggccgccgagcggcccgtctccggacgcctctccggaggcc420 cggggggccacgttctttggggagctggaagcgcgccttggcgggggcgataacatctgc480 attttttcgtcgacggtctccttcgcggagatcgtggcccggttctgccgtcagtttacg540 gaccgcgtgctgttgcttcactcgctcacccccctcggggacgtgaccacgtggggccaa600 taccgcgtggttatatacacgacggtcgtaaccgtgggcctcagcttcgatcccctgcac660 tttgatggcatgttcgcctacgtgaaacccatgaactacggaccggacatggtgtccgtg720 taccagtccctgggacgggtgcgcaccctccgcaagggggagctactgatttacatggac780 ggctccggggcgcgctcggagcccgtctttacgcccatgctccttaatcacgtggtcagt840 tcctgcggccagtggcccgcgcagttctcccaggtcacaaacctgctgtgtcgccggttc900 aaggggcgctgtgacgcgtcggcatgcgacacgtcgctggggcgggggtcgcgcatctac960 aacaaattccgttacaaacactactttgagagatgcacgctggcgtgtctctcggacagc1020 cttaacatccttcacatgctgctgaccctaaactgcatacgcgtgcgcttctggggacac1080 gacgataccctgaccccaaaggacttctgtctgtttttgcggggcgtacatttcgacgcc1140 ctcagggcccagcgcgatctacgggagctgcggtgccgggatcccgaggcgtcgctgccg1200 gcccaggccgccgagacggaggaggtgggtcttttcgtcgaaaaatacctccggtccgat1260 gtcgcgccggcggaaattgtcgcgctcatgcgcaacctcaacagcctgatgggacgcacg1320 cggtttatttacctggcgttgctggaggcctgtctccgcgttcccatggccacccgcagc1380 agcgccatatttcggcggatctatgaccactacgccacgggcgtcatccccacgatcaac1440 gtcaccggagagctggagctcgtggccctgccccccaccctgaacgtaacccccgtctgg1500 gagctgttgtgcctgtgcagcaccatggccgcgcgcctgcattgggactcggcggccggg1560 ggatctgggaggaccttcggccccgatgacgtgctggacctactgaccccccactacgac1620 cgctacatgcagctggtgttcgaactgggccactgtaacgtaaccgacggacttctgctc1680 tcggaggaagccgtcaagcgcgtcgccgacgccctaagcggctgtcccccgcgcgggtcc1740 gttagcgagacggaccacgcggtygcgctgttcaagataatctggggcgaactgtttggc1800 gtgcagatggccaaaagcacgcagacgtttcccggggcggggcgcgttaaaaacctcacc1860 aaacagacaatcgtggggttgttggacgcccaccacatcgaccacagcgcctgccggacc1920 cacaggcagctgtacgccctgcttatggcccacaagcgggagtttgcgggcgcgcgcttc1980 aagctacgcgtgcccgcgtgggggcgctgtttgcgcacgcactcatccagcgccaacccc2040 aacgctgacatcatcctggaggcggcgctgtcggagctccccaccgaggcctggcccatg2100 atgcagggggcggtgaactttagcaccctataa <210> 2 <211> 710 <212> PRT
<213> Unknown <400> 2 Met Asn Asp Arg Pro Phe His Arg Leu Ile Val Gln Val Glu Ser Leu His Arg Val Gly Pro Asn Leu Leu Asn Asn Tyr Asp Val Leu Val Leu Asp Glu Val Met Ser Thr Leu Gly Gln Leu Tyr Ser Pro Thr Met Gln Gln Leu Gly Arg Val Asp Ala Leu Met Leu Arg Leu Leu Arg Ile Cys Pro Arg Ile Ile Ala Met Asp Ala Thr Ala Asn Ala Gln Leu Val Asp Phe Leu Cys Gly Leu Arg Gly Glu Lys Asn Val His Val Val Val Gly Glu Tyr Ala Met Pro Gly Phe Ser Ala Arg Arg Cys Leu Phe Leu Pro Arg Leu Gly Thr Glu Leu Leu Gln Ala Ala Leu Arg Pro Pro Gly Pro Pro Ser Gly Pro Ser Pro Asp Ala Ser Pro Glu Ala Arg Gly Ala Thr Phe Phe Gly Glu Leu Glu Ala Arg Leu Gly Gly Gly Asp Asn Ile Cys Ile Phe Ser Ser Thr Val Ser Phe Ala Glu Ile Val Ala Arg Phe Cys Arg Gln Phe Thr Asp Arg Val Leu Leu Leu His Ser Leu Thr Pro Leu Gly Asp Val Thr Thr Trp Gly Gln Tyr Arg Val Val Ile Tyr Thr 'I'hr Val Val Thr Val Gly Leu Ser Phe Asp Pro Leu His Phe Asp Gly Met Phe Ala Tyr Val Lys Pro Met Asn Tyr Gly Pro Asp Met Val Ser Val Tyr Gln Ser Leu Gly Arg Val Arg Thr Leu Arg Lys Gly Glu Leu Leu Ile Tyr Met Asp Gly Ser Gly Ala Arg Ser Glu Pro Val Phe Thr Pro Met Leu Leu Asn His Val Val Ser Ser Cys Gly Gln Trp Pro Ala Gln Phe Ser Gln Val Thr Asn Leu Leu Cys Arg Arg Phe Lys Gly Arg Cys Asp Ala Ser Ala Cys Asp Thr Ser Leu Gly Arg Gly Ser Arg Ile Tyr Asn Lys Phe Arg Tyr Lys His Tyr Phe Glu Arg Cys Thr Leu Ala Cys Leu Ser Asp Ser Leu Asn Ile Leu His Met Leu Leu Thr Leu Asn Cys Ile Arg Val Arg Phe Trp Gly His Asp Asp Thr Leu Thr Pro Lys Asp Phe Cys Leu Phe Leu Arg Gly Val His Phe Asp Ala Leu Arg Ala Gln Arg Asp Leu Arg Glu Leu Arg Cys Arg Asp Pro Glu Ala Ser Leu Pro Ala Gln Ala Ala Glu Thr Glu Glu Val Gly Leu Phe Val Glu Lys Tyr Leu Arg Ser Asp Val Ala Pro Ala Glu Ile Val Ala Leu Met Arg Asn Leu Asn Ser Leu Met Gly Arg Thr Arg Phe Ile Tyr Leu Ala Leu Leu Glu Ala Cys Leu Arg Val Pro Met Ala Thr Arg Ser Ser Ala Ile Phe Arg Arg Ile Tyr Asp His Tyr Ala Thr Gly Val Ile Pro Thr Ile Asn Val Thr Gly Glu Leu Glu Leu Val Ala Leu Pro Pro Thr Leu Asn Val Thr Pro Val Trp Glu Leu Leu Cys Leu Cys Ser Thr Met Ala Ala Arg Leu His Trp Asp Ser Ala Ala Gly Gly Ser Gly Arg Thr Phe Gly Pro Asp Asp Val Leu Asp Leu Leu Thr Pro His Tyr Asp Arg Tyr Met Gin Leu Val Phe Glu Leu Gly His Cys Asn Val Thr Asp Gly Leu Leu Leu Ser Glu Glu Ala Val Lys Arg Val Ala Asp Ala Leu Ser Gly Cys Pro Pro Arg Gly Ser Val Ser Glu Thr Asp His Ala Val Ala Leu Phe Lys Ile Ile Trp Gly Glu Leu Phe Gly Val Gln Met Ala Lys Ser Thr Gln Thr Phe Pro Gly Ala Gly Arg Val Lys Asn Leu Thr Lys Gln Thr Ile Val Gly Leu Leu Asp Ala His His Ile Asp His Ser Ala Cys Arg Thr His Arg Gln Leu Tyr Ala Leu Leu Met Ala His Lys Arg Glu Phe Ala Gly Ala Arg Phe Lys Leu Arg Val Pro Ala Trp Gly Arg Cys Leu Arg Thr His Ser Ser Ser Ala Asn Pro Asn Ala Asp Ile Ile Leu Glu Ala Ala Leu Ser Glu Leu Pro Thr Glu Ala Trp Pro Met Met Gln Gly Ala Val Asn Phe Ser Thr Leu <210> 3 <211> 2127 <212> DNA
<213> Unknown <400> 3 atgaacgaccgccccttccaccgtctcatcgtgcaggtggaaagtcttcatcgcgtgggc60 ccgaacctgttgaacaactacgacgtgctcgtcacgaagtcactcatgtcgacgttgggc120 caactgtactcgccgacgatgcagcagctgggccgcgtcgacgcgttgatgctgcgcctg180 ctgcgcacgtgcccgcggatcatcgccatggacgccaccgccaacgcgcagctggtggac240 tttctgtgcagcctccggggcgaaaagaacgttcacgtggtcatcggggagtacgccatg300 cccggattttcggcgcgccgttgtctgtttctcccgcgcctggggcccgaggtcctgcag360 gcggccctgcgccgccgggggccggcgggcggggcgccccccccggacgcccccccggac420 gccaccttcttcggggagctggaggcgcgcctggccggcggagataacgtctgcatcttc480 tcgtcgacggtctccttcgcggaggtcgttgccaggttctgccggcagtttacggaccgc540 gtgctgctgctccactcgctcaccccgcccggcgacgtgaccacatggggccggtaccgg600 gtggtcatctacacaacggtcgtgacggtgggccttagcttcgatccgccgcactttgac660 agcatgttcgcctacgtgaaacccatgaactacgggccggacatggtgtccgtgtaccag720 tcgctggggcgggtacggactctccgcaagggggagctgctgatctacatggacgggtcc780 ggggcgcgctcggagcccgtctttacgcccatgctgctcaaccacgtggtgagcgccagc840 gggcagtggccggcacagttttcccaggtgacgaacctgttgtgccgccggttcaaaggg900 cgctgcgacgcgtcgcacgccgacgcggcgcaggcgcgggggtcgcgcatctacagcaaa960 ttccggtacaagcactacttcgagaggtgcacgctggcgtgcctcgcggacagtcttaac1020 atcctccacatgcttctgaccctcaactgcatgcacgtgcggttctggggccacgacgcc1080 gcgctgaccccgaggaacttttgtctgtttttgcgggggatacattttgatgccctgagg1140 gcccagcgggatctgcgggagctgcgctgccaggaccccgacacgtccctgtcggcccag1200 gccgccgagacggaggaggtgggccttttcgtcgaaaagtacctccggccggacgtcgcg1260 ccggccgaggtggtcgcgctcatgcgcggcctcaacagcctggtcggccgcacgcggttc1320 atctacctggtgctgctggaggcctgtcttcgcgtccccatggccgcccatagcagcgcc1380 atcttccggcggctttacgaccactacgccacgggcgtcatccccacgatcaacgccgcc1440 ggagagctggagcttgtggccctacaccccaccctaaacgtcgcccccgtctgggagctg1500 ttccgtctgtgcagcaccatggccgcgtgcctgcagtgggactcgatggccggggggtcg1560 gggcgaacctttagccccgaggacgtgctggagctgctgaacccccactacgaccgctac1620 atgcagctggtgttcgaactgggccactgtaacgtgaccgacggccccttgctgtcggag1680 gacgcggttaagcgcgtggccgacgccctgagcggctgccccccgcgcgggtccgtgagc1740 gagacggagcacgcgctgtcgctgttcaagatcatctggggcgaactgttcggggtgcag1800 ctggccaagagcacgcagacgtttcccggggcggggcgcgttaaaaacctcaccaagcga1860 gccatcgtggagctgctggacgcccaccgcatcgaccacagcgcctgccggacgcacaga1920 cagctgtacgcgctgctgatggcccataagcgggagtttgcgggcgcgcgcttcaagctg1980 cgcgcgcccg cgtgggggcg ctgcttgcgc acgcacgcct ccggcgccca gcccaacact 2040 gacatcattc tcgaggcggc tctgtcggag cttcccaccg aggcctggcc catgatgcag 2100 ggggcggtga actttagcac cctataa 2127 <210> 4 <211> 708 <212> PRT
<213> Unknown <400> 4 Met Asn Asp Arg Pro Phe His Arg Leu Ile Val Gln Val Glu Ser Leu His Arg Val Gly Pro Asn Leu Leu Asn Asn Tyr Asp Val Leu Val Thr Lys Ser Leu Met Ser Thr Leu Gly Gln Leu Tyr Ser Pro Thr Met Gln Gln Leu Gly Arg Val Asp Ala Leu Met Leu Arg Leu Leu Arg Thr Cys Pro Arg Ile Ile Ala Met Asp Ala Thr Ala Asn Ala Gln Leu Val Asp 65 70 75 gp Phe Leu Cys Ser Leu Arg Gly Glu Lys Asn Val His Val Val Ile Gly Glu Tyr Ala Met Pro Gly Phe Ser Ala Arg Arg Cys Leu Phe Leu Pro Arg Leu Gly Pro Glu Val Leu Gln Ala Ala Leu Arg Arg Arg Gly Pro Ala Gly Gly Ala Pro Pro Pro Asp Ala Pro Pro Asp Ala Thr Phe Phe Gly Glu Leu Glu Ala Arg Leu Ala Gly Gly Asp Asn Val Cys Ile Phe Ser Ser Thr Val Ser Phe Ala Glu Val Val Ala Arg Phe Cys Arg Gln Phe Thr Asp Arg Val Leu Leu Leu His Ser Leu Thr Pro Pro Gly Asp 180 1$5 190 Val Thr Thr Trp Gly Arg Tyr Arg Val Val Ile Tyr Thr Thr Val Val Thr Val Gly Leu Ser Phe Asp Pro Pro His Phe Asp Ser Met Phe Ala Tyr Val Lys Pro Met Asn Tyr Gly Pro Asp Met Val Ser Val Tyr Gln Ser Leu Gly Arg Val Arg Thr Leu Arg Lys Gly Glu Leu Leu Ile Tyr Met Asp Gly Ser Gly Ala Arg Ser Glu Pro Val Phe Thr Pro Met Leu Leu Asn His Val Val Ser Ala Ser Gly Gln Trp Pro Ala Gln Phe Ser Gln Val Thr Asn Leu Leu Cys Arg Arg Phe Lys Gly Arg Cys Asp Ala Ser His Ala Asp Ala Ala Gln Ala Arg Gly Ser Arg Ile Tyr Ser Lys Phe Arg Tyr Lys His Tyr Phe Glu Arg Cys Thr Leu Ala Cys Leu Ala Asp Ser Leu Asn Ile Leu His Met Leu Leu Thr Leu Asn Cys Met His Val Arg Phe Trp Gly His Asp Ala Ala Leu Thr Pro Arg Asn Phe Cys Leu Phe Leu Arg Gly Ile His Phe Asp Ala Leu Arg Ala Gln Arg Asp Leu Arg Glu Leu Arg Cys Gln Asp Pro Asp Thr Ser Leu Ser Ala Gln Ala Ala Glu Thr Glu Glu Val Gly Leu Phe Val Glu Lys Tyr Leu Arg Pro Asp Val Ala Pro Ala Glu Val Val Ala Leu Met Arg Gly Leu Asn Ser Leu Val Gly Arg Thr Arg Phe Ile Tyr Leu Val Leu Leu Glu Ala Cys Leu Arg Val Pro Met Ala Ala His Ser Ser Ala Ile Phe Arg Arg Leu Tyr Asp His Tyr Ala Thr Gly Val Ile Pro Thr Ile Asn Ala Ala Gly Glu Leu Glu Leu Val Ala Leu His Pro Thr Leu Asn Val Ala Pro Val Trp Glu Leu Phe Arg Leu Cys Ser Thr Met Ala Ala Cys Leu Gln Trp Asp Ser Met Ala Gly Gly Ser Gly Arg Thr Phe Ser Pro Glu Asp Val Leu Glu Leu Leu Asn Pro His Tyr Asp Arg Tyr Met Gln Leu Val Phe Glu Leu Gly His Cys Asn VaI Thr Asp Gly Pro Leu Leu Ser Glu Asp Ala Val Lys Arg Val Ala Asp Ala Leu Ser Gly Cys Pro Pro Arg Gly Ser Val Ser Glu Thr Glu His Ala Leu Ser Leu Phe Lys Ile Ile Trp Gly Glu Leu Phe Gly Val Gln Leu Ala Lys Ser Thr Gln Thr Phe Pro Gly Ala Gly Arg Val Lys Asn Leu Thr Lys Arg Ala Ile Val Glu Leu Leu Asp Ala His Arg Ile Asp His Ser Ala Cys Arg Thr His Arg Gln Leu Tyr Ala Leu Leu Met Ala His Lys Arg Glu Phe Ala Gly Ala Arg Phe Lys Leu Arg Ala Pro Ala Trp Gly Arg Cys Leu Arg Thr His Ala Ser Gly Ala Gln Pro Asn Thr Asp Ile Ile Leu Glu Ala Ala Leu 6?5 Fan Ser Glu Leu Pro Thr Glu Ala Trp Pro Met Met Gln Gly Ala Val Asn Phe Ser Thr Leu <210> 5 <211> 2130 <212> DNA
<213> Unknown <400> 5 atgggttttaaacgtttgattgtgcaacttgaaagcctacaccgcgtatccagcgaagct 60 atcgacagctacgacgtattaatactggatgaggtaatgtcagtgattggacaattatac 120 tcccccacaatgagacgtctttccgcggttgatagcctattatatcgtcttttaaatcgc 180 tgttctcaaattatcgcgatggatgctacagtaaactcgcagtttattgatttaatctcc 240 ggattgcgtggagatgaaaacatacacacaattgtgtgtacatacgcgggagttgggttc 300 tccggaagaacttgcacgatcctgcgtgatatgggcatcgacacgcttgtgcgagtcatt 360 aaacgatctcctgaacacgaggatgtacgtaccatacaccaactacgtggaacatttttt 420 gacgaactagcactacgattacaatgtgggcataacatctgtatattttcatcaacttta 480 tcgttttcggagctagttgctcagttttgtgcaatatttacagactctattcttatttta 540 aactcaactcggcccctatgtaatgtaaacgaatggaaacattttcgcgtgttggtgtac 600 actaccgtcgtgaccgttggattgagttttgacatggctcattttcatagcatgtttgct 660 tacataaagccaatgtcatatgggccggatatggtatcggtctaccagtcattagggcgt 720 gtacgtttattgctacttaatgaagttttgatgtacgtcgatggctcaaggaccagatgc 780 ggacccctgttctcgccaatgttactaaactttaccatcgcaaataaatttcaatggttt840 cctacacacacccaaataactaacaaactgtgctgtgcatttaggcaacgatgtgcaaat900 gcatttacacgctcgaacacccatctcttctcaagatttaaatacaaacaccttttcgag960 agatgctctctttggagtttagccgatagcattaatatcttacaaactcttttggcctct1020 aaccaaattttggttgtattggatggcatgggtccaataacggacgtttccccagttcaa1080 ttttgtgcatttatacacgatctcagacatagcgctaacgccgtagcttcctgtatgcgt1140 tctcttagacaggacaatgacagctgcttgaccgattttggcccttccggatttatggcc1200 gataacattaccgcgtttatggaaaagtatcttatggagtcaattaataccgaagaacaa1260 attaaagtatttaaagcccttgcatgtccaatagaacagcctagactagtcaatacggca1320 atattgggggcgtgtatacgaatacctgaagcgttggaagcatttgacgtatttcaaaaa1380 atatacacgcactacgcttccggttggtttcccgtcctggacaaaaccggggaatttagc1440 atcgcgactataactaccgccccaaatttaaccacacattgggagctgtttcgccgttgt1500 gcctatattgcaaaaacactcaagtggaatccgtccaccgaaggctgtgtaacacaagtt1560 ttggatacggacattaatacacttttcaatcaacacggggattcgctggctcaactaata1620 tttgaggttatgcgctgtaacgttactgacgctaagattatattaaaccgcccggtttgg1680 cgaacaaccggattcttagatggatgccataatcaatgcttccgtccaatccctacaaaa1740 cacgaatataacattgctctatttcgtttaatttgggaacaattatttggcgcccgcgta1800 actaaaagtacccagacctttccgggaagtactcgtgtgaaaaacctaaaaaaaaaagat1860 ctagaaactttacttgattcaattaacgtggatcgttctgcatgtcgtacctaccgccag1920 ttgtataacctgcttatgagccagcgccattcgttctctcaacagcgttacaaaattact1980 gcccccgcttgggcacgccacgtgtattttcaagcacatcaaatgcacttggccccgcat2040 gccgaagccatgctacaattagcgctatcggaactgtccccgggatcgtggccgcggata2100 aacggggcggtaaattttgaaagtttataa 2130 <210> 6 <211> 709 <212> PRT
<213> Unknown <400> 6 Met Gly Phe Lys Arg Leu Ile Val Gln Leu Glu Ser Leu His Arg Val Ser Ser Glu Ala Ile Asp Ser Tyr Asp Val Leu Ile Leu Asp Glu Val Met Ser Val Ile Gly Gln Leu Tyr Ser Pro Thr Met Arg Arg Leu Ser Ala Val Asp Ser Leu Leu Tyr Arg Leu Leu Asn Arg Cys Ser Gln Ile Ile Ala Met Asp Ala Thr Val Asn Ser Gln Phe Ile Asp Leu Ile Ser Gly Leu Arg Gly Asp Glu Asn Ile His Thr Ile Val Cys Thr Tyr Ala Gly Val Gly Phe Ser Gly Arg Thr Cys Thr Ile Leu Arg Asp Met Gly Ile Asp Thr Leu Val Arg Val Ile Lys Arg Ser Pro Glu His Glu Asp Val Arg Thr Ile His Gln Leu Arg Gly Thr Phe Phe Asp Glu Leu Ala Leu Arg Leu Gln Cys Gly His Asn Ile Cys Ile Phe Ser Ser Thr Leu Ser Phe Ser Glu Leu Val Ala Gln Phe Cys Ala Ile Phe Thr Asp Ser Ile Leu Ile Leu Asn Ser Thr Arg Pro Leu Cys Asn Val Asn Glu Trp Lys His Phe Arg Val Leu Val Tyr Thr Thr Val Val Thr Val Gly Leu Ser Phe Asp Met Ala His Phe His Ser Met Phe Ala Tyr Ile Lys Pro Met Ser Tyr Gly Pro Asp Met Val Ser Val Tyr Gln Ser Leu Gly Arg Val Arg Leu Leu Leu Leu Asn Glu Val Leu Met Tyr Val Asp Gly Ser Arg Thr Arg Cys Gly Pro Leu Phe Ser Pro Met Leu Leu Asn Phe Thr Ile Ala Asn Lys Phe Gln Trp Phe Pro Thr His Thr Gln Ile Thr Asn Lys Leu Cys Cys Ala Phe Arg Gln Arg Cys Ala Asn Ala Phe Thr Arg Ser Asn Thr His Leu Phe Ser Arg Phe Lys Tyr Lys His Leu Phe Glu Arg Cys Ser Leu Trp Ser Leu Ala Asp Ser Ile Asn Ile Leu Gln Thr Leu Leu Ala Ser Asn Gln Ile Leu Val Val Leu Asp Gly Met Gly Pro Ile Thr Asp Va1 Ser Pro Val Gln Phe Cys Ala Phe Ile His Asp Leu Arg His Ser Ala Asn Ala Val Ala Ser Cys Met Arg Ser Leu Arg Gln Asp Asn Asp Ser Cys Leu Thr Asp Phe Gly Pro Ser Gly Phe Met Ala Asp Asn Ile Thr Ala Phe Met Glu Lys Tyr Leu Met Glu Ser Ile Asn Thr Glu Glu Gln Ile Lys Val Phe Lys Ala Leu Ala Cys Pro Ile Glu Gln Pro Arg Leu Val Asn Thr Ala Ile Leu Gly Ala Cys Ile Arg Ile Pro Glu Ala Leu Glu Ala Phe Asp Val Phe Gln Lys Ile Tyr Thr His Tyr Ala Ser Gly Trp Phe Pro Val Leu Asp Lys Thr Gly Glu Phe Ser Ile Ala Thr Ile Thr Thr Ala Pro Asn Leu Thr Thr His Trp Glu Leu Phe Arg Arg Cys Ala Tyr Ile Ala Lys Thr Leu Lys Trp Asn Pro Ser Thr Glu Gly Cys Val Thr Gln Val Leu Asp Thr Asp Ile Asn Thr Leu Phe Asn Gln His Gly Asp Ser Leu Ala Gln Leu Ile Phe Glu Val Met Arg Cys Asn Val Thr Asp Ala Lys Ile Ile Leu Asn Arg Pro Val Trp Arg Thr Thr Gly Phe Leu Asp Gly Cys His Asn Gln Cys Phe Arg Pro Ile Pro Thr Lys His Glu Tyr Asn Ile Ala Leu Phe Arg Leu Ile Trp Glu Gln Leu Phe Gly Ala Arg Val Thr Lys Ser Thr Gln Thr Phe Pro Gly Ser Thr Arg Val Lys Asn Leu Lys Lys Lys Asp Leu Glu Thr Leu Leu Asp Ser Ile Asn Val Asp Arg Ser Ala Cys Arg Thr Tyr Arg Gln Leu Tyr Asn Leu Leu Met Ser Gln Arg His Ser Phe Ser Gln Gln Arg Tyr Lys Ile Thr Ala Pro Ala Trp Ala Arg His Val Tyr Phe Gln Ala His Gln Met His Leu Ala Pro His Ala Glu Ala Met Leu Gln Leu Ala Leu Ser Glu Leu Ser Pro Gly Ser Trp Pro Arg Ile Asn Gly Ala Val Asn Phe Glu Ser Leu

Claims (16)

What is claimed is:
1. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
2. An isolated polynucleotide comprising a polynucleotide having at least a 70% identity to a polynucleotide encoding the same polypeptide expressed by the OBP-NT
gene contained in the Herpes simplex virus type 1 (HSV-1).
3. An isolated polynucleotide comprising a polynucleotide encoding a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO:2.
4 The polynucleotide of Claim 1 comprising a sequence selected from SEQ ID
NO:1, SEQ ID NO: 3 and SEQ ID NO: 5.
5. The polynucieotide of Claim 1 which encodes a polypeptide comprising the amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
6. A vector comprising the polynucleotide of Claim 1.
7. A host cell comprising the vector of Claim 6.
8. A process for producing a OBP-NT polypeptide or fragment comprising culturing a host cell of claim 7 under conditions sufficient for the production of said polypeptide or fragment.
9. A polypeptide comprising an amino acid sequence which 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.
11. An antagonist which inhibits the activity, binding or expression of the polypeptide of Claim 10.
12. A method for the treatment of an individual in need of herpes anti-viral treatment comprising: administering to the individual a therapeutically effective amount of the polypeptide of Claim 11.
13. A process for diagnosing a disease related to expression or activity of the polypeptide of Claim 9 in an individual comprising:
(a) analyzing for the presence or amount of said polypeptide in a sample derived from the individual.
14. A method for identifying compounds which interact with and inhibit or activate an activity of the polypeptide of Claim 9 comprising:

contacting a composition comprising the polypeptide with the compound to be screened under conditions to permit interaction between the compound and the polypeptide to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of the polypeptide with the compound;
and determining whether the compound interacts with and activates or inhibits an activity of the polypeptide by detecting the presence or absence of a signal generated from the interaction of the compound with the polypeptide.
15. A method for inducing an immunological response in a mammal which comprises inoculating the mammal with an OBP-NT polypeptide of Claim 9, or a fragment or variant thereof, adequate to produce antibody and/or T cell immune response to protect said animal from disease.
16. A method of inducing immunological response in a mammal which comprises delivering a nucleic acid vector to direct expression of OBP-NT polypeptide of Claim 9, or fragment or a variant thereof, for expressing said OBP-NT polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response.
CA002330230A 1998-06-10 1999-06-10 Herpes simplex virus origin binding protein n-terminal truncation (obp-nt) Abandoned CA2330230A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US8877098P 1998-06-10 1998-06-10
US60/088,770 1998-06-10
PCT/US1999/013140 WO1999064633A1 (en) 1998-06-10 1999-06-10 Herpes simplex virus origin binding protein n-terminal truncation (obp-nt)

Publications (1)

Publication Number Publication Date
CA2330230A1 true CA2330230A1 (en) 1999-12-16

Family

ID=22213347

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002330230A Abandoned CA2330230A1 (en) 1998-06-10 1999-06-10 Herpes simplex virus origin binding protein n-terminal truncation (obp-nt)

Country Status (4)

Country Link
EP (1) EP1086252A4 (en)
JP (1) JP2002517261A (en)
CA (1) CA2330230A1 (en)
WO (1) WO1999064633A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042918A (en) * 2022-11-15 2023-05-02 圣湘生物科技股份有限公司 Five virus joint inspection compositions, kit, method and application thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6821519B2 (en) 2000-06-29 2004-11-23 Corixa Corporation Compositions and methods for the diagnosis and treatment of herpes simplex virus infection

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042918A (en) * 2022-11-15 2023-05-02 圣湘生物科技股份有限公司 Five virus joint inspection compositions, kit, method and application thereof
CN116042918B (en) * 2022-11-15 2024-04-05 圣湘生物科技股份有限公司 Five virus joint inspection compositions, kit, method and application thereof

Also Published As

Publication number Publication date
EP1086252A4 (en) 2005-01-26
JP2002517261A (en) 2002-06-18
EP1086252A1 (en) 2001-03-28
WO1999064633A1 (en) 1999-12-16

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