WO1999053043A2

WO1999053043A2 - Herpes simplex virus latency associated transcript polypeptides

Info

Publication number: WO1999053043A2
Application number: PCT/GB1999/001111
Authority: WO
Inventors: Robert Stuart Coffin; David Seymour Latchman; Suzanne Kristina Thomas
Original assignee: University College London; Glaxo Group Limited
Priority date: 1998-04-09
Filing date: 1999-04-09
Publication date: 1999-10-21
Also published as: GB9807865D0; JP2002511254A; WO1999053043A3; EP1070123A2; AU3433999A

Abstract

A polynucleotide in substantially isolated form consisting essentially of an open reading frame encoded by a herpes simplex virus (HSV) latency-associated transcript (LAT) operably linked to a homologous or heterologous regulatory sequence permitting expression of a LAT ORF polypeptide in a cell.

Description

HERPES SIMPLEX VIRUS LATENCY ASSOCIATED TRANSCRIPT POLYPEPTIDES

Field of the Invention

The present invention relates to polynucleotide constructs that allow for the expression of heφes simplex virus latency associated transcript polypeptides under the control of heterologous regulatory sequences. It also relates to cell lines and viruses comprising these polynucleotide constructs.

Background to the invention Herpes simplex virus (HSV) 1 and 2 establish and reactivate from latency in sensory neurons following an initial peripheral infection. During latency the virus genome is transcriptionally inactive other than within a small region of the genome from which the latency associated transcripts (LATs) are transcribed (reviewed by Latchman, 1990; Fraser et al., 1992; Ho, 1992). The LATs are transcribed from the long repeats of the genome generating a 8.5 kb primary transcript of low abundance and a highly abundant 2 kb species which is probably a stable intron derived from a larger transcript (Spivack and Fraser, 1987; Stevens et al, 1987; Farrel et al, 1991). Smaller species probably resulting from further splicing of the 2 kb transcript are also generated in neurons (see Spivack et al, 1991). The molecular mechanisms governing the establishment and reactivation from latency are not well understood, although it is thought that interactions with host transcription factors of virus immediate early (IE) gene promoters plays a role in both establishing latency (repressing factors) and initiating the reactivation process (activating factors), particularly affecting the activity of the IE1 promoter. The role of the LATs in these processes are not known, as various mutations or deletion of some or all of the LAT locus from the wild type virus results in only very subtle effects on the kinetics of latency in various animal models. Results have however consistently suggested that a possible role for LATs is in reactivation from latency, as a reduction in reactivation efficiency has been detected with a number of different LAT mutants in a number of different animal models. Recently a role in the establishment of latency has also been demonstrated, as LAT (-) mutants establish latency at a reduced efficiency (Thomson and Sawtell, 1997). LAT thus appears to play a subtle role in influencing the kinetics of latency, although its mechanism of action is unknown.

Various functions have been proposed for the LATs, for example repression of

IE1 gene expression as the LAT transcripts are antisense to the IE1 gene (Stevens et al, 1987). It has also been suggested that transcription of the LAT locus may keep the genome in a transcriptionally competent state during latency such that reactivation is possible, or that a protein encoded by the LAT may somehow influence virus latency

(Spivack et al, 1991 ; Wechsler et al., 1988). However even though a LAT encoded protein has been reportedly detected in latency infected cells (Doerig et al, 1991), this latter hypothesis has been rejected due to the lack of a detectable phenotype when LAT

ORFs are mutated (Fareed and Spivack, 1994), and due to a lack of reproducibility in detection of the protein.

Indeed, recent work has shown that the 2 kb LAT as well as being non- polyadenylated, is also in a lariat structure from which translation would not be expected to be possible (Wu et al, 1996; Rodahl and Haarr, 1996). This and the observation that mutation of the individual ORFs results in no detectable effect in an explant reactivation model of HSV latency (Fareed and Spivack, 1994) has suggested that the LAT ORFs are unlikely to encode functional proteins.

Summary of the invention

We have found that de-regulated expression of an HSV LAT ORF, both from within the virus genome and in a number of stably transfected cell lines, gives greatly enhanced growth of the virus in cells of both neuronal and non-neuronal origin which are otherwise relatively non-permissive for HSV. Moreover, an HSV mutant deleted for ICPO (IE1) grows to nearly wild type levels in cells constitutively expressing the LAT

ORF. ICPO mutants usually show greatly reduced growth in culture as compared to wild type virus.

ICPO is thought to be necessary for the reactivation of HSV latency in vivo.

Indeed it is thought that ICPO expression is one of the first events to occur during reactivation. Thus, since we have shown that LAT ORF expression overcomes ICPO deletion, it would be expected that if the LAT ORF were expressed during HSV latency in vivo, such expression would be likely to result in virus reactivation. Thus the HSV 2 kb LAT encodes a protein which is likely to function during reactivation from latency and provides a potential drug target for HSV during latency as inhibition of HSV LAT ORF activity would be likely to reduce the efficiency of reactivation. Consistent with an activity of the HSV LAT ORF in reactivation, we have also found that viruses constitutively expressing the LAT ORF are of considerably greater virulence in mice than an otherwise identical control virus not constitutively expressing the LAT ORF, probably due to enhanced virus growth preventing the virus entering latency.

Viruses in which a frame shift mutation has been introduced into the ORF show none of the LAT ORF associated effects. This confirms that these effects result from LAT ORF protein expression, rather than an activity of the LAT RNA itself. LAT ORF expression was also demonstrated in cells in culture infected with the LAT ORF- expressing virus showing efficient translation of the LAT ORF when it is present in a normally processed mRNA instead of in the 2 kb LAT in which it is usually positioned. Thus the HSV LATs encode a protein which can greatly enhance virus growth and which is likely to function in aiding the reactivation of the virus from latency. The HSV LAT ORF thus provides a novel drug target for the prevention of reactivation by HSV.

Accordingly the present invention provides a polynucleotide in substantially isolated form consisting essentially of an open reading frame encoded by a herpes simplex virus (HSV) latency-associated transcript (LAT) operably linked to a regulatory sequence permitting expression of a LAT ORF polypeptide in a cell. The regulatory sequence may thus be a homologous or heterologous regulatory sequence.

Generally, the heterologous regulatory sequence is a non-LAT promoter, preferably a viral promoter or a mammalian promoter permitting expression of said LAT in a mammalian cell. Such promoters are preferably inducible promoters or constitutive promoters.

The present invention also provides a nucleic acid vector comprising a polynucleotide of the invention and a viral vector comprising a polynucleotide of the invention. Especially preferred viral vectors are strains of HSV, in particular HSV1 and HSV2.

The polynucleotides of the invention may also be introduced into a cell or cell line. Thus the present invention also provides a cell or cell line comprising a polynucleotide of the invention. Preferably the cell line is a mammalian cell line. The cell may be a eukaryotic cell, for example a yeast or mammalian cell, or it may be a prokaryotic cell, for example a bacterial cell. Cell lines or cells of the invention may be used in several ways. For example, cells or cell lines of the invention may be used to produce an HSV LAT polypeptide when cultured under suitable conditions. Accordingly the present invention also provides an HSV LAT polypeptide in substantially isolated form obtainable by incubating a cell or cell line of the invention under conditions that allow for expression of the HSV LAT ORF contained within the cell or cell line. Polypeptides of the invention can also be produced by incubating a polynucleotide of the invention in a cell-free protein expression system under conditions that allow for expression of the LAT ORFcontained within the polynucleotide.

Cell lines of the invention, expressing an HSV LAT polypeptide may also be used to propagate HSV strains, in particular an HSV strain lacking a functional ICPO gene.

The polynucleotide constructs and polypeptides of the invention can also be used in methods of scientific research. Polypeptides of the invention may be used in in vitro or in vivo cell culture systems to study the role of HSV LAT ORFs in viral disease. Such cell culture systems in which polypeptide of the invention are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides of the invention in the cell.

Thus the present invention further provides a method for identifying a substance capable of interacting with an HSV LAT polypeptide, which method comprises incubating a candidate substance with an HSV LAT polypeptide and determining whether the candidate substance interacts with the LAT polypeptide. Such a method will also aid in identifying a substance capable of reducing or preventing reactivation of a heφes simplex virus from latency.

A substance identified by the method of the invention as interacting with an HSV LAT polypeptide may be able to prevent reactivation of HSV from latency in a mammalian, for example human, host. Thus the present invention also provides a substance identified by the method of the invention for use in a method of treating HSV infection in a mammal, preferably a human.

Detailed description of the invention

A. Polynucleotide constructs

Polynucleotide constructs of the invention consist essentially of a HSV latency associated transcript (LAT) open reading frame (ORF) operably linked to a regulatory sequence permitting expression of said LAT ORF-encoded polypeptide in a cell. The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. Thus, for example, a promoter operably linked to polynucleotide sequence encoding an HSV LAT ORF is ligated in such a way that expression of the LAT ORF is achieved under conditions which are compatible with the activation of expression from the promoter.

Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the puφoses of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of polynucleotides of the invention.

The polynucleotides of the invention can be constructed using routine cloning techniques known to persons skilled in the art (see, for example, Sambrook et al, 1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

1. HSV LAT ORF

An HSV LAT ORF is here defined as a open reading frame present in the nucleotide sequence of an HSV strain, the expression of the open reading frame being under the transcriptional regulatory control of a polynucleotide sequence (for example promoter) allowing transcription of the ORF during latency. HSV LAT ORFs in the context of the present invention thus include the three ORFs encoded by the 2 kb HSV1 LAT (for example, corresponding to approximately nucleotides 119,461 to 121,416 of strain 17+, GenBank HE ICG (SEQ ID No.l)) These three ORFs contain 274 (ORF1 - SEQ ID. No. 2), 108 (ORF2) and 174 (ORF3) amino acids, respectively (see Wechsler et al, 1994). A polynucleotide of the invention may comprise one or more of any and each of these three ORFs.

Suitable HSV LAT ORFs also include the single large ORF of 419 amino acids (SEQ ID. No. 4) encoded by the 2 kb HSV2 LAT corresponding to approximately nucleotides 6593 to 8807 of accession no. D10471 (SEQ ID. No. 3) (part of strain HG52, EMBL accession no. Z86099). Other suitable HSV LAT ORFs include homologous sequences present in other

HSV strains. Preferably the homologous sequence are at least 70% homologous, more preferably at least 80, 90 or 95% homologous at the nucleotide or polypeptide level to any of the sequences described above. HSV LAT ORFs also include polynucleotides encoding the HSV LAT polypeptides described below. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code.

2. Regulatory sequences

Regulatory sequences are polynucleotide sequences that regulate the levels of expression of a polynucleotide sequence to which they are operably linked. The term

"expression" in the context of the present invention preferably includes both transcription and translation to produce a polypeptide. Regulatory sequences include, for example, promoters and enhancers, as well as translational control sequences. In the context of the present invention, a heterologous regulatory sequence specifically excludes the complete regulatory sequences which are naturally associated with a LAT ORF within the genome of an HSV strain, but does not exclude component parts of a naturally occurring HSV LAT regulatory sequence, for example the LAP1 promoter or the LAP2 promoter.

The term promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.

The promoter may be a prokaryotic promoter, for example a bacterial promoter, or a eukaryotic promoter, for example a yeast or mammalian promoter. The promoter is preferably selected from promoters which are functional in mammalian, more preferably human, cells. The promoter may be derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression of the HSV LAT ORF is to occur, preferably a mammalian cell line. With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of α-actin, β-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). Promoters which are active in only certain neuronal cell types are especially preferred (for example the tyrosine hydroxylase (TH), L7, or neuron specific enolase (NSE) promoters). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the HSV1 or HSV2 LAP1 promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.

Suitable prokaryotic (for example bacterial) or yeast promoters include the E. coli lac promoter. S. cerevisiae GAL4 and ADH promoters and the S. pombe nmtl and adh promoters.

It may also be advantageous for the promoters to be inducible so that the levels of expression of the HSV LAT ORF can be regulated. Inducible means that the levels of expression obtained using the promoter can be regulated. Particular examples of inducible promoters include steroid hormone or glucocorticoid responsive promoters (for example comprising progesterone, oestrogen and ecdysone response elements), the lac promoter, antibiotic responsive promoters (for example the tet promoter) and T7 promoters which require co-transfection of a plasmid construct encoding T7 RNA polymerase, expression of which is induced with IPTG.

In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above. B. Vectors

The polynucleotide construct of the invention may be used in the form of a naked nucleic acid construct. Polynucleotides of the invention can also be incoφorated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells.

Such vectors may be transformed or transfected into a suitable host cell as described below to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of the HSV LAT polypeptides, and optionally recovering the expressed polypeptides. Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the vector is designed (see above).

The vectors may be for example, plasmid or virus vectors provided with an origin of replication. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector (which, for example, allows for the selection of stably transfected cell lines).

Vectors may further include sequences flanking the polynucleotide construct which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of polynucleotide construct into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the polynucleotide construct flanked by viral sequences, preferably HSV1 or HSV2 sequences, can be used to prepare a viral vector, preferably an HSV vector, suitable for delivering the polynucleotide construct of the invention to a mammalian cell. The techniques employed are well-known to a skilled person and will be suitable for other viruses such as adenoviruses. Other examples of suitable viral vectors include viral vectors able to integrate their genomes into the host cell genome, for example retroviruses, including lenti viruses, and adeno-associated virus. C. Heφes Simplex Virus Vectors

1. Viral Strains

The HSV strains of the invention comprising the polynucleotide construct may be derived from, for example, HSV1 or HSV2 strains, or derivatives thereof, preferably HSV1. Derivatives include inter-type recombinants containing DNA from HSV1 and HSV2 strains. Derivatives preferably have at least 70% sequence homology to either the HSV1 or HSV2 genomes, more preferably at least 90%, even more preferably 95%.

2. HSV strains comprising the polynucleotide construct The polynucleotide construct may be inserted into the HSV genome at any location provided that the virus can still be propagated, which may require the use of a cell line carrying another HSV essential gene if the heterologous gene is inserted into an essential gene. For example, if the polynucleotide construct gene is inserted into the ICP27 gene of the HSV strain, then a cell-line expressing ICP27 would be needed. Preferably the polynucleotide construct is inserted into a non-essential gene, for example UL43 or US5.

The polynucleotide may be inserted into the HSV genome by homologous recombination of HSV strains with, for example, plasmid vectors carrying the expression cassette flanked by HSV sequences, as described above for introducing mutations. The expression cassette may be introduced into a suitable plasmid vector comprising HSV sequences using cloning techniques well-known in the art.

D. Polypeptides

HSV LAT polypeptides of the invention comprise polypeptides encoded by an HSV LAT ORF (as defined above) and derivatives thereof. Particular examples of polypeptides of the invention include the amino acid sequence set out in SEQ ID Nos. 2 and 4 or a substantially homologous sequence, or of a fragment of either of these sequences. In general, the naturally occurring HSV LAT polypeptide sequences shown in SEQ ID Nos 2 and 4, are preferred. However, the polypeptides of the invention include derivatives of the natural occurring HSV LAT polypeptides. The term derivative is taken to encompass all homologues and allelic variants having substantial homology to the HSV LAT polypeptide sequences set out in SEQ ID. Nos. 2 and 4. In particular, it is preferred that a derivative is capable of improving the growth of ICPO^" HSV strains in a cell line expressing the derivative.

Homologues of the HSV1 and HSV2 LAT polypeptide sequences set out in SEQ ID Nos. 2 and 4 include the functionally equivalent polypeptide which occurs naturally in other strains. An allelic variant includes a variant that occurs naturally in an HSV strain which will function in a substantially similar manner to the polypeptides of SEQ ID Nos. 2 and 4.

Allelic variants and homologues in different HSV strains can be obtained using standard cloning procedures. In particular, it will be possible to use an HSV LAT ORF nucleotide sequence to probe libraries made from HSV genomes to obtain clones encoding the allelic or strain variants. The clones can be manipulated by conventional techniques to identify an HSV LAT polypeptide of the invention which can then be produced by recombinant or synthetic techniques known per se. An HSV LAT polypeptide is preferably at least 70% homologous to the polypeptide of SEQ ID Nos. 2 or 4, more preferably at least 80, 90 or 95% homologous thereto over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. Methods of measuring polypeptide homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").

Further, the sequence of the polypeptide of SEQ ID Nos. 2 and 4 and of allelic variants and strain homologues can be modified to provide polypeptides of the invention. Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions provided that the modified polypeptide retains biological activity.

Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

In addition, polypeptides of the invention may be produced as fusion proteins wherein an HSV LAT polypeptide, a homologue or derivative thereof is fused, using standard cloning techniques, to another polypeptide which may, for example, comprise a DNA-binding domain, a transcriptional activation domain or a ligand suitable for affinity purification (for example glutathione-S-transferase or six consecutive histidine residues).

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended puφose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be made by synthetic means or recombinantly using techniques well known to skilled persons.

The use of mammalian host cells is expected to provide for such post-translational modifications (e.g. myristolation, glycosylation, truncation, lapidation and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on HSV LAT polypeptides of the invention. However, yeast or prokaryotic (for example bacterial) host cells may also be used to produce polypeptides of the invention. In addition, HSV LAT polypeptides of the invention may be produced using cell free expression systems, for example a rabbit reticulocyte expression system (for example TnT™ - Promega).

E. Cell lines

The polynucleotide construct of the invention may be introduced into a cell line using standard techniques to produce a cell line of the invention. A cell line of the invention may be used for example to propagate HSV viruses defective in ICPO. A cell line of the invention may also be used to produce an HSV LAT polypeptide of the invention which may then be subsequently purified. Cell lines can be produced by co- transfecting, for example, mammalian cells, with a vector, preferably a plasmid vector, comprising a polynucleotide construct of the invetion, and a vector, preferably a plasmid vector, encoding a selectable marker, for example neomycin resistance. Clones possessing the selectable marker are then screened further to determine which clones also express the HSV LAT polypeptide, for example on the basis of their ability to enhance the growth of HSV strains (for example wild-type or ICPO^" mutant strains), using methods known to those skilled in the art.

Particularly include preferred cell lines mammalian cell lines, for example neuronal fibroblast and epithelial cell lines, for example BHK, Vero or ND7 cells.

F. Assay Methodologies

The polynucleotide constructs and polypeptides of the invention can also be used in methods of scientific research. Polypeptides of the invention may be used in in vitro or in vivo cell culture systems to study the role of HSV LAT ORFs in viral disease. Such cell culture systems in which polypeptide of the invention are expressed may be used in assay systems to identify candidate substances which interfere with or enhance the functions of the polypeptides of the invention in the cell. Thus another aspect of the present invention provides a method for identifying a substance capable of interacting with an HSV LAT polypeptide, which method comprises incubating a candidate substance with an HSV LAT polypeptide and determining whether the candidate substance interacts with the LAT polypeptide. In a preferred assay method, the HSV LAT polypeptide is immobilised on beads such as agarose beads. Typically this is achieved by expressing the HSV LAT polypeptide as a GST-fusion protein in bacteria, yeast or higher eukaryotic cell lines and purifying the GST-fusion protein from crude cell extracts using glutathione-agarose beads (Smith and Johnson, 1988). The binding of the candidate sustance to the HSV LAT polypeptide is then evaluated. This type of assay is known in the art as a GST pulldown assay.

It is also possible to perform this type of assay using different affinity purification systems for immobilising the HSV LAT polypeptide, for example Ni-NTA agarose and histidine-tagged HSV LAT polypeptides.

Binding of the candidate substance to the HSV LAT polypeptide (and vice-versa) may be determined by a variety of methods well-known in the art. For example, the non- immobilised component may be labelled (with for example, a radioactive label, an epitope tag or an enzyme-antibody conjugate). Alternatively, binding may be determined by immunological detection techniques.

For example, the reaction mixture can be Western blotted and the blot probed with an antibody that detects the non-immobilised component. ELISA techniques may also be used.

Another method contemplated by the invention for identifying a substance that disrupt an interaction between the first component and the second component involves immobilising the HSV LAT polypeptide on a solid support coated (or impregnated with) a fluorescent agent, labelling the candidate substance with a compound capable of exciting the fluorescent agent, contacting the immobilised HSV LAT polypeptide with the labelled candidate substance and detecting light emission by the fluorescent agent to determine binding. In all the above methods, alternatively, the candidate substance may be immobilised and HSV LAT polypeptide labelled in the assay.

Assays for identifying polypeptide compounds that interact with a polypeptide of the invention may also conveniently be performed by the two-hybrid screen technique which typically involves:

(a) transforming or transfecting an appropriate host cell with a nucleic acid construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain;

(b) expressing in the host cell a first hybrid DNA sequence encoding a fusion protein of all or part of an HSV LAT polypeptide and the DNA binding domain or the activating domain of the transcription factor; expressing in the host cells a second hybrid DNA sequence encoding all or part of the candidate polypeptide; and

(c) evaluating the binding of the candidate polypeptide to the HSV LAT polypeptide by measuring the production of reporter gene product in the host cell in the presence or absence of the test compound.

The host cell may be a bacterium or other microbial cell. It may also be a yeast or mammalian cell. Presently preferred for use in such an assay are a lexA promoter to drive expression of the reporter gene, the lacZ reporter gene, a transcription factor comprising the lexA DNA domain and the GAL4 transactivation domain and yeast host cells. The two-hybrid screen technique can conveniently be used to screen entire libraries of sequences for interacting polypeptides.

Another assay system which may be used to identify substances which interact with an HSV LAT polypeptide and inhibit its biological function involves using a cell line of the invention which expresses an HSV LAT polypeptide. The assay method comprises:

(i) infecting a cell line of the invention with an HSV strain capable of growing in the cell line and determining the ability of the HSV strain to grow in the cell line in the absence of a candidate substance, (ii) infecting a cell line of the invention with an HSV strain capable of growing in the cell line and determining the ability of the HSV strain to grow in the cell line in the presence of a candidate substance. (iii) determining whether the candidate substance interacts with and inhibits the activity of the HSV LAT polypeptide by whether the growth rate in (ii) is lower than the growth rate in (i).

Preferably said HSV strain lacks a functional ICPO gene and therefore has a slow growth rate in the absence of functional HSV LAT polypeptide expressed by a cell line of the invention.

Candidate substances that are identified by the method of the invention as interacting with an HSV LAT polypeptide of the invention may be tested for their ability to, for example, reduce the ability of HSV to reactivate from latency. Such compounds could be used therapeutically to prevent or treat viral infection.

Typically, an assay to determine the effect of a candidate substance identified by the method of the invention on the ability of HSV to reactivate from latency comprises:

(a) administering a virus, for example HSV1 , to an animal cell, in the absence of the candidate substance;

(b) administering the virus to the cell in the presence of the candidate substance; and

(c) determining if the candidate substance reduces or abolishes the ability of the virus to reactivate.

The candidate substance may be administered before, after or concomitant with, the virus to establish if infection is prevented. Administration of candidate substances to cells may be performed, for example by direct injection. Alternatively, polynucleotides/vectors encoding polypeptide candidate substances can be adminstered by lipofection, viral vector or a variety of other techniques known to skilled persons.

The assay is typically carried out using an animal model . Techniques for assaying for reactivation of latent viruses in animal models (for example mice) are well-known in the art.

G. Administration of compounds in therapy

Compounds identified by the method of the invention as interacting with HSV L AT polypeptides may be used to disrupt the normal function of HSV LAT polypeptides in reactivation of HSV from latency. Thus the present invention also provides a substance identified by the method of the invention for use in a method of treating HSV infection in a human. The formulation of a substance according to the invention will depend upon the nature of the substance identified but typically a substance may be formulated for clinical use with a pharmaceutically acceptable carrier or diluent. For example it may formulated for oral, topical, parenteral, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration. A physician will be able to determine the required route of administration for any particular patient and condition.

Preferably, the substance is used in an orally administrable or injectable form. It may therefore be mixed with any vehicle which is pharmaceutically acceptable for an orally administrable or injectable formulation, for example for a direct injection at the site to be treated. The pharmaceutically carrier or diluent may be, for example, sterile or isotonic solutions. It is also preferred to formulate that substance in an orally active form. Typically, said substance may be a polypeptide, an antibody or a nucleic acid construct. Nucleic acid constructs may be administered by various well-known techniques including lipofection, biolistic transformation or the use of viral vectors.

The dose of substance used may be adjusted according to various parameters, especially according to the substance used, the age, weight and condition of the patient to be treated, the mode of administration used and the required clinical regimen. A physician will be able to determine the required route of administration and dosage for any particular patient and condition.

The invention will be described with reference to the following Examples which are intended to be illustrative only and not limiting. EXAMPLES

Example 1. BHK cells expressing the HSVl LAT ORF show enhanced growth of HSVl BHK 21 C13 fibroblast cells (ECACC no. 8501143) were stably transfected with a construct expressing the HSVl LAT ORF. This construct (pGKLAT) was prepared by digestion of pGKneo with EcoRV and insertion of the HSVl LAT ORF as a Sapl-Pstl fragment from pJ7LAT. pJ7LAT was prepared by the insertion of the HSVl LAT ORF excised from pNOT3.5 with BstXI nd BspMI and insertion into the Smal site of pJ7 (Morgenstern and Land, 1990). pNot3.5 consists of HSVl nts 118,439 (GenBank

HE ICG) as a Notl fragment inserted into the Not I site of pGem5 (Promega). pGKneo contains the neomycin resistance gene under the control of a constitutively active promoter (GK), pGKLAT thus provides a plasmid with a neomycin resistance selection marker and in which the LAT ORF is driven by the CMV IE promoter and terminated by an SV40 poly-A sequence. pGKLAT was transfected into BHK cells, cultured in DMEM + 10% fetal calf serum (both GIBCO) and following G418 selection (Gibco) 24 stably transfected colonies were picked and each of these independant clones compared with a pGKneo transfected control cell line for their ability to support the growth of HSV.

Six representative clones from above and a pGKneo clone were seeded into 24 well plates and infected with HSVl (strain 17+) at multiplicities of infection of 0.1, 0.01 and 0.001. 36hrs later the contents of each well were harvested and titrated onto standard BHK cells and the final virus yield achieved on the BHK-LAT cells and BHK-neo control cells calculated. Each experiment was performed in duplicate and repeated three times. The average of each duplicate experiment is shown, giving three results at each MOI. Results:

MOI 0.1 0.01 0.001

BHK Cell line Final Yield (pfu x l0^: ³)

GK-neo 16 14 17 4 5 4 0.1 0.3 0.3

LAT-1-1 70 90 60 7 8 9 4 3.5 5

LAT- 1-2 360 440 320 9 10 10 1 3 3

LAT- 1-3 70 70 70 8 6 7 0.5 1 2

LAT- 1-4 50 70 50 10 10 9 4 3 ND

LAT- 1-5 30 20 20 9 7 9 4 1 4

LAT- 1-6 80 90 100 25 20 20 3 4 1

These results show that constitutive expression of the HSVl LAT ORF in BHK cells enhances the growth of HSV 1 in these cells.

Example 2 ND7 cells expressing the HSVl LAT ORF show greatly enhanced growth of HSVl Stably transfected ND7 cells (Wood et al., 1990) constitutively expressing the LAT ORF were prepared as above for BHK cells using pGKLAT, and also pGKneo as a control plasmid. ND7 cells are a cell line which show characteristics of sensory neurons and were prepared by fusion of primary rat DRG neurons with mouse C1300 neuroblastoma cells. ND7 cells are non-premissive for HSV due to a specfic repression of IE genes (Kemp and Latchman, 1989) and provide a model for the study of HSV in neurons. For these experiments ND7 cells were cultured in RPMI + 10% foetal calf serum (both GIBCO).

6 representative clones and a pGKneo clone from above were again seeded into 24 well plates and infected with HSVl (strain 17+), here at multiplicities of infection of 1, 0.1 and 0.01. 72hrs later the contents of each well were harvested and titrated onto standard BHK cells and the final virus yield from the ND7-LAT cells and ND7-neo control cells calculated. Experiments were again performed in duplicate and repeated three times. Averages of each duplicate experiment are shown.

Results:

MOI 1 0.1 0.01

ND7 Cell line Final Yield (pfuxlO⁴)

GK-neo 50 40 40 2 1 1 0.4 0.8 0.5

LAT-3-4 800 700 800 1500 1000 1300 20 20 20

LAT-3-5 1000 1000 700 800 700 800 4 3 2

LAT-3-6 20 30 50 40 40 40 0.8 0.4 1

LAT-3-9 600 500 700 500 700 300 20 50 20

LAT-3-16 400 400 500 80 70 90 8 10 10

LAT-4-3 1200 1500 1000 600 500 600 20 7 20

These results demonstrate that constitutive expression of the HSVl LAT ORF in ND7 cells greatly enhances the growth of HSVl in these cells.

Example 3 HSV constitutively expressing the HSVl LAT ORF shows enhanced growth on ND7 and BHK cells An HSVl strain 17+ derivative constitutively expressing the LAT ORF was prepared. Here the LAT ORF was under the control of the CMV IE promoter, terminated by a bovine growth hormone poly-A sequence from pcDNA3 (Invitrogen™), and inserted into the non-essential US5 gene together with a lacZ gene driven by an RSV promoter. A control virus expressing green fluorescent protein (GFP) in place of the LAT ORF was also constructed.

Plasmid pR20.5 consists of an RSV/LacZ/pA and a CMV/GFP/pA cassette in opposite (back-to-back) orientations separated by a central region derived from the HSV genome (nts 118,866-120,219) which allows expression of both the RVS and CMV driven genes during HSV latency. These sequences can be excised from the pGEM5 (Promega) plasmid backbone with Srfl as an oligonucleotide containing an Srfl site was inserted on either side of this cassette. The RSV promoter was excised from pRc/RSV (Invitrogen), lacZ/pA from pCHl 10 (Pharmacia), CMV/pA from pcDNA3 (Invitrogen) and GFP from pEGFP-Nl (Clontech) for the construction of plasmid pR20.5. The HSVl LAT ORF was inserted into plasmid pR20.5 in place of GFP gene.

The GFP gene was excised from pR20.5 with Xho I and Hind III and replaced by the HSV 1 LAT ORF as a Bgl II-Hind III fragment from PSP72LAT. The pR20.5 cassette was excised from the pGEM5 plasmid backbone with Srf l and inserted into pUS5, containing US5 flanking regions (nts 136,289-139,328) between the BamHI and EcoNI sites of pAT153 at a unique Xbal site previously inserted as an oligonucleotide at the unique Sad site of US5, resulting in plasmid pR20.5/US5. The CMV/LAT cassette from pR20.5/LAT was inserted into pR20.5 US5 by digestion of both plasmids with Spel and Xhol and ligation so as to replace CMV/GFP in pR20.5/US5 with CMV/LAT from pR20.5/LAT, giving pR20.5 US5/LAT. This plasmid was co-transfected together with purified HSVl strain 17+ genomic DNA into BHK cells allowing homologous recombination of the pR20.5/LAT cassette into the US5 gene giving virus strain 17+/pR20.5/US5/LAT.

Three recombinant, X-gal staining 17+/pR20.5/US5 LAT plaques were picked and plaque purified 5 times, providing three independently generated virus clones. These were tested for their ability to grow on BHK cells, ND7 cells, and ND7 cells constitutively expressing the LAT ORF (ND7-LAT-3-5 from Example 2 above), in comparison to the growth characteristics of a 17+/pR20.5/US5 control virus which had previously been produced. 17+/pR20.5 US5 is identical to 17+/pR20.5 US5/LAT, except with a GFP gene in place of the LAT ORF. Experiments were performed as before, in each case at three MOI's and harvesting 24 well plates 72 hours after infection. Final yields/per well are shown, and each experiment was performed in duplicate. The average of each duplicate experiment is shown. Results:

MOI 0.1 0.01 0.001 1 0.1 0.001 1 0.1 0.001

Final Yield (pfu) BHK (xl0⁶) ND7 (x 10⁵) ND7-Lat x (xlO⁵)

Virus

17+/pR20.5/US5 20 15 0.7 11 2 0.2 110 15 12

17+/pR20.5/US5/LAT-l 110 100 9 20 1 10 200 80 13

17+/pR20.5/US5/LAT-2 300 100 12 90 9 4 310 100 90

17+/pR20.5/US5/LAT-3 300 75 19 200 10 4 130 500 98

Constitutive expression of the HSVl LAT ORF from the HSVl genome results in a virus which shows enhanced growth in BHK cells and ND7 cells as compared to a control virus not constitutively expressing the LAT ORF. The comparative difference in growth between viruses which do and do not constitutively express the LAT ORF is less marked in ND7 cells which themselves already constitutively express the LAT ORF although enhanced growth of both types of virus is shown.

Example 4 HSV constitutively expressing the HSVl LAT ORF shows enhanced virulence in mice in vivo

17+/pR20.5/US5 and 17+/pR20.5/US5/LAT-l were intracranially inoculated into 3 week old female BALB/c mice as described previously (Coffin et al., 1996) at titres ranging from lO'-lO⁵ pfu in 20 μl, with 2 mice inoculated with each concentration of each virus. Mice were killed if showing signs of severe distress. Results show surviving mice at daily timepoints after inoculation with the LAT ORF expressing and control viruses.

Results: 17+/pR20.5/US5

Time after inoculation (days)

1 1 2 2 3 4 5 6 7

Titre Number of mice surviving l x lO⁵ 1 0 0 0 0 0 0 l x lO⁴ 1 1 0 0 0 0 0 l x lO³ 2 2 2 2 1 1 1

1 x lO² 2 2 2 2 2 2 2 l x lO¹ 2 2 2 2 2 2 2 17+/pR20.5/US5/LAT

Time after inoculation (days^')

Titre Number of mice surviving l x lO⁵ 0 0 0 0 0 0 0

1 x lO⁴ 0 0 0 0 0 0 0 l x lO³ 1 0 0 0 0 0 0 l x lO² 1 1 1 1 1 1 1

1 x lO¹ 2 2 2 2 2 2 1

HSVl constitutively expressing the LAT ORF shows significantly enhanced virulence after intracranial inoculation of mice as compared to a control virus in which the LAT ORF is not constitutively expressed.

Example 5 An HSV 1 ICPO deletion mutant shows enhanced wild-type growth kinetics on ND7 or BHK cells constitutively expressing the HSVl LAT ORF An HSVl strain 17+ ICPO deletion mutant (dI1403; Stow and Stow, 1986) was tested for growth on BHK-GKneo control cells, ND7-GKneo control cells, two representative BHK-LAT cell lines (BHK-LAT- 1-2 and BHK-LAT- 1-7 from example 1 above), and two representative ND7-LAT cell lines (ND7-LAT-3-5 and ND7-LAT-3-9 from example 2 above) at various MOIs. Results are shown as before as the total yield/well from a 24 well plate for the ICPO deleted virus at each MOI on each cell line after 36 or 72hrs respectively. Experiments were conducted in duplicate, and averages of each duplicate experiment are shown. Results

MOI 1 0.1 0.01 0.00

Cell line Final yield (pfu x 10²)

ND7-GK-neo 3.5 0.5 0.3 -

ND7-LAT-3-5 225 300 20 -

ND7-LAT-3-9 1250 120 9 -

BHK-GK-neo _ 40 3.2 0.6

BHK-LAT- 1-2 - 180 80 18

BHK-LAT- 1-7 - 450 200 10 An HSVl strain deleted for ICPO shows enhanced growth on ND7 or BHK cells constitutively expressing the LAT ORF as compared to control cells in which the LAT ORF is not expressed. ICPO deleted viruses otherwise show very poor growth on ND7 and BHK cells. At low MOI the enhanced growth on BHK and ND7 cells constitutively expressing the LAT ORF is sufficient to give growth levels similar to wild type HSVl on ND7 and BHK cells not expressing the LAT ORF at similar MOI. Expression of the LAT ORF can thus functionally replace ICPO in aiding virus growth under these circumstances.

Example 6 HSVl constitutively expressing a mutated LAT ORF shows no enhancement in growth

An HSVl strain 17+/pR20.5/US5/LATΔSal was produced which was identical to 17+/pR20.5/US5/LAT-l except here a frame shift was inserted into the LAT ORF in US5 by digestion of pSP72LAT with Sail, treatment with T4 DNA polymerase, and religation prior to insertion of the LAT ORF into pR20.5 as in Example 3. The HSVl LAT ORF contains a single Sail site near the centre of the ORF, and such treatment inserts a frame shift which would be expected to prevent the expression of the full length LAT ORF. However little effect on RNA structure would be expected. Thus if the LAT ORF functions as RNA the phenotypes of 17+/pR20.5 US5/LAT-l and 17+/pR20.5 US5/LATDSal would be expected to be similar, whereas if the LAT ORF functions as a protein, none of the LAT ORF associated effects in examples 1-5 with

17+/pR20.5 US5/LAT-l would be expected with 17+/pR20.5/US5/LATDSal, as here no functional LAT ORF protein would be expected to be produced. For these experiments 3 independently produced recombinant virus plaques were picked and plaque purified, providing three independent virus clones.

Results

17+/pR20.5/US5/LAT-l and the three isolates of 17+/pR20.5/US5/LATDSal were compared for growth on ND7 and BHK cells as in Eexample 3, but here only at a single MOI. Experiments were performed in duplicate and averages of each duplicate experiment are shown. The effect of enhanced growth which was seen in Example 3 with viruses constitutively expressing the LAT ORF was not seen with 17+/pR20.5 US5/LATΔSal. In fact all three isolates of 17+/pR20.5/US5/LATΔSal were found to grow particularly poorly on both cell types. This was assumed to be due to the high level expression of RNA antisense to ICPO in the 17+/pR20.5/US5/LATΔSal viruses, which without the effect of functional LAT ORF expression, resulted in a virus with reduced growth kinetics on ND7 and BHK cells due to reduced expression of ICPO.

MOI 0.001 0.01 Virus Final yield (pfu) BHK (xl0⁴) ND7 (xl0³)

17+/pR20.5/US5 70 30

17+/pR20.5/US5/LAT-l 900 800

17+/pR20.5/US5/LATDSal-l 0.6 0.3 17+/pR20.5/US5/LATDSal-2 1 0.1

17+/pR20.5/US5/LATDSal-3 1.2 0.4

The effects of enhanced virus growth resulting from constitutive expression of the LAT ORF in Examples 1-5 are due to the translation of a protein from the LAT ORF encoding RNA, rather than an effect mediated by the LAT ORF RNA itself.

Example 7 Detection of the LAT ORF protein product in cells infected with HSV constitutively expressing the HSVl LAT ORF The HSVl strain 17+ LAT ORF contains 46 proline residues out of a total of 274 amino acids, and is thus a particularly proline rich protein. On the other hand it contains only 4 methionine residues. It would thus be expected to label efficiently with 14C- proline but less efficiently with 35S-methionine. Thus to detect the protein in vitro BHK cells were infected at an MOI of 1 in a twenty four well plate with either 17+/pR20.5/US5, 17+/pR20.5/US5/LAT-l, or mock infected, in duplicate. Either ³⁵S- methionine (50 μCi/well) or ¹⁴C-proline (2.5 μCi/well; both Amersham) were included in the media after inoculation with the virus, and cells were harvested for SDS polyacrylamide gel electrophoresis after 24hrs. Results

Gels were dried, and after autoradiography a very distinct band at about 25 kDa (around the predicted size for the LAT ORF from its deduced amino acid sequence) could be seen in the ¹⁴C-proline labelled sample infected with 17+/pR20.5/US5/LAT-l. This band was not present in the ¹⁴C-proline labelled sample infected with 17+/pR20.5/US5, indicating efficient transcription and translation of the LAT ORF in cells infected with 17+/pR20.5/US5/LAT-l. A slightly larger and considerably fainter band corresponding to expression of the GFP protein in 17+/pR20.5/US5 could be seen in 17+/pR20.5 US5 infected samples labelled with ¹⁴C-proline which was not present in the samples infected with 17+/pR20.5/US5/LAT- 1. GFP has been replaced by the LAT ORF in

17+/pR20.5 US5/ LAT-1, and thus the presence of this band was expected. No novel, distinct band present in only one or other of the virus infected samples could be seen after labelling with ³⁵S-methionine. The LAT ORF would be expected not to label efficiently with ⁵S-methionine, as discussed above. Thus results indicative of efficient expression of the LAT ORF in 17+/pR20.5 US5/LAT- 1 , similar to the expression of GFP in 17+/pR20.5/US5, has been demonstrated here by these means.

These results show that a novel protein product can be detected in cells infected with an HSV strain constitutively expressing the HSVl LAT ORF which is not present in cells infected with an HSV strain not constitutively expressing the LAT ORF. Thus, when present in an RNA processed as a poly-adenylated mRNA the LAT ORF can be efficiently translated producing a protein product which can easily be detected.

Example 8 HSV grown on LAT ORF-containing cells give enhanced immediate early gene expression as compared to HSV grown on non LAT ORF containing cells or cells containing a mutated LAT ORF.

To explore the level at which the LAT ORF-encoded polypeptide might function, effects on HSV IE gene expression were explored. We have found that the LAT ORF does not directly trans-activate the promoters controlling the expression of the HSV IE proteins in co-transfection assays where reporter constructs with the respective IE promoters driving a CAT reporter gene were transfected together with a plasmid encoding the LAT ORF under CMV promoter control in ND7 and BHK cells (Coffin et al, Journal of General Virology 1998, vol 79, pp 3019-3026).

However further work has demonstrated an effect of LAT ORF expression on HSV IE gene expression: ND7 cells, ND7 cells containing the LAT ORF, or ND7 cells containing the mutated LAT ORF (as in Examples 2 and 6 above) were infected with HSVl strain 17+ at MOI=l and cells harvested 8hrs after infection. Western blots of cell extracts were then performed and probed with either anti-ICPO, anti-ICP27 or anti-ICP4 antibodies. This experiment showed that while no ICPO, ICP27 or ICP4 could be detected in cell extracts from the ND7 cells at this time point after infection, each of the proteins ICPO, ICP27 and ICP4 could easily be detected in the LAT ORF-containing ND7 cells (strong bands detected at the expected molecular weight with each of the antibodies), and ICP27 and ICP4 could just be detected in the mutated LAT ORF containing cells (faint bands could be detected at the expected molecular weight with each of the antibodies).

From this it can be concluded that while the LAT ORF probably does not directly trans-activate HSV IE promoters (Coffin et al, Journal of General Virology 1998, vol 79, pp 3019-3026), LAT ORF expression does result in increased HSV IE gene expression. Thus the mechanism by which the LAT ORF functions must result in increased induction of expression of these HSV proteins at least, which if this were to occur during HSV latency would be expected to result in virus reactivation. It can also be concluded that the mutated LAT ORF used here probably still retains some residual LAT ORF activity as IE gene expression is slightly enhanced in mutant LAT ORF containing ND7 cells as compared to control ND7 cells, even though no effects on virus growth (Example 6) could be seen.

References

Coffin R.S., Maclean A.R., Latchman D.S., Brown S.M. (1996) Gene Therapy 3, 886- 891.

Coffin et al, Journal of General Virology 1998, vol 79, pp 3019-3026

Doerig, C, Pizer, L. I. & Wilcox, C. L. (1991), Journal of Virology 65, 2724-2727.

Fareed, M. U. and Spivack, J. G. (1994), Journal of Virology 68, 8071-8081. Farrel, M. J., Dobson, A. T. & Feldman, L. T. (1991). Proceedings of the National

Academy of Sciences, USA 88, 790-794.

Fraser, N. W., Block, T. M. & Spivack, J. G. (1992). Virology 191, 1-8.

Ho, D. Y. (1992). Progress in Medical Virology 39, 76-115.

Kemp, L. M. and Latchman, D. S. (1989). Virology 11 , 607-610. Latchman, D.S. (1990). International Journal of Experimental Pathology 71, 133-141.

Morgenstern, J. P. and Land, H. (1990). Nucleic Acids Research 18, 1068.

Rodahl, E. and Haarr, L. (1997). Journal of Virology 71, 1703-1707.

Spivack, J. G. and Fraser N. W. (1987). Journal of Virology 61, 3841-3847.

Spivack, J. G., Woods, G. M. & Fraser N. W. (1991). Journal of Virology 65, 6800-6810. Thompson, R. L. and Sawtell, N. M. (1997), Journal of Virology 71, 5432-5440.

Stevens, J. G., Wagner, E. K., Devi-Rao, G. B., Cook, M. L. & Feldman L. T. (1987).

Science 235, 1056-1059

Stow, N. D. and Stow, E. C. (1986) Journal of General Virology, 61: 2571-2585.

Wechsler, S. L., Nesburn, A. B., Watson, R., Slanina, S. N. & Ghiasi, H. (1988). Journal of Virology 62, 4051 -4058.

Woods, J. N., Bevan, S. J., Coote, P. R., Dunn, P. M., Harmer, A., Hogen, P., Latchman,

D. S., Morrison, C, Rougon, G., Theveniou, M & Wheatley, S. (1990). Proceedings of the Royal Society London (B) 241, 187-194.

Claims

1. A polynucleotide in substantially isolated form consisting essentially of an open reading frame encoded by a he╧åes simplex virus (HSV) latency-associated transcript (LAT) operably linked to a homologous or heterologous regulatory sequence permitting expression of a LAT ORF polypeptide in a cell.

2. A polynucleotide according to claim 1 wherein said heterologous regulatory sequence is a non-LAT promoter.

3. A polynucleotide according to claim 1 or 2 wherein said heterologous regulatory sequence is a viral promoter.

4. A polynucleotide according to claim 1 or 2 wherein said heterologous regulatory sequence is a mammalian promoter permitting expression of said LAT in a mammalian cell.

5. A polynucleotide according to any one of claims 2 to 4 wherein said promoter is an inducible promoter.

6. A polynucleotide according to any one of claims 2 to 4 wherein said promoter is a constitutive promoter.

7. A polynucleotide according to claim 5 or 6 wherein said promoter is a tissue-specific promoter.

8. A nucleic acid vector comprising a polynucleotide as defined in any one of the preceding claims.

9. A vector according to claim 8 further comprising mammalian genomic sequences flanking said polynucleotide.

10. A vector according to claim 8 or 9 further comprising HSV genomic sequences flanking said polynucleotide.

11. A viral strain comprising a polynucleotide as defined in any one of claims

1 to 7.

12. A viral strain according to claim 11 which is an HSV strain.

13. An HSV strain according to claim 13 which is HSV-1 or HSV-2.

14. A cell line comprising a polynucleotide according to any one of claims 1 to

15. A cell line according to claim 14 which is a mammalian cell line.

16. A non-mammalian cell comprising a polynucleotide according to claim 1.

17. A cell according to claim 16 which is a prokaryotic cell or a yeast cell

18. A HSV LAT polypeptide in substantially isolated form obtainable by incubating a cell or cell line according to any one of claims 14 to 17 under conditions that allow for expression of said LAT ORF.

19. A HSV LAT polypeptide in substantially isolated form obtainable by incubating a polynucleotide according to any one of claims 1 to 7 in a cell-free protein expression system under conditions that allow for expression of said LAT ORF.

20. A method for identifying a substance capable of interacting with an HSV LAT polypeptide, which method comprises incubating a candidate substance with an

HSV LAT polypeptide and determining whether the candidate substance interacts with the LAT polypeptide.

21. A method for identifying a substance capable of reducing or preventing reactivation of a he╧åes simplex virus from latency, which method comprises incubating a candidate substance with an HSV LAT polypeptide and determining whether the candidate substance interacts with the LAT polypeptide.

22. A method according to claim 30 or 31 wherein the LAT polypeptide is as defined in claim 18 or 19.

23. A substance identified by the method of any one of claims 20 to 22 for use in a method of treating HSV infection in a human.

24. A method of producing an HSV strain according to claim 22 which method comprises introducing a polynucleotide as defined in any one of claims 1 to 7 into the genome of a he╧åes simplex virus.

25. A method for propagating in vitro an HSV strain lacking a functional ICPO gene which method comprises using a cell line according to claim 15 as a complementing cell line.

26. An HSV strain obtainable by the method of claim 25.