WO2004009027A2

WO2004009027A2 - Method and composition for treating and preventing herpesvirus infection

Info

Publication number: WO2004009027A2
Application number: PCT/US2003/022828
Authority: WO
Inventors: Scott Morham; Kenton Zavitz; Adrian Hobden
Original assignee: Myriad Genetics, Inc
Priority date: 2002-07-19
Filing date: 2003-07-21
Publication date: 2004-01-29
Also published as: AU2003254089A8; WO2004009027A3; AU2003254089A1

Abstract

The invention provides compositions and methods for treating HSV infection. The compositions and methods of the invention target the HSV budding pathway.

Description

METHOD AND COMPOSITION FOR TREATING AND PREVENTING

HERPESVΓRUS INFECTION

Field of the invention The present invention relates generally to treating and preventing viral infections and associated diseases, and particularly to methods and compositions for treating and preventing herpesvirus infection.

Background of the Invention Herpesviruses are one of the most common human pathogens. Members of the herpesvirus family include herpesvirus- 1, herpesvirus-2, Varcella-zoster virus

(herpesvirus-3, chicken pox), and Epstein-Barr virus (herpesvirus-4). Herpesvirus- 1 (hereafter referred to as "HSV-1") commonly causes herpes labialis (also called oral herpes), which are highly infectious open sores that crust over before healing. HSV-1 causes cold sores, fever blisters, and eye and brain infection. Latent infections of HSV-1 are without signs of disease and are characterized by establishment of latency in sensory neurons. Reactivation can cause recurrent lesions close or identical to the initially infected area. HSV-1 can also cause genital herpes, although this is less probable. In addition to cold sores and genital herpes, HSV-1 and HSV-2 can cause other diseases. Examples of such diseases include herpes simplex encephalitis, a rare but potentially fatal herpetic infection of the brain; neonatal herpes, a rare but potentially severe HSV infection in a newborn (resulting from transmission of the virus from the mother to the baby during delivery); herpetic whitlow, an HSV infection of the finger (acquired either from transfer of the infection from another part of the body or from direct contact with another party having an HSV infection); and herpes keratitis - an HSV infection of the eye (one of the most common causes of blindness). Thus, herpesvirus infection of humans is a significant health problem.

Genital herpes is primarily treated with suppressive and episodic therapies. Suppressive therapy is used to treat outbreaks before they occur, while episodic therapy treats outbreaks when they occur. Treatment with valacyclovir HCl, acyclovir, and famciclovir, can be used in both suppressive and episodic therapies.

Currently there is no known cure for HSV-1 infection. The available antiviral therapies are not completely effective and there is a chance that the virus will become resistant to the treatment. Thus, there is a clear need for improved methods and compositions for treating HSV-1. Epstein-Barr virus (human herpesvirus-4), hereafter referred to as "EBV", occurs worldwide. In fact, most people become infected with EBV during their lives. A large percentage of adults in the United States have been infected. Infants are susceptible to EBV as soon as maternal antibody protection present at birth disappears. Many children become infected with EBV, and these infections usually cause no symptoms. The symptoms of EBV infection in children can be indistinguishable from the symptoms of other typical childhood illnesses. Individuals not infected as a child have a risk of being infected during adolescence or young adulthood, which often causes infectious mononucleosis (mono). Symptoms of infectious mononucleosis include fever, sore throat, and swollen lymph glands, less often a swollen spleen or liver involvement may develop. Rarely, heart problems or involvement of the central nervous system occur. Infectious mononucleosis is almost never fatal. The symptoms of infectious mononucleosis usually resolve in 1 or 2 months, but EBV remains dormant or latent in a few cells in the throat and blood for the rest of the infected person's life. Periodically, the virus can reactivate and is commonly found in the saliva of infected persons. Reactivation usually occurs without symptoms of illness. EBV is thought to be associated with a number of other diseases including Burkitt's lymphoma, nasopharyngeal carcinoma, and Hodgkin's disease. Diseases caused by EBV are particularly common among people with reduced immunity. EBV is associated with a tumor often found in organ transplant patients which is referred to as post-transplant lymphoproliferative disease. The immune systems of such patients are usually artificially suppressed by drug therapy to help prevent the body from rejecting the new organ. Individuals infected with HIV, and have AIDS, also have reduced immunity and commonly suffer from oral hairy leukoplakia, a condition involving considerable replication of EBV in cells along the edge of the tongue. It has also been suggested that the high incidence of malaria in countries where Burkitt's lymphoma is prevalent may also play a role in the disease by suppressing the body's immune system.

Scientists are finding it difficult to explain why the virus causes a relatively mild disease like glandular fever in some people and malignant tumors in others. Genetic factors may play a role. Regardless, treatments are needed to combat EBV. The mechanisms that viruses use to depart an infected cell are only beginning to be understood on a general level. Recently several proteins involved in the egress of certain viruses from infected cells have been discovered. Studies with the HIV-1 gag protein have demonstrated an interaction between a human protein, tsglOl, and gag which is critical for viral budding from the host cell (Garrus et al. Cell 107:55-65 (2001)). Related studies have established a common budding theme among retrovirus. It appears that protein-protein interactions between specific host cell proteins and viral proteins are critical for viral budding. Rous sarcoma virus (RSV), equine infectious anemia virus (ELAN), as well as other retroviruses, were shown to have late domains with characteristic amino acid sequence motifs; the late-domain motifs are critical binding motifs that facilitate the interaction of the viral proteins with host cell proteins. See, e.g., Puffer et al. J. Virol. 72:10218-10221 (1998) and Campbell et al. J. Virol. 71:4425-35 (1997). Unexpectedly, it was discovered that specific herpes virus proteins have late- domain motifs which can be manipulated or modulated to affect viral budding and egress. This discovery now provides an entirely new class of HSV antivirals.

Summary of the Invention The present invention provides for the treatment and prevention of Herpesvirus (HSV) infection. The invention generally relates to treating and preventing HSV infection by inducing an altered budding phenotype. Ln particular, the invention provides compositions and methods that affect the ability of HSV, or a variant thereof, to utilize the host's cellular machinery for viral budding and egress. The invention relates to the discovery that interfering with the normal ability of viruses to utilize the host cell's vesicular trafficking, recycling, and/or vacuolar sorting machinery for viral propagation can reduce the infectivity of the virus. Accordingly, the invention provides HSV treatment methods and compositions based on the modulation of viral budding. Modulation of the normal HSV budding mechanism can enhance the host's immune response against the virus. The invention therefore provides compositions and methods for enhancing an immune response against HSV.

In one embodiment, the invention provides a method of affecting the ability of HSV to bud. According to this method, an effective amount of a composition capable of modulating the budding of the virus is administered to an individual in need of treatment. The composition can be provided as a nucleic acid, a polypeptide, a peptide, a vaccine, a small molecule therapeutic, and the such, as long as the budding modulation composition affects the ability of the virus to bud and/or induces an altered budding phenotype. In one aspect of this embodiment, a HSV protein that participates in the normal budding pathway of the virus can be provided in a mutant form, e.g., a nucleic acid encoding a mutant protein or a mutant protein itself, to an infected host cell, in order to affect the budding of the virus or induce an altered budding phenotype. Ln an aspect of this embodiment, the budding of HSV is inhibited partially or completely. In another aspect of this embodiment, the budding of HSV is rendered dysfunctional or abnormal. In another embodiment, the invention provides a method of treating an individual infected with HSV, or individual in need of prophylactic treatment, by enhancing or stimulating an immune response against HSV. According to one aspect of this embodiment, the method of includes the step of administering to an individual an effective amount of a composition capable of affecting HSV budding. According to another aspect of this embodiment, the method of includes the step of administering to an individual an effective amount of a composition capable of inducing an HSV altered budding phenotype. The composition can be provided as a nucleic acid, a polypeptide, a peptide, a vaccine, a small molecule therapeutic, and the such, as long as the budding modulation composition affects the ability of the virus to bud and/or induces an altered budding phenotype. The administered composition is capable of modulating the normal pathway which HSV utilizes for budding and egress. Modulation of the normal HSV pathway for budding and egress can lead to the aberrant production of viral particles. The aberrant production of viral particles can cause an altered budding phenotype. The altered budding phenotype can stimulate/enhance an immune response in the host against HSV and HSV infected cells. In yet another embodiment, the present invention provides a method of treating an individual infected with HSV by modulating the ability of a virus to bud from a cellular membrane in a host cell by administering, to a patient in need of therapeutic or prophylactic treatment, a mutant HSV protein encoding a late-domain motif that can affect the ability of the virus to bud or by administering to the patient a nucleic acid encoding such a mutant HSV protein. Such treatments can be used to suppress the symptoms of HSV, i.e., suppressive therapy, or for treating episodic outbreaks, i.e., episodic therapy. For example, a mutant HSV protein that interacts with a host cell's protein(s) to affect budding from a particular membrane, can be provided by nullifying the late-domain motif of a wild-type or modified HSV protein that is capable of mediating the budding process. Preferably, a nucleic acid encoding a mutant HSV protein sufficient for viral particle assembly but devoid of late-domain motifs is incorporated into a plasmid vector which is injected directly into a patient. The nucleic acid can also be delivered into a patient by infecting the patient with a recombinant live vector (e.g., recombinant viral or bacterial vector) carrying the nucleic acid. Preferably, the nucleic acid is administered to a patient by gene gun or powder jet or an equivalent device thereof.

The invention further provides a method of identifying compounds that modulate the activity of a viral protein host cell protein protein-protein interaction that is involved in a viral egress and/or budding pathway. More particularly, the method involves identifying compounds that modulate an interaction between a viral protein having a late- domain motif and a host cell protein that interacts with the late-domain motif containing protein. According to this embodiment an assay system is employed to detect compounds and/or compositions that affect the ability of the virus to propagate itself. The assay system targets host cell proteins that are utilized by the virus for budding that interact with the viral late-domain motif containing protein(s). Ln one aspect of this embodiment, the assay is configured to identify compounds that modulate the activity of a specific protein involved in a viral budding and/or egress pathway. In another aspect of this embodiment, the assay is configured to identify compounds that modulate a protein-protein interaction that is involved in the viral budding and/or egress pathway. In one aspect of this embodiment, the host protein that is modulated is selected from the group consisting of TSG 101, NEDD4, a NEDD4-like protein, ALP 1 , a host cell protein containing a WW domain, or fragments, derivatives, or homologs thereof. Ln another aspect of this embodiment the viral late-domain motif containing protein has a YXXL, PXXP, or PPXY late-domain motif, or fragments, derivatives, or homologs thereof. As the skilled artisan is aware, the screening assays of the invention can be accomplished in any manner as long as they examine the affect of a particular treatment on a host cell viral protein protein-protein interaction involved in viral budding (or egress) or a host cell biochemical pathway involved in viral egress.

Detailed Description of the Invention The present invention provides methods for treating and preventing HSV infection. Furthermore, the invention provides compositions for use in the methods for treating and preventing HSV infection. Included within the scope of the invention are methods for identifying compositions useful for treating and preventing HSV infection. In particular, the invention provides compositions and methods that affect the ability of HSV or a variant thereof to utilize the host's cellular machinery for viral budding and egress. The invention relates to the discovery that interfering with the normal ability of viruses to utilize the host cell's vesicular trafficking, recycling, and vacuolar sorting machinery for viral budding and egress can reduce the infectivity of the virus. Additionally, interfering with the viral budding/egress pathway can enhance and stimulate the host's immune response against the virus. HSV infects a host by first entering the cell through the plasma membrane. The virus makes it way eventually to the cell's nucleus where it begins to replicate itself. After infection of a host cell, HSV is propagated to other cells through a budding/egress mechanism. Immature HSV particles initially appear to bud from the nuclear membrane. Maturation of the immature HSV particles occurs in several of the host cell's organelles. The details of the maturation pathway are not completely understood. Eventually the mature particles egress from the plasma membrane where the mature virus can go on to infect other cells. Achievement of egress is certainly a complex orchestrated event involving the interplay of viral and host proteins. It was discovered that HSV has and encodes a number of proteins that have a late- domain motif. Late-domain motif containing proteins are known to be central in viral budding pathways. Proteins that have late-domain motifs interact with specific proteins of the host cell during viral budding. HSV has proteins and genes encoding proteins that fall within the YXXL, PPXY, and PT(S)AP amino acid motifs which are hereafter collectively referred to as "late-domain motifs" (where Y is tyrosine, P is proline, T is threonine, S is serine, A is alanine and X stands for any amino acid). As used herein, the term "late-domain motif refers to the above-mentioned motifs as well as functional homologs which interact with host cell proteins to affect viral budding. Examples of functional homologs of late-domain motifs include ΘXXΨ, PPX , and PXXP, wherein θ can be any aromatic amino acid (including tyrosine, phenylalanine (F), tryptophan (W) and histidine(H)), can be any bulky hydrophobic amino acid, particularly large aliphatic amino acids (leucine, isoleucine (I), and valine (V)). Non-limiting examples of the ΘXXΨ late-domain motif include FXXL, YXXI, YXXV, WXXL, and the such. Non-limiting example of the PPX late-domain motif include PPXF, PPXW, PPXH, and the such. As will be understood by one of skill in the art, the term "late-domain motif encompasses any variation or homolog of these motifs that binds a host cell protein during the viral budding process. Table 1 below lists a number HSV-1 proteins or genes encoding late-domain motifs.

Table 1:

Late Domain Motifs in HSV-1 GenBank Accession Number NC 001806

Table 2:

Late Domain Motifs in HS V-2

GenBank Accession Number NC 001798

Table 3:

Late Domain Motifs in ΕBV

GenBank Accession Number NC 001345

Late-domain motifs appear to specifically bind several proteins from the host cell. The PTAP/PSAP motif binds the protein TSGIOI, and it is thought that similar motifs falling within the PXXP motif in proteins can bind to SH3 domain containing partners. The YXXL motif binds to the AP-50 subunit of the AP-2 complex. Although it is believed that the YXXL motif binds AP-50, it is contemplated that the motif can bind ALPl and the methods of the invention apply to the use of this protein (Vincent et al. Mol. Cell Biol. 23:1647-1655 (2003). The PPXY motif binds to NEDD4. The PPXY motif is thought to bind to proteins having WW domains. The invention contemplates affecting an interaction between any HSV protein and its interacting partner as long as the interaction is related to the viral budding pathway. As used herein, the term "late- domain motif binding partner" refers to the host cell protein(s) that binds to the viral protein containing the late-domain motif and affects viral budding. Late-domain motif binding partners include TSGIOI, NEDD4, AP-50, as well as other host cell proteins which bind a viral late-domain motif containing protein and affect viral budding. The invention provides a method of affecting the ability of HSV to bud.

According to this method, an effective amount of a composition capable of modulating the budding of the virus is administered to an individual in need of such treatment. The composition can be provided as a nucleic acid, a polypeptide, a peptide, a vaccine, a small molecule therapeutic, and the such, as long as the budding modulation composition affects the ability of the virus to bud. Administration of an effective amount of a composition capable of modulating viral budding can cause an altered budding phenotype. In a preferred embodiment, the altered budding phenotype can enhance an immune response against the virus or virus infected cells. For example, a HSV protein which participates in the normal budding pathway for the virus can be provided in a mutated form, e.g., a nucleic acid encoding a mutant protein or a mutant protein itself, to an infected host cell, in order to affect the budding of the virus. Ln another preferred embodiment, the budding of HSV is inhibited partially or completely. In another preferred embodiment, the budding of HSV is rendered dysfunctional or abnormal. In another embodiment the composition induces an HSV altered budding phenotype that can stimulate or enhance an immune response against HSV and/or HSV infected cells.

As used herein, the term "altered budding phenotype" or "HSV altered budding phenotype" refers to the phenotype that a host cell or tissue displays upon modulation of a viral-protein host-protein protein-protein interaction involved in viral budding. The term "HSV altered budding phenotype" is in comparison to the normal HSV budding phenotype which is one characterized by normal viral budding and extracellular virus release. Thus, in a cell infected with a wild-type HSV strain, the virus will bud normally. In an "HSV altered budding phenotype" cell, viral particles may never reach maturity (i.e., never form wild-type viral particles or virus), and/or have an accumulation of virus in a particular cellular compartment. Cells having an HSV altered budding phenotype may also display different markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses. The difference in markers serves as a tag for the immune system to recognize these cells or tissues with the altered phenotype. The HSV altered budding phenotype is readily determined by one of skill in the art. Markers in control host cells, wild-type HSV infected cells, and cells which have had a viral-protein host-protein protein-protein interaction modulated, can be quantitated using standard techniques to determine the altered budding phenotype. A non-limiting example is a comparison of the type and number of viral particles found in, e.g. , the cytoplasm, the uninfected host is expected to have no viral particles in the cytoplasm, the wild-type HSV infected host cell is expected to have a given amount of virus in the cytoplasm, and an altered budding phenotype cell is expected to have a substantially different amount of virus in the cytoplasm. Electron microscopy is another useful tool for determining a HSV altered budding phenotype by examination of the cellular localization of viral particles and proteins. Thus, an altered budding phenotype is recognizable by one of ordinary skill in the art by making such comparisons using art- known techniques. As used herein, the term "altered egress phenotype" refers to the phenotype that the host cell displays upon modulation of a host cell protein-protein interaction involved in viral egress. The term "altered egress phenotype" is in comparison to the normal egress phenotype which is one characterized by normal viral egress and extracellular virus release. Thus, in a cell infected with a wild-type strain, the virus will egress normally. In a cell having an "altered egress phenotype", the virus may never reach maturity, i.e., form wild-type virus, or alternatively, have an accumulation of virus in a particular cellular compartment. Altered egress phenotype displaying cells may also display different molecular markers on the cell surface or within the cell as compared to normal uninfected cells or cells infected with wild-type viruses. The difference in markers can serve as a signal for the immune system to recognize the cells or tissues with the altered phenotype. The altered egress phenotype is readily determined by one of skill in the art. For example, markers in uninfected control host cells, virus infected cells, and infected cells which have had a protein-protein interaction modulated, or a protein modulated, can be quantitated using standard techniques to determine the altered egress phenotype. A non-limiting example is a comparison of the type and number of viral particles found in, e.g., the cytoplasm, the uninfected host is expected to have no viral particles in the cytoplasm, the wild-type virus infected host cell is expected to have a given amount of virus in the cytoplasm, and an altered egress phenotype cell is expected to have a substantially different amount of virus in the cytoplasm. Electron microscopy is another useful tool for determining an altered egress phenotype by examination of the cellular localization of viral particles and proteins. Thus, an altered egress phenotype is recognizable by one of ordinary skill in the art by making such comparisons.

As used herein, the term "HSV" or "Herpesvirus" refers generally to any of the herpesvirus group of viruses. Particular members of HSV include, but are not limited to, hepersirus-1, herpesvirus-2. and hepervirus-4 (Epstein-Barr virus). As used herein, the term "viral-like particle" refers to nucleocapsids or any HSV protein containing particles which in their wild-type form are capable of being secreted or are capable of budding.

As used herein, the term "homologue," when used in connection with a first native protein or fragment thereof that is discovered, according to the present invention, to interact with a second native protein or fragment thereof, means a polypeptide that exhibits an amino acid sequence homology and/or structural resemblance to the first native interacting protein, or to one of the interacting domains of the first native protein such that it is capable of interacting with the second native protein. Typically, a protein homologue of a native protein may have an amino acid sequence that is at least 50%, preferably at least 75%, more preferably at least 80%, 85%, 86%, 87%, 88% or 89%, even more preferably at least 90%, 91%, 92%, 93% or 94%, and most preferably 95%, 96%, 97%, 98% or 99% identical to the native protein. Examples of homologues may be the ortholog proteins of other species including animals, plants, yeast, bacteria, and the like. Homologues may also be selected by, e.g., mutagenesis in a native protein. For example, homologues may be identified by site-specific mutagenesis in combination with assays for detecting protein-protein interactions, e.g., the yeast two-hybrid system described below, as will be apparent to skilled artisans apprised of the present invention. Other techniques for detecting protein-protein interactions include, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, and the like. As used herein, the term "derivative," when used in connection with a first native protein (or fragment thereof) that is discovered, according to the present invention, to interact with a second native protein (or fragment thereof), means a modified form of the first native protein prepared by modifying the side chain groups of the first native protein without changing the amino acid sequence of the first native protein. The modified form, i. e. , the derivative should be capable of interacting with the second native protein. Examples of modified forms include glycosylated forms, phosphorylated forms, myristylated forms, ribosylated forms, ubiquitinated forms, prenylated forms, and the like. Derivatives also include hybrid or fusion proteins containing a native protein or a fragment thereof. Methods for preparing such derivative forms should be apparent to skilled artisans. The prepared derivatives can be easily tested for their ability to interact with the native interacting partner using techniques known in the art, e.g., protein affinity chromatography, affinity blotting, in vitro binding assays, yeast two-hybrid assays, and the like.

As used herein, the term "interacting" or "interaction" means that two protein domains, fragments or complete proteins exhibit sufficient physical affinity to each other so as to bring the two "interacting" protein domains, fragments or proteins physically close to each other. An extreme case of interaction is the formation of a chemical bond that results in continual and stable proximity of the two entities. Interactions that are based solely on physical affinities, although usually more dynamic than chemically bonded interactions, can be equally effective in co-localizing two proteins. Examples of physical affinities and chemical bonds include but are not limited to, forces caused by electrical charge differences, hydrophobicity, hydrogen bonds, van der Waals force, ionic force, covalent linkages, and combinations thereof. The state of proximity between the interaction domains, fragments, proteins or entities may be transient or permanent, reversible or irreversible. In any event, it is in contrast to and distinguishable from contact caused by natural random movement of two entities. Typically, although not necessarily, an "interaction" is exhibited by the binding between the interaction domains, fragments, proteins, or entities. Examples of interactions include specific interactions between antigen and antibody, ligand and receptor, enzyme and substrate, and the like.

An "interaction" between two protein domains, fragments or complete proteins can be determined by a number of methods. For example, an interaction can be determined by functional assays such as the two-hybrid systems. Protein-protein interactions can also be determined by various biophysical and biochemical approaches based on the affinity binding between the two interacting partners. Such biochemical methods generally known in the art include, but are not limited to, protein affinity chromatography, affinity blotting, immunoprecipitation, and the like. The binding constant for two interacting proteins, which reflects the strength or quality of the interaction, can also be determined using methods known in the art. See Phizicky and Fields, Microbiol. Rev., 59:94-123 (1995).

The term "protein fragment" as used herein means a polypeptide that represents a portion of a protein. When a protein fragment exhibits interactions with another protein or protein fragment, the two entities are said to interact through interaction domains that are contained within the entities.

As used herein, the term "domain" means a functional portion, segment or region of a protein, or polypeptide. "Interaction domain" refers specifically to a portion, segment or region of a protein, polypeptide or protein fragment that is responsible for the physical affinity of that protein, protein fragment or isolated domain for another protein, protein fragment or isolated domain.

A method of treating an individual infected with HSV is also provided. The method involves enhancing an immune response against HSV in an individual in need of such treatment. The method of this embodiment includes the step of administering an effective amount of a composition capable of affecting HSV budding to an individual infected with HSV. The composition can be provided as a nucleic acid, a polypeptide, a peptide, a vaccine, a small molecule therapeutic, and the such, as long as the composition affects the ability of the virus to bud. The administered composition interferes with the viral budding pathway and is capable of modulating the normal pathway which HSV utilizes for budding and egress. Modulation of the normal HSV pathway for budding and egress can lead to the aberrant production of viral particles. It is contemplated that enhancement of the host's immune response can occur through a variety of mechanisms. For example, the aberrant production of viral particles can cause lysis of the host cell and phagocytosis by the host's immune systems cells which can lead to an enhanced the immune response against HSV.

The invention provides a method of screening for modulators of the HSV budding process. According to this method, a protein-protein interaction assay system is provided which allows for the detection of modulators of an interaction between a HSV late- domain motif containing protein and its late-domain motif binding partner. In a preferred aspect of this embodiment, the assay is used to identify a compound or composition that inhibits viral budding by disrupting the protein-protein interaction. The assay system employed for detecting the protein-protein interaction can be any assay capable of detecting protein-protein interactions and/or a lack of such an interaction. For example, the protein-protein interaction detection assays of the invention can be based on a yeast two-hybrid based system. Another system capable of detecting protein-protein interactions is FRET (Fluorescence Resonance Energy Transfer) based. The FRET based system involves labeling the test proteins (or fragments thereof) with a matched pair of fluorophores wherein irradiation of one of the members of the pair with its characteristic wavelength of energy will allow the transmission of part of this energy to the second member of the pair which can then be detected, the transfer of energy only occurs when both members of the pair are in close proximity to one another, i.e., at the interface of a protein-protein interaction.

1. General Methods

The treatment methods of the invention involve administering to an individual an effective amount of a compound or composition capable of affecting the ability of HSV to bud. The step of "administering" can be accomplished by any method capable of delivering the composition or compound to the cells of an individual to be treated. Examples of such administering steps include gene therapy methods where a nucleic acid encoding a HSV protein, a mutant HSV protein, a peptide, a mutant HSV virus (as in a vaccine), and the such, is administered to an individual in need of treatment. The nucleic acid is designed to have a gene encoding an activity that affects HSV budding. Described in the following section are some methods that may be employed for delivering compositions to a cell to affect HSV budding. Any suitable gene therapy methods may be used for purposes of the present invention. Generally, the exogenous nucleic acid of the present invention is incorporated into a suitable expression vector and is operably linked to a promoter in the vector such that the promoter can drive the transcription from the exogenous nucleic acid. Suitable promoters include, but are not limited to, viral transcription promoters derived from adenovirus, simian virus 40 (SV40) (e.g., the early and late promoters of SV40), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) (e.g., CMV immediate-early promoter), human immunodeficiency virus (HIV) (e.g., long terminal repeat (LTR)), vaccinia virus (e.g., 7.5K promoter), and herpes simplex virus (HSV-1) (e.g., thymidine kinase promoter). Where tissue-specific expression of the exogenous gene is desirable, tissue-specific promoters may be operably linked to the exogenous gene. In this regard, a CD4+ T cell-specific promoter will be most desirable.

In one embodiment, the exogenous nucleic acid is incorporated into a plasmid DNA vector. Many commercially available expression vectors may be useful for the present invention, including, e.g., pCEP4, pcDNAI, pIND, pSecTag2, pVAXl, pcDNA3.1 , pBl-EGFP, pBlueScript, and pDisplay.

Various viral vectors may also be used. Typically, in a viral vector, the viral genome is engineered to eliminate the disease-causing capability, e.g., the ability to replicate in the host cells. The exogenous nucleic acid to be introduced into a patient may be incoφorated into the engineered viral genome, e.g., by inserting it into a viral gene that is non-essential to viral infectivity. Viral vectors are convenient to use as they can be easily introduced into tissue cells by way of infection. Once in the host cell, the recombinant virus typically is integrated into the genome of the host cell. In rare instances, the recombinant virus may also replicate and remain as extrachromosomal elements. A large number of retroviral vectors have been developed for gene therapy.

These include vectors derived from oncoretroviruses (e.g., MLV), lentiviruses (e.g., HLV and SLV) and other retroviruses. For example, gene therapy vectors have been developed based on murine leukemia virus (See, Cepko, et al., Cell, 37:1053-1062 (1984), Cone and Mulligan, Proc. Natl. Acad. Sci. U.S.A., 81:6349-6353 (1984)), mouse mammary rumor virus (See, Salmons et ah, Biochem. Biophys. Res. Commun.,l59:ll91-ll98 (1984)), gibbon ape leukemia virus (See, Miller et al, J. Virology, 65:2220-2224 (1991)), HIV, (See Shimada et al, J. Clin. Invest., 88: 1043-1047 (1991)), and avian retroviruses (See Cosset et al, J. Virology, 64:1070-1078 (1990)). In addition, various retroviral vectors are also described in U.S. Patent Nos. 6,168,916; 6,140,111; 6,096,534; 5,985,655; 5,911,983; 4,980,286; and 4,868,116, all of which are incorporated herein by reference. Adeno-associated virus (AAV) vectors have been successfully tested in clinical trials. See e.g., Kay et al, Nature Genet. 24:257-61 (2000). AAV is a naturally occurring defective virus that requires other viruses such as adenoviruses or herpes viruses as helper viruses. See Muzyczka, Curr. Top. Microbiol. Immun., 158:97 (1992). A recombinant AAV virus useful as a gene therapy vector is disclosed in U.S. Patent No. 6, 153 ,436, which is incorporated herein by reference.

Adenoviral vectors can also be useful for purposes of delivering the exogenous nucleic acid in accordance with the present invention. For example, U.S. Patent No. 6,001,816 discloses an adenoviral vector, which is used to deliver a leptin gene intravenously to a mammal to treat obesity. Other recombinant adenoviral vectors may also be used, which include those disclosed in U.S. Patent Nos. 6,171,855; 6,140,087; 6,063,622; 6,033,908; and 5,932,210, and Rosenfeld et al, Science, 252:431-434 (1991); and Rosenfeld et al, Cell, 68:143-155 (1992).

Other useful viral vectors include recombinant hepatitis viral vectors (See, e.g., U.S. Patent No. 5,981,274), and recombinant entomopox vectors (See, e.g., U.S. Patent Nos. 5,721,352 and 5,753,258). In addition, alphavirus vectors may also be desirable. Alphaviruses are positive stranded RNA viruses. The sequence encoding replica structural proteins can be replaced by the exogenous nucleic acid. The alphavirus vectors carrying the exogenous nucleic acid can be targeted to dendritic cells to facilitate the stimulation of cytotoxic T lymphocytes. See Polo et al, Proc. Natl. Acad. Sci. USA, 96:4598-4603 (1996). In addition to viral vectors, bacterial vectors can also be used. That is, the exogenous nucleic acid to be delivered can be incorporated into attenuated bacteria such as attenuated salmonella and shigella. See Levine et al, J. Biotechnol, 44:193-196 (1996); Tacket et al, Infect. Immun., 65:3381-3385 (1997); and Sizemore et al, Vaccine, 15:804-807 (1997).

The exogenous nucleic acid itself or a plasmid or viral or other expression vector carrying the exogenous nucleic acid can be introduced into a patient by various methods known in the art. For example, as will apparent to skilled artisans, the exogenous nucleic acid incorporated into a viral or bacterial vector can be administered to patients by direct infection. The nucleic acid carried by plasmid vectors can be used as DNA or RNA vaccines, i.e., in the form of naked DNA or RNA, that are administered directly into an appropriate tissue or organ of a patient. The naked DNA or RNA can be delivered by injection into skin, muscle or other tissues. Alternatively, a gene gun or an equivalent device thereof can be used for delivery into skin or mucous membrane or other tissues. Some success has been achieved using such genetic vaccines in the art. The use of this approach for purposes of the present invention will be apparent to a skilled artisan apprised of the present disclosure. See Ulmer et al, Science, 259:1745 (1993); Weiner & Kennedy, Scientific American, July, 1999; Barouch et al, Science, 290:486-492 (2000), all of which are incorporated herein by reference. Alternatively, catheters or like devices may be used for delivery into a target organ or tissue. Suitable catheters are disclosed in, e.g., U.S. Patent Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705, all of which are incorporated herein by reference.

Other non-traditional vectors may also be used for purposes of this invention. For example, International Publication No. WO 94/18834 discloses a method of delivering DNA into mammalian cells by conjugating the DNA to be delivered with a polyelectrolyte to form a complex. The complex may be microinjected into or taken up by cells.

The exogenous nucleic acid or plasmid DNA vector containing the exogenous gene may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Patent No. 6,090,619; Wu and Wu, J. Biol. Chem., 263:14621 (1988); Curiel et al, Proc. Natl. Acad. Sci. USA, 88:8850 (1991). For example, U.S. Patent No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety (e.g., poly-L-lysine, having 3-100 lysine residues), which is itself coupled to an integrin receptor binding moiety (e.g., a cyclic peptide having the amino acid sequence RGD).

Alternatively, the exogenous nucleic acid or vectors containing it can also be delivered into cells via amphiphiles. See e.g., U.S. Patent No. 6,071,890. Typically, the exogenous nucleic acid or a vector containing the nucleic acid forms a complex with the cationic amphiphile. Cells in a patient's body contacted with the complex can readily absorb the complex.

Many molecules and structures known in the art can be used as "transporter." Ln one embodiment, a penetratin is used as a transporter. For example, the homeodomain of Antennapedia, a Drosophila transcription factor, can be used as a transporter to deliver a polypeptide of the present invention. Indeed, any suitable member of the penetratin class of peptides can be used to carry a polypeptide of the present invention into cells.

Penetratins are disclosed in, e.g., Derossi et al, Trends Cell Biol, 8:84-87 (1998), which is incorporated herein by reference. Penetratins transport molecules attached thereto across cytoplasm membranes or nucleus membranes efficiently in a receptor- independent, energy-independent, and cell type-independent manner. Methods for using a penetratin as a carrier to deliver oligonucleotides and polypeptides are also disclosed in U.S. Patent No. 6,080,724; Pooga et al, Nat. Biotech., 16:857 (1998); and Schutze et al, J. Immunol, 157:650 (1996), all of which are incoφorated herein by reference. U.S. Patent No. 6,080,724 defines the minimal requirements for a penetratin peptide as a peptide of 16 amino acids with 6 to 10 of which being hydrophobic. The amino acid at position 6 counting from either the N- or C-terminal is tryptophan, while the amino acids at positions 3 and 5 counting from either the N- or C-terminal are not both valine. Preferably, the helix 3 of the homeodomain of Drosophila Antennapedia is used as a transporter. More preferably, a peptide having a sequence of the amino acids 43-58 of the homeodomain Antp is employed as a transporter. In addition, other naturally occurring homologs of the helix 3 of the homeodomain of Drosophila Antennapedia can also be used. For example, homeodomains of Fushi-tarazu and Engrailed have been shown to be capable of transporting peptides into cells. See Han et al, Mol. Cells, 10:728-32 (2000). As used herein, the term "penetratin" also encompasses peptoid analogs of the penetratin peptides. Typically, the penetratin peptides and peptoid analogs thereof are covalently linked to a compound to be delivered into cells thus increasing the cellular uptake of the compound.

Formation of Immunogenic Cells In vitro

Ln accordance with another aspect of the present invention, a method for treating and/or preventing HSV infection and symptoms thereof is provided comprising administering to a patient in need of treatment an effective amount of cells (or a portion or fraction thereof) displaying HSV altered budding phenotype. That is, cells displaying HSV altered budding phenotype are prepared in vitro and delivered to a patient in need of treatment. Although cells from a variety of sources may be employed to prepare cells displaying HSV altered budding phenotype, mammalian cells including human cells and others may be preferable. It one aspect, the cells to be administered can be a particular fraction or portion thereof, prepared by methods known to an ordinary skilled artisan.

As will be apparent to skilled artisans, the methods described above for causing, in the body, the formation of cells displaying HSV altered budding phenotype can be used in in vitro procedures to create cells displaying HSV altered budding phenotype. For example, a mutant HSV protein sufficient for viral particle assembly but devoid of a late-domain motif may be introduced into cells in vitro to initiate the assembly of viruslike particles and form cells displaying HSV altered budding phenotype. Alternatively, a nucleic acid encoding a mutant HSV protein devoid of the late-domain motif or devoid of the domain containing the late-domain motif is introduced into cells in vitro to express the mutant HSV protein mutant in the cells.

Ln another embodiment, a wild-type or mutant HSV protein sufficient for viral particle assembly or a nucleic acid encoding such a polypeptide can be administered to cells defective in respect to one or more proteins required for HSV budding. That is, the host cell late-domain binding partner involved in viral budding can be rendered defective. For example, human cells with the AP-2 complex, particularly the AP-50 subunit gene being knocked out may be used for this puφose. Also, dominant-negative mutations may also be used to prevent HSV budding from the cells and cause altered budding phenotype when a wild-type or mutant HSV protein sufficient for viral particle assembly is produced in the cells. Methods for creating AP-2 complex, particularly the AP-50 subunit-deficient cells or cells with dominant-negative mutations are apparent to skilled artisans.

A nucleic acid encoding a wild-type or mutant HSV late-domain containing protein or vectors containing the nucleic acid can be introduced into cells in vitro using any known techniques such as calcium phosphate precipitation, microinjection, lipofection, electroporation, gene gun, receptor-mediated endocytosis, and the like. The wild-type or mutant HSV protein can be introduced into cells in vitro by attaching the polypeptide to a transporter as described above capable of increasing the cell uptake of the polypeptide. The cells displaying or capable of displaying HSV altered budding phenotype can be administered to a patient by, e.g., injection or cell transplantation. The appropriate amount of cells delivered to a patient will vary with patient conditions, and desired effect, which can be determined by a skilled artisan. See e.g., U.S. Patent Nos. 6,054,288; 6,048,524; and 6,048,729.

2. HSV Budding Modulation Compounds and Methods of Detecting

The present invention includes compounds and compositions that affect the ability of HSV to bud. Furthermore, the invention provides methods of detecting compounds that affect the ability of HSV to bud. The compounds and methods of detecting them are based on the ability to affect an interaction between a viral late-domain motif containing protein and the host cell partner protein(s) with which it interacts during viral budding. Any compounds capable of interfering with the protein-protein interaction between a HSV late-domain motif containing protein and its interacting partner may be used. This means useful compounds include those that interfere with, block, disrupt or destabilize the protein-protein interaction; block or interfere with the formation of a protein complex by a HSV late-domain motif containing protein and its interacting partner; or destabilize, disrupt or dissociate an existing protein complex comprising a HSV late-domain motif containing protein and its interacting partner. Such compounds can be selected by various screening assays known in the art. For example, test compounds may be screened in an in vitro assay to select interaction antagonists. A HSV late-domain motif containing protein and its interacting partner protein complex can be contacted with a test compound and disruption or destabilization of the protein complex can be detected. For example, the presence or absence of the protein complex can be detected by an antibody selectively immunoreactive with the protein complex. Thus, after incubation of the protein complex with a test compound, an immunoprecipitation assay can be conducted with the antibody. If the test compound disrupts the protein complex, then the amount of immunoprecipitated protein complex in this assay will be significantly less than that in a control assay in which the same protein complex is not contacted with the test compound. Various other detection methods may be suitable in the dissociation assay, as will be apparent to skilled artisan apprised of the present disclosure. In one embodiment, one of the interacting partners with a detectable marker fused thereto is fixed to a solid support. For example, a GST-HSV late-domain motif containing fusion protein is attached to a solid support. Then the other interacting partner with a detectable marker fused thereto (e.g. , a myc-tagged late-domain motif binding partner) is contacted with the immobilized first interacting partner in the presence of one or more test compounds. If binding between the two interacting partners occurs, the myc-tagged late-domain motif binding partner fragment is also immobilized, which can be detected using an anti-myc antibody after the binding reaction mixture is washed to remove unbound myc-tagged late-domain binding partner fragment.

Alternatively, test compounds can also be screened in any in vivo assays to select compounds capable of interfering with the interaction between a HSV late-domain motif containing protein and its interacting partner. Any in vivo assays known in the art useful in selecting compounds capable of interfering with the stability of the protein complexes of the present invention may be used.

In a preferred embodiment, one of the yeast two-hybrid systems or their analogous or derivative forms is used. Examples of suitable two-hybrid systems known in the art include, but are not limited to, those disclosed in U.S. Patent Nos. 5,283,173; 5,525,490; 5,585,245; 5,637,463; 5,695,941; 5,733,726; 5,776,689; 5,885,779; 5,905,025; 6,037,136; 6,057,101; 6,114,111; and Bartel and Fields, eds., The Yeast Two-Hybrid System, Oxford University Press, New York, NY, 1997, all of which are incoφorated herein by reference.

Typically, in a classic transcription-based two-hybrid assay, two chimeric genes are prepared encoding two fusion proteins: one contains a transcription activation domain fused to an interacting protein member of a protein complex of the present invention or an interacting domain of the interacting protein member, while the other fusion protein includes a DNA binding domain fused to another interacting protein member of the protein complex or an interacting domain thereof.

In a screening assay for dissociators, a host cell late-domain interacting protein partner, a mutant form or a binding domain thereof, and a HSV protein containing late- domain motif, or a mutant form or a binding domain thereof, are used as test proteins expressed in the form of fusion proteins as described above for puφoses of a two-hybrid assay. The fusion proteins are expressed in a host cell and allowed to interact with each other in the presence of one or more test compounds. In a preferred embodiment, a counterselectable marker is used as a reporter such that a detectable signal (e.g., appearance of color or fluorescence, or cell survival) is present only when the test compound is capable of interfering with the interaction between the two test proteins. In this respect, the reporters used in various "reverse two- hybrid systems" known in the art may be employed. Reverse two-hybrid systems are disclosed in, e.g., U.S. Patent Nos. 5,525,490; 5,733,726; 5,885,779; Vidal et al, Proc. Natl Acad. Sci. USA, 93:10315-10320 (1996); and Vidal et al, Proc. Nat Acad. Sci. USA, 93:10321-10326 (1996), all of which are incoφorated herein by reference. Examples of suitable counterselectable reporters useful in a yeast system include the URA3 gene (encoding orotidine-5'-decarboxylase, which converts 5-fluroorotic acid (5- FOA) to the toxic metabolite 5-fluorouracil), the CAN1 gene (encoding arginine permease, which transports toxic arginine analog canavanine into yeast cells), the GAL1 gene (encoding galactokinase, which catalyzes the conversion of 2-deoxygalactose to toxic 2-deoxygalactose- 1 -phosphate), the LYS2 gene (encoding α-aminoadipate reductase, which renders yeast cells unable to grow on a medium containing a- aminoadipate as the sole nitrogen source), the MET15 gene (encoding O- acetylhomoserine sulfhydrylase, which confers on yeast cells sensitivity to methyl mercury), and the CYH2 gene (encoding L29 ribosomal protein, which confers sensitivity to cycloheximide). In addition, any known cytotoxic agents including cytotoxic proteins such as the diphtheria toxin (DTA) catalytic domain can also be used as counterselectable reporters. See U.S. Patent No. 5,733,726. DTA causes the ADP- ribosylation of elongation factor-2 and thus inhibits protein synthesis and causes cell death. Other examples of cytotoxic agents include ricin, Shiga toxin, and exotoxin A of Pseudomonas aeruginosa.

For example, when the URA3 gene is used as a counterselectable reporter gene, yeast cells containing a mutant URA3 gene can be used as host cells (Ura- FoaR phenotype) for the in vivo assay. Such cells lack URA3-encoded functional orotidine-5'- phosphate decarboxylase, an enzyme required for the biosynthesis of uracil. As a result, the cells are unable to grow on media lacking uracil. However, because of the absence of a wild-type orotidine-5 '-phsphate decarboxylase, the yeast cells cannot convert non-toxic 5-fluoroorotic acid (5-FOA) to a toxic product, 5-fiuorouracil. Thus, such yeast cells are resistant to 5-FOA and can grow on a medium containing 5-FOA. Therefore, for example, to screen for a compound capable of disrupting interaction between a HSV late- domain motif containing protein and its interacting partner, the late-domain binding partner can be expressed as a fusion protein with a DNA-binding domain of a suitable transcription activator while the HSV late-domain motif containing protein is expressed as a fusion protein with a transcription activation domain of a suitable transcription activator. In the host strain, the reporter URA3 gene may be operably linked to a promoter specifically responsive to the association of the transcription activation domain and the DNA-binding domain. After the fusion proteins are expressed in the Ura- FoaR yeast cells, an in vivo screening assay can be conducted in the presence of a test compound with the yeast cells being cultured on a medium containing uracil and 5-FOA. If the test compound does not disrupt the interaction between a HSV late-domain motif containing protein and its interacting partner, active URA3 gene product, i.e., orotidine- 5 '-decarboxylase, which converts 5-FOA to toxic 5-fluorouracil, is expressed. As a result, the yeast cells cannot grow. On the other hand, when the test compound disrupts the interaction between a HSV late-domain motif containing protein and its interacting partner, no active orotidine-5 '-decarboxylase is produced in the host yeast cells. Consequently, the yeast cells will survive and grow on the 5-FOA-containing medium. Therefore, compounds capable of interfering with or dissociating the interaction between a HSV late-domain motif containing protein and its interacting partner can thus be identified based on colony formation. As will be apparent, the screening assay of the present invention can be applied in a format appropriate for large-scale screening. For example, combinatorial technologies can be employed to construct combinatorial libraries of small organic molecules or small peptides. See generally, e.g., Kenan et al, Trends Biochem. Sc, 19:57-64 (1994); Gallop et al, J. Med. Chem., 37:1233-1251 (1994); Gordon et al, J. Med. Chem., 37:1385-1401 (1994); Ecker et al, Biotechnology, 13:351-360 (1995). Such combinatorial libraries of compounds can be applied to the screening assay of the present invention to isolate specific modulators of particular protein-protein interactions. Ln the case of random peptide libraries, the random peptides can be co-expressed with the fusion proteins of the present invention in host cells and assayed in vivo. See e.g., Yang et al, Nucl Acids Res., 23:1152-1156 (1995). Alternatively, they can be added to the culture medium for uptake by the host cells.

Conveniently, yeast mating is used in an in vivo screening assay. For example, haploid cells of a-mating type expressing one fusion protein as described above is mated with haploid cells of alpha-mating type expressing the other fusion protein. Upon mating, the diploid cells are spread on a suitable medium to form a lawn. Drops of test compounds can be deposited onto different areas of the lawn. After culturing the lawn for an appropriate period of time, drops containing a compound capable of modulating the interaction between the particular test proteins in the fusion proteins can be identified by stimulation or inhibition of growth in the vicinity of the drops. The screening assays for selecting compounds capable of interfering with protein- protein interactions can also be fine-tuned by various techniques to adjust the thresholds or sensitivity of the positive and negative selections. Mutations can be introduced into the reporter proteins to adjust their activities. The uptake of test compounds by the host cells can also be adjusted. For example, yeast high uptake mutants such as the erg6 mutant strains can facilitate yeast uptake of the test compounds. See Gaber et al, Mol. Cell. Biol, 9:3447-3456 (1989). Likewise, the uptake of the selection compounds such as 5-FOA, 2-deoxygalactose, cycloheximide, α-aminoadipate, and the like can also be ' fine-tuned.

Any test compounds may be screened in the screening assays of the present invention to select compounds capable of interfering with the interaction between a HSV- 1 late-domain motif containing protein and its interacting partner. The test compounds may include, by way of example, proteins (e.g., antibodies, small peptides, artificial or natural proteins), nucleic acids, and derivatives, mimetics and analogs thereof, and small organic molecules having a molecular weight of no greater than 10,000 daltons, more preferably less than 5,000 daltons. Preferably, the test compounds are provided in library formats known in the art, e.g., in chemically synthesized libraries, recombinant expression libraries (e.g., phage display libraries), and in vitro translation-based libraries

(e.g., ribosome display libraries).

Ln preferred embodiments, the compound capable of interfering with the interaction between a HSV late-domain motif containing protein and its interacting partner used in the methods of the present invention comprise a late-domain motif amino acid sequence motif capable of binding the late-domain motif binding partner.

Preferably, the amino acid sequence comprises a YXXL, PXXP, or PPXY motif. The compound which comprises the amino acid sequence YXXL, PXXP, or PPXY motif and is capable of binding the late-domain motif binding partner can be of any type of chemical compounds so long as the compound is capable of binding the late-domain motif binding partner. For example, the compound can be a peptide, a modified peptide, an oligonucleotide-peptide hybrid (e.g., PNA), etc.

3. Late-Domain Motif Mimetics The present invention includes late-domain motif mimetics for use in treating

HSV infection. These mimetics are based on the naturally occurring peptide sequences of the proteins encoding the HSV late-domain motif containing protein.

YXXL Motif Mimetics Ln one aspect of the invention, a compound that affects viral budding is provided which comprises an amino acid sequence motif YXiX L and is capable of binding the AP-50 subunit of AP-2 complex (or AIPl), wherein

is selected from the group consisting of glutamine (Q), arginine (R), serine (S), leucine (L), and alanine (A). In another embodiment, the X₂ in the motif is selected from the group consisting of R, G, P L, F, and D. In a more preferred embodiment, the compound administered comprising the amino acid sequence motif of YXιX₂L, where X₁ and X₂ are the corresponding amino acids in the HSV late-domain motif containing protein involved in the budding process. In a preferred embodiment, the compound capable of binding AP-50 (or ALPl) comprises an amino acid sequence motif X_IX^ _J, wherein Xi is Y or W or an analog thereof, X is either L or I or an analog thereof. In one embodiment, X in the motif is a basic residue, has a NH₂ group, or is an analog thereof. In another embodiment, X₃ is a basic residue, has a NH₂ group, or is an analog thereof. In another preferred embodiment, the compound administered has the amino acid sequence motif of YX₂X₃L. In a more preferred embodiment, X₂ in the YX₂X₃L motif is a basic residue, has a NH₂ group, or is an analog thereof, and X₃ is a basic residue, has a NH group, or is an analog thereof. In a highly preferred embodiment, Xi in the XiX₂X₃ motif is Y or an analog thereof, X₂ is a basic residue, has a NH group, or is an analog thereof, and X₃ is a basic residue, has a NH₂ group, or is an analog thereof, X₄ is L or an analog thereof.

The compound can also include a longer peptide comprising the amino acid sequence motif of YXXL and is capable of binding the AP-50 subunit of the AP-2 complex (or AIPl). For example, the compound may include a peptide of 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids.

In a preferred embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids of a naturally occurring late-domain motif containing protein sequence. However, generally speaking, the contiguous span has less than 50, preferably less than 40, more preferably less than 30 amino acids. The contiguous span should span the HSV late domain motif containing protein which can be the YXXL motif or a variation thereof. Preferably, the late domain motif in the contiguous span is the YXXL motif.

PXXP Motif Mimetics In one aspect of the invention, a compound that affects viral budding is provided which comprises an amino acid sequence motif PX]X₂P and is capable of binding the UEV domain of TSGIOI (or alternatively a protein containing a SH3 domain that binds the HSV late-domain motif containing protein involved in viral budding), wherein X₁ is selected from the group consisting of threonine (T), serine (S), and isoleucine (I , and X₂ is not R. In another embodiment, the X₂ in the motif is alanine (A) or threonine (T). In a preferred embodiment, the compound administered has the amino acid sequence motif of PX₁X₂P, wherein X_\ is selected from the group consisting of T, S, and I, and X₂ is A or T. In a more preferred embodiment, Xi in the PXiX₂P motif is T or S, and X₂ is A. Thus, the compound can be a tetrapeptide having an amino acid sequence of PX^P, wherein X is an amino acid or an amino acid analog other than arginine. In one embodiment, the tetrapeptide has an amino acid sequence of P(T/S/I)(A/T)P In a preferred embodiment, the tetrapeptide has the sequence of PTAP another preferred embodiment, the tetrapeptide has the sequence of PSAP. The compound can also include a longer peptide comprising the amino acid sequence motif of PXιX₂P and capable of binding the UEV domain of TSGIOI (or alternatively a protein containing a SH3 domain that binds the HSV late-domain motif containing protein involved in viral budding). For example, the compound may include a peptide of 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids.

In one embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of a naturally occurring HSV late-domian motif containing sequence that is involved in viral budding. The contiguous span should span the HSV late domain motif which can be the P(T/S/I)(A/T)P motif or a variation thereof. Preferably, the late-domain motif in the contiguous span is the P(T/S)AP motif. In another embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of one of the proteins in Tables 1-3. The contiguous span should span the late-domain motif P(T/S)AP sequence in that particular protein in Tables 1-3. PPXY Motif Mimetics

In one aspect of the invention, a compound that affects viral budding is provided which comprises an amino acid sequence motif PX_tX₂X₃ and is capable of binding NEDD4 protein (or a NEDD4-like protein or alternatively a protein containing a WW domain that is involved in viral budding), wherein X₃ is Y or W or an analog thereof. In another embodiment, the Xi in the motif is P or an analog thereof. In a preferred embodiment, the compound administered has the amino acid sequence motif of PX₁X₂X₃, wherein X₁ is P or an analog thereof, and X₃ is Y or W or an analog thereof. In a more preferred embodiment, Xi in the PXnX₂X₃ motif is P or an analog thereof, and X₂ is P or an analog thereof, and X₃ is Y or W or an analog thereof. In a most preferred embodiment, Xi in the PXiX₂X₃ motif is P or an analog thereof, and X₂ is P or an analog thereof, and X₃ is Y or an analog thereof.

In one embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of a naturally occurring HSV late-domian motif containing sequence that is involved in viral budding. The contiguous span should span the HSV late domain motif which can be the PPXY motif or a variation thereof. Preferably, the late-domain motif in the contiguous span is the PPXY motif. In another embodiment, the compound includes a peptide that contains a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids of one of the proteins in Tables 1-3. The contiguous span should span the late domain motif PPXY in that particular protein in Tables 1-3.

Modification of Late-Domain Motif Mimetics In another embodiment, the motif in the compound according to the present invention is within an amino acid sequence that is at least 70 percent, preferably at least 80 percent or 85 percent, more preferably at least 90 percent or 95 percent identical to a contiguous span of at least 5, 6, 7, 8 or 9 amino acids, preferably 10, 11, 12, 13, 14, 15 or more amino acids, but preferably less than 50 or 40, more preferably less than 30 amino acids, of a naturally occurring HSV- 1 late-domain motif containing amino acid sequence, which contiguous span of amino acids spans the HSV-1 late domain motif. In this respect, the percentage identity is determined by the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-77 (1993), which is incoφorated into the various BLAST programs. Specifically, the percentage identity is determined by the "BLAST 2 Sequences" tool, which is available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html. See Tatusova and Madden, FEMS Microbiol. Lett., 174(2):247-50 (1999). For pairwise protein-protein sequence comparison, the BLASTP 2.1.2 program is employed using default parameters (Matrix: BLOSUM62; gap open: 11; gap extension: 1; x_dropoff: 15; expect: 10.0; and wordsize: 3, with filter). It should be understood that such homolog peptides should retain the ability to bind their corresponding host cell late-domain motif binding partner.

The motif mimetics can be made by site-directed mutagenesis based on a late domain motif-containing HSV protein sequence. The site-directed mutagenesis can be designed to generate amino acid substitutions, insertions, or deletions. Methods for conducting such mutagenesis should be apparent to skilled artisans in the field of molecular biology. The resultant homologues can be tested for their binding affinity to the late-domain motif binding partner.

The peptide portion in the compounds according to the present invention can also be in a modified form. Various modifications may be made to improve the stability and solubility of the compound, and/or optimize its binding affinity to the late-domain motif binding partner. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, prenylated forms, etc. Modifications also include intramolecular crosslinking and covalent attachment to various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, etc. In addition, modifications may also include cyclization, and branching. Amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide sequence in the compound of the present invention. For example, the compounds may include D-amino acids in place of L-amino acids.

To increase the stability of the peptidic compounds according to the present invention, various protection groups can also be incoφorated into the amino acid residues of the compounds. In particular, terminal residues are preferably protected. Carboxyl groups may be protected by esters (e.g., methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl or t- amyl esters, etc.), lower alkoxyl groups (e.g., methoxy, ethoxy, propoxy, butoxy, etc.), aralkyloxy groups (e.g., benzyloxy, etc.), amino groups, lower alkylamino or di(lower alkyl)amino groups. The term "lower alkoxy" is intended to mean an alkoxy group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. Protection groups for amino groups may include lower alkyl, benzyloxycarbonyl, t- butoxycarbonyl, and sobornyloxycarbonyl. "Lower alkoxy" is intended to mean an alkyl group having a straight, branched or cyclic hydrocarbon moiety of up to six carbon atoms. In one example, a 5-oxo-L-prolyl residue may be used in place of a prolyl residue. A 5-oxo-L-prolyl residue is especially desirable at the N-terminus of a peptide compound.

Additionally, as will be apparent to skilled artisans apprised of the present disclosure, peptide mimetics can also be used. For example, peptoid analogs of the late- domain motif can be prepared using known methods. Peptoids are oligomeric N- substituted glycines. Typically, various side chain groups can be included when forming an N-substituted glycine (peptoid monomer) that mimics a particular amino acid. Peptoid monomers can be linked together to form an oligomeric N-substituted glycines - peptoid. Peptoids are easy to synthesize in large amounts. In contrast to peptides, the backbone linkage of peptoids are resistant to hydrolytic enzymes. In addition, since a variety of functional groups can be presented as side chains off of the oligomeric backbone, peptoid analogs corresponding to any peptides can be produced with improved characteristics. See Simon et al, Proc. Natl. Acad. Sci. USA, 89:9367-9371 (1992); Figliozzi et al, Methods Enzymol, 267:437-447 (1996); Horwell, Trends Biotechnol, 13:132-134 (1995); and Horwell, DrugDes. Discov., 12:63-75 (1994), all of which are incoφorated herein by reference.

Thus, peptoid analogs of the above-described late-domain motif-containing compounds of the present invention can be made using methods known in the art. The prepared peptoid analogs can be tested for their binding affinity to a late-domain motif binding partner. They can also be tested in anti- viral assays for their ability to inhibit virus budding from infected host cells and ability to inhibit virus propagation. In particular, they can be tested for ability to suppress HSV budding from infected cells and inhibit HSV propagation.

Mimetics of the compounds of the present invention can also be selected by rational drug design and/or virtual screening. Methods known in the art for rational drug design can be used in the present invention. See, e.g., Hodgson et al, Bio/Technology, 9:19-21 (1991); U.S. Patent Nos. 5,800,998 and 5,891,628, all of which are incoφorated herein by reference. Structural information on the late-domain motif binding partner and/or the binding complex formed by the late-domain motif binding partner and the HSV late-domain motif containing protein are obtained. The interacting complex can be studied using various biophysics techniques including, e.g., X-ray crystallography, NMR, computer modeling, mass spectrometry, and the like. Likewise, structural information can also be obtained from protein complexes formed by the late-domain motif binding partner and a variation of the late domain YXXL, PXXP, or PPXY motif.

Computer programs are employed to select compounds based on structural models of the binding complex formed by the late-domain motif binding partner and the HSV YXXL, PXXP, or PPXY motif or polypeptides containing the motif. Ln addition, once an effective compound is identified, structural analogs or mimetics thereof can be produced based on rational drug design with the aim of improving drug efficacy and stability, and reducing side effects. In addition, understanding of the interaction between the late-domain motif binding partner and compounds of the present invention can also be derived from mutagenesis analysis using yeast two-hybrid system or other methods for detection protein-protein interaction. In this respect, various mutations can be introduced into the interacting proteins and the effect of the mutations on protein-protein interaction is examined by a suitable method such as in vitro binding assay or the yeast two-hybrid system.

Various mutations including amino acid substitutions, deletions and insertions can be introduced into the protein sequence of the late-domain motif binding partner and/or a compound of the present invention using conventional recombinant DNA technologies. Generally, it is particularly desirable to decipher the protein binding sites. Thus, it is important that the mutations introduced only affect protein-protein interaction and cause minimal structural disturbances. Mutations are preferably designed based on knowledge of the three-dimensional structure of the interacting proteins. Preferably, mutations are introduced to alter charged amino acids or hydrophobic amino acids exposed on the surface of the proteins, since ionic interactions and hydrophobic interactions are often involved in protein-protein interactions. Alternatively, the "alanine scanning mutagenesis" technique is used. See Wells, et al., Methods Enzymol., 202:301-306 (1991); Bass et al, Proc. Natl. Acad. Sci. USA, 88:4498-4502 (1991); Bennet et al, J. Biol. Chem., 266:5191-5201 (1991); Diamond et al, J. Virol, 68:863-876 (1994). Using this technique, charged or hydrophobic amino acid residues of the interacting proteins are replaced by alanine, and the effect on the interaction between the proteins is analyzed using e.g., an in vitro binding assay. In this manner, the domains or residues of the proteins important to compound-target interaction can be identified.

Based on the structural information obtained, structural relationships between the late-domain motif binding partner and a compound of the present invention are elucidated. The moieties and the three-dimensional structures critical to the interaction are revealed. Medicinal chemists can then design analog compounds having similar moieties and structures.

The residues or domains critical to the modulating effect of the identified compound constitute the active region of the compound known as its "pharmacophore." Once the pharmacophore has been elucidated, a structural model can be established by a modeling process that may incoφorate data from NMR analysis, X-ray diffraction data, alanine scanning, spectroscopic techniques and the like. Various techniques including computational analysis, similarity mapping and the like can all be used in this modeling process. See e.g., Perry et al, in OSAR: Quantitative Structure- Activity Relationships in Drug Design, pp.189- 193, Alan R. Liss, Inc., 1989; Rotivinen et al, Acta Pharmaceutical Fennica, 97:159-166 (1988); Lewis et al, Proc. R. Soc. Lond., 236:125-140 (1989); McKinaly et al, Annu. Rev. Pharmacol Toxiciol., 29: 111-122 (1989). Commercial molecular modeling systems available from Polygen Coφoration, Waltham, MA, include the CHARMm program, which performs the energy minimization and molecular dynamics functions, and QUANTA program which performs the construction, graphic modeling and analysis of molecular structure. Such programs allow interactive construction, visualization and modification of molecules. Other computer modeling programs are also available from BioDesign, Inc. (Pasadena, CA.), Hypercube, Inc. (Cambridge, Ontario), and Allelix, Inc. (Mississauga, Ontario, Canada).

A template can be formed based on the established model. Various compounds can then be designed by linking various chemical groups or moieties to the template. Various moieties of the template can also be replaced. These rationally designed compounds are further tested. In this manner, pharmacologically acceptable and stable compounds with improved efficacy and reduced side effect can be developed. The compounds identified in accordance with the present invention can be incoφorated into a pharmaceutical formulation suitable for administration to an individual.

The mimetics including peptoid analogs can exhibit optimal binding affinity to the late-domain motif binding partner or animal orthologs thereof. Various known methods can be utilized to test the late-domain binding partner binding characteristics of a mimetics. For example, the entire late-domain motif binding partner protein or a fragment thereof containing the binding domain may be recombinantly expressed, purified, and contacted with the mimetics to be tested. Binding can be determined using a surface plasmon resonance biosensor. See e.g., Panayotou et al, Mol Cell Biol, 13:3567-3576 (1993). Other methods known in the art for estimating and determining binding constants in protein-protein interactions can also be employed. See Phizicky and Fields, et al, Microbiol Rev., 59:94-123 (1995). For example, protein affinity chromatography may be used. First, columns are prepared with different concentrations of an interacting member, which is covalently bound to the columns. Then a preparation of its interacting partner is run through the column and washed with buffer. The interacting partner bound to the interacting member linked to the column is then eluted. Binding constant is then estimated based on the concentrations of the bound protein and the eluted protein. Alternatively, the method of sedimentation through gradients monitors the rate of sedimentation of a mixture of proteins through gradients of glycerol or sucrose. At concentrations above the binding constant, the two interacting members sediment as a complex. Thus, binding constant can be calculated based on the concentrations. Other suitable methods known in the art for estimating binding constant include but are not limited to gel filtration column such as nonequilibrium "small-zone" gel filtration columns (See e.g., Gill et al, J. Mol Biol, 220:307-324 (1991)), the Hummel-Dreyer method of equilibrium gel filtration (See e.g., Hummel and Dreyer, Biochim. Biophys. Acta, 63:530-532 (1962)) and large-zone equilibrium gel filtration (See e.g., Gilbert and Kellett, J. Biol. Chem., 246:6079-6086 (1971)), sedimentation equilibrium (See e.g., Rivas and Minton, Trends Biochem., 18:284-287 (1993)), fluorescence methods such as fluorescence spectrum (See e.g., Otto-Bruc et al, Biochemistry, 32:8632-8645 (1993)) and fluorescence polarization or anisotropy with tagged molecules (See e.g., Weiel and Hershey, Biochemistry, 20:5859-5865 (1981)), and solution equilibrium measured with immobilized binding protein (See e.g., Nelson and Long, Biochemistry, 30:2384-2390 (1991)).

The compounds capable of interfering with the interaction between a HSV late- domain motif containing protein and its interacting partner can be delivered into cells by direct cell internalization, receptor mediated endocytosis, or via a "transporter." It is noted that a compound preferably is delivered into patient's cells in order to achieve optimal results. Preferably, peptidic transporters as described above such as penetratins and HLV tat protein are employed. Fusion proteins can be conveniently made by recombinant expression to contain a transporter peptide covalently linked by a peptide bond to a peptide having the late-domain motif. Alternatively, conventional methods can be used to chemically synthesize a transporter peptide or a peptide of the present invention or both.

4. Adjuvants

Although not required, it is preferred that an adjuvant capable of stimulating immune response is also administered to a patient who is treated with cells displaying HSV altered budding phenotype or with a compound (nucleic acids, polypeptides or small organic compounds) capable of causing the formation, in a patient's body, of cells displaying HSV altered budding phenotype. As used herein, the term "adjuvant" means any substance that is not a component of HSV but is capable of stimulating or enhancing immune responses to an immunogen administered to a patient. The disruption or interference with the protein-protein interaction between the host cellular protein a HSV late-domain motif containing protein and its interacting partner is contemplated in itself to be effective in treating and/or preventing HSV infection and symptoms thereof by way of inhibiting HSV viral budding and propagation. However, the disruption or interference with the protein-protein interaction between the host cellular late-domain motif binding partner and a HSV late-domain motif containing protein may also induce the formation of cells displaying HSV altered budding phenotype which act as immunogens in eliciting immune responses in the patient. Therefore, the compounds administered to a patient capable of disrupting or interfering with the protein- protein interaction between a HSV late-domain motif containing protein and its interacting partner can also stimulate immune responses in the patient against HSV viruses. In this respect, the administration of an adjuvant capable of enhancing the patient's immune response, along with the administration to the patient of a compound capable of disrupting or interfering with the protein-protein interaction between a HSV late-domain motif containing protein and its interacting partner will significantly bolster the anti-HSV immune response in the patient and result in a treatment efficacy significantly greater than the administration of the compound alone.

Similarly, an adjuvant capable of enhancing a patient's immune response can also significantly boost the prophylactic and/or therapeutic effect when coupled with a mutant HSV late-domain containing protein or polypeptide that modulates viral budding in cells, or a nucleic acid encoding a mutant HSV late-domain motif containing protein or polypeptide.

Any adjuvant may be used so long as the adjuvant is capable of stimulating immune response against HSV or against the HSV viral proteins in such cells, and thus can enhance immune response against HSV viruses in a patient. For example, alum has long been used as an adjuvant for human use. Another example of suitable adjuvant is MF59. See Minutello et al, Vaccine, 17:99-104 (1999). Another useful adjuvant in development that may also be used is LTR72, a mutant of E. coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity. See Giuliani et al, J. Exp. Med., 187:1123-1132 (1998). Polyphosphazine adjuvant may also be used. In addition, cytokines and lymphokines may also be used as adjuvants along with the cells, polypeptides, nucleic acids and/or a compound capable of disrupting or interfering with the protein-protein interaction between a HSV late-domain motif containing protein and its interacting partner. Examples of cytokines and lymphokines include, but are not limited to, interleukins such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, interferons such as alpha-interferon and gamma-interferon, tumor necrosis factor (TNF), platelet derived growth factor (PDGF), GCSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF), and the like.

Many adjuvants capable of stimulating cellular immune responses are known in the art. For example, cytokines secreted by helper T cells called Thl cells have been shown to promote cellular responses. Such Thl type cytokines include interleukin-2 (IL- 2), interleukin-4, and interleukin-12 (IL-12), interleukin-18, among others. In addition, fusion proteins having one of such Thl type cytokines (e.g., IL-2) fused to the Fc portion of immunoglobulin G (IgG) may also be used. See e.g., Barouch et al, Science, 290:486- 492 (2000), which is incoφorated herein by reference. Other preferred adjuvants capable of stimulating cell-mediated immune responses include interferons such as alpha- interferon, beta-interferon and gamma-interferon. Certain chemokines may also be used to enhance cellular immune responses. Such chemokines are small molecules that attract T cells to infected tissues. Additionally, certain short bacterial immunostimulatory DNA sequences (LSSs) have also been discovered as CTL response-promoting adjuvants that potently stimulate immune responses to co-administered antigens. See Roman et al, Nat. Med., 3:849-854 (1997), which is incoφorated herein by reference. Thus, noncoding, ISS-enriched plasmid DNAs or ISS oligonucleotides (ISS-ODNs) can also be used in the present invention as adjuvants to enhance cellular immunity against cells displaying HSV altered budding phenotype.

One or more adjuvants can be used for puφoses of the present invention. The adjuvant(s) can be administered in the same composition as the cells or compounds as described above. Alternatively, the adjuvant(s) can also be given to a patient separately. As is apparent from the above description, many of the suitable adjuvants are proteins or peptides. Such adjuvants can be administered to patients in the form of nucleic acids (e.g., DNA, RNA, and the like) so long as the administered nucleic acids are capable of expressing the adjuvants they encode. Thus, the term "administering an adjuvant to a patient" or a paraphrase thereof also encompasses administering a nucleic acid encoding a protein adjuvant and capable of expressing the adjuvant in a patient's body. Methods for administering nucleic acids are described above.

Accordingly, the present invention provides a composition comprising at least (1) a first nucleic acid encoding a mutant HSV late-domain motif containing protein or polypeptide, and (2) a second nucleic acid encoding an adjuvant capable of stimulating an immune response. The two nucleic acids can be incoφorated into separate expression vectors. Preferably, the two nucleic acids are carried within the same expression vector. In one embodiment, each of the two nucleic acids is operably linked to a transcriptional promoter, which drives the transcription from the nucleic acid linked to the promoter. In preferred embodiments, the nucleic acid encoding a mutant HSV late-domain motif is capable of expressing the mutant HSV late-domain motif containing polypeptide. For example, the nucleic acid may contain one or more mutations that decrease the effect of an inhibitory/instability sequence that is present in the corresponding nucleotide sequence of the native HSV late-domain motif containing protein encoded by the nucleic acid. Methods for making the expression vectors and compositions and for administering them to a patient are as described elsewhere in the present disclosure.

5. Combination Therapy

The methods and compositions according to the present invention are distinct from other known or commercially available approaches for treating or preventing HSV infection and symptoms thereof. Therefore, it may be desirable to employ combination therapies to combine a method of the present invention with other prophylactic or treatment methods so as to increase the prophylactic or therapeutic effect.

Thus, any other methods useful in treating or preventing HSV infection and symptoms combined with the methods of the present invention. The therapeutic and/or prophylactic materials in the methods of the present invention and in other methods may be combined in the same pharmaceutical composition or administered separately. In addition, one or more adjuvants described above may be included in the same composition or administered separately in the combination therapy approaches. However, it is to be understood that such other anti-HSV agents should be pharmaceutically compatible with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention. By "pharmaceutically compatible" it is intended that the other anti-viral agent(s) will not interact or react with the cells, nucleic acids, polypeptides or other active agents, and/or adjuvants of the present invention, directly or indirectly, in such a way as to substantially adversely affect the effect of the treatment, or to cause any significant adverse side reaction in the patient.

6. Dosage, Formulation and Administration

Typically, the active agents (cells, polypeptides, small organic molecules, nucleic acids in plasmid vectors or recombinant vectors) of the present invention are administered to a patient in a pharmaceutical composition, which typically includes one or more pharmaceutically acceptable carriers that are inherently nontoxic and non- therapeutic.

The pharmaceutical composition according to the present invention may be administered to a subject needing treatment or prevention through any appropriate routes such as parenteral, oral, mucosal or topical administration. The active agents of this invention are administered at a therapeutically or prophylactically effective amount to achieve the desired therapeutic and/or prophylactic effect without causing any serious adverse effects in the patient treated. Generally, the toxicity profile and therapeutic or prophylactic efficacy of the active agents can be determined by standard pharmaceutical procedures in suitable cell models or animal models or human clinical trials. As is known in the art, the LD₅₀ represents the dose lethal to about 50% of a tested population. The ED₅₀ is a parameter indicating the dose therapeutically or prophylactically effective in about 50% of a tested population. Both LD₅₀ and ED₅₀ can be determined in cell models and animal models. In addition, the IC₅₀ may also be obtained in cell models and animal models, which stands for the circulating plasma concentration that is effective in achieving about 50% of the maximal inhibition of the symptoms of a disease or disorder. Such data may be used in designing a dosage range for clinical trials in humans. Typically, as will be apparent to skilled artisans, the dosage range for human use should be designed such that the range centers around the ED₅₀ and/or IC₅₀, but significantly below the LD50 obtained from cell or animal models. When a nucleic acid (either alone or in a plasmid vector or a recombinant live vector) is used as an active agent in the present invention, the nucleic acid can be administered in an amount of from about 0.1 microgram to about 5000 milligram, preferably from about 1 microgram to about 500 milligram per dosage. The appropriate amount can be administered daily, weekly, monthly, bimonthly, semi-annually or annually. The nucleic acid can be administered to a patient in a manner as described above in Section 1 or by a standard procedure known in the art, as will be apparent to skilled artisans.

Typically, the mutant HSV late-domain motif containing polypeptides and the other active peptidic or small organic compounds of the present invention capable of modulating viral budding can be effective at an amount of from about 0.01 microgram to about 5000 mg per day, preferably from about 1 microgram to about 2500 mg per day. However, the amount can vary with the body weight of the patient treated and the state of disease conditions. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time. The suitable dosage unit for each administration of the compounds of the present invention can be, e.g., from about 0.01 microgram to about 2000 mg, preferably from about 1 microgram to about 1000 mg.

Ln the case of combination therapy, a therapeutically or prophylactically effective amount of another anti-HSV compound can be administered in a separate pharmaceutical composition, or alternatively included in the pharmaceutical composition that contains a compound according to the present invention. The pharmacology and toxicology of many of such other anti-HSV compounds are known in the art. See e.g., Physicians Desk Reference, Medical Economics, Montvale, NJ; and The Merck Index, Merck & Co., Rahway, NJ. The therapeutically or prophylactically effective amounts and suitable unit dosage ranges and route of administration of such compounds used in art can be equally applicable in the present invention.

It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. The therapeutically or prophylactically effective amount for each active agent can vary with factors including but not limited to the activity of the agent used, stability of the active agent in the patient's body, the severity of the conditions to be alleviated, the puφose of the treatment (prophylactic vs. therapeutic), the total weight of the patient treated, the route of administration, the ease of absoφtion, distribution, and excretion of the active agent by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan. The amount of administration can also be adjusted as the various factors change over time.

The active agents according to this invention (cells, nucleic acids, polypeptides and small organic compounds) can be administered to patients to be treated through any suitable routes of administration. Advantageously, the active agents are delivered to the patient parenterally, i.e., by intravenous, intramuscular, intraperiotoneal, intracisternal, subcutaneous, or intraarticular injection or infusion.

For parenteral administration, the non-cell active agents can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use. Lyophilized compositions may include pharmaceutically acceptable carriers such as gelatin, DL-lactic and glycolic acids copolymer, D-mannitol, etc. To convert the lyophilized forms into solutions or suspensions, diluent containing, e.g., carboxymethylcellulose sodium, D-mannitol, polysorbate 80, glycerine, and water may be employed. Lyophilized forms may be stored in, e.g., a dual chamber syringe with one chamber containing the lyophilized composition and the other chamber containing the diluent. In addition, the active ingredient(s) can also be incoφorated into sterile lyophilized microspheres for sustained release. Methods for making such microspheres are generally known in the art. See U.S. Patent Nos. 4,652,441 ; 4,728,721; 4,849,228; 4,917,893; 4,954,298; 5,330,767; 5,476,663; 5,480,656; 5,575,987; 5,631,020; 5,631,021; 5,643,607; and 5,716,640. In a solution or suspension form suitable for parenteral administration, the pharmaceutical composition can include, in addition to a therapeutically or prophylactically effective amount of an active agent of the present invention, a buffering agent, an isotonicity adjusting agent, a preservative, and/or an anti-absorbent. Examples of suitable buffering agent include, but are not limited to, citrate, phosphate, tartrate, succinate, adipate, maleate, lactate and acetate buffers, sodium bicarbonate, and sodium carbonate, or a mixture thereof. Preferably, the buffering agent adjusts the pH of the solution to within the range of 5-8. Examples of suitable isotonicity adjusting agents include sodium chloride, glycerol, mannitol, and sorbitol, or a mixture thereof. A preservative (e.g., anti-microbial agent) may be desirable as it can inhibit microbial contamination or growth in the liquid forms of the pharmaceutical composition. Useful preservatives may include benzyl alcohol, a paraben and phenol or a mixture thereof.

Materials such as human serum albumin, gelatin or a mixture thereof may be used as anti- absorbents. n addition, conventional solvents, surfactants, stabilizers, pH balancing buffers, and antioxidants can all be used in the parenteral formulations, including but not limited to dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, ascorbic acid, sodium bisulfite, and the like. The parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes.

The active agents (particularly nucleic acids, polypeptides and small organic compounds) can also be delivered orally in enclosed gelatin capsules or compressed tablets. Capsules and tablets can be prepared in any conventional techniques. For example, the active agents can be incoφorated into a formulation which includes pharmaceutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum tragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets. In addition, liquid carriers such as fatty oil can also be included in capsules.

Other forms of oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing the active compounds used in this invention. Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included. In addition, for convenient administration by enteral feeding tube in patients unable to swallow, the active agents can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil.

The active agents (e.g., nucleic acids in recombinant live vectors, polypeptides and small organic compounds) can also be administered topically through rectal, vaginal, nasal, bucal, or mucosal applications. Topical formulations are generally known in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, drops and aerosols. Typically, topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like. A special form of topical administration is delivery by a transdermal patch.

Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al, Annual Review of Medicine, 39:221-229 (1988), which is incoφorated herein by reference. The active agents (e.g., cells, nucleic acids, polypeptides and small organic molecules) can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant the active agents in any suitable formulation into the subcutaneous space of the anterior abdominal wall. See, e.g., Wilson et al, J. Clin. Psych. 45:242-247 (1984). Sustained release can be achieved by incoφorating the active ingredients into a special carrier such as a hydrogel. Typically, a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material. Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co-L-lactic acid) are suitable for this invention. See, e.g., Phillips et al, J. Pharmaceut. Sci., 73:1718-1720 (1984).

Alternatively, other forms controlled release or protection including microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the active agents.

Another preferable delivery form is using liposomes as carrier. Liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Active compounds can be enclosed within such micelles. Methods for preparing liposomal suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, and Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., both of which are incoφorated herein by reference. Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomes can reduce the toxicity of the active agents, and increase their stability.

7. Examples

Example 1: Analysis of the PPXY Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PPXY motif of an HSV-1 protein containing the motif on the interaction between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain, or fragments thereof) and the HSV-1 protein or fragments thereof. A yeast two- hybrid activation domain-NEDD4 construct is prepared from a DNA fragment encompassing the full-length coding sequence (or fragment thereof) for NEDD4 (GenBank Accession No. D42055) by PCR from an appropriate human cDNA library and is subsequently cloned into selected sites of an activation domain parent plasmid, such as GADpN2 (LEU2, CEN4, ARS1, ADHlp-SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-HSV-1 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV-1 protein derived from a selected HSV-1 strain. In the context of this example the strain corresponds to GenBank accession number NC_001806. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the gene encoding the HSV- 1 late-domain motif sequence in the yeast two-hybrid DNA binding domain-construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of proteins and mutations that can be introduced into the DNA binding domain-HSV-1 construct are summarized in Tables 4-6 below.

Table 4: Mutations in Heφesvirus RL2 Protein

The effect of the wild-type and mutants can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tipl-901 leu2-3,112 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-NEDD4 construct (or a NEDD4 like protein, or a protein containing a WW domain) and one of the binding domain-mutant HSV-1 constructs or the binding domain- wild type construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain). All binding domain constructs are also tested for self-activation of β-Gal activity.

Interactions between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) and HSV-1 wild-type (WT) or mutant late-domain motif containing proteins are further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HP04, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) that binds to a HSV-1 wild-type binding partner in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10- fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV-1 late-domain motif containing protein point mutants can be used to test whether the NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) binding interaction requires the late domain motif within the particular HSV- 1 protein, and will result in reduced (3-galactosidase activity.

Example 2: Yeast Screen To Identify Small Molecule Inhibitors Of The Interaction Between a HSV- 1 late-domain motif containing protein And NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain). -galactosidase can be used as a reporter enzyme to signal the interaction between yeast two-hybrid protein pairs expressed from plasmids in Saccharomyces cerevisiae.

Yeast strain MY209 (ade2 his3 leu2 tφl cyh2 ura3::GALlp-lacZ gal4 gal80 lys2: :GALlp-HIS3) bearing the plasmids Mp364 (LEU2 CEN4 ARS 1 ADHlp-

SV40NLS-GAL4 (768-881)- gene encoding NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain)-PGKlt AmpR ColEl_ori) and Mp206 (TRP1 CEN4 ARS ADHlp-GAL4(l-147)-gene encoding HSV-1 late domain motif containing protein - ADHlt AmpR ColEl_ori) is cultured in synthetic complete media lacking leucine and tryptophan (SC -Leu -Tip) overnight at 30°C. This culture is diluted to 0.01 OD630 units/ml using SC -Leu -Trp media. The diluted MY209 culture is dispensed into 96- well microplates. Compounds from a library of small molecules are added to the microplates; the final concentration of test compounds is approximately 60μM. The assay plates are incubated at 30°C overnight.

The following day an aliquot of concentrated substrate/lysis buffer is added to each well and the plates incubated at 37°C for 1-2 hr. At an appropriate time an aliquot of stop solution is added to each well to halt the beta-galactosidase reaction. For all microplates an absorbance reading is obtained to assay the generation of product from the enzyme substrate. The presence of putative inhibitors of the interaction between HSV-1 late-domain motif containing protein and NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) results in inhibition of the beta-galactosidase signal generated by MY209. Additional testing eliminates compounds that decreased expression of beta- galactosidase by affecting yeast cell growth and non-specific inhibitors that affected the beta-galactosidase signal generated by the interaction of an unrelated protein pair. Once a hit, i.e., a compound which inhibits the interaction between the viral and cellular proteins, is obtained, the compound is identified and subjected to further testing wherein the compounds are assayed at several concentrations to determine an IC50 value, this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor.

Example 3: Analysis of the PXXP Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PXXP motif of a HSV-1 protein containing the motif on the interaction between TsglOl (or fragments thereof) (or another late-domain motif binding partner) and the HSV-1 protein (or fragments thereof). A yeast two-hybrid activation domain-TsglOl construct is prepared from a DNA fragment encompassing the full-length coding sequence for TsglOl (GenBank Accession No. U82130) by PCR from a human fetal brain cDNA library and is subsequently cloned into the EcoRI/Pstl sites of the activation domain parent plasmid GADpN2 (LEU2, CEN4, ARS1, ADHlp-SV40NLS- GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-HSV-1 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV protein derived from a selected HSV strain. In the context of this example the strain corresponds to GenBank accession number NC_001806. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the HSV-1 late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- HSV-1 late domain motif containing protein construct described above. The mutations can be verified by DNA sequence analysis. Non-limiting examples of HSV- 1 proteins and mutations that can be introduced into the DNA binding domain- HSV-1 late domain motif containing protein construct are summarized in Tables 7-9 below.

The effect of the mutations (and the WT) can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tipl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-TsglOl construct and one of the binding domain-mutant HSV-1 protein constructs or the binding domain- wild type protein construct. Filter lift assays for β-Gal activity can be conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild-type or mutant protein interacts with TsglOl. All binding domain constructs can also be tested for self-activation of β-Gal activity.

Interactions between TSGIOI and wild-type HSV-1 late domain motif containing protein (WT) or the protein mutants can be further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, - Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length TsglOl bound to wild- type HSV-1 late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV-1 late domain motif containing protein point mutants can be used to test whether the TsglOl binding interaction requires the HSV-1 late domain motif within HSV-1 late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 4: In vitro Binding Assay.

A fusion protein with a GST tag fused to the HSV late domain motif containing protein can be recombinantly expressed and purified by chromatography. In addition, a HSV-1 late domain motif containing protein fragment peptide containing can be synthesized chemically by standard peptide synthesis methods. The peptide can be purified by conventional protein purification techniques, e.g., by chromatography. Nunc/Nalgene Maxisoφ plates are incubated overnight at 4°C or for 1-2 hrs at room temperature in 100 μl of a protein coupling solution containing purified GST-HSV late- domain motif containing protein and 50mM Carbonate, pH=9.6. This allows the attachment of the GST-HSV- 1 late-domain motif containing protein fusion protein to the plates. Liquids in the plates are then emptied and the wells are filled with 400 μl/well of a blocking buffer (SuperBlock; Pierce-Endogen, Rockford, IL). After incubation for 1 hr at room temperature, 100 μl of a mixture containing Drosophila S2 cell lysate myc- tagged TsglOl (residues 1-207) and a specific amount of the HSV late domain motif containing protein peptide fragment is applied to the wells of the plate. This mixture is allowed to react for 2 hr at room temperature to form HSV late domain motif containing protein:Tsgl01 protein-protein complexes (other HSV late-domain containing proteins and late-domain binding proteins can be used according to this procedure)..

Plates are then washed 4 x lOOμl with 1 x PBST solution (Lnvitrogen; Carlsbad, CA). After washing, lOOμl of 1 μg/ml solution of anti-myc monoclonal antibody (Clone 9E10; Roche Molecular Biochemicals; Indianapolis, LN) in 1 x PBST is added to the wells of the plate to detect the myc-epitope tag on the TsglOl protein. Plates are then washed again with 4 x lOOμl with 1 x PBST solution and lOOμl of 1 μg/ml solution of horseradish peroxidase (HRP) conjugated Goat anti-mouse IgG (Jackson Immunoresearch Labs; West Grove, Pennsylvania) in 1 x PBST was added to the wells of the plate to detect bound mouse anti-myc antibodies. The plates are then washed again with 4 x lOOμl with 1 x PBST solution and 100 μl of fluorescent substrate (QuantaBlu; Pierce-Endogen, Rockford, IL) is added to all wells. After 30 min, 100 μl of stop solution is added to each well to inhibit the function of HRP. Plates are then analyzed on a Packard Fusion instrument at an excitation wavelength of 325nm and an emission wavelength of 420nm. The presence of fluorescent signals indicates binding of TsglOl to the fixed GST-HSV- 1 protein. In contrast, the absence of fluorescent signals indicates that the PXXP-containing short peptide is capable of disrupting the interaction between Tsg 101 and HSV- 1 late domain motif containing protein.

Different concentrations of HSV-1 late domain motif containing protein peptide can be tested, and the relative intensities of the fluorescence signals obtained at different concentrations are plotted against the peptide concentrations.

Example 5 : Analysis of the YXXL Motif.

A yeast two-hybrid assay is utilized to determine the effect of amino acid substitution mutations in the YXXL motif of a HSV-1 late domain motif containing protein on the interaction between AP-2 complex, particularly the AP-50 subunit (or AIPl) and the HSV-1 protein. A yeast two-hybrid activation domain- AP-50 construct is prepared from a DNA fragment encompassing the full-length coding sequence for AP-50 (GenBank Accession No. D63475)(or AIPl GenBank Accession No. AF151793) by PCR from a human cDNA library and is subsequently cloned into an appropriate site of an activation domain parent plasmid such as GADpN2 (LEU2, CEN4, ARS1, ADHlp- SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori). The yeast two-hybrid DNA binding domain-HSV-1 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV protein derived from a selected HSV strain. In the context of this example the strain corresponds to GenBank accession number NC_001806. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q. Any amino acid substitution mutations can be introduced by PCR into the HSV-1 late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- HSV-1 late domain motif containing protein construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of a protein and mutations that can be introduced into the DNA binding domain- HSV-1 late domain motif containing protein construct are summarized in Table 8 below.

The effect of the mutations can be tested in yeast cells of the strain Yl 89 purchased from Clontech (ura3-52 his3*200 ade2-101 tipl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain- AP-50 construct (or AIPl) and one of the binding domain-mutant HSV-1 late domain motif containing protein constructs or the binding domain- wild type HSV-1 late domain motif containing protein construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with AP-50. All binding domain constructs are also tested for self-activation of β-Gal activity. Interactions between AP-50 subunit and wild-type HSV-1 late domain motif containing protein (WT) or the YXXL HSV-1 late domain motif containing protein mutants are further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length AP-50 subunit bound wild-type HSV-1 late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of β- galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV- 1 late domain motif containing protein point mutants can be used to test whether the AP-50 binding interaction requires the YXXL late domain motif within the HSV-1 late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 6: Examination of Antiviral Effects.

The following examples demonstrate the anti-viral effect of the proteins and peptides according to the present invention which are tested in the Examples. Approximately 1 million PC 12 cells in a T25 flask are either mock-infected or infected with HSV-1 in 1.5 ml of Medium 199 at a multiplicity of infection of 0.1. In addition, the test peptides according to the present invention are also added to the medium at various concentrations. At 8 hr after infection, cells are harvested and RNA and protein extracts are prepared as described in Liu & Roizman, J. Virol, 65:5149-5156 (1991). A PCR protocol (see Koenig et al, Diagn. Microbiol. Infect. Dis., 40(3): 107- 10 (2001) or enzyme-linked immunoassay can then be used to detect the extent of viral infection and determine the efficacy of the test peptides.

Example 7: Analysis of the PPXY Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PPXY motif of an HSV-2 protein containing the motif on the interaction between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain, or fragments thereof) and the HSV-2 protein (or fragments thereof). A yeast two-hybrid activation domain-NEDD4 construct is prepared from a DNA fragment encompassing the full-length coding sequence (or fragment thereof) for NEDD4 (GenBank Accession No. D42055) by PCR from an appropriate human cDNA library and is subsequently cloned into selected sites of an activation domain parent plasmid, such as GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-HSV-2 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV-2 protein derived from a selected HSV-2 strain. In the context of this example the strain corresponds to GenBank accession number NC_001798. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the gene encoding the HSV-2 late-domain motif sequence in the yeast two-hybrid DNA binding domain-construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of proteins and mutations that can be introduced into the DNA binding domain-HSV-2 construct are summarized in Tables 11-13 below.

Table 11 : Mutations in Heφesvirus-2 RL2 Protein

Table 12: Mutations in Heφesvirus-2 Major Capsid Protein (UL19)

The effect of the wild-type and mutants can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tφl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-NEDD4 construct (or a NEDD4 like protein, or a protein containing a WW domain) and one of the binding domain-mutant HSV-2 constructs or the binding domain-wild type construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain). All binding domain constructs are also tested for self-activation of β-Gal activity. Interactions between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) and HSV-2 wild-type (WT) or mutant late-domain motif containing proteins are further quantitated by performing liquid culture 3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) that binds to a HSV-2 wild-type binding partner in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV-2 late-domain motif containing protein point mutants can be used to test whether the NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) binding interaction requires the late domain motif within the particular HSV-2 protein, and will result in reduced /3-galactosidase activity.

Example 8: Yeast Screen To Identify Small Molecule Inhibitors Of The interaction Between a HSV-2 late-domain motif containing protein And NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain).

/3-galactosidase can be used as a reporter enzyme to signal the interaction between yeast two-hybrid protein pairs expressed from plasmids in Saccharomyces cerevisiae. Yeast strain MY209 (ade2 his3 leu2 frpl cyh2 ura3::GALlρ-lacZ gal4 gal80 lys2::GALlp-HIS3) bearing the plasmids Mp364 (LEU2 CEN4 ARSl ADHlp-

SV40NLS-GAL4 (768-881)- NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain)-PGKlt AmpR ColEl_ori) and Mp206 (TRP1 CEN4 ARS ADHlp- GAL4(1-147)-HSV-XX- (448-500)-ADH It AmpR ColEl_ori) is cultured in synthetic complete media lacking leucine and tryptophan (SC -Leu -Tφ) overnight at 30°C. This culture is diluted to 0.01 OD630 units/ml using SC -Leu -Tφ media. The diluted MY209 culture is dispensed into 96-well microplates. Compounds from a library of small molecules are added to the microplates; the final concentration of test compounds is approximately 60μM. The assay plates are incubated at 30°C overnight. The following day an aliquot of concentrated substrate/lysis buffer is added to each well and the plates incubated at 37°C for 1-2 hr. At an appropriate time an aliquot of stop solution is added to each well to halt the beta-galactosidase reaction. For all microplates an absorbance reading is obtained to assay the generation of product from the enzyme substrate. The presence of putative inhibitors of the interaction between HSV late- domain motif containing protein and NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) results in inhibition of the beta-galactosidase signal generated by MY209. Additional testing eliminates compounds that decreased expression of beta- galactosidase by affecting yeast cell growth and non-specific inhibitors that affected the beta-galactosidase signal generated by the interaction of an unrelated protein pair. Once a hit, i.e., a compound which inhibits the interaction between the viral and cellular proteins, is obtained, the compound is identified and subjected to further testing wherein the compounds are assayed at several concentrations to determine an IC50 value, this being the concentration of the compound at which the signal seen in the two-hybrid assay described in this Example is 50% of the signal seen in the absence of the inhibitor.

Example 9: Analysis of the PXXP Motif. A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PXXP motif of a HSV-2 protein containing the motif on the interaction between TsglOl (or a late-domain motif binding partner)(or a fragment thereof) and the HSV-2 protein (or fragments thereof). A yeast two-hybrid activation domain-TsglOl construct is prepared from a DNA fragment encompassing the full-length coding sequence for TsglOl (GenBank Accession No. U82130) by PCR from a human fetal brain cDNA library and is subsequently cloned into the EcoRI/Pstl sites of the activation domain parent plasmid GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS- GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-HSV-2 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV protein derived from a selected HSV strain. In the context of this example the strain corresponds to GenBank accession number NC_001798. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q. Any amino acid substitution mutations can be introduced by PCR into the HSV-2 late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- HSV-2 late domain motif containing protein construct described above. The mutations can be verified by DNA sequence analysis. Non-limiting examples of HSV-2 proteins and mutations that can be introduced into the DNA binding domain- HSV-2 late domain motif containing protein construct are summarized in Tables 14-16 below.

The effect of the mutations (and the WT) can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tφl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-TsglOl construct and one of the binding domain-mutant EBV protein constructs or the binding domain-wild type protein construct. Filter lift assays for β-Gal activity can be conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild- type or mutant protein interacts with TsglOl . All binding domain constructs can also be tested for self-activation of β-Gal activity.

Interactions between TSGIOI and wild-type HSV-2 late domain motif containing protein (WT) or the protein mutants can be further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, - Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM

Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length TsglOl bound to wild- type HSV-2 late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV-2 late domain motif containing protein point mutants can be used to test whether the TsglOl binding interaction requires the HSV-2 late domain motif within HSV-2 late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 10: Analysis of the YXXL Motif. A yeast two-hybrid assay is utilized to determine the effect of amino acid substitution mutations in the YXXL motif of a HSV-2 late domain motif containing protein on the interaction between AP-2 complex, particularly the AP-50 subunit (or ALPl) and the HSV-2 protein. A yeast two-hybrid activation domain- AP-50 construct is prepared from a DNA fragment encompassing the full-length coding sequence for AP-50 (GenBank Accession No. D63475) by PCR from a human cDNA library and is subsequently cloned into an appropriate site of an activation domain parent plasmid such as GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori). The yeast two-hybrid DNA binding domain-HSV-2 late domain motif containing protein construct can be prepared from a fragment corresponding to the HSV protein derived from a selected HSV strain. In the context of this example the strain corresponds to GenBank accession number NC_001798. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the HSV-2 late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- HSV-2 late domain motif containing protein construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of a protein and mutations that can be introduced into the DNA binding domain- HSV-2 late domain motif containing protein construct are summarized in Table 17 below.

Table 17: Mutations in Heφesvirus-2 UL31 Protein

The effect of the mutations can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tipl-901 leu2-3,112 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-AP-50 construct and one of the binding domain-mutant HSV-2 late domain motif containing protein constructs or the binding domain- ild type HSV-2 late domain motif containing protein construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with AP-50. All binding domain constructs are also tested for self- activation of β-Gal activity.

Interactions between AP-50 subunit (or API) and wild-type HSV-2 late domain motif containing protein (WT) or the YXXL HSV-2 late domain motif containing protein mutants are further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mMNaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length AP-50 subunit bound wild-type HSV-2 late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of D- galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different HSV-2 late domain motif containing protein point mutants can be used to test whether the AP-50 binding interaction requires the YXXL late domain motif within the HSV-2 late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 11: Examination of Antiviral Effects The following examples demonstrate the anti- viral effect of the proteins and peptides according to the present invention which are tested in the Examples. Approximately 1 million PC 12 cells in a T25 flask are either mock-infected or infected with HSV-2 in 1.5 ml of Medium 199 at a multiplicity of infection of 0.1. In addition, the test peptides according to the present invention are also added to the medium at various concentrations. At 8 hr after infection, cells are harvested and RNA and protein extracts are prepared as described in Liu & Roizman, J. Virol, 65:5149-5156 (1991). A PCR protocol (see Koenig et al, Diagn. Microbiol Infect. Dis., 40(3): 107-10 (2001) or enzyme-linked immunoassay can then be used to detect the extent of viral infection and determine the efficacy of the test peptides.

Example 12: Analysis of the PPXY Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PPXY motif of an EBV protein containing the motif on the interaction between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain, or fragments thereof) and the EBV protein (or fragments thereof). A yeast two- hybrid activation domain-NEDD4 construct is prepared from a DNA fragment encompassing the full-length coding sequence (or fragment thereof) for NEDD4 (GenBank Accession No. D42055) by PCR from an appropriate human cDNA library and is subsequently cloned into selected sites of an activation domain parent plasmid, such as GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-EBV late domain motif containing protein construct can be prepared from a fragment corresponding to the EBV protein derived from a selected EBV strain. Ln the context of this example the strain corresponds to GenBank accession number NC_001345. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q. Any amino acid substitution mutations can be introduced by PCR into the gene encoding the EBV late-domain motif sequence in the yeast two-hybrid DNA binding domain-construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of proteins and mutations that can be introduced into the DNA binding domain-EBV construct are summarized in Tables 18-20 below.

The effect of the wild-type and mutants can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 trpl-901 leu2-3,112 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-NEDD4 construct (or a NEDD4 like protein, or a protein containing a WW domain) and one of the binding domain-mutant EBV constructs or the binding domain-wild type construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain). All binding domain constructs are also tested for self-activation of β-Gal activity.

Interactions between NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) and EBV wild-type (WT) or mutant late-domain motif containing proteins are further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) that binds to a EBV wild-type binding partner in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different EBV late-domain motif containing protein point mutants can be used to test whether the NEDD4 (or a NEDD4 like protein, or a protein containing a WW domain) binding interaction requires the late domain motif within the particular EBV protein, and will result in reduced /3-galactosidase activity.

Example 13: Analysis of the PXXP Motif.

A yeast two-hybrid assay can be utilized to determine the effect of amino acid substitution mutations in the PXXP motif of an EBV protein containing the motif on the interaction between TsglOl (or a late-domain motif binding partner) and the EBV protein. A yeast two-hybrid activation domain-TsglOl construct is prepared from a DNA fragment encompassing the full-length coding sequence for TsglOl (GenBank Accession No. U82130) by PCR from a human fetal brain cDNA library and is subsequently cloned into the EcoRL/Pstl sites of the activation domain parent plasmid GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS-GAL4 (768-881)-MCS (multiple cloning site)-PGKlt, AmpR, ColEl_ori).

The yeast two-hybrid DNA binding domain-EBV late domain motif containing protein construct can be prepared from a fragment corresponding to the EBV protein derived from a selected EBV strain. Ln the context of this example the strain corresponds to GenBank accession number NC_001345. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the EBV late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- EBV late domain motif containing protein construct described above. The mutations can be verified by DNA sequence analysis. Non-limiting examples of EBV proteins and mutations that can be introduced into the DNA binding domain- EBV late domain motif containing protein construct are summarized in Tables 21-23 below.

Table 21 : Mutations in Epstein-Barr Virus BPLF1 Protein

Table 23 : Mutations in Epstein-Barr Virus EBNA2 Protein

The effect of the mutations (and the WT) can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3-52 his3*200 ade2-101 tφl-901 leu2-3,l 12 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-TsglOl construct and one of the binding domain-mutant EBV protein constructs or the binding domain- wild type protein construct. Filter lift assays for β-Gal activity can be conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the protein wild-type or mutant protein interacts with TsglOl. All binding domain constructs can also be tested for self-activation of β-Gal activity.

Interactions between TSGIOI and wild-type EBV late domain motif containing protein (WT) or the protein mutants can be further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, - Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length TsglOl bound to wild- type EBV late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of /3-galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different EBV late domain motif containing protein point mutants can be used to test whether the TsglOl binding interaction requires the EBV late domain motif within EBV late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 14: Analysis of the YXXL Motif.

A yeast two-hybrid assay is utilized to determine the effect of amino acid substitution mutations in the YXXL motif of an EBV late domain motif containing protein on the interaction between AP-2 complex, particularly the AP-50 subunit (or AIPl) and the EBV protein. A yeast two-hybrid activation domain-AP-50 (or AIPl) construct is prepared from a DNA fragment encompassing the full-length coding sequence for AP-50 (GenBank Accession No. D63475) by PCR from a human cDNA library and is subsequently cloned into an appropriate site of an activation domain parent plasmid such as GADpN2 (LEU2, CEN4, ARSl, ADHlp-SV40NLS-GAL4 (768-881)- MCS (multiple cloning site)-PGKlt, AmpR, ColE l_ori).

The yeast two-hybrid DNA binding domain-EBV late domain motif containing protein construct can be prepared from a fragment corresponding to the EBV protein derived from a selected EBV strain. In the context of this example the strain corresponds to GenBank accession number NC_001345. The gene or a fragment thereof that encodes the late-domain motif containing protein can be obtained by PCR from the selected strain, followed by cloning into the selected restriction sites of a binding domain parent plasmid such as pGBT.Q.

Any amino acid substitution mutations can be introduced by PCR into the EBV late domain motif containing protein sequence in the yeast two-hybrid DNA binding domain- EBV late domain motif containing protein construct described above. The mutations are verified by DNA sequence analysis. Non-limiting examples of a protein and mutations that can be introduced into the DNA binding domain-EBV late domain motif containing protein construct are summarized in Table 24 below.

Table 24: Mutations in Epstein-Barr Virus BFLF2 Protein

The effect of the mutations can be tested in yeast cells of the strain Y189 purchased from Clontech (ura3 -52 his3 *200 ade2- 101 tφ 1 -901 leu2-3 , 112 met gal4 gal80 URA3::GALlp-lacZ) by co-transforming with the activation domain-AP-50 construct and one of the binding domain-mutant EBV late domain motif containing protein constructs or the binding domain- wild type EBV late domain motif containing protein construct. Filter lift assays for β-Gal activity are conducted by lifting the transformed yeast colonies with filters, lysing the yeast cells by freezing and thawing, and contacting the lysed cells with X-Gal. Positive β-Gal activity indicates that the wild type or mutant protein interacts with AP-50 (or AIPl). All binding domain constructs are also tested for self-activation of β-Gal activity.

Interactions between AP-50 subunit (or AIPl) and wild-type EBV late domain motif containing protein (WT) or the YXXL EBV late domain motif containing protein mutants are further quantitated by performing liquid culture /3-galactosidase assays. Cultures are grown overnight in synthetic media (-Leu, -Tφ, + glucose) in 96 well plates, normalized for optical density, and lysed by addition of 6X lysis/substrate solution in 6X Z-buffer (60mM KC1, 6mM MgSO4, 360mM Na2HPO4, 240 mM NaH2PO4, 6mg/ml CPRG, 0.12U/ml lyticase, 0.075% NP-40). Cultures are incubated for 2 hr at 37°C, clarified by centrifugation, and the optical absorbance of each supernatant is measured (575 nm). Full length AP-50 subunit bound wild-type EBV late domain motif containing protein in the two-hybrid liquid culture assay, will result in higher levels of β- galactosidase activity (e.g., greater than 10-fold, more preferably greater than 100-fold, more preferably greater than 300-fold over background). Different EBV late domain motif containing protein point mutants can be used to test whether the AP-50 binding interaction requires the YXXL late domain motif within the EBV late domain motif containing protein, and will result in reduced /3-galactosidase activity.

Example 15: Examination of Antiviral Effects

The following examples demonstrate the anti-viral effect of the proteins and peptides according to the present invention which are tested in the Examples. Approximately 1 million PC 12 cells in a T25 flask are either mock-infected or infected with EBV in 1.5 ml of Medium 199 at a multiplicity of infection of 0.1. In addition, the test peptides according to the present invention are also added to the medium at various concentrations. At 8 hr after infection, cells are harvested and RNA and protein extracts are prepared as described in Liu & Roizman, J. Virol, 65:5149-5156 (1991). A PCR protocol (see Koenig et al, Diagn. Microbiol. Infect. Dis., 40(3):107-10 (2001) or enzyme-linked immunoassay can then be used to detect the extent of viral infection and determine the efficacy of the test peptides.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incoφorated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incoφorated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. Use of cells displaying HSV altered budding phenotype for the manufacture of a medicament useful for treating HSV infection.

2. The use of Claim 1, wherein said medicament further comprises an adjuvant capable of stimulating cellular immune response.

3. The use of Claim 2, wherein said adjuvant is selected from the group consisting of IL-2, IL-4, IL- 12, IL- 18, and gamma-interferon.

4. A composition comprising: cells capable of displaying HSV altered budding phenotype; and an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

5. The composition of Claim 4, wherein said cells are human cells and contain a nucleic acid encoding a mutant HSV protein sufficient for virus particle assembly but devoid of late-domain motifs.

6. The composition of Claim 4 or 5, wherein said cells contain a mutant HSV polypeptide sufficient for virus-like particle assembly but devoid of late-domain motifs.

7. The composition of any one of Claims 4-7, wherein said adjuvant is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, and gamma-interferon.

8. Use of a composition for the manufacture of a medicament useful for the treatment of HSV infection, said composition comprising a compound capable of causing the formation of cells displaying HSV altered budding phenotype, and an adjuvant capable of stimulating cytotoxic T lymphocytes (CTL) response.

9. The use of Claim 8, wherein said adjuvant is a cytokine protein or a nucleic acid encoding the same.

10. The use of Claim 9, wherein said cytokine is selected from the group consisting of IL-2, IL-4, IL-12, IL-18, gamma-interferon, and a fusion protein thereof.

11. The use of any one of Claims 8-11, wherein said composition comprises a compound capable of interfering with the protein-protein interaction between a host cell protein capable of binding a late domain motif and a HSV protein containing a late domain motif.

12. The use of any one of Claims 8-11, wherein said composition comprises a mutant HSV polypeptide devoid of late domain motifs and/or a nucleic acid encoding said mutant HSV polypeptide.

13. The use of Claim 12, wherein said nucleic acid is within an HSV genome.

14. The use of Claim 13 wherein the genome is devoid of late domain motifs capable of effecting viral budding.

15. The use of any one of Claim 8-14, wherein said composition further comprises another HSV protein or an immunogenic fragment thereof, and/or a nucleic acid encoding said other HSV protein or said immunogenic fragment.

16. The use of Claim 15, wherein said other HSV protein is an envelop protein.

17. A method for identifying modulators of HSV budding comprising: providing a HSV protein comprising a late domain motif, or a fragment, derivative, or homologue thereof of said HSV protein; providing a host cell protein capable of binding a late domain motif binding, or a fragment, derivative or homologue thereof of said host cell protein; contacting said HSV protein and said host cell protein in the presence and absence of a test compound; wherein a modulator of HSV budding is identified if the amount of said HSV protein bound to said host cell protein is more or less in the presence as compared to the amount of said HSV protein bound to said host cell protein in the absence of said test compound.

18. The method of claim 17 wherein said HSV protein is HSV core protein.

19. The method of claim 17 wherein said host cell protein is selected from the group consisting of NEDD4, a NEDD4-like protein, or a protein having a WW domain, TSGIOI, AIPl, and AP-50.

20. The method of claim 17 wherein said amount of said HSV protein bound to said host cell protein is less in the presence as compared to the amount of said HSV protein bound to said host cell protein in the absence of said test compound.