WO2001017552A1

WO2001017552A1 - Kaposi's sarcoma-associated herpesvirus ks-sm gene product and uses thereof

Info

Publication number: WO2001017552A1
Application number: PCT/US2000/024698
Authority: WO
Inventors: Sankar Swaminathan
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1999-09-09
Filing date: 2000-09-08
Publication date: 2001-03-15
Also published as: AU7361500A; WO2001017552A8

Abstract

A novel gene encoding a Kaposi's Sarcoma-Associated Herpesvirus KS-SM gene and gene product and uses thereof is disclosed.

Description

KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS KS-SM GENE PRODUCT AND USES THEREOF

TECHNICAL FIELD OF THE INVENTION The present invention relates in general to the field of gene expression, and more particularly, to the isolation and characterization of a novel Kaposi's Sarcoma-Associated Herpesvirus (KSHV) encoded protein that activates cellular growth factor receptors and promotes gene expression.

BACKGROUND OF THE INVENTION Without limiting the scope of the invention, its background is described in connection with Kaposi's Sarcoma and related health problems, as an example. Heretofore, in this field, the Kaposi's has been identified in the most common neoplasm occurring in persons with acquired immunodeficiency syndrome (AIDS). Kaposi's sarcoma (KS) affects approximately 15-20% of AIDS patients, which develop a neoplasm that rarely occurs in immunocompetent individuals. Epidemiologic studies suggest that AIDS-associated KS (AIDS-KS) has an infectious etiology because gay and bisexual AIDS patients are approximately twenty times more likely than hemophiliac AIDS patients to develop KS. Therefore, KS may be associated with specific sexual practices among gay men with AIDS. Furthermore, KS is uncommon in adult AIDS patients infected through heterosexual or parenteral HIV transmission, in pediatric AIDS parenteral HIV transmission, or in pediatric AIDS patients infected through vertical HIV transmission.

Agents previously suspected of causing KS include cytomegalovirus, hepatitis B virus, human papillomavirus, Epstein-Barr virus, human herpesvirus 6, human immunodeficiency virus (HIV), and Mycoplasma penetrans. Non-infectious environmental agents, such as nitrite inhalants, may also play a role in KS tumorigenesis.

Kaposi's sarcoma-associated herpesvirus (KSHV) is also known as human herpesvirus (HHV8). What is needed is the isolation and characterization of genes and gene products that are involved in KSHV -mediated transformation. Also needed are recombinant genes and protein products that may be used to identify therapeutic agents for the treatment and cure of KSHV associated malignancies and infectious disease.

SUMMARY OF THE INVENTION

The present invention is directed to the isolation and characterization of novel gene and gene products associated with KSHV gene activation, angiogenesis, transformation and oncogenesis. More particularly, the present inventor has discovered a new gene cloned from human cells infected with human herpes virus (HHV8), which will be used interchangeably herein with Kaposi's sarcoma-associated herpes virus (KSHV).

The gene, described herein as KS-SM, localizes to the nucleus of infected cells and was found to enhance the level of mRNA expression of certain co- transfected reporter genes, such as a receptor for vascular endothelial growth factor, known as KDR/flk-l. Expression of other transfected genes (luciferase) did not seem to be affected. These effects seem to be mediated at the post- transcriptional level, based on nuclear run-on experiments.

Importantly, the discovery of the KS-SM gene of the present invention provides a target for pharmaceutical agents for treatment of Kaposi's sarcoma, in particular those that block the sarcoma's effects on angiogenesis. Furthermore, transfection or gene delivery of the gene itself might be used to promote angiogenesis, at appropriate locations by inducing VEGF receptor, enhancing the sensitivity of tissue to endogenous VEGF. Also, KS-SM may be used as an activator of gene expression, at the post-transcriptional level, or as a post- transcriptional activator of the VEGF -receptor gene, flk-1.

In one aspect, the invention provides a novel isolated DNA molecule encoding an KS-SM gene product, which product is associated with the infection of HHV-8, and is referred to herein by SEQ ID NO.:l. The cell may be any mammalian cell that is infected by HHV-8 or any cell containing and expressing the isolated DNA molecule of the invention including, but not limited to, body cavity based lymphoma cells, cells from a Kaposi's sarcoma lesion, and cells from primary endothelial tissues. The nucleic acid sequence of the isolated DNA molecule contains the DNA sequence designated herein as SEQ ID NO:l or a portion thereof, which portion is at least 10 nucleotides in length.

Another aspect of the invention is an antisense RNA molecule of at least 10 nucleotides in length capable of hybridizing to sense transcript encoding the expressed KS-SM protein (SEQ ID NO.:2) and having a sequence which is encoded by SEQ ID NO.:l or a portion thereof. In an embodiment of the invention in which the antisense nucleic acid is RNA, thymine residues of SEQ ID NO: 1 are replaced by uracil residues.

An embodiment of the invention is a cell containing the isolated DNA molecule encoding the KS-SM, HHV-8 gene product, which product is localized to the cell membrane. The isolated DNA molecule may be operably attached to exogenous transcription and translational control sequences in an expression cassette for expression in the cell.

Yet another aspect of the invention is a substantially pure polypeptide having an amino acid sequence (SEQ ID NO.:2) deduced from SEQ ID NO.:l. Also comprehended are polypeptide fragments duplicating only a part of the continuous amino acid sequence or secondary conformations within the KS-SM expressed polypeptide predicted to be localized in the cytoplasm and nucleus.

In another embodiment of the invention, polypeptide analogs useful in the practice of this invention include those having one or more amino acid additions, substitutions, and/or deletions as compared to either naturally-derived or recombinant proteins, e.g., the KS-SM protein in the cytoplasm or nucleus. Substitution analogs may be particularly useful in that such analogs may enable improved purification, stability or other useful features as compared to either naturally-derived or recombinant proteins.

Another aspect of the invention is an antibody that binds specifically to a polypeptide having an amino acid sequence of SEQ ID NO:2 or to an immunogenic epitope defined by a portion of SEQ ID NO:2. Techniques for producing polyclonal and monoclonal antibodies for a compound are well known to those skilled in the art. An example of a reference in which such techniques are described is: ANTIBODIES: A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor Press (1988).

An embodiment of the invention is also a cell containing the isolated DNA molecule encoding a KS-SM gene product, which product is localized to the cell cytoplasm and/or nucleus. The isolated DNA molecule is operably attached to exogenous transcription and translational control sequences in, e.g., an expression cassette for expression in the cell.

Yet another aspect of the invention is a method of detecting the presence of HHV-8 in a patient. The detection method of the invention includes the steps of contacting a biological sample from a patient suspected of being infected with HHV-8 with an antibody that binds specifically to a KS-SM polypeptide, which may be produced in a cell infected with HHV-8 and predicted to be localized to the nucleus. Following the contacting step, binding of the antibody to a KS-SM polypeptide of the biological sample is detected by standard immunochemical methods well known to one of ordinary skill in the art. The biological sample of the patient includes, but is not limited to, body fluids such as blood and serum, tissue samples, lysates of cells within a tissue sample, a histological preparation of a tissue, extracts of such samples,, and the like. An alternative aspect of the invention is a method for detecting the presence of KS-SM transcripts expressed in latently infected cells of a patient, which method includes the steps of detecting the presence of nucleic acids produced during infection and encoded by SEQ ID NO:l or a portion thereof. A further aspect of the invention is a method for detecting the presence of KS-SM transcripts expressed in latently infected cells of a patient, the method including the steps of: obtaining a nucleic acid sample and detecting the presence of mRNA complementary to the isolated DNA encoding a KS-SM 8 polypeptide produced during infection and localized to the nucleus. In one embodiment, the isolated nucleic acid is detectable and has the sequence of SEQ ID NO:l or a portion thereof.

Still another aspect of the invention is a method for inhibiting expression of HHV-8 genes in a cell, which DNA encodes a polypeptide produced during infection with HHV-8, the method includes providing antisense nucleic acids complementary to the mRNA encoding the polypeptide. The antisense RNA is at least 10 nucleotides in length and, when annealed to the mRNA, is capable of inhibiting translation of the mRNA into a functional KS-SM polypeptide. The step of providing an antisense RNA is accomplished by introducing into the cell the antisense RNA or an isolated DNA molecule which, when transcribed within the cell, encodes the antisense RNA. The invention includes a method of antisense treatment that includes administering to a mammalian (human) cell in vitro or in vivo a pharmaceutical formulation that includes a pharmaceutically acceptable carrier having dispersed therein a therapeutically effective amount of a compound of a nucleic acid capable of hybridizing to the sense RNA, which nucleic acid may be antisense RNA.

An immunoassay may employ monoclonal or polyclonal antibodies to detect the KS-SM gene product. The immunoassay may also include a solid or semi-solid support having sample-receiving areas. Exemplary sample-receiving supports include test tubes, microtiter plates, dipsticks, membranes, lateral flow devices, resins, PVC or latex beads, and nitrocellulose. The sample receiving areas of the support have surface-bound capture reagent (e.g., recombinantly expressed KS-SM protein) capable of binding to a specific binding member (e.g., an antibody specific for KS-SM). A method of detection for monitoring the result of the immunoassay may be, for example, means to produce and/or measure a detectable reaction. Thus, a method of detection may include an enzyme and a substrate for the enzyme. Examples of detectable labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase), chemiluminescent enzymes (e.g., lucifease), and chromophores (e.g., dyes, colored latex beads, dyed particles, pigments, metal particles (e.g. gold or silver metal particles), dye encapsulated liposomes, carbon). The detectable label may be attached to the KS-SM protein, the anti-KS-SM protein antibody, or a second antibody directed against the anti-KS-SM protein antibody. Examples of substrates for the detection of the label include o-phenylenediamine dihydrochloride; Amerlite Signal Reagent (available from Amersham International PLC); p-nitrophenol phosphate; and luciferin. It will be appreciated that an external detection device such as a spectrophotometer, luminometer or fluorimeter may be employed.

Still another embodiment of the invention is a histological method of detecting the presence of a KS-SM protein produced in cells infected with HHV-8, and which polypeptide is predicted to be localized to the cytoplasm and the nucleus of the infected cell. According to a method of the present invention, a tissue specimen of a patient suspected of being infected with HHV-8 is prepared for analysis by standard histological techniques well known to those of ordinary skill in the art. The histological sample is then contacted with an antibody that binds specifically to the KS-SM polypeptide of the invention. The KS-SM polypeptide-antibody complex is then detected by standard immunochemical techniques such as radioimmunolabelling or immunofluorescence. An embodiment of the invention features a kit for qualitatively or quantitatively detecting 1) an expressed KS-SM protein and/or 2) an antibody to the KS-SM protein. The kit includes reagents that may be used as an assay for detecting the protein or antibody by immunochemical techniques described above and well known in the art.

Alternatively, the kit may include a container containing an expressed KS- SM protein-specific antibody and a device, molecule or compound for concentrating (e.g. immunoprecipitating) antibody-KS-SM protein complexes formed by contacting a biological sample with the anti-KS-SM antibody. It is understood that the alternative reaction of detecting an anti-KS-SM protein-specific antibody in a body fluid is also within the scope of the invention.

The present invention will also find application as a target for the isolation, identification and improvement of pharmaceutical agents that affect the activity of the KS-SM gene and protein product. One such use is as as a tool to find cellular interactors whose interaction with KS-SM can be then specifically inhibited by agents either pharmaceutical or designed chemical compounds, e.g., nucleic acid or peptides, for the treatment of, e.g., Kaposi's Sarcoma.

The KS-SM gene or gene product may also be used directly as a tool for promoting angiogenesis in vivo and in vitro. Such an activity may be used to mimick or enhance the effects of tumor-induced angiogenesis in vivo or in vitro in systems that would benefit from the enhanced expression of, e.g., flk-1. The use of KS-SM in an induced angiogenesis system may include its use as an induced angiogenesis model system for isolating pharmaceutical agents that inhibit angiogenesis and/or neoplastic growth. Finally, the present invention may be used to enhance the activation of genes, gene products or other cellular structures on which KS-SM may have a positive or negative effect. For example, KS-SM may be used to enhance the activation, expression, enzymatic activity or interaction of cellular genes that are otherwise deficiently expressed or activated in order to a disease condition that may be positively affected by KS-SM.

BRIEF DESCRIPTION OF THE DRAWINGS For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures in which corresponding numerals in the different figures refer to corresponding parts and in which:

Figure 1 is a nucleic acid sequence (SEQ ID NO:l) of the KS-SM gene of the present invention;

Figure 2 is a single-letter amino acid sequence of the protein product of the KS-SM gene of the present inveniton;.

Figure 3 is a graph showing the relative levels of CAT expression activated by the KS-SM protein in a promoter-independent manner; Figure 4 is a nuclear-run on assay showing that KS-SM activates gene expression at the post-transcriptional level; and

Figure 5 shows that KS-SM increases expression of the human flk-1 gene.

DETAILED DESCRIPTION OF THE INVENTION While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that may be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention. DEFINITIONS

To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

As used throughout the present specification the following abbreviations are used: ORF, open reading frame; kb, kilobase (pairs); UTR, untranslated region; kD, kilodalton; PCR, polymerase chain reaction; RT, reverse transcriptase; KSHV (Kaposi's Sarcoma Associated Herpervirus); Human Herpes Virus (HHV8); Vascular Endothelial Growth Factor (VEGF); VEGF Receptor (flk-1); mRNA (messenger ribonucleic acid); chloramphenicol acetyl transferase (CAT); and Epstein-Barr Virus (EB V).

The term "KS-SM" refers to the nucleotides essentially as set forth (SEQ ID NO.: 1) or the amino acid sequence essentially as set forth (SEQ ID NO.: 2).

The terms "a sequence essentially as set forth in SEQ ID NO. (#)", "a sequence similar to", "nucleotide sequence" and similar terms, with respect to nucleotides, refers to sequences that substantially correspond to any portion of the sequence identified herein as SEQ ID NO.: 1. These terms refer to synthetic as well as naturally-derived molecules and includes sequences that possess biologically, immunologically, experimentally, or otherwise functionally equivalent activity, for instance with respect to hybridization by nucleic acid segments, or the ability to encode all or portions of the upregulation of flk-1 gene expression activities. Naturally, these terms are meant to include information in such a sequence as specified by its linear order.

The terms "a sequence essentially as set forth in SEQ ID NO.: 2", "a sequence similar to", "amino acid sequence" and similar terms, with respect to amino acids, refers to peptides, polypeptides, proteins, fragments, fusions, derivatives and alterations thereof that substantially correspond to the sequences of SEQ ID NO.: 2. These terms refer to synthetic as well as naturally-derived molecules and includes sequences that possess biologically, immunologically, experimentally, or otherwise functionally equivalent activities, for instance, segments of amino acids which possess immunological activity as an antigenic determinant. Naturally, these terms are meant to include information in such a sequence as specified by its linear order.

The term "homology" refers to the extent to which two nucleic acids are complementary. There may be partial or complete homology. A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially homologous." The degree or extent of hybridization may be examined using a hybridization or other assay (such as a competitive PCR assay) and is meant, as will be known to those of skill in the art, to include specific interaction even at low stringency.

The inhibition of hybridization of the completely complementary sequence to the target sequence may also be examined using a hybridization assay involving a solid support (e.g., Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. Low stringency conditions may be used to identify the binding of two sequences to one another while still being specific (i.e., selective). The absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 40% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target and the original interaction will be found to be selective.

Low stringency conditions are generally conditions equivalent to binding or hybridization at 42 degrees Centigrade in a solution that includes: 5XSSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH 7.4), 0.1% SDS, 5X Denhardt's reagent (50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma) and 100 micrograms/ml denatured salmon sperm DNA); followed by washing in a solution comprising 5X SSPE, 0.1 % SDS at 42 degrees Centigrade when a probe of about 500 nucleotides in length is employed.

The art knows that numerous equivalent conditions may be employed to achieve low stringency conditions. Factors that affect the level of stringency include: the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., formamide, dextran sulfate, polyethylene glycol). Likewise, the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, inclusion of formamide, etc.).

An oligonucleotide sequence that is "substantially homologous to the KS- SM gene of SEQ ID NO.:l" is defined herein as an oligonucleotide sequence that exhibits greater than or equal to 75% identity to the sequence of SEQ ID NO.:l when sequences having a length of 100 bp or larger are compared.

The term "gene" is used to refer to a functional protein, polypeptide or peptide-encoding unit. As will be understood by those in the art, this functional term includes both genomic sequences, cDNA sequences, or fragments or combinations thereof, as well as gene products, including those that may have been altered by the hand of man. Purified genes, nucleic acids, protein and the like are used to refer to these entities when identified and separated from at least one contaminating nucleic acid or protein with which it is ordinarily associated.

As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The vector may be further defined as one designed to propagate KS-SM sequences, or as an expression vector that includes a promoter operatively linked to the KS-SM sequence, or one designed to cause such a promoter to be introduced. The vector may exist in a state independent of the host cell chromosome, or may be integrated into the host cell chromosome.

The term "host cell" refers to cells that have been engineered to contain KS-SM nucleic acid segments, or altered segments, whether archeal, prokaryotic, or eukaryotic. Thus, engineered, or recombinant cells, are distinguishable from naturally occurring cells that do not contain recombinantly introduced genes through the hand of man.

The term "agonist" refers to a molecule that enhances either the strength or the time of an effect of KS-SM on, for example, flk-1 gene expression and encompasses small molecules, proteins, nucleic acids, carbohydrates, lipids, or other compounds. The term "antagonist" refers to a molecule that decreases either the strength or the time of an effect of KS-SM and encompasses small molecules, proteins, nucleic, acids, carbohydrates, lipids, or other compounds.

The term "altered", or "alterations" or "modified" with reference to nucleic acid or polypeptide sequences is meant to include changes such as insertions, deletions, substitutions, fusions with related or unrelated sequences, such as might occur by the hand of man, or those that may occur naturally such as polymorphisms, alleles and other structural types. Alterations encompass modifications of the KSHV genome and RNA sequences that may differ with respect to their hybridization properties using a given hybridization probe. Alterations of polynucleotide sequences for KS-SM, or fragments thereof, include those that increase, decrease, or have no effect on functionality. Alterations of polypeptides refer to those that have been changed by recombinant DNA engineering, chemical, or biochemical modifications, such as amino acid derivatives or conjugates, or post-translational modifications. The term "promoter" shall mean a nucleotide sequence that, when operably linked to a DNA sequence of interest, promotes transcription of that DNA sequence. By "substantially inhibit" or "substantial reduction" is meant a decrease or reduction in expression, transcription, translation, or a measurable phenotypic characteristic that is approximately 80% of the wild type level, preferably reduced to approximately 50% of the wild type level, or more preferably reduced to approximately 10% or less of the wild type level.

KS-SM The present inventor has cloned and characterized a novel gene from the human herpes virus HHV8, also known as Kaposi's sarcoma-associated herpes virus (KSHV). The entire coding region was cloned from an HHV8-infected primary effusion lymphoma cell line using anchored PCR (5' RACE). The cDNA sequence for the KS-SM gene is depicted in Figure 1 (SEQ ID NO:l) and includes a short first exon spliced to a long second exon at canonical splice donor and acceptor sites. Upstream and downstream noncoding regions were thus identified. Figure 2 is a single-letter amino acid sequence for the HHV8 gene KS- SM protein. The protein is predicted to encode a 455 amino acid protein with approximately 35% overall similarity to the EBV SM and HSV ICP27 proteins. Expression of KS-SM was observed to increase by chemical induction of lytic HHV8 replication (data not shown).

The present inventor has expressed the HHV8 protein in cells by transfection. Tagged KS-SM is expressed in the nucleus of transfected cells. Cotransfection of KS-SM with reporter gene constructs indicated that KS-SM is an activator of gene expression. Activation by KSSM led to increased accumulation of target gene mRNA in both cytoplasm and nucleus. KS-SM also displays gene-dependent activity. One of the genes that KS-SM activates is the receptor for vascular endothelial growth factor (VEGF), known as flk-1, KDRT and VEGFR-2. KS-SM is thus a gene that may be involved in activation of other HHV8 genes. Importantly, KS-SM is an attractive target for antiviral therapy as it is does not resemble known human genes and is likely to be important in lytic replication of HHV8. Drugs or other specific agents developed to target KS-SM have the potential to be generalized and be applicable to the treatment of other herpes viruses.

Even more significantly, KS-SM has the potential to be used as a therapeutic agent that induces expression of VEGF receptors on specific tissues, endogenous or transplanted. Such a use has potential applications in the fields of surgery, wound healing, cancer, cardiovascular disease and others. Finally, as other target genes that are upregulated by KS-SM are identified, further uses for KS-SM as an enhancer of gene expression for therapeutic application, including gene therapy are encompassed.

Cloning techniques are generally known to those of skill in the art, e.g., Current Protocols in Molecular Biology (F.M. Ausbel, R. Brent, R.E. Kingston, D.D. Publishing Associates and Wiley-Interscience, John Wiley & Sons, New York), which reference is specifically incorporated herein by reference for this purpose. The uses for the KS-SM gene includes as a reagent to develop antiviral and anticancer treatments and as a stimulator of specific cell genes, including, but not limited to, a receptor for human endothelial growth factor known as flk-1, KDR or VEGFR 2.

It was also found that the coding sequences and surrounding non-coding sequences (promoter and three prime untranslated region) of KS-SM have not been previously identified. The use of the KS-SM promoters, enhancers and pre and post-translational control sequences are also encompassed herein.

The KS-SM gene encodes a protein (KS-SM protein) that can increase expression of other genes, that is, it appears to provide for the initiation or control of gene expression. KS-SM is express by HHV8, which is causally associated with Kaposi's sarcoma and primary effusion lymphomas. KS-SM is, therefore, a target for antiviral and anti-cancer therapies. Knowledge of the KS-SM sequence, as disclosed herein, allows structure modeling and rational design of therapeutic agents against HHV8.

Furthermore, KS-SM is a potent inducer of human flk-^ which is a receptor for human vascular endothelial growth factor (VEGF). Thus, KS-SM has potential as a pro-angiogenic agent, promoting and stimulating the growth of vascular endothelium. Such an application has great utility in many areas, including surgery, wound healing, transplantation and treatments for cardiovascular disease. As KS-SM enhances gene expression of infected and/or transfected cells, control over the expression of cellular genes permits the use of KS-SM as a stimulator of other human genes. Thus, KS-SM may be of potential utility in areas besides angiogenesis and may be useful in gene therapy strategies.

KS-SM is an activator of gene expression

The present inventor has found that KS-SM, when transfected into B lymphocytes, strongly activates chloramphenicol acetyl transferase (CAT) reporter gene expression (Figure 3). This effect of KS-SM on CAT expression was shown to be post-transcriptional, as KS-SM did not enhance transcription when measured in a nuclear run-on transcription assay (Figure 4). It is important to note that the effect of KS-SM is gene-dependent. In other words, KS-SM like EBV SM, does not stimulate expression of all genes. For example, KS-SM does not enhance expression of the luciferase reporter gene (data not shown).

KS-SM leads to post-transcriptional activation of the KDR/flk-l gene.

Several lines of evidence support the role of the VEGF/VEGF -receptor axis in KS lesion formation. KS cells express VEGF at higher levels than primary endothelial cells. KS lesions are angiomatous and the VEGF receptor flk-1 is upregulated in KS cell culture and primary tumor tissues. The present inventor determined that KS-SM affects the expression of flk-1 in transfection experiments. As shown in figure 5, expression of KS-SM dramatically enhanced expression of flk-1 mRNA. The levels of flk-1 mRNA were increased in both nucleus and cytoplasm, but particularly in the latter, suggesting that KS-SM may be involved in nuclear export of flk-1 mRNA.

The KS-SM gene and gene product of the present invention finds particular utility as a tool for developing antiviral drugs or treatments. KS-SM may also be used as a potentiator or simulator of angiogenesis. By blocking the activity of KS-SM using, for example, molecular antagonists or inhibitors, angiogenesis and tumor growth in KS and other HHV8 associated diseases, as well as potentially other herpes virus diseases, may be caused by blocking the activity of KS-SM. Blocking the activity of KS-SM in vivo or in vitro may be used to identify anti-neoplastic agents that control the angiogenesis required to support solid tumors, e.g., Kaposi's Sarcoma lesions. On the other hand, delivery of the KS-SM gene or derivatives to areas where neo-angiogenesis would be beneficial may have broad use as a heterologous promoter of angiogenesis. For example, expression of KS-SM in skin or other tissue could promote engraftment after transplantation. Similarly, expression in areas of compromised vasculature and ischemia could promote wound healing, oxygen delivery and endothelialization.

The identification of KS-SM also provides a valuable tool for the enhancement of VEGF activity. KS-SM is the first genes product that has been shown to have specific effects on vascular endothelial growth factor receptors such as flk-1. For example, the KS-SM gene and its gene product has the advantage that it may make a target tissue more responsive to certain stimuli such as VEGF (and potentially other cytokines), avoiding the systemic effects and difficulties of administration of large amounts of cytokines.

Figure 3 is a graph that shows the relative levels of CAT expression activated by the KS-SM protein in a promoter-independent manner. BJAB cells were contransfected with KS-SM (shaded bars) or antisense SM control (aSM, open bars) and CAT plasmid. CAT was transcribed from either the EBV latent promoted (Wp) or the CMV IE promoter (CMV). CAT assay was performed with lysates from cells harvested 18 hours after transfection. Results are the mean of three independent experiments.

Figure 4 is a nuclear-run on assay showing that KS-SM activates gene expression at the post-transcriptional level. Nuclear run-on assay was performed with labeled nuclear transcripts from KSSM or control (aSM) transfected BJAB cell nuclei, which were hybridized to immobilized cDNA corresponding to either 18S RNA or the CAT gene. The rate of CAT transcript initiation was not significantly different in the presence of KS-SM as compared to control.

Figure 5 shows that KS-SM increases expression of the human flk-1 gene. BJAB cells were transfected with human flk-1 plasmid and either control plasmid or plasmid expressing the HHV8 KS-SM gene. RNA from cytoplasmic (C) or nuclear (N) fractions was harvested 18 hours after transfection, electrophoresed, blotted and probed with human-flk-1 cDNA. The blot was stripped and probed with human GAPDH cDNA as a control for RNA loading.

Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials or methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the particular information for which the publication was cited

Before the methods and compositions of the present invention are described and disclosed it is to be understood that this invention is not limited to the particular methods and compositions described as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims.

While this invention has been described in reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass any such modifications or embodiments.

Claims

What is claimed is:

1. A purified nucleic acid segment comprising a nucleic acid sequence substantially homologous to the gene sequence in SEQ ID NO:l.

2. The nucleic acid segment of claim 1, further defined as encoding KS-SM, wherein the segment:

(a) encodes the amino acid sequence of SEQ ID NO:2; or

(b) is capable of hybridizing to the nucleotide sequence of SEQ ID NO:l under standard hybridization conditions.

3. The nucleic acid segment of claim 1, which comprises the sequence of SEQ ID NO: 1.

4. The nucleic acid segment of claim 2, defined further as consisting essentially of the nucleotide sequence of SEQ ID NO: 1.

5. The nucleic acid segment of claim 1, wherein said gene sequence further comprises 5' and 3' flanking regions.

6. The nucleic acid segment of claim 1 , wherein said gene further comprises intervening sequences.

7. A recombinant DNA vector comprising the oligonucleotide of claim 1.

8. A host cell comprising the recombinant DNA vector of claim 7.

9. The recombinant host cell of claim 7, defined further as a prokaryotic cell.

10. The recombinant host cell of claim 7, defined further as a eukaryotic cell.

11. A nucleic acid segment comprising at least a 14 nucleotide sequence corresponding to, or complementary to, the nucleic acid sequence of SEQ ID NO:l.

12. A purified KS-SM protein encoded by an oligonucleotide comprising a nucleic acid sequence substantially homologous to the coding strand of the gene sequence set forth in SEQ ID NO:l.

13. The purified protein of claim 11, wherein said protein is a human KSHV KS-SM protein.

14. The purified protein of claim 11, wherein said protein is the KS-SM protein of SEQ ID NO. :2.

15. A fusion protein comprising a portion of the KS-SM of claim 11 and a non- viral protein sequence.

16. The fusion protein of claim 14, wherein said the KS-SM protein is the protein of SEQ ID NO. :2.

17. The fusion protein of claim 14, wherein said protein is contacted to a cell in order to stimulate the expression of a vascular endothelial growth factor receptor.

18. A method for detecting a nucleic acid segment encoding an KS-SM protein comprising the steps of: providing in any order: a sample suspected of containing an KS-SM protein encoding nucleic acid; and a nucleic acid segment comprising 15 or more contiguous bases of SEQ ID NO:l; mixing said sample with said nucleic acid segment under conditions wherein said segment can bind to said KS-SM protein encoding nucleic acid; and detecting said binding.

19. The method of claim 18, wherein said nucleic acid segment comprises a detectable label.

20. The method of claim 18, wherein said sample is of mammalian origin.

21. A method of enhancing VEGF receptor expression comprising: expression the KS-SM gene in a cell; and monitoring the expression of the flk-1 gene or protein expression, wherein an increase in flk-1 gene expression leads to increases surface expression of flk-1 protein on the cell surface.

22. The method of claim 21, further comprising the steps of: contacting a cell expressing KS-SM with a test compound; and comparing the expression levels of flk-1 in cells expressing KS-SM that were contacted with the test compound and cells expressing KS-SM not exposed to the test compound; wherein a change in the expression levels of flk-1 is indicative that the test compound affects the cellular response to KS-SM expression.