WO1995010288A1 - Hepatitis b virus interacts with cellular dna repair processes - Google Patents

Abstract

A method of treating viral disease in an animal or human, comprising the step of interfering with the interaction of the viral protein with a DNA repair complex in the animal or human to be treated. Specific examples include inhibition of the interaction of the X protein from HBV and the XAP-1 protein of the DNA repair complex. The method can also be used for the treatment of cancer secondary to viral infection. Also, there is a nucleic acid sequence encoding the XAP-1 protein and the amino acid sequence of the XAP-1 protein. Antibodies to the protein can be used for diagnostic purposes and the XAP-1 gene can be used as the object of screening assays to detect genetic alterations.

Description

HEPATITIS B VIRUS INTERACTS WITH CELLULAR DNA REPAIR PROCESSES

This application is a Continuation-in-Part of United States Application Serial No. 08/133,932 filed October 11, 1993.

The present invention was made utilizing funds of the United States Government. The United States Government is entitled to certain rights under this invention.

Field of the Invention This invention relates to methods of treating viral diseases, liver cancer secondary to viral diseases, diagnosing viral diseases and specific antibodies against antigens involved in interactions with proteins of viral origin. More particularly it relates to the nucleic acid sequence for XAP-1 and its amino acid sequence of the human XAP-1 protein, which is part of the DNA repair complex.

Background Hepatitis B Virus (HBV): Clinical Importance

HBV is worldwide in distribution. It causes acute and chronic liver cell damage and hepatocellular carcinoma (HCC; liver cancer). HBV may cause long-term persistent infections, with the frequency depending on the age and immunologic status of the host at the time of infection, and genetic factors of both the virus and host. Persistent infections often result in serious liver disease, including cirrhosis and cancer. The prevalence rates of persistent infections range from as high as 20% in certain regions of China, Southeast Asia, and subsaharan Africa to less than 0.5% in North America. It is estimated there are approximately 300 million persistently infected carriers of HBV worldwide. An estimated 1 million deaths annually are attributable to the harmful effects of HBV infection. At this time, no clinically useful antivirals against HBV are available.

HBV is transmitted in various ways, including from mother to offspring, by contact, and by parenteral and sexual routes. High-risk groups include parenteral drug abusers, institutionalized persons, health care personnel (surgeons, pathologists, and other physicians, dentists, nurses, laboratory technicians, and blood bank personnel), individuals who have recently received blood transfusions, hemodialysis patients and staff, highly promiscuous persons, and newborn infants born to mothers with hepatitis B.

A variety of modes of HBV transmission and rates of persistent infections exist between endemic countries with high prevalence rates and nonendemic countries. In endemic areas, the major routes of infection are perinatal transmission from infected mothers to offspring and contact-associated transmission during the first years of life.

It is the chronic infection with HBV that is a major risk factor of HCC. Viral infection usually persists for several decades before the emergence of HCC, and the risk of tumor development increases with the duration of chronic infection. Chronic carriers are about 200 times more likely to develop HCC than uninfected persons living in the same area. Nearly 50% of Chinese males with chronic HBV infections will eventually suffer HCC. Analyses of liver tissue from patients with chronic HBV infection have identified both replicative intermediates (indicative of active viral replication) and integrated forms of HBV DNA.

HBV Genome and Gene .Products

HBV is a member of the hepadnavirus family, a group of hepatotropic, DNA-containing viruses. Additional animal hepadnaviruses include the woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus, and a virus of tree squirrels. Avian hepadnaviruses include the duck hepatitis B virus (DHBV) and DHBV-related viruses from geese, grey herons, and other species of ducks. Although minor differences exist, hepadnaviruses are similar in morphology, genome structure, and pathogenesis. All hepadnaviruses are characterized by a restrictive host and tissue tropism and are associated with both acute and chronic liver disease. Infectious HBV is present mainly in the blood of infected patients, but it is also found in other body fluids, such as saliva, urine, and semen. The liver is the target organ, but blood cells may also be infected. The HBV genome consists of partially double-stranded DNA, approximately 3,200 base pairs (bp) in length; it represents the smallest genome of any virus known to infect man. DNA sequence analyses have demonstrated 90—98% nucleotide sequence homology among different HBV isolates, and the genome structures of the HBV isolates share many features in common with other hepadnaviruses. The full-length (i.e., 3,200 bp) DNA strand of the HBV genome is of minus polarity and is complementary to all HBV mRNAs. In contrast, the positive strand is complementary at its 5' end to the first 224 bp of the negative strand, but has a variable 3' end (ranging from 53 to 88% of unit length). The organization of the HBV genome is remarkably efficient. Analysis of different HBV isolates has revealed the conservation of four long open reading frames (ORFs) within the genome which encode specific viral proteins, including the virus nucleocapsid (core antigen or HBcAg), envelope glycoproteins (surface antigen or HBsAg), polymerase (product of the P gene), and a protein from the X gene.

HBV Replication

Replication of HBV begins with the attachment of the virus particle to susceptible cells, such as hepatocytes. Following entry into hepatocytes, the core component is released from the virion, and the partially double-stranded DNA genome is converted to a covalently closed circular (CCC) form that can be detected in the cell nucleus. The CCC DNA then serves as template for the production of HBV mRNAs. The 3.5-kb pre-genome transcript is encapsidated, via a packaging signal located near the 5' end of the RNA, into newly synthesized core particles where it serves as template for the HBV reverse transcriptase. An RNase-H-like activity within the HBV polymerase removes the RNA template, as the negative-strand DNA is synthesized. Viral replicative intermediates, consisting of full-length minus-strand DNA plus variable-length (20—40%) positive-strand DNA, are encapsidated within the core particle during normal virus replication. Virus replication is completed as the DNA-containing core particles bud from the cell surface.

The integration of HBV DNA into chromosomal DNA, although not a part of normal viral replication, occurs during chronic infection with HBV. Integrated HBV can be detected in most but not all HCCs that arise in

HBV-positive individuals and is usually present in 1 — genome copies per cell. The percentage of liver tumors that are HBV positive approaches 95% in high-HBV-endemic areas such as China, and viral DNA may be detected in approximately 80—85% of HBV-related HCCs from other regions of the world. The analysis of many HBV inserts cloned from HCCs has established that a preferred site for recombination of viral DNA with cellular DNA is located near the DR region of the viral genome. As replicative intermediates share structural features with many integrated HBV forms (i.e., one end near the DR region, with variable lengths of the positive-strand DNA), it is possible that replicative intermediates may serve as the template for integration of viral DNA.

Integration of HBV DNA is not required for viral replication, so the viral DNA insertions detected in the chromosomal DNA of HCCs most probably occur via illegitimate recombination. Such recombinational events are thought to occur at random locations. Gross chromosomal alterations are frequently observed at the site of viral integration and include deletions, duplications, and translocations. The effect of these chromosomal alterations on the hepatocyte will depend on the identity of genes nearby the insertion site and the extent to which gene expression is altered. Although HBV integration occurs at random within chromosomes, there is a subsequent nonrandom selection for cells containing viral integration events in specific chromosomes during the evolution of a tumor. Of 28 HBV inserts cloned from HCCs and mapped to human chromosomes, 61% have been assigned to chromosomes 3, 11, 17, and 18. When considering the mutagenic consequence of HBV integration, the data suggest that the disruption of genes on chromosomes 3, 11, 17, and 18 may be particularly important in the genesis of subsets of HCCs. HBV X Protein

The X ORF represents the smallest ORF of the HBV genome and has the potential to encode a protein 146 to 154 amino acids in length, depending on the isolate of HBV. The X ORF overlaps with parts of the P and pre-C ORFs and encompasses several regulatory regions of the genome, including both DRs, enhancer II, and the C gene promoter. The X ORF peptide sequence is highly conserved among different viral isolates. Although detection of the X protein within HBV-infected cells remains difficult, the presence of anti-X protein antibodies in the sera of HBV-infected patients provides evidence that the X ORF is expressed during natural infection.

Antibodies to X are detected most often in patients with chronic hepatitis, liver cirrhosis, and/or hepatoma.

DNA transfection approaches using the cloned X gene have clearly demonstrated that the X gene product can transactivate a wide variety of viral and cellular promoters. The heterogeneity of the elements responsive to X suggests that the X gene product exerts its effect by a mechanism other than direct binding to a specific DNA sequence, probably functioning via effects on cellular factors. The ability of the X gene to transactivate the expression of HBV RNA suggests an important regulatory role for X during viral replication. Indeed, animal studies have indicated the requirement for

X expression for virus growth in vivo.

Specific functions attributable to X protein have been difficult to pinpoint. In addition to being a transactivator, X protein has been reported to be a serine protease inhibitor, and to activate the cellular protein kinase C signalling pathway. It has been suggested that the X protein may affect other cellular processes, besides transcription, that are regulated by cellular kinases.

Cellular DNA Repair Mechanisms

Accidental lesions occur continually in the DNA of eucaryotic cells. DNA damaging agents include thermal fluctuations, ultraviolet (UV) irradiation, environmental toxins, and man-made chemicals. It is estimated that thousands of DNA nucleotides are damaged in a cell every day by such chemical processes. If unattended, this would quickly lead to unacceptably high rates of mutations in germ cells (which would affect maintenance of the species) and in somatic cells (which would adversely affect the individual). Uncontrolled cell proliferation (i.e., cancer) is one deleterious outcome of the accumulation of genetic changes in a cell. Cells have a complex system for recognizing and repairing damage in the DNA, in order to maintain the fidelity of the genome. This process is called DNA repair. Based on yeast genetic studies, it is estimated that eucaryotic cells contain more than 50 genes involved in DNA repair functions, reflecting the great importance of this process to the cell. Individuals with the autosomal recessive genetic disease called xeroderma pigmentosum (XP) have a defect in the DNA repair system. Such individuals are sun-sensitive (displaying an abnormal sensitivity to UV radiation) and have a marked predisposition toward skin cancer (2000-fold increased frequency). Somatic cell fusion experiments using cells derived from XP patients have defined seven complementation groups (A— G), suggesting that a multienzyme complex is involved in efficient DNA repair. XP patients are defective in the "bulky lesion" repair mechanism that is believed to be responsible for scanning and repairing large changes in the structure of the DNA double helix. Two groups have described a protein that binds to damaged DNA that may represent the defective protein in XP group E patients (Takao et al., Nucleic Acid Res. 21:4111-4118, 1993; Hwang and Chu, Biochemistry 32:1657-1666, 1993). The monkey gene "UV-DDB" was cloned by Takao et al; the human cognate of the UV-DDB gene was mapped to chromosome 11. Hwang and Chu purified the protein (XPE-binding factor) from human placenta. Both proteins are about the same size (125—127 kDa) and have a high binding affinity for damaged DNA. Both groups conclude their isolate is probably involved in the recognition step of the excision repair pathway. However, as the proteins differ in some biochemical properties, it is not yet clear if the two groups have recovered the same protein.

A number of yeast excision repair genes (RAD) and rodent DNA-repair genes (ERCC) have been isolated. It has recently been shown that the RAD2, ERCC5, and XPGC genes are equivalent. This emphasizes the striking homology between eukaryotic nucleotide-excision repair genes. Of interest is the recent observation that repair protein XPBC/ERCC3 is part of the basal transcription factor TFIIH. This suggests that the same cellular proteins are involved in two different aspects of DNA metabolism: initiation of transcription and excision repair.

Summary of the Invention

An object of the present invention is a method for diagnosing viral infection.

An object of the present invention is a method for diagnosing cancer secondary to viral infection. A further object of the present invention is the provision of a nucleic acid sequence for XAP-1.

Another aspect of the present invention is the provision of an amino acid sequence for the XAP-1 protein.

Another object of the present invention is a method for monitoring viral infection or cancer secondary to viral infection.

Another aspect of the present invention is the use of the XAP-1 gene as the object of screening assays to detect genetic alterations indicating a possible elevated risk of developing cancer.

Thus, in accomplishing the foregoing objects, there is provided in accordance with one aspect of the present invention a method of treating viral diseases in an animal or human, comprising the step of interfering with the interaction of a viral protein with a DNA repair complex in the animal or human to be tested. This same procedure can be used also to treat liver cancer secondary to viral infection. Other aspects of the invention include a nucleic acid sequence coding for the XAP-1 protein as well as the amino acid sequence for the XAP-1 protein and antibodies which bind to the XAP-1 protein or bind to the separate peptides of the XAP-1 protein.

The antibodies can be used to detect the infection, detect the cancer, monitor the effectiveness of therapy or monitor the stages of the cancer or infection.

Other objects, features and advantages will be apparent and more readily understood from reading the following specification and by references to the accompanying drawings forming a part thereof. Examples of the presently preferred embodiments of the invention are given for the purpose of disclosure.

Brief Description of the Drawings

Figure 1 shows the transcriptional activation by reconstitution of GAL4 activity. (A) Transcription of ZαcZ (detected using chromogenic substrate

X-GAL) does not occur when GAL4 does not bind GAL4 upstream activator sequence (UAS_G). (B) GAL4, which contains a DNA-binding domain (BIND) and a transcription activation domain (ACT), binds to UAS_G which results in activation of transcription. Bait (C) or prey (D) plasmids alone do not induce transcription. (E) Interaction of bait protein X and prey protein Y results in transcriptional activity.

Figure 2 shows the nucleic acid sequence which encodes the human XAP-1 protein.

Figure 3 shows the amino acid sequence of the XAP-1 protein. Figure 4 shows the plasmid precursor to the bait plasmid pAS-l-X.

The GAL4 DNA-binding domain (amino acids Nos. 1-147) are fused in frame with X ORF. The Trp gene is used as a selective marker.

Figure 5 shows the prey plasmid pACT. The GAL4 activation domain

(amino acid Nos. 768-881) is fused with a cDNA library made from EBV-transformed human lymphocytes. The Leu gene is used as a selective marker. The host cell is the yeast strain Y153 and the LacZ gene and His3 gene are controlled by GAL4 responsive elements.

Figure 6 shows the immunoprecipitation of labeled yeast extracts with anti-X antiserum. Lane one is the negative control. Lane two is the immunoprecipitate from yeast cells co-expressing X + LBP (Laminin-binding protein). Lane three is the immunoprecipitate from yeast cells co-expressing

X and XAP-1. Anti-X antibody co-precipitated a 140 KD protein.

Figure 7 shows the hydrophilicity plot for XAP-1. Underlined regions 1 and 2 refer to peptides 1 and 2 used to generate antipeptide antibodies in rabbits.

Figure 8 shows that XAP-1 synthesized in vitro in rabbit reticulocyte lysates primed with XAP-1 mRNA is recognized by anti-peptide antisera in immunoprecipitation tests. Lane 1 is a sample of the radiolabeled lysate; XAP-1 migrates at about 127 kD. Lane 2 is a sample of lysate reacted with control serum. Lanes 3 and 4 are samples of lysate reacted with anti-peptide 2 serum and anti-peptide 1 serum, respectively.

Figure 9 shows the expression of XAP-1 in HEpG2 cells (human heptoma-derived cells). Panel A is a Northern blot showing that XAP-1 mRNA is 4.4 kb in size. XAP-1 protein is expressed in the HEpG2 cells (Panels B and C). Cells were metabolically labeled with [³⁵S]methionine, extracted, and immunoprecipitated with anti-peptide 2 antiserum (Panel B). XAP-1 (127 kD) plus several associated cellular proteins were recovered. When unlabeled HEpG2 cells were extracted, reacted with anti-peptide serum, and then analyzed in an immunoblot reaction using anti-peptide serum, XAP-1 protein was detected (Panel C).

Figure 10 shows the association between X and XAP-1 when X is expressed as a GST fusion protein. Lane 1 is radiolabeled in vitro translation of XAP-1. The translation mixtures containing labeled XAP-1 were applied to gluthathione-sepharose beads containing immobilized control GST protein (lane 2) or GST-X fusion protein (lane 3). Bound protein was eluted and analyzed by SDS-PAGE. Note that XAP-1 was recovered using the GST-X fusion protein. Figure 11 shows the procedure used in the experiment in Figure 10 for verifying the interaction between X and XAP-1.

Detailed Description

It will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

As used herein, DNA repair function refers to the general steps of DNA repair, including: (1) recognition of a lesion in the DNA; (2) incision of the damaged strand on both sides of the lesion; (3) excision of the damaged nucleotides; (4) synthesis of new DNA by copying the "good" complementary strand as template; and (5) ligation (sealing) of the nick left in the repaired strand. The end result of DNA repair is the restoration of two good copies of the DNA sequence. As used herein, DNA repair complex refers to the chemical components involved in the intricate system of recognizing and repairing damage to DNA, particularly the multienzyme complexes that have been found to be associated with the recognition of a lesion in DNA, incision and removal of damaged nucleotides, synthesis of new DNA and ligation of the nick in the repaired strand.

Gene therapy, as used herein, refers generally to the method of treating an individual with genetic material to treat a disease or other pathophysiological condition. As used herein, cancer secondary to HBV infection refers to the occurrence of carcinomas and other cancers in an individual as the result of infection with Hepatitis B Virus.

One aspect of the present invention is a method of treating viral disease in a non-human animal or a human, comprising the step of interfering with the interaction of a viral protein with a DNA repair complex in the animal or human to be treated. One specific example would be the treatment of Hepatitis B Virus (HBV) viral disease in non-human animals and humans. In this procedure, the interaction of the X protein of HBV with the XAP-1 protein of the DNA repair complex is inhibited. Thus, acute and chronic infections may be treated and possibly cured by the use of inhibitory substances to block the interaction of X protein with the cellular DNA repair machinery. Such an inhibitory substance and approaches can include, but are not limited to: (a) Antiviral drugs to reduce expression of X protein. (b) A decoy synthetic peptide mimicking the interactive domain of either

X or XAP-1 (or related proteins) to block their interaction.

(c) Gene therapy vectors encoding such blocking peptides.

(d) Antisense constructs or inhibitory oligonucleotides directed against HBV sequences that would block the synthesis of X protein. (e) Intracellular antibodies able to block the interaction of X with XAP-1 or other components of the repair complex, (f) Chemicals to enhance the level of nuclease activity or other activities of the repair process so that the repair pathway would not be overwhelmed by HBV DNA and X protein. (g) Substances that would prevent the migration of HBV cores to the intracellular location where they contact the DNA repair mechanism.

One skilled in the art readily recognizes that these approaches can be used to inhibit the interaction of a viral protein with the DNA repair complex. Another alternative embodiment includes the method of treating cancer secondary to viral infection in an animal or human, comprising the steps of interfering with the interaction of a viral protein with a DNA repair complex in the animal or human to be treated. One skilled in the art readily understands that in treating the cancer, one can reduce, delay or prevent the effects of viral protein on the cellular DNA repair system. This would allow the repair process to monitor and repair chromosomal DNA damage, preventing the accumulations of mutations in the cell. In one specific example of the present invention, liver cancer secondary to HBV infection can be treated by inhibiting the interaction of the X protein of HBV with the XAP-1 protein of DNA repair complex.

Another embodiment is the use of the XAP-1 gene in diagnostic tests to identify those at increased risk of developing cancer. If the XAP-1 gene were to be mutated spontaneously or by exposure to viral infection or environmental factors, the loss of the normal DNA repair function might predispose cells to carcinogenic changes. Genetic errors would rapidly accumulate in those cells, some of which would affect important growth-regulatory genes. Similarly, individuals with inherited mutations in the XAP-1 gene might be cancer-prone. Diagnostic tests based on the detection of altered forms of the XAP-1 gene or gene product would be useful to identify those at increased risk of cancer development. At-risk individuals could then be monitored to detect cancers at their earliest stage, when treatment is most successful.

Although the specific examples above for treatment of disease and for treatment of cancer involve the HBV, other viral infections that induce cellular DNA damage or that involve the DNA repair system at some stage in the virus host interaction may be prevented, suppressed, modulated and/or cured using an inhibitory substance to block the interaction of specific viral proteins with the DNA repair mechanisms of the host cell, and similar to that described for HBV and HBV X protein. This can include members of the adeno-, arena-, bunya-, calici-, corona-, flavi-, hepadna-, herpes-, ortho-, papova-, paramyxo-, parvo-, picorna-, pox-, reo-, retro-, rhabdo-, and togavirus families, viroids, and agents not yet classified, and specifically would include hepatitis C virus. It would include target tissues in any organ system in the body, including but not limited to liver, gastrointestinal tract, respiratory system, skin, blood and blood-forming tissues, and the brain and nervous system.

Another approach is the use of gene therapy. These approaches include blocking HBV X function and its interactions with cellular DNA repair components. One skilled in the art also readily recognizes that gene therapy approaches can be directed to XAP-1, related proteins, and other components of the multienzyme DNA repair complex to modulate its ability to correct DNA damage induced by toxic chemicals, environmental toxins, viruses or other infectious agents, pharmaceuticals used to treat other conditions, or inherited genetic defects including mutator phenotypes.

Another embodiment of the present invention is the XAP-1 gene nucleic acid sequence which encodes for the XAP-1 protein, shown in Figure 2.

Another embodiment of the present invention is the amino acid sequence of the XAP-1 protein which is shown in Figure 3.

Polyclonal or monoclonal antibodies can be made against two synthetic peptides derived from the sequences of XAP-1, as well as to the whole XAP-1 protein or other fragments of the XAP-1. This procedure includes preparing the polyclonal or monoclonal antibodies against the synthetic peptides from the sequence, against authentic intact protein purified from eucaryotic cells or protein expressed using various express systems, including not only modified or unmodified intact protein, but the fragments of the protein also. Examples of these antibodies are shown in the examples of antibodies prepared against peptide 1 and peptide 2. In a specific embodiment of the present invention, the antibodies are used for diagnostic purposes. These antibodies can be used to identify defects in xeroderma. pigmentosum patients, stage HBV infections or liver cancer development and to monitor the effectiveness of various therapeutic treatments using immunohistochemical, immunofluorescence, immunoprecipitation, immunoblot, enzyme-linked immunosorbent assays, or other immunological methods.

Example 1 Identification of HBV X Protein Interactive Cellular Proteins Using the Yeast Two-Hybrid System

Background about system: Understanding the functions of the HBV X protein requires a knowledge of the protein-protein interactions that occur in different target cells and at different stages in the viral life cycle. The difficulty encountered so far in detecting X protein synthesized in HBV-infected human cells or in transgenic mouse cells has precluded studies of cellular proteins able to complex with the viral polypeptide. Even when found, X may well be an unstable, low-abundance protein which will further complicate studies of protein-protein interactions. The present invention utilizes the yeast two-hybrid system to circumvent these problems. The two-hybrid system was described by Fields and Song, Nature

340:245-246, 1989, and modified by Elledge, [Durfee et al. Genes and Dev. 7:555-569, 1993]. The procedure is a genetic method to identify and clone genes that interact with a protein of interest using in vivo complementation in yeast. The system relies on the properties of the yeast GAL4 protein, which contains two separable domains responsible for transcriptional activation and DNA binding. Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein of interest X (bait) and the other consisting of the GAL4 transcription activator domain fused to test protein Y (prey), are introduced into yeast. Interaction between bait and prey proteins leads to transcriptional activation of a reporter gene containing a binding site for GAL4. In principle, a mammalian cDNA library (prey plasmids) can be quickly screened provided neither participant (bait or prey) is self-activating. A schematic depicting transcription activation using the two-hybrid system, with ZαcZ as reporter gene, is shown in Figure 1. The two-hybrid system has several advantages over other commonly used methods to study protein-protein interactions: It is more sensitive than immunoprecipitation; purified proteins are not required; and it can be used to isolate genes encoding proteins that are normally expressed at a low level. The bait component of the two-hybrid system: The cloning vector was pASl, which carries as a selective marker TRPl (Figure 4). A bait plasmid that expressed GAL4 DNA-binding domain— X fusion protein was constructed using the HBV X gene. Saccharomyces cerevisiae host strain Y153 was used. Transformed cells were plated on synthetic complete (SC) media lacking tryptophan. As stable propagation of bait plasmids in yeast does not guarantee expression of the cloned test gene, HBV X protein expression was verified by immunoprecipitation of ^S-labeled proteins from sonically lysed yeast cells, using our X antisera. Bait proteins that self-activate reporter genes cannot be used. The suitability of the X bait plasmids for the two-hybrid system was tested using a ZαcZ reporter assay and a 3-aminothiazole (3-AT) susceptibility assay. The ZαcZ reporter assay checks for activation of the yeast -galactosidase reporter gene. Transformed yeast cells were bound to a reinforced nitrocellulose filter, permeabilized in liquid nitrogen, and then incubated overnight in buffer containing X-gal; the colonies turn blue if ZαcZ is activated. The X plasmid did not display self-activation.

The prey component of the two-hybrid system: To search for interactive proteins, an activation-domain tagged cDNA expression library was co-transformed into yeast expressing the HBV X bait plasmid. Prey plasmids were derived from a lambda-phage (λ) cDNA expression vector (λ-ACT) (Figure 5). λ-ACT encodes a plasmid that automatically excises, recircularizes, and propagates when grown in the proper E. coli strain, obviating the need for subcloning λ-amplified cDNAs. The cDNA library used was derived from Epstein-Barr virus-immortalized human lymphocytes.

Recipient yeast containing bait plasmids were transformed with library DNA using a lithium sorbitol transformation protocol. Library transformed cells were placed for 3 hr in liquid SC media lacking His, Trp, and Leu to establish transformants and activate HIS3 transcription and then plated onto solid selective media. Colonies that grew after 3 — 5 days were tested using the

ZαcZ reporter assay. Blue colonies were kept for further analysis.

Screens to rule out false positives: Some blue colonies able to grow on selective media may not be true positives due to specific bait and prey protein interactions. Approaches to rule out false positives included the nonspecific interactor test. In this test, false-positives were ruled out by determining if they reacted nonspecifically with a panel of indicators expressed as bait fusion proteins. The panel contained lamin c, human cyclin D, truncated p53, and yeast protein SNFl. Prey fusion proteins that interacted with some or all panel proteins were considered to be false positives. One true positive that presumably encoded an X-associated protein was designated XAP-1 (X- associated protein 1). The laminin-binding protein and the beta subunit of G protein are partially X-specific.

Identification of HBV X interactive proteins: True positive prey plasmids were DNA sequenced and an identity scan performed using a databank search. Although the XAP-1 gene was not represented on the first screen, it later appeared in GenBank (accession no. L20216) as a related sequence for a UV-damaged DNA-binding (UV-DDB) protein recovered from a monkey cell cDNA library. The monkey UV-DDB gene is about 98% homologous to the human XAP-1 gene at both the nucleotide and amino acid levels. (Fifty-one nucleotide mismatches and one amino acid mismatch.)

Preparation of immunological reagents against XAP-1: We selected hydrophilic sequences from XAP-1, based on analysis of the gene sequence, and prepared anti-peptide antisera to be used for subsequent protein characterization (Figure 7). The peptides used were the following: peptide

1 - REKEFNKGPWKQENVE (amino acids 198-213); peptide 2 - QYDDGSGMKREATA (amino acids 1113-1126). Antibodies were raised that recognized XAP-1 synthesized in vitro in rabbit reticulocyte lysates primed with XAP-1 mRNA, as well as endogenous protein synthesized in human hepatoma-derived HEpG2 cells (Figures 8 and 9).

Proof of protein-protein interaction: Two approaches were used to verify HBV X protein interaction with XAP-1. Yeast cells expressing the two proteins were labeled with [^SJmethionine, extracted, and the lysate precipitated with anti-X antibody; GAL4— XAP-1 fusion protein was co-immunoprecipitated (Figure 6). In a second approach to prove binding, X was expressed as a glutathione S-transferase (GST) fusion protein in E. coli. The GST-fusion protein was immobilized on glutathione-sepharose beads, and ^S-labeled in vitro translation mixtures containing the XAP-1 protein were applied to the column. Bound protein was eluted and analyzed by SDS-PAGE. XAP-1 was recovered (Figure 10). An outline of the procedure is shown in Figure 11.

-Example 2 Model of Significance to HBV Infections And Disease Pathogenesis

The model is that HBV usurps a normal DNA repair pathway in the cell to convert its partially double-stranded DNA genome to a covalently closed circular form, a necessary step in the virus life cycle. This step must occur with each progeny DNA molecule that is generated, either in newly infected cells, or as new rounds of replication are initiated in persistently infected cells. A cellular process presumably accomplishes this step; the viral polymerase functions to reverse transcribe pregenomic RNA to a partial double-stranded DNA, and inhibition of viral polymerase activity reportedly had no effect on generation of CCC DNA. Primer removal, and plus-strand completion must be early steps in the initiation of new rounds of replication.

The steps necessary to convert the incomplete HBV genome to an intact infectious form are reminiscent of the repair process designed to correct damaged cellular DNA.

It is believed that the HBV X protein is required for the recruitment and/or functioning of the cellular repair proteins to repair the HBV genome or to trigger a series of changes that makes the hepatocyte more permissive for viral replication. The involvement of the X protein is to direct the cellular complex from a cellular site to HBV DNA, to stabilize the interaction of the multienzyme complex with HBV DNA, to displace or inhibit one or more enzymatic processes that are part of the normal DNA repair process that might be deleterious to HBV DNA, to enhance the activity of the complex to make it more beneficial for HBV, and/or to alter the function of the repair process in some other way.

Therefore, if the involvement of the cellular DNA repair mechanism in the repair and completion of the HBV genome is blocked, the replication of

HBV is inhibited. It is thus possible to eliminate chronic infections with HBV, as well as to treat acute infections. This model helps explain the role of HBV in liver cancer. The DNA repair proteins are in close proximity to chromosomal DNA and, as the genomic HBV DNA is being repaired, the viral DNA is, on occasion, accidentally integrated into the chromosome. If an important cellular gene were interrupted or affected, this could have profound effects on the cell.

Inhibiting this on-going repair process of HBV DNA would decrease the number of integration events of HBV DNA and reduce the likelihood of eventual development of HCC. In addition, the involvement of X protein in redirecting the DNA repair process may be efficient enough that it interferes with normal repair of chromosomal DNA, resulting in the accumulation of mutations and genetic instability, eventually leading to cancer development. Therefore, blocking the interaction of X with the cellular repair mechanism will decrease the development of HCC.

This model explains several other poorly understood observations in the HBV system. The proposed function of X as a protease inhibitor might serve to dysregulate the cellular DNA repair complex by inhibiting a specific cleavage of a member of the complex in order to accentuate a function especially beneficial to HBV repair. As it now appears that some of the same cellular proteins are involved in initiation of transcription, excision repair, and DNA replication, this could explain the function of X protein as a promiscuous transcriptional activator. The X protein may be altering to a greater or lesser extent components of the transcription complex at the same time that it is modifying the DNA repair mechanism to meet the needs for HBV DNA repair. These effects on cellular processes would occur in cells expressing X protein from an integrated HBV genome, even in the absence of replicating HBV, because of the ability of X protein to interact with the cellular target proteins. The X protein has never been observed to exhibit DNA-binding activity. It is probable that X protein interaction with XAP-1 and possibly other members of the repair complex is necessary to enable X to affect the DNA-based processes.

Although the involvement of the X protein is required for the recruitment of the repair complex to repair HBV DNA, it is not unexpected that X has been reported to be dispensable for virus replication in tissue culture. Established cells in culture frequently contain mutations and display altered patterns of gene expression; redundant biochemical pathways are expressed that would be normally repressed in hepatocytes in vivo. There are many examples of viral genes that are essential for viral growth in a host organism that are not necessary for viral growth in tissue culture cells. All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods, procedures and techniques described herein, as well as the antibodies, nucleic acid sequence and amino acid sequences are presently representative of the preferred embodiments, are intended to be exemplary, and are not intended as limitations on the scope. Changes therein and other uses which are encompassed within the spirit of the invention or defined by the scope of the appended claims will occur to those skilled in the art.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Butel, Janet S. Lee, Teh-Hsiu Elledge, Stephen J.

(ii) TITLE OF INVENTION: Hepatitis B Virus Interacts With Cellular DNA Repair Processes

(iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fulbright & Jaworski

(B) STREET: 1301 McKinney, Suite 5100

(C) CITY: Houston

(D) STATE: Texas (E) COUNTRY: U.S.A.

(F) ZIP: 77010-3095

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/238,396

(B) FILING DATE: 05-May-1994 (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Paul, Thomas D.

(B) REGISTRATION NUMBER: 32,714

(C) REFERENCE/DOCKET NUMBER: D-5628-C1 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 713/651-5151

(B) TELEFAX: 713/651-5246

(C) TELEX: 762829

(2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATGTCGTACA ACTACGTGGT AACGGCCCAG AAGCCCACCG CCGTGAACGG CTGCGTGACC 60 GGACACTTTA CTTCGGCCGA AGACTTAAAC CTGTTGATTG CCAAAAACAC GAGATTAGAG 120 ATCTATGTGG TCACCGCCGA GGGGCTTCGG CCTGTCAAAG AGGTGGGCAT GTATGGGAAG 180 ATTGCGGTCA TGGAGCTTTT CAGGCCCAAG GGGGAGAGCA AGGACCTGCT GTTTATCTTG 240 ACAGCGAAGT ACAATGCCTG CATCCTGGAG TATAAACAGA GTGGCGAGAG CATTGACATC 300 ATTACGCGAG CCCATGGCAA TGTCCAGGAC CGCATTGGCC GCCCCTCAGA GACCGGCATT 360 ATTGGCATCA TTGACCCTGA GTGCCGGATG ATTGGCCTGC GTCTCTATGA TGGCCTTTTC 420 AAGGTTATTC CACTAGATCG CGATAATAAA GAACTCAAGG CCTTCAACAT CCGCCTGGAG 480 GAGCTGCATG TCATTGATGT CAAGTTCCTA TATGGTTGCC AAGCACCTAC TATTTGCTTT 540 GTCTACCAGG ACCCTCAGGG GCGGCACGTA AAAACCTATG AGGTGTCTCT CCGAGAAAAG 600 GAATTCAATA AGGGCCCTTG GAAACAGGAA AATGTCGAAG CTGAAGCTTC CATGGTGATC 660 GCAGTCCCAG AGCCCTTTGG GGGGGCCATC ATCATTGGAC AGGAGTCAAT CACCTATCAC 720 AATGGTGACA AATACCTGGC TATTGCCCCT CCTATCATCA AGCAAAGCAC GATTGTGTGC 780 CACAATCGAG TGGACCCTAA TGGCTCAAGA TACCTGCTGG GAGACATGGA AGGCCGGCTC 840 TTCATGCTGC TTTTGGAGAA GGAGGAACAG ATGGATGGCA CCGTCACTCT CAAGGATCTC 900 CGTGTAGAAC TCCTTGGAGA GACCTCTATT GCTGAGTGCT TGACATACCT TGATAATGGT 960 GTTGTGTTTG TCGGGTCTCG CCTGGGTGAC TCCCAGCTTG TGAAGCTCAA CGTTGACAGT 1020 AATGAACAAG GCTCCTATGT AGTGGCCATG GAAACCTTTA CCAACTTAGG ACCCATTGTC 1080 GATATGTGCG TGGTGGACCT GGAGAGGCAG GGGCAGGGGC AGCTGGTCAC TTGCTCTGGG 1140 GCTTTCAAGG AAGGTTCTTT GCGGATCATC CGGAATGGAA TTGGAATCCA CGAGCATGCC 1200 AGCATTGACT TACCAGGCAT CAAAGGATTA TGGCCACTGC GGTCTGACCC TAATCGTGAG 1260 ACTGATGACA CTTTGGTGCT CTCTTTTGTG GGCCAGACAA GAGTTCTCAT GTTAAATGGA 1320 GAGGAGGTAG AAGAAACCGA ACTGATGGGT TTCGTGGATG ATCAGCAGAC TTTCTTCTGT 1380 GGCAACGTGG CTCATCAGCA GCTTATCCAG ATCACTTCAG CATCGGTGAG GTTGGTCTCT 1440 CAAGAACCCA AAGCTCTGGT CAGTGAATGG AAGGAGCCTC AGGCCAAGAA CATCAGTGTG 1500 GCCTCCTGCA ATAGCAGCCA GGTGGTGGTG GCTGTAGGCA GGGCCCTCTA CTATCTGCAG 1560 ATCCATCCTC AGGAGCTCCG GCAGATCAGC CACACAGAGA TGGAACATGA AGTGGCTTGC 1620 TTGGACATCA CCCCATTAGG AGACAGCAAT GGACTGTCCC CTCTTTGTGC CATTGGCCTC 1680 TGGACGGACA TCTCGGCTCG TATCTTGAAG TTGCCCTCTT TTGAACTACT GCACAAGGAG 1740 ATGCTGGGTG GAGAGATCAT TCCTCGCTCC ATCCTGATGA CCACCTTTGA GAGTAGCCAT 1800 TACCTCCTTT GTGCCTTGGG AGATGGAGCG CTTTTCTACT TTGGGCTCAA CATTGAGACA 1860 GGTCTGTTGA GCGACCGTAA GAAGGTGACT TTGGGCACCC AGCCCACCGT ATTGAGGACT 1920 TTTCGTTCTC TTTCTACCAC CAACGTCTTT GCTTGTTCTG ACCGCCCCAC TGTCATCTAT 1980 AGCAGCAACC ACAAATTGGT CTTCTCAAAT GTCAACCTCA AGGAAGTGAA CTACATGTGT 2040 CCCCTCAATT CAGATGGCTA TCCTGACAGC CTGGCGCTGG CCAACAATAG CACCCTCACC 2100 ATTGGCACCA TCGATGAGAT CCAGAAGCTG CACATTCGCA CAGTTCCCCT CTATGAGTCT 2160 CCAAGGAAGA TCTGCTACCA GGAAGTGTCC CAGTGTTTCG GGGTCCTCTC CAGCCGCATT 2220 GAAGTCCAAG ACACGAGTGG GGGCACGACA GCCTTGAGGC CCAGCGCTAG CACCCAGGCT 2280 CTGTCCAGCA GTGTAAGCTC CAGCAAGCTG TTCTCCAGCA GCACTGCTCC TCATGAGACC 2340 TCCTTTGGAG AAGAGGTGGA GGTGCACAAC CTACTTATCA TTGACCAACA CACCTTTGAA 2400 GTGCTTCATG CCCACCAGTT TCTGCAGAAT GAATATGCCC TCAGTCTGGT TTCCTGCAAG 2460 CTGGGCAAAG ACCCCAACAC TTACTTCATT GTGGGCACAG CAATGGTGTA TCCTGAAGAG 2520 GCAGAGCCCA AGCAGGGTCG CATTGTGGTC TTTCAGTATT CGGATGGAAA ACTACAGACT 2580 GTGGCTGAAA AGGAAGTGAA AGGGGCCGTG TACTCTATGG TGGAATTTAA CGGGAAGCTG 2640 TTAGCCAGCA TCAATAGCAC GGTGCGGCTC TATGAGTGGA CAACAGAGAA GGAGCTGCGC 2700 ACTGAGTGCA ACCACTACAA CAACATCATG GCCCTCTACC TGAAGACCAA GGGCGACTTC 2760 ATCCTGGTGG GCGACCTTAT GCGCTCAGTG CTGCTGCTTG CCTACAAGCC CATGGAAGGA 2820 AACTTTGAAG AGATTGCTCG AGACTTTAAT CCCAACTGGA TGAGTGCTGT GGAAATCTTG 2880 GATGATGACA ATTTTCTGGG GGCTGAAAAT GCCTTTAACT TGTTTGTGTG TCAAAAGGAT 2940 AGCGCTGCCA CCACTGACGA GGAGCGGCAG CACCTCCAGG AGGTTGGTCT TTTCCACCTG 3000 GGCGAGTTTG TCAATGTCTT TTGCCACGGC TCTCTGGTAA TGCAGAATCT GGGTGAGACT 3060 TCCACCCCCA CACAAGGCTC GGTGCTCTTC GGCACGGTCA ACGGCATGAT AGGGCTGGTG 3120 ACCTCACTGT CAGAGAGCTG GTACAACCTC CTGCTGGACA TGCAGAATCG ACTCAATAAA 3180 GTCATCAAAA GTGTGGGGAA GATCGAGCAC TCCTTCTGGA GATCCTTTCA CACCGAGCGG 3240 AAGACAGAAC CAGCCACAGG TTTCATCGAC GGTGACTTGA TTGAGAGTTT CCTGGATATT 3300 AGCCGCCCCA AGATGCAGGA GGTGGTGGCA AACCTACAGT ATGACGATGG CAGCGGTATG 3360 AAGCGAGAGG CCACTGCAGA CGACCTCATC AAGGTTGTGG AGGAGCTAAC TCGGATCCAT 3420 TAG 3423

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1140 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ser Tyr Asn Tyr Val Val Thr Ala Gin Lys Pro Thr Ala Val Asn 1 5 10 15

Gly Cys Val Thr Gly His Phe Thr Ser Ala Glu Asp Leu Asn Leu Leu 20 25 30 lie Ala Lys Asn Thr Arg Leu Glu lie Tyr Val Val Thr Ala Glu Gly 35 40 45 Leu Arg Pro Val Lys Glu Val Gly Met Tyr Gly Lys lie Ala Val Met

50 55 60 Glu Leu Phe Arg Pro Lys Gly Glu Ser Lys Asp Leu Leu Phe lie Leu 65 70 75 80

Thr Ala Lys Tyr Asn Ala Cys lie Leu Glu Tyr Lys Gin Ser Gly Glu 85 90 95 Ser lie Asp lie lie Thr Arg Ala His Gly Asn Val Gin Asp Arg lie

100 105 110

Gly Arg Pro Ser Glu Thr Gly lie lie Gly lie lie Asp Pro Glu Cys 115 120 125

Arg Met lie Gly Leu Arg Leu Tyr Asp Gly Leu Phe Lys Val lie Pro 130 135 140

Leu Asp Arg Asp Asn Lys Glu Leu Lys Ala Phe Asn lie Arg Leu Glu 145 150 155 160

Glu Leu His Val lie Asp Val Lys Phe Leu Tyr Gly Cys Gin Ala Pro 165 170 175 Thr lie Cys Phe Val Tyr Gin Asp Pro Gin Gly Arg His Val Lys Thr

180 185 190

Tyr Glu Val Ser Leu Arg Glu Lys Glu Phe Asn Lys Gly Pro Trp Lys 195 200 205

Gin Glu Asn Val Glu Ala Glu Ala Ser Met Val lie Ala Val Pro Glu 210 215 220

Pro Phe Gly Gly Ala He He He Gly Gin Glu Ser He Thr Tyr His 225 230 235 240

Asn Gly Asp Lys Tyr Leu Ala He Ala Pro Pro He He Lys Gin Ser 245 250 255 Thr He Val Cys His Asn Arg Val Asp Pro Asn Gly Ser Arg Tyr Leu

260 265 270

Leu Gly Asp Met Glu Gly Arg Leu Phe Met Leu Leu Leu Glu Lys Glu 275 280 285

Glu Gin Met Asp Gly Thr Val Thr Leu Lys Asp Leu Arg Val Glu Leu 290 295 300

Leu Gly Glu Thr Ser He Ala Glu Cys Leu Thr Tyr Leu Asp Asn Gly 305 310 315 320

Val Val Phe Val Gly Ser Arg Leu Gly Asp Ser Gin Leu Val Lys Leu 325 330 335 Asn Val Asp Ser Asn Glu Gin Gly Ser Tyr Val Val Ala Met Glu Thr

340 345 350

Phe Thr Asn Leu Gly Pro He Val Asp Met Cys Val Val Asp Leu Glu 355 360 365

Arg Gin Gly Gin Gly Gin Leu Val Thr Cys Ser Gly Ala Phe Lys Glu 370 375 380

Gly Ser Leu Arg He He Arg Asn Gly He Gly He His Glu His Ala 385 390 395 400

Ser He Asp Leu Pro Gly He Lys Gly Leu Trp Pro Leu Arg Ser Asp 405 410 415 Pro Asn Arg Glu Thr Asp Asp Thr Leu Val Leu Ser Phe Val Gly Gin 420 425 430

Thr Arg Val Leu Met Leu Asn Gly Glu Glu Val Glu Glu Thr Glu Leu 435 440 445 Met Gly Phe Val Asp Asp Gin Gin Thr Phe Phe Cys Gly Asn Val Ala

450 455 460

His Gin Gin Leu He Gin He Thr Ser Ala Ser Val Arg Leu Val Ser 465 470 475 480

Gin Glu Pro Lys Ala Leu Val Ser Glu Trp Lys Glu Pro Gin Ala Lys 485 490 495

Asn He Ser Val Ala Ser Cys Asn Ser Ser Gin Val Val Val Ala Val 500 505 510

Gly Arg Ala Leu Tyr Tyr Leu Gin He His Pro Gin Glu Leu Arg Gin 515 520 525 He Ser His Thr Glu Met Glu His Glu Val Ala Cys Leu Asp He Thr

530 535 540

Pro Leu Gly Asp Ser Asn Gly Leu Ser Pro Leu Cys Ala He Gly Leu 545 550 555 560

Trp Thr Asp He Ser Ala Arg He Leu Lys Leu Pro Ser Phe Glu Leu 565 570 575

Leu His Lys Glu Met Leu Gly Gly Glu He He Pro Arg Ser He Leu 580 585 590

Met Thr Thr Phe Glu Ser Ser His Tyr Leu Leu Cys Ala Leu Gly Asp 595 600 605 Gly Ala Leu Phe Tyr Phe Gly Leu Asn He Glu Thr Gly Leu Leu Ser

610 615 620

Asp Arg Lys Lys Val Thr Leu Gly Thr Gin Pro Thr Val Leu Arg Thr 625 630 635 640

Phe Arg Ser Leu Ser Thr Thr Asn Val Phe Ala Cys Ser Asp Arg Pro 645 650 655

Thr Val He Tyr Ser Ser Asn His Lys Leu Val Phe Ser Asn Val Asn 660 665 670

Leu Lys Glu Val Asn Tyr Met Cys Pro Leu Asn Ser Asp Gly Tyr Pro 675 680 685 Asp Ser Leu Ala Leu Ala Asn Asn Ser Thr Leu Thr He Gly Thr He

690 695 700

Asp Glu He Gin Lys Leu His He Arg Thr Val Pro Leu Tyr Glu Ser 705 710 715 720

Pro Arg Lys He Cys Tyr Gin Glu Val Ser Gin Cys Phe Gly Val Leu 725 730 735

Ser Ser Arg He Glu Val Gin Asp Thr Ser Gly Gly Thr Thr Ala Leu 740 745 750

Arg Pro Ser Ala Ser Thr Gin Ala Leu Ser Ser Ser Val Ser Ser Ser 755 760 765 Lys Leu Phe Ser Ser Ser Thr Ala Pro His Glu Thr Ser Phe Gly Glu 770 775 780

Glu Val Glu Val His Asn Leu Leu He He Asp Gin His Thr Phe Glu 785 790 795 800 Val Leu His Ala His Gin Phe Leu Gin Asn Glu Tyr Ala Leu Ser Leu

805 810 815

Val Ser Cys Lys Leu Gly Lys Asp Pro Asn Thr Tyr Phe He Val Gly 820 825 830

Thr Ala Met Val Tyr Pro Glu Glu Ala Glu Pro Lys Gin Gly Arg He 835 840 845

Val Val Phe Gin Tyr Ser Asp Gly Lys Leu Gin Thr Val Ala Glu Lys 850 855 860

Glu Val Lys Gly Ala Val Tyr Ser Met Val Glu Phe Asn Gly Lys Leu 865 870 875 880 Leu Ala Ser He Asn Ser Thr Val Arg Leu Tyr Glu Trp Thr Thr Glu

885 890 895

Lys Glu Leu Arg Thr Glu Cys Asn His Tyr Asn Asn He Met Ala Leu 900 905 910

Tyr Leu Lys Thr Lys Gly Asp Phe He Leu Val Gly Asp Leu Met Arg 915 920 925

Ser Val Leu Leu Leu Ala Tyr Lys Pro Met Glu Gly Asn Phe Glu Glu 930 935 940

He Ala Arg Asp Phe Asn Pro Asn Trp Met Ser Ala Val Glu He Leu 945 950 955 960 Asp Asp Asp Asn Phe Leu Gly Ala Glu Asn Ala Phe Asn Leu Phe Val

965 970 975

Cys Gin Lys Asp Ser Ala Ala Thr Thr Asp Glu Glu Arg Gin His Leu 980 985 990

Gin Glu Val Gly Leu Phe His Leu Gly Glu Phe Val Asn Val Phe Cys 995 1000 1005

His Gly Ser Leu Val Met Gin Asn Leu Gly Glu Thr Ser Thr Pro Thr 1010 1015 1020

Gin Gly Ser Val Leu Phe Gly Thr Val Asn Gly Met He Gly Leu Val 1025 1030 1035 1040 Thr Ser Leu Ser Glu Ser Trp Tyr Asn Leu Leu Leu Asp Met Gin Asn

1045 1050 1055

Arg Leu Asn Lys Val He Lys Ser Val Gly Lys He Glu His Ser Phe 1060 1065 1070

Trp Arg Ser Phe His Thr Glu Arg Lys Thr Glu Pro Ala Thr Gly Phe 1075 1080 1085

He Asp Gly Asp Leu He Glu Ser Phe Leu Asp He Ser Arg Pro Lys 1090 1095 1100

Met Gin Glu Val Val Ala Asn Leu Gin Tyr Asp Asp Gly Ser Gly Met 1105 1110 1115 1120 Lys Arg Glu Ala Thr Ala Asp Asp Leu He Lys Val Val Glu Glu Leu 1125 1130 1135

Thr Arg He His 1140

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating viral disease in an animal or human, comprising the step of interfering with the interaction of a viral protein with a DNA repair complex in the animal or human to be treated.

2. The method of claim 1, wherein the viral disease is caused by the Hepatitis B Virus (HBV) and the interaction of the X protein of HBV with XAP-1 of the DNA repair complex is inhibited.

3. A method of treating cancer secondary to viral infection in an animal or human, comprising the steps of interfering with the interaction of a viral protein with a DNA repair complex in the animal or human to be treated.

4. The method of claim 3, wherein the liver cancer is secondary to HBV infection and the interaction of the X protein of HBV with XAP-1 of the DNA repair complex is inhibited.

5. A method of identifying persons at increased risk of developing cancer, comprising detecting alterations in the XAP-1 gene of an individual.

6. The method of claim 5, wherein said XAP-1 gene alterations are detected by one or more of the following techniques: restriction enzyme analysis, polymerase chain reaction assays, nucleic acid hybridization, and/or using any of various immunological assays.

7. The nucleic acid sequence of XAP-1 protein shown in Figure 2.

8. The amino acid sequence of XAP-1 protein shown in Figure 3.

9. An antibody which binds to the protein of claim 6.

10. The antibody of claim 7 which binds to peptide 1, or peptide 2 or both.

11. A method of detecting patients with HBV infection, comprising the step of combining a sample from said patient with the antibody of claim 7 and measuring the amount of antibody-antigen interaction.

12. A method of monitoring the effectiveness of therapeutic treatments, comprising the step of combining a sample from said patient with the antibody of claim 9 and measuring the amount of antibody-antigen interaction.

13. A method of determining the stage of liver cancer secondary to

HBV infection, comprising the step of combining a sample from said patient with the antibody of claim 9 and measuring the amount of antibody-antigen interaction.