WO2010037083A1

WO2010037083A1 - Screen for anti-viral compounds against hepatitis b virus

Info

Publication number: WO2010037083A1
Application number: PCT/US2009/058727
Authority: WO
Inventors: John E. Tavis; Matthew P. Badtke
Original assignee: Saint Louis University
Priority date: 2008-09-29
Filing date: 2009-09-29
Publication date: 2010-04-01

Abstract

Methods of identifying compounds having anti-viral activity against Hepatitis B Virus (HBV) and other hepadnavirυses are disclosed. In these methods, a mixture is formed comprising a candidate compound, a T3 domain of a hepadnavirus polymerase and/or an RT-1 domain of a hepadnavirus polymerase, and a nucleobase polymer. When more than one polymerase domain is used, the domains can be from the same species of hepadnavirus, such as Duck Hepatitis B Virus (DHBV), or from different species of hepadnavirus. Compounds having anti-viral activity against HBV are identified as compounds which interfere with the binding between T3 domain and a nucleobase polymer, or between an RT-1 domain and a nucleobase polymer.

Description

SCREEN FOR ANTI-VIRAL COMPOUNDS AGAINST HEPATITIS B VIRUS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This research was funded by Grant # ROl AI38447 awarded by the National Institutes of Health. The Government may have certain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCES SUBMITTED IN ELECTRONIC FILES

[0002] The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising amino acid sequences and nucleic acid sequences of the present disclosure. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

INTRODUCTION

[0003] Hepatitis B virus (HBV) is believed to infect approximately 400,000 people worldwide. Currently available pharmaceutical compounds for treating HBV infection are generally regarded as unsatisfactory for a variety of reasons such as side effects, efficacy and drug resistance that can arise when anti-viral therapeutics are administered to a subject having an HBV infection.

[0004] HBV is the prototypic member of the family Hepadnaviridae, hepatotropic double stranded (ds) DNA viruses (Seeger, C, F. Zoulim, and W. S. Mason.. Hepadnaviruses, p. 2977-3029, 2007, in Knipe, D.M., et al. (ed.), Fields Virology. Lippincott Williams & Wilkins, Philadelphia). An example of a hepatitis B virus DNA sequence is provided herein as SEQ ID NO: 29. HBV is a small DNA virus that replicates by reverse transcription (Summers, J. et al., Cell 29: 403-415, 1982). The virion has a lipid envelope studded with viral glycoproteins that surrounds an icosahedral core particle. Within the core particle are the viral nucleic acids and a virally encoded reverse transcriptase, called the polymerase. Hepadnaviruses have been found in numerous birds and mammals, such as ducks, herons, geese, ground squirrels, woodchucks and woolly monkeys (Guo, H., et al., J. Virol. 79: 2729-2742, 2005; Lanford, R. E., et al., Proc. Nat'l Acad. Sci. USA 95: 5757-5761, 1998; Schoedel, F., et al., Advances in Viral Oncology 8: 73-102, 1989). Hepadnaviruses viruses all have ~3 kb partially dsDNA genomes. Duck hepatitis B virus (DHBV) is a common model for HBV. An example of a sequence of a DHBV DNA is provided herein as SEQ ID NO: 30.

[0005] Reverse transcription occurs in cytoplasmic subviral capsids and is catalyzed by the viral polymerase. Reverse transcription is initiated by binding of the polymerase to the viral pregenomic RNA template at a 54-nucleotide stem loop called epsilon (ε) (Beck, J., et al., J. Virol. 71 : 4971-4980, 1997; Hirsch, R. C, et al., Nature (London) 344: 552-555, Junker-Niepmann, M., et al., EMBO Journal 9: 3389-3396, 1990; Pollack, J. R., et al., J. Virol. 68: 5579-5587, 1994). Binding to epsilon induces a conformational change in the polymerase that is necessary to trigger its enzymatic activity (Tavis, J. E., et al., J. Virol. 70: 5741-5750, 1996; Tavis, J. E., et al., J. Virol. 72: 5789-5796, 1998). The polymerase:RNA complex is then encapsidated through polymerization of the viral core protein around it to form the capsid. Reverse transcription is templated by a bulge in ε (Tavis, J. E., et al., J. Virol. 68: 3536-3543, 1994; Wang, G.H., et al., Cell 71 : 663-670, 1992) and is primed by a tyrosine residue on the polymerase itself. The result of this unique protein-priming mechanism is that the minus-strand DNA is covalently attached to the polymerase (Lanford, R. E., et al., J. Virol. 71: 2996-3004, 1997; Weber, M., et al., J. Virol. 68: 2994-2999, 1994; Zoulim, F., et al., J. Virol. 68: 6-13, 1994). Binding of the polymerase to ε and reverse transcription are dynamic processes involving several conformational changes by the polymerase that are mediated by essential interactions with host chaperone proteins, including HSP90, HSP70, HSP40, p23 and HOP (Beck, J., et al., J. Biol. Chem. 178: 3612836138, 2003; Hu, J., et al., Proc. Nat'l. Acad. Sci. USA 93: 1060-1064, 1996; Hu, J., et al., J. Virol. 76: 269-279, 2002; Hu, J., et al., EMBO J.16: 59-68, 1997). Therefore, an HBV polymerase functions in complex with viral nucleic acids and cellular chaperones.

[0006] A hepadnaviral polymerase has 4 domains (Chang, L.J., et al., J. Virol. 64: 5553-5558, 1990; Radziwill, G., et al., Virology 163: 123-132, 1988). These domains are illustrated in FIG. IA. The terminal protein domain contains a tyrosine residue that primes DNA synthesis and covalently links the polymerase to the viral DNA (Y96 in DHBV, Y63 in HBV) (Lanford, R. E., et al., J. Virol. 71: 2996-3004, 2007; Weber, M., et al., J. Virol. 68: 2994-2999, 1994; Zoulim, F., et al., J. Virol. 68: 6-13, 1994). The spacer domain has no known function other than to link the terminal protein domain to the rest of the polymerase. The reverse transcriptase domain contains the conserved YMDD motif in the active site for reverse transcription, and the RNase H domain degrades the RNA template during reverse transcription. The structure of the polymerase has not been solved due to an inability to crystallize the protein, but the HBV reverse transcriptase and RNase H domains have been modeled based on structures of the Moloney murine leukemia virus- 1 and human immunodeficiency virus-1 reverse transcriptases (Bartholomeusz, A., et al., Antivir. Ther. 9: 149-160, 2004; Das, K., et al., J. Virol. 75: 4771-4779, 2001; Langley, D. R., et al., J. Virol. 81: 3992-4001, 2007; Potenza, N., et al., Protein Expr. Purif. 55: 93-99, 2007). However, there is no model for the terminal protein domain as it has no homology to non- hepadnaviral proteins. Complementation studies with recombinant fragments of HBV polymerase imply that there are multiple contacts between the terminal protein domain and the reverse transcriptase/RNase H domains (Lanford, R. E., et al., J. Virol. 73: 1885-1893, 1999; Lanford, R. E., et al., J. Virol. 71 : 2996-3004, 1997), but the relative arrangement and contact points between the domains are unknown. [0007] We previously described a motif in the terminal protein domain of DHBV polymerase that is essential for DNA synthesis (Badtke, M.P., et al., Biol. Proced. Online. 8: 77-86, 2006; Cao, F., et al., J. Virol. 79: 10164-10170, 2005). We found that six monoclonal antibodies (mAb) against the terminal protein domain were able to immunoprecipitate enzymatically active polymerase translated in vitro in a partially denaturing buffer (RIPA), but only three were able to immunoprecipitate the polymerase in a physiological buffer (IPP 150). Epitope mapping revealed that the epitopes obscured in IPP 150 flanked a highly conserved region (aa 176-183), which we named T3. Mutations to T3 exposed the obscured mAb epitopes while simultaneously inhibiting DNA priming by the polymerase, and mutant genomes with lesions in T3 of both HBV and DHBV failed to synthesize DNA within cells. Importantly, synthetic peptides containing T3 sequences specifically inhibited priming in a dose-dependent manner. SUMMARY

[0008] The present inventors realized that there is an unmet need for new pharmaceutical agents for treating Hepatitis B virus (HBV) infection, and have developed methods of identifying anti-viral compounds against Hepatitis B Virus. In some aspects of the present teachings, the inventors disclose methods of identifying an anti-viral compound against HBV, wherein the methods comprise a) forming a mixture comprising i) a candidate compound, ii) a polypeptide comprising A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase, and iii) at least one nucleobase polymer; and b) detecting a decrease in binding between the nucleobase polymer and the polypeptide in comparison to a mixture comprising the polypeptide and the nucleobase polymer in the absence of the candidate compound. In these methods, a decrease in the binding compared to a mixture comprising the polypeptide and the nucleobase polymer in the absence of the candidate compound can indicate anti-viral activity of the compound. In some configurations, the polypeptide can comprise a full-length hepadnavirus polymerase. In some configurations, a polypeptide can comprise A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase. In other configurations, a polypeptide can consist essentially of A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase. In yet other configurations, a polypeptide can consist of a T3 domain of a hepadnavirus polymerase, and an RT-I domain of a hepadnavirus polymerase.

[0009] In some aspects of the present teachings, the inventors disclose methods of identifying an anti-viral compound against HBV, wherein the methods comprise a) forming a mixture comprising i) a candidate compound, ii) at least one polypeptide comprising a T3 domain of a hepadnavirus polymerase or an RT- 1 domain of a hepadnavirus polymerase, and iii) at least one nucleobase polymer; and b) detecting a decrease in binding between the nucleobase polymer and the polypeptide(s) compared to a mixture comprising the polypeptide(s) and the nucleobase polymer in the absence of the candidate compound. In some configurations, at least one polypeptide can comprise a T3 domain of a hepadnavirus polymerase or an RT-I domain of a hepadnavirus polymerase. In other configurations, at least one polypeptide can consist essentially of a T3 domain of a hepadnavirus polymerase or an RT- 1 domain of a hepadnavirus polymerase. In yet other configurations, at least one polypeptide can consist of a T3 domain of a hepadnavirus polymerase, or an RT- 1 domain of a hepadnavirus polymerase.

[0010] In various configurations of these aspects, a hepadnavirus polymerase can be a polymerase from any known hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase. In yet other configurations, the polypeptide can be immobilized on a solid support. [0011] In addition, in other configurations of these methods, a T3 domain and an RT- 1 domain can be of a polymerase from the same species of hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase, or of polymerases from different hepadnavirus species, i.e., a T3 domain of a polymerase of one hepatitis B virus species and an RT-I domain of a polymerase of a different hepatitis B virus species, such as, without limitation, a T3 domain of a DHBV polymerase and an RT-I domain of an HBV.

[0012] Furthermore, a hepadnavirus polymerase of these methods or a portion thereof such as a T3 domain or an RT-I domain, can be a polymerase or portion thereof from any hepadnavirus known to skilled artisans, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase. [0013] In various configurations of these methods, at least one of a nucleobase polymer or a polypeptide can comprise a label. In these configurations, detecting binding between a nucleobase polymer and a polypeptide can comprise detecting presence and/or quantity of the label. A label of these configurations can be any label known to skilled artisans, such as, without limitation, a chromophore, a fluorophore, a radioisotope, an enzyme, or a probe-binding target. When the label is a chromophore, the label can be any chromophore known to skilled artisans. When the label is a fluorophore, the label can be any fluorophore known to skilled artisans. Furthermore, when the label is a fluorophore, detecting a decrease in binding between the nucleobase polymer and the polypeptide(s) can utilize any fluorescence assay known to skilled artisans, such as, without limitation, a fluorescence resonance energy transfer (FRET) assay or a fluorescence anisotropy polarization assay. When the label is a radioisotope, the radioisotope can be any radioisotope known to skilled artisans. [0014] In some configurations, when the label is an enzyme, a method of these aspects can further comprise adding to the mixture a substrate of the enzyme, and detecting presence and/or quantity of a product of a reaction between the enzyme and the substrate.

[0015] In other aspects, methods of identifying an anti-viral compound against HBV comprise a) forming a mixture comprising i) a candidate compound, ii) at least one polypeptide comprising a T3 domain of a hepadnavirus polymerase or an RT-I domain of a hepadnavirus polymerase; b) adding to the mixture at least one nucleobase polymer; and c) detecting a decrease in binding compared to a mixture comprising the at least one polypeptide and the nucleobase polymer in the absence of the candidate compound, whereby a decrease in the binding indicates anti-viral activity of the compound. In various configurations of these methods, a T3 domain of a hepadnavirus polymerase can be a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, and a woodchuck hepatitis B virus polymerase. In addition, an RT-I domain of a hepadnavirus polymerase can be a RT- 1 domain of a polymerase of a hepadnavirus selected from a duck hepatitis B virus polymerase, a human hepatitis B virus polymerase, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, and a woodchuck hepatitis B virus polymerase. In addition, in various configurations of these methods, a T3 domain and an RT-I domain can be of a polymerase from the same species of hepadnavirus, such as, without limitation, a HBV or a DHBV, or of polymerases from different hepadnavirus species, such as, without limitation, a T3 domain of a HBV polymerase and an RT-I domain of a DHBV polymerase. [0016] In various configurations of these methods, a nucleobase polymer can be a nucleic acid, or nucleic acid analogue such as a peptide-nucleic acid (PNA; Ray, A., and Norden, B., FASEB J. 14: 1041-1060, 2000). In various configurations, a nucleic acid can be an RNA or a DNA. Furthermore, a nucleobase polymer can be single- stranded or double-stranded, such as a single-stranded nucleic acid or a double- stranded nucleic acid. In some configurations, a nucleobase polymer can be other than an epsilon RNA of a hepadnavirus, such as an HBV epsilon RNA. In some configurations, a nucleobase polymer can be an epsilon RNA of a hepadnavirus, such as an HBV epsilon RNA.

[0017] In various configurations of these aspects, a nucleobase polymer can comprise a label, such as, without limitation, a radioisotope, a fluorophore, an enzyme, and a probe-binding target. When the label is an enzyme, a method can further comprise adding to the mixture a substrate of the enzyme, and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate. In various configurations, the enzymes and substrates can be, without limitation, those enzymes and substrates described herein with regard to detecting binding between at least one antibody and at least one epitope. In various configurations, the enzymes and substrates can be, without limitation, those enzymes and substrates described herein with regard to detecting binding between the nucleobase polymer and a polypeptide comprising a T3 motif and/or a RT 1 motif.

[0018] In yet other configurations of these aspects, a method can further comprise adding oligonucleotide primers to the mixture. The oligonucleotides primers of these configurations can hybridize to the at least one nucleobase polymer or its complement. In these configurations, the detecting binding can comprise performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of a reaction product.

[0019] In various configurations of these teachings, either a nucleobase polymer component of a mixture described herein or a polypeptide component of a mixture described herein can be immobilized on a solid support while the other component is not immobilized on the solid support. In these configurations, an assay can comprise detecting a reduction in binding between the immobilized and non-immobilized components in the presence of a candidate compound. Detecting a reduction in binding can comprise detecting presence, absence or quantity of the component not immobilized on a solid support, and comparing the amount of binding to that of a mixture not comprising the candidate compound, whereby a decrease in the binding compared to a mixture not comprising the candidate compound indicates anti-viral activity of the compound. In various configurations, the component not immobilized on a solid support can comprise a label, and the detecting can comprise detecting the label. A label of these configurations can be any label known to skilled artisans, such as, without limitation, a chromophore, a fluorophore, a radioisotope, an enzyme, or a probe-binding target. When the label is a chromophore, the label can be any chromophore known to skilled artisans. When the label is a fluorophore, the label can be any fluorophore known to skilled artisans. Furthermore, when the label is a fluorophore, detecting a decrease in binding can utilize any fluorescence assay known to skilled artisans, such as, without limitation, a fluorescence resonance energy transfer (FRET) assay or a fluorescence anisotropy polarization assay. When the label is a radioisotope, the radioisotope can be any radioisotope known to skilled artisans, such

32 33 35 14 125 131 3 as, without limitation, a P, a P, a S, a C, an I, an I, or a H. When the label is an enzyme, a method of these aspects can further comprise adding to the mixture a substrate of the enzyme, and detecting presence and/or quantity of a product of a reaction between the enzyme and the substrate. When the label is a probe-binding target, the probe-binding target can be any molecular target for a probe, such as, without limitation, a ligand to which a probe binds, such as, without limitation, an antigen to which an antibody binds.

[0020] In some configurations of these aspects, an assay can comprise a fluorescence resonance energy transfer (FRET) assay. In various configurations, either a nucleobase polymer component of a mixture or a polypeptide component of a mixture can further comprise a fluorophore, while the other component can comprise a fluorescence quencher which quenches the fluorescence of the fluorophore when the two components are bound to each other. In some configurations, a first component of a mixture comprising a fluorescence quencher can be immobilized on a solid support, and a second component (its binding partner) can comprise a fluorophore. Alternatively, a first component comprising a fluorophore can be immobilized on a solid support, and a second component (its binding partner) can comprise a fluorescence quencher. In these configurations, detecting a reduction in binding between a fluorophore labeled component and a component comprising a fluorescence quencher can comprise detecting an increase in fluorescence intensity of the fluorophore compared to a mixture comprising the components in the absence of the candidate compound. In some configurations, all components can be in solution. In addition, in some configurations, fluorescence-based detection can utilize fluorescence anistropy polarization (see, e.g., Lakowicz, J.R., et al, Biophys. J. 24: 213-231, 1978; Blesbois, E., et al., Reproduction 129: 371-378, 2005).

[0021] In some configurations of these aspects, a polypeptide can consist essentially of a T3 domain of a hepadnavirus polymerase, or can consist of a T3 domain of a hepadnavirus polymerase.

[0022] In some configurations of these aspects, a polypeptide can consist essentially of an RT- 1 domain of a hepadnavirus polymerase, or can consist of an RT- 1 domain of a hepadnavirus polymerase.

[0023] In various aspects of the present teachings, a T3 domain can have an amino acid sequence of a T3 domain of any hepadnavirus, such as, without limitation, amino acids 176-183 of DHBV, EAGILYKR (SEQ ID NO: 31) or amino acids 155-162 of a HBV strain AD W2 polymerase, KAGILYKR (SEQ ID NO: 16). In addition, in various configurations, an RT-I domain can have an amino acid sequence of an RT-I domain of any hepadnavirus, such as, without limitation, amino acids 383-415 of DHBV polymerase, PNRITGKLFLVDKNSRNTTEARLVVDFSQFSKG (SEQ ID NO: 17); or amino acids 368-400 of a HBV strain AD W2 polymerase, PARVTGGVFLVDKNPHNTAESRLVVDFSQFSRG (SEQ ID NO: 18). [0024] In various configurations, a sequence of a hepadnavirus polymerase, a domain or a subsequence of a hepadnavirus polymerase such as a T3 domain, an RT- 1 domain or a subsequence comprising an epitope, can be a naturally occurring variant of a hepadnavirus sequence or subsequence set forth herein.

[0025] In various configurations, a sequence of a hepadnavirus polymerase, a domain or a subsequence of a hepadnavirus polymerase such as a T3 domain, an RT-I domain or a subsequence comprising an epitope, can comprise one or more amino acid insertions, deletions, and/or substitutions which do not substantially alter binding interactions between a T3 domain and an RT-I domain compared to a naturally occurring hepadnavirus polymerase, a T3 domain thereof or an RT-I domain thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 illustrates structural organization of DHBV polymerase.

[0027] FIG. 2 illustrates binding of ε by wild-type and T3-mutant polymerases.

[0028] FIG. 3 illustrates that synthetic peptides containing T3 sequences can inhibit purified polymerase in the absence of the cellular chaperones that are normally needed for P to function.

[0029] FIG. 4. This figure illustrates that T3 and RT-I are conserved among the hepadnavirus es.

[0030] FIG. 5. This figure illustrates that RT-I is predicted to be on the surface of the reverse transcriptase domain.

[0031] FIG. 6. This figure illustrates that T3 and RTl peptides bind nucleic acids in vitro.

[0032] FIG. 7. This figure illustrates that miniRT2 can bind RNA in vitro.

DETAILED DESCRIPTION

[0033] The methods and compositions described herein utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0034] The present inventors disclose herein methods of identifying anti-viral compounds against Hepatitis B Virus (HBV).

[0035] Without being limited by theory, analyses by the present inventors revealed that T3 cooperates with a motif on the polymerase, named RT-I (DHBV aa 383-415; SEQ ID NO: 17), to form an RNA binding site that mediates the initial interaction between the polymerase and ε, and hence the inventors concluded that binding between the T3 and RT-I motif and ε constitutes the initial, essential step in the reverse transcription pathway.

[0036] A candidate compound which can be tested for anti-HBV activity by the present methods can be any compound. Initial selection of a compound can be through methods known to skilled artisans. For example, and without limitation, one such method of selecting a candidate compound can comprise using a digital computer comprising the coordinates of an HBV or a reverse transcriptase domain such as illustrated in FIG. 5, and applying an algorithm to select a structure that would be expected to bind a T3 domain, an RT-I domain, or a region of interaction between the T3 and RT-I domains.

[0037] Because of the sequence diversity known to occur within hepadnaviruses, even within the same species or strain of a hepadnavirus, sequences disclosed herein are intended to be understood as exemplary; i.e., the sequence of a polymerase or a polymerase domain from a different isolate than that use to determine a sequence presented herein can be substituted and still provide a skilled artisan with sequence data for practicing the disclosed methods. For example, although the sequence designated SEQ ID NO: 2 for a HBV polymerase is that of an HBV subtype AYR, there are numerous other non-limiting examples of sequences of an HBV polymerases, such as those set forth in table 1 below for polymerases from HBVs of subtype adw2. These polypeptide sequences have accession numbers which can be accessed on the internet at websites such as http://www.ncbi.nlm.nih.gov/, and are set forth in the sequence listings. Table 1 : Examples of HBV polymerase sequences

[0038] In some configurations, a polypeptide or nucleobase polymer of the various assays of the present teachings can be immobilized on a solid support. In these configurations, a solid support can be any kind of immobilizing support known to skilled artisans, such as an ELISA plate, a protein-adsorbent bead, or a protein- adsorbent membrane such as a nitrocellulose or nylon membrane. In a binding assay, the binding partner of an immobilized component of an assay mixture can be soluble and furthermore can carry a label, such as a fluorophore or a radioisotope. [0039] In various configurations, a hepadnavirus polymerase can be a polymerase from any known hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase (e.g., SEQ ID NO: 1), a human hepatitis B virus (HBV) polymerase (e.g., SEQ ID NO: 2), a crane hepatitis B virus polymerase (e.g., SEQ ID NO: 3), a heron hepatitis B virus polymerase (e.g., SEQ ID NO: 4), a sheldgoose hepatitis B virus polymerase (e.g., SEQ ID NO: 5), a Ross's goose hepatitis B virus polymerase (e.g., SEQ ID NO: 6), a snow goose hepatitis B virus polymerase (e.g., SEQ ID NO: 7), a stork hepatitis B virus polymerase (e.g., SEQ ID NO: 8), a chimpanzee hepatitis B virus polymerase (e.g., SEQ ID NO: 9), a woolly monkey hepatitis B virus polymerase (e.g., SEQ ID NO: 10), a ground squirrel hepatitis B virus polymerase (SEQ ID NO: 32), an arctic ground squirrel hepatitis B virus polymerase (e.g., SEQ ID NO: 11) or a woodchuck hepatitis B virus polymerase (e.g., SEQ ID NO: 12). [0040] In methods that involve detecting binding of a probe such as a nucleobase polymer to a complex comprising T3 and RT-I, the detecting can, in some configurations, utilize a polypeptide immobilized on a solid support. These methods can also comprise removal of unbound probe by, e.g., washing a solid support, then detecting probe remaining on the solid support. The detecting can comprise determining the presence, absence, and/or quantity of the probe remaining on the solid support using any method and label known to skilled artisans, such as, without limitation, an ELISA, a radioimmunoassay, radiography, scintillation, a PCR assay, or a blotting assay such as a Southern blot assay or a Western blot assay. [0041] In addition, in other configurations of these methods, a T3 domain and an RT- 1 domain can be of a polymerase from the same species of hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase, or of polymerases from different hepadnavirus species, i.e., a T3 domain of a polymerase of one hepatitis B virus species and an RT- 1 domain of a polymerase of a different hepatitis B virus species, such as, without limitation, a T3 domain of a DHBV polymerase and an RT-I domain of an HBV.

[0042] In some configurations, when the label is a chromophore, the label can be any chromophore known to skilled artisans, such as, without limitation, a dichlorotriazine dye such as l-Amino-4-[3-(4,6-dichlorotriazin-2-ylamino)-4-

® sulfophenylamino]anthraquinone-2-sulfonic acid (Procion Blue MX-R (Fluka AG,

Switzerland)). Such labels can be detected by methods known to skilled artisans, such as measurement of optical absorbance using a spectrophotometer. [0043] In some configurations, when the label is a fluorophore, the label can be any fluorophore known to skilled artisans, such as, without limitation, a fluorescein, a rhodamine, an Alexa Fluor® (Invitrogen Corporation, Carlsbad, California) a coumarin, an indocyanine or a quantum dot (Colton, H.M., et al, Toxicological Sciences 80: 183-192, 2004) . Such labels can be detected by methods known to skilled artisans, such as measurement of fluorescence using a fluorometer.

[0044] In some configurations, when the label is a radioisotope, the radioisotope can

32 33 be any radioisotope known to skilled artisans, such as, without limitation, a P, a P,

35 14 125 131 3

S, a C, an I, an I, or a H.

[0045] In some configurations, when the label is an enzyme, a method of these aspects can further comprise adding to the mixture a substrate of the enzyme, and detecting presence and/or quantity of a product of a reaction between the enzyme and the substrate.

[0046] An enzyme of these configurations can be any enzyme for which a substrate is available. Examples of such enzymes include, without limitation, a peroxidase such as a horseradish peroxidase, a phosphatase such as an alkaline phosphatase, a galactosidase such as a β-galactosidase, and a luciferase, such as a firefly luciferase. In some configurations, a substrate can be a chromogen or a fluorogen, or can yield a chemiluminescent product. If the substrate is a chemiluminescent substrate, qualitative and/or quantitative detection of the enzyme can comprise visual assessment, and/or measuring light produced as a product of a reaction between the substrate and the enzyme. For example, if the enzyme is an alkaline phosphatase, the substrate can be a chemiluminescent substrate such as CDP-Star® (Sigma-Aldrich Chemical Co., St. Louis, MO). In another example, if the enzyme is a luciferase, the substrate can be a luciferin.

[0047] If the substrate is a chromogenic substrate, qualitative and/or quantitative detection of the enzyme can comprise visual assessment, and/or measuring optical absorbance of the reaction product, such as, without limitation, measuring absorbance at 400 nm when the enzyme is an alkaline phosphatase and the substrate is dinitrophenyl phosphate. If the substrate is a fluorogenic substrate, qualitative and/or quantitative detection of the enzyme can comprise visual assessment, and/or measuring fluorescent light intensity using a fluorometer.

[0048] In some configurations, when the label is a probe-binding target, the probe- binding target can be any molecular target for a probe, such as, without limitation, a ligand to which a probe binds, such as, without limitation, an antigen which an antibody binds. In various configurations of these methods, a probe-binding target can be, without limitation, a biotin, a digoxygenin, or a peptide, and a probe for the probe- binding target can be, without limitation, an avidin, a streptavidin, an anti-biotin antibody, an anti-digoxygenin antibody, or a peptide antibody directed against a peptide. Accordingly, in various configurations of these methods, a label and a probe can be, without limitation, a) a biotin and an avidin, b) a biotin and a streptavidin, c) a biotin and an anti-biotin antibody, d) a digoxygenin and an anti-digoxygenin antibody, or e) a peptide and an antibody directed against the peptide. [0049] In some aspects of the present teachings, the inventors disclose methods of identifying an anti-viral compound against Hepatitis B Virus (HBV), wherein the methods comprise a) forming a mixture comprising i) a candidate compound, ii) a polypeptide comprising A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase, iii) at least one nucleobase polymer; and b) detecting binding between the nucleobase polymer and the polypeptide. In these methods, a decrease in the binding compared to a mixture comprising the polypeptide and the nucleobase polymer in the absence of the candidate compound can indicate anti-viral activity of the compound. In some configurations, the polypeptide can comprise a full-length hepadnavirus polymerase. In other configurations, the polypeptide can consist essentially of A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase. In yet other configurations, the polypeptide can consist of A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase. In various configurations of these aspects, a hepadnavirus polymerase can be a polymerase from any known hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase. In yet other configurations, the polypeptide can be immobilized on a solid support.

[0050] In yet other aspects, methods of identifying an anti-viral compound against HBV comprise a) forming a mixture comprising i) a candidate compound, ii) a first polypeptide comprising a T3 domain of a hepadnavirus polymerase iii) a second polypeptide comprising an RT-I domain of a hepadnavirus polymerase; b) adding to the mixture at least one nucleobase polymer; and c) detecting binding between the nucleobase polymer and the polypeptides, wherein a decrease in the binding compared to a mixture comprising the polypeptides and the nucleobase polymer in the absence of the candidate compound indicates anti-viral activity of the compound. In various configurations of these methods, a T3 domain of a hepadnavirus polymerase can be a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, and a woodchuck hepatitis B virus polymerase. In addition, an RT-I domain of a hepadnavirus polymerase can be a RT-I domain of a polymerase of a hepadnavirus selected from a duck hepatitis B virus polymerase, a human hepatitis B virus polymerase, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, and a woodchuck hepatitis B virus polymerase. In addition, in various configurations of these methods, a T3 domain and an RT-I domain can be of a polymerase from the same species of hepadnavirus, such as, without limitation, a HBV or a DHBV, or of polymerases from different hepadnavirus species, such as, without limitation, a T3 domain of a HBV polymerase and an RT-I domain of a DHBV polymerase. [0051] In some aspects of the present teachings, the inventors disclose methods of identifying an anti-viral compound against Hepatitis B Virus (HBV), wherein the methods comprise a) forming a mixture comprising i) a candidate compound, ii) a polypeptide comprising a T3 domain of a hepadnavirus polymerase or an RT-I domain of a hepadnavirus polymerase, iii) at least one nucleobase polymer; and b) detecting binding between the nucleobase polymer and the polypeptide. In these methods, a decrease in the binding compared to a mixture comprising the polypeptide and the nucleobase polymer in the absence of the candidate compound can indicate anti-viral activity of the compound. In some configurations, the polypeptide can comprise a full- length hepadnavirus polymerase. In other configurations, the polypeptide can consist essentially of a T3 domain of a hepadnavirus polymerase or an RT-I domain of a hepadnavirus polymerase. In yet other configurations, the polypeptide can consist of a T3 domain of a hepadnavirus polymerase or an RT-I domain of a hepadnavirus polymerase. In various configurations of these aspects, a hepadnavirus polymerase can be a polymerase from any known hepadnavirus, such as, without limitation, a duck hepatitis B virus (DHBV) polymerase, a human hepatitis B virus (HBV) polymerase, a crane hepatitis B virus polymerase, a heron hepatitis B virus polymerase, a sheldgoose hepatitis B virus polymerase, a Ross's goose hepatitis B virus polymerase, a snow goose hepatitis B virus polymerase, a stork hepatitis B virus polymerase, a chimpanzee hepatitis B virus polymerase, a woolly monkey hepatitis B virus polymerase, a ground squirrel hepatitis B virus polymerase, an arctic ground squirrel hepatitis B virus polymerase, or a woodchuck hepatitis B virus polymerase. In yet other configurations, the polypeptide can be immobilized on a solid support.

[0052] In various configurations of these methods, a nucleobase polymer can be a nucleic acid, or nucleic acid analogue such as a peptide-nucleic acid (PNA; Ray, A., and Norden, B., FASEB J. 14: 1041-1060, 2000). In various configurations, a nucleic acid can be an RNA or a DNA. Furthermore, a nucleobase polymer can be single- stranded or double-stranded, such as a single-stranded nucleic acid or a double- stranded nucleic acid. In various configurations, any nucleobase polymer can be used for these methods. In some configurations, the nucleobase polymer can have any sequence other than the sequence of an epsilon sequence of a hepadnavirus, such as an HBV epsilon RNA of sequence

UGUACAUGUCCCACUGUUCAAGCCUCCAAGCUGUGCCUUGGGUGGCUUU GGGGCAUGGACA (SEQ ID NO: 15) (Knaus and Nassal, 1993, Nucleic Acids Research 21: 3967-3975).

[0053] In these aspects, the methods can comprise separating unbound nucleobase from a mixture. Separating can achieved by any method known to skilled artisans, such as, for example, forming a mixture with at least one component immobilized on a solid substrate, and washing unbound nucleobase polymer from the mixture. In various configurations of these aspects, a nucleobase polymer can comprise a label, such as, without limitation, a radioisotope, a fluorophore, an enzyme, and a probe-binding target as described herein for an antibody label, and detecting the nucleobase polymer can comprise detecting the label. When the label is an enzyme, a method can further comprise adding to the mixture a substrate of the enzyme, and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate. When the label is a fluorophore the fluorophore can be a fluorophore that binds nucleic acids with high affinity, such as, for example, 4',6-diamidino-2-phenylindole (DAPI), or bis- benzimides such as Hoechst compound 33258 or Hoechst compound 33342. In various configurations, the enzymes and substrates can be, without limitation, those enzymes and substrates described herein with regard to detecting binding between at least one antibody and at least one epitope. When the label is a radioisotope, the radioisotope

32 can be any radioisotope known to skilled artisans, such as, without limitation, a P, a

33 35 14 125 131 3

P, S, a C, an I, an I, or a H. A label can be added to a nucleobase polymer by any method known to skilled artisans, such as, for example, nick translation of double

32 stranded DNA using a P-labeled dNTP.

[0054] In yet other configurations of these aspects, a method can further comprise adding oligonucleotide primers to the mixture. The oligonucleotides primers of these configurations can hybridize to the at least one nucleobase polymer or its complement. In some configurations, the primers are added after unbound nucleobase polymer is separated from the mixture. In these configurations, the detecting binding can comprise performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of reaction product. Detection of a PCR product can comprise any detection method known to skilled artisans, such as, for example, quantification of DNA comprising a PCR reaction product, or measurement of radioactivity incorporated from a radiolabeled nucleoside triphosphate. In some configurations, a PCR amplification can be a quantitative amplification, such as a TaqMan® assay (Applied Biosystems, Foster City, CA).

[0055] In various configurations of the methods disclosed herein, a component of a method, either a polypeptide or a nucleobase polymer, can be immobilized on a solid support while its binding partner is not immobilized on the solid support. In these configurations, detecting a reduction in binding comprises detecting presence, absence or quantity of the component not immobilized on a solid support in a mixture comprising a candidate compound, wherein a decrease in binding compared to a mixture comprising the a polypeptide and a nucleobase polymer in the absence of the candidate compound indicates anti-viral activity of the compound. In various configurations, the component not immobilized on a solid support can comprise a label, and the detecting can comprise detecting the label. A label of these configurations can be any label known to skilled artisans, such as, without limitation, a chromophore, a fluorophore, a radioisotope, an enzyme, or a probe-binding target. When the label is a chromophore, the label can be any chromophore known to skilled artisans. When the label is a fluorophore, the label can be any fluorophore known to skilled artisans. When the label is a radioisotope, the radioisotope can be any

32 33 35 radioisotope known to skilled artisans, such as, without limitation, a P, a P, S, a

14 125 131 3

C, an I, an I, or a H. When the label is an enzyme, a method of these aspects can further comprise adding to the mixture a substrate of the enzyme, and detecting presence and/or quantity of a product of a reaction between the enzyme and the substrate. When the label is a probe-binding target, the probe-binding target can be any molecular target for a probe, such as, without limitation, a ligand to which a probe binds, such as, without limitation, an antigen to which an antibody binds. [0056] In some configurations of these aspects, a component, either a polypeptide or a nucleobase polymer, can further comprises a fluorophore label, and the first polypeptide can further comprises a fluorescence quencher which quenches the fluorescence of the fluorophore when the first polypeptide and the second polypeptide are bound to each other. In these configurations, detecting a reduction in binding between the first polypeptide and the second polypeptide can comprise detecting an increase in fluorescence intensity of the fluorophore compared to a mixture comprising the first polypeptide and the second polypeptide in the absence of the candidate compound. In these configurations, one of these components can be immobilized on a solid support while the other can be added in solution.

[0057] In some configurations of these aspects, a component, either a polypeptide or a nucleobase polymer, can further comprise a fluorophore label, and binding between components can be detected using fluorescence anisotropy polarization. A binding assay utilizing fluorescence anisotropy polarization can be performed using methods well known to skilled artisans, such as, for example, methods described in LeTiIIy, V., et al., Biochemistry 32: 7753-7758, 1993.

[0058] In some configurations of these aspects, the first polypeptide can consist essentially of a T3 domain of a hepadnavirus polymerase, or can consist of a T3 domain of a hepadnavirus polymerase. [0059] In some configurations of these aspects, the second polypeptide can consist essentially of an RT-I domain of a hepadnavirus polymerase, or can consist of an RT-I domain of a hepadnavirus polymerase.

[0060] In various aspects of the present teachings, a T3 domain can have an amino acid sequence of a T3 domain of any hepadnavirus, such as, without limitation, amino acids 176-183 of DHBV, EAGILYKR (SEQ ID NO: 31) or amino acids 155-162 of a HBV strain AD W2 polymerase, KAGILYKR (SEQ ID NO: 16). In addition, in various configurations, an RT- 1 domain can have an amino acid sequence of an RT- 1 domain of any hepadnavirus, such as, without limitation, amino acids 383-415 of DH BV polymerase, PNRITGKLFLVDKNSRNTTEARLVVDFSQF SKG (SEQ ID NO: 17); or amino acids 368-400 of a HBV strain AD W2 polymerase, PARVTGGVFLVDK NPHNTAESRLVVDFSQFSRG (SEQ ID NO: 18).

[0061] In various configurations, a sequence of a hepadnavirus polymerase, a domain or a subsequence of a hepadnavirus polymerase such as a T3 domain, an RT-I domain or a subsequence comprising an epitope, can be a naturally occurring variant of a hepadnavirus sequence or subsequence set forth herein.

[0062] In various configurations, a sequence of a hepadnavirus polymerase, a domain or a subsequence of a hepadnavirus polymerase such as a T3 domain, an RT-I domain or a subsequence comprising an epitope, can comprise one or more amino acid insertions, deletions, and/or substitutions which do not substantially alter binding interactions between a T3 domain and an RT-I domain compared to a naturally occurring hepadnavirus polymerase, a T3 domain thereof or an RT-I domain thereof.

EXAMPLES

[0063] The following examples, while illustrative of various aspects of the present teachings, are not intended to be limiting of any claim. Unless explicitly stated in past tense, an example may or may not have been reduced to practice. [0064] The methods and compositions described in the following examples utilize laboratory techniques well known to skilled artisans, and can be found in laboratory manuals such as Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; and Harlow, E., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In addition, some of Examples may utilize the following materials and methods. [0065] The four domains of the polymerase and their boundaries are shown along with the location of T3, RT-I and the mAb epitopes which flank T3 in the DHBV polymerase are illustrated in FIG. IA. Black bars represent the polymerase sequences employed as synthetic peptides. FIG. IB illustrates miniRT2, in which an active truncation of the DHBV polymerase is shown with the boundaries of the truncations.

[0066] Viruses and DNA clones

[0067] pT7BDPol contains DHBV strain 3 (Sprengel, R., et al., J. Med. Virol. 15: 323-333, 1985) nucleotides (nt) 170 to 3021 encoding the polymerase within pBluescript (Stratagene Corp., La Jolla, CA); the construct contains a 33-nt insertion at DHBV nt 901 encoding the influenza virus hemagglutinin epitope (Kolodziej, P. A., et al., Methods in Enzymology 194: 508-519, 1991) as well as leader sequences from Brome mosaic virus to promote translation in vitro. Mutations (Table 2) were inserted into pT7BDPol. The plasmid pdε contains DHBV nt 2526 to 2845 encoding ε within pBluescript. pdε-dl bulge is pdε with a deletion of nt 2571-2576. pDRF-BS contains DHBV nt 2401-2605 in pBluescript.

Table 2. Effects of mutations to T3 and RT-I on priming, exposure of an occluded mAb epitope and epsilon binding.

Values normalized to the activity of wild-type P, set to 100

[0068] Bioinformatics

[0069] Amino acid sequences were aligned using ClustalW. Accession numbers for the sequences employed are DQ195079 (DHBV) (SEQ ID NO: 30; genomic sequence encoding DHBV polymerase), CAC80820 (SHV) (SEQ ID NO: 8), AAA45738 (HHV) (SEQ ID NO: 4), AAA45748 (RGHV) (SEQ ID NO: 6), AAA46767 (WHV) (SEQ ID NO: 12), P03161 (GSHV) (SEQ ID NO: 32), AAC16908 (WMHV) (SEQ ID NO: 10) and AM282986 (HBV) (SEQ ID NO: 2). The coordinates for the model of the HBV RT domain (Das, K., J. Virol. 75: 4771-4779, 2001) were displayed using Pymol (DeLano Scientific).

[0070] In vitro transcription and translation

[0071] mRNAs for DHBV polymerase were transcribed with T7 RNA polymerase from pT7BDPol. DRF+ and ε RNAs were transcribed with T3 RNA polymerase from pDRF-BS and pdε, respectively. All RNAs were transcribed using Megascript kits

32

(Ambion) according to the manufacturer's instructions. In some cases P-labeled

32

RNAs were transcribed by including 25 μCi of [α P]UTP (3000 Ci/mmole, GE

35

Healthcare) in the reaction. S-labeled DHBV polymerase was translated in vitro by employing rabbit reticulocyte lysate (Promega) in 10 or 20 μl total volume containing

35

[ S]methionine (1,000 Ci/mmol; GE Healthcare) at 30⁰C for 1.5 h according to the manufacturer's instructions.

[0072] Polymerase:RNA binding assay [0073] The polymerase:RNA binding assay described previously (Beck, J., et al, J. Virol. 71 : 4971-4980, 1997) was used with minor modifications. WT or mutant

32 polymerase was translated in vitro in the presence of 250 ng of P-labeled RNA Translation was stopped by addition of cycloheximide (80 μM), and then the polymerase:RNA complex was immunoprecipitated using the anti-DHBV polymerase polyclonal antibodies R2B2 or R2B3 in the presence of IPP150+ (10 mM Tris pH 7.5,

150 mM NaCl, 0.1% NP40 and 100 μg/mL yeast tRNA) at 4° for two hours. Following binding, the complex was washed with IPP 150+ four times. Radiolabeled polymerase and RNA were resolved with by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and detected by phosphorimager analysis. Where

35 necessary, the S signal of the polymerase was blocked with a sheet of exposed autoradiography film. In some experiments, the polymerase was translated without labeled RNA and synthetic peptides were added following termination of translation. o 32

Following incubation for 20 minutes at 30 , P-labeled RNA was added and incubated for a further 20 minutes prior to immunoprecipitation.

[0074] MiniRT2 purification and refolding

[0075] MiniRT2 containing a six-histidine tag on the C-terminus was expressed and purified as previously described (Wang, X., et al., J. Virol. 77: 4471-4480, 2003), with minor changes. Briefly, the bacterial pellet was resuspended in 6 M guanidineHCl along with 20 mM imidazole and 0.1% NP40. After cell lysis the protein was bound to Ni-NTA agarose beads (Qiagen) and washed with 8 M urea. While still attached to the beads, the protein was refolded by adding refolding buffer (Wang, X., et al., J. Virol. 77: 4471-4480, 2003) along with decreasing amounts of urea. Elution buffer (350 mM imidazole, 300 mM NaCl, 0.1% NP40 and 27.5% glycerol) was used to elute the protein from the beads, followed by dialyzing overnight in 50 mM Hepes (pH 8), 0.1 M NaCl, 20% glycerol, 0.2% NP40, 50 mM dithiothreitol and protease inhibitor cocktail (Sigma). In addition, native purification of miniRT2 employed conditions established for hexa-histidine tagged Hepatitis C virus NS5B RNA polymerase as described in Ferrari et al., J. Virol. 73: 1649-1654, 1999.

[0076] Priming assay [0077] The polymerase was translated in vitro and an aliquot was removed to measure

32 translation efficiency. 10 μCi [α P]dGTP (3000 Ci/mmole, GE Healthcare) along with

4 mM MgC12 and 0.25 μg ε were added and the mixture was incubated at 30 for 30 minutes. The samples were then resolved by SDS-PAGE and the translation and priming signals were quantitated using a phosphorimager. The priming signal was normalized to translation efficiency. DNA priming was also assessed using 200 ng purified miniRT2 as described previously (Wang, X., et al., J. Virol. 77: 4471-4480, 2003). Synthetic peptides (Genscript) were included in some experiments after translation.

[0078] Example 1

[0079] This example illustrates that mutations at T3 ablate polymerases binding. [0080] Function of T3 (aa 176-183, EAGILYKR, SEQ ID NO: 31) is needed for the polymerase to prime DNA synthesis, but T3 could contribute to ε binding, enzymatic activation of the polymerase or priming itself. To understand the mechanism by which T3 contributes to priming we first tested the ability of T3 mutants to bind ε. In these

32 experiments, the polymerase was translated in vitro, P-labeled RNA was added, and the mixture was incubated at 30 for 60 minutes. The complexes were immunoprecipitated using an anti-polymerase polyclonal antibody, and then the polymerase and the co-precipitated RNA were resolved by SDS-PAGE. The RNAs employed were ε, ε-dl Bulge (a biologically inactive mutant form of ε that binds the polymerase very poorly) (Pollack, J. R., et al., J. Virol. 68: 5579-5587, 1994) and DRF+, an irrelevant RNA.

[0081] FIG. 2 illustrates binding of ε by wild-type and T3-mutant polymerases.

As shown in FIG. 2A, the polymerase was translated in vitro with the specified radiolabeled RNAs. The polymerase was then immunoprecipitated, and the polymerase and the co-precipitating RNAs were resolved by SDS-PAGE. Lanes 1-3 show the migration of the RNAs, lanes 4-6 show the relative binding of the RNAs to wild-type

35 polymerase and lane 7 is the polymerase alone. The S signal of immunoprecipitated polymerase is shown at the bottom. In experiments illustrated in FIG. 2B, wild-type and T3 -mutant polymerase molecules were assayed for ε binding as in FIG. 2A, and the RNA binding signal was then normalized to the immunoprecipitation signal for each protein and the specific activities of the RNAs. Error bars are the Standard deviation from three experiments. Unless indicated, ε RNA was used. In experiments illustrated in FIG. 2C, LMH cells were transfected with CDNA3.1 as a negative control or with wild-type or P(K182E/K183E) overlength DHBV expression vectors. Four days post-transfection intracellular DHBV core particles were harvested, the endogenous nucleic acids were removed, and the polymerase was detected by western blot.

[0082] Following normalization of the data to the amount of polymerase precipitated and the specific activities of the RNAs, the polymerase was found to bind ε seven- fold better than ε-dl Bulge and DRF+ (Fig. 2A Lanes 4-6). We also tested several T3 mutants, some of which have been described previously (Cao, F., et al, J. Virol. 79: 1016410170, 2005), for their ability to bind ε (Fig. 2B). P(Y170A/L171A), a mutant polymerase with lesions upstream of T3, bound ε only -50% as well as wild-type, and was also deficient in the priming assay. P(Y181F), a mutant polymerase we had shown previously to be wild-type in its priming ability (Cao, F., et al., J. Virol. 79: 10164- 10170, 2005), bound ε slightly better than wild-type polymerase. However, two mutants that were unable to prime DNA synthesis (Cao, F., et al., J. Virol. 79: 10164- 10170, 2005), P(I179D/L180D) and P(K182E/K183E) were severely reduced in ε binding (Fig. 2B). Therefore, there was a correlation between ε binding and priming of mutants to the T3 domain. We have shown previously that the polymerase undergoes an activation step after binding to ε, which can be detected by the appearance of a partially protease-resistant band (Tavis, J. E., et al., J. Virol. 70: 5741-5750, 1996; Tavis, J. E., et al., J. Virol. 72: 5789-5796, 1998). The polymerase with mutations at T3 did not undergo this post-binding activation, as addition of ε to P(K182E/K183E) did not induce the characteristic 36 KDa papain-resistant fragment (data not shown). This suggests that the polymerase is inhibited prior to this step in the reverse transcription pathway.

[0083] If T3 is needed for the polymerase to bind ε, then mutating T3 should block encapsidation of the polymerase because polymerases binding is essential for encapsidation of both the polymerase and the pgRNA (Bartenschlager, R., et al., J. Virol. 64: 5324-5332, 1990; Pollack, J. R., et al., J. Virol. 68: 5579-5587, 1994). Therefore, overlength WT and T3-mutant P(K182E/K183E) DHBV genomes were transfected into LMH cells. Viral core particles were isolated (Tavis, J. E., et al., J. Virol. 72: 5789-5796, 1998), the cores were permeablized by low pH treatment (Radziwill, G., et al., J. Virol. 163: 123-132, 1988) and the covalently-attached nucleic acids were degraded with micrococcal nuclease. The polymerase was then resolved by SDS-PAGE and detected by western blot with mAb 11. WT viral cores contained the polymerase, but the T3 mutant did not (Fig. 2C). Therefore, mutations at T3 ablated encapsidation as was predicted by their inability to bind ε in vitro. Together, the correlation between ε binding and priming activity for the T3 mutants, the failure of ε to induce the protease resistant conformation of P(K182E/K183E), and the failure of P(K182E/K183E) to promote ε-dependent encapsidation indicate that T3 functions to promote binding to ε.

[0084] Example 2

[0085] This example illustrates a prediction of the putative partner for T3. [0086] In these experiments, we examined the sequence of the polymerase for potential partner for T3. The spacer and RNase H domains were excluded because these regions are not present in miniRT2, an active truncation derivative of the polymerase that can bind ε. Motifs in the TP domain that we had previously screened for their ability to inhibit priming as synthetic peptides (M.P. Badtke and J.E. Tavis, unpublished data) were considered unlikely to be the ligand. Sequences that contained the universally-conserved motifs in the reverse transcriptase domain (Delarue, M., et al., Protein Engineering 3: 461-467, 1990; Poch, O., et al., EMBO J. 8: 3867-3874, 1989; Xiong, Y., et al., EMBO J. 9: 3353-3362, 1990) were also excluded because their molecular function is well characterized. Finally, we hypothesized that the partner would be conserved among the avian and mammalian hepadnaviruses because T3 is highly conserved (Fig. 4A). The region that fit these criteria best was aa 385-415 in the N-terminal portion of the reverse transcriptase domain (Fig. 4B), which we named RT- 1.

[0087] FIG. 4 illustrates that T3 and RT-I are conserved among the hepadnaviruses. Multiple sequence alignments of regions flanking T3 and RT-I are shown in FIG. 4A and FIG. 4B, respectively.

[0088] DHBV3 to RGHBV are avian hepadnaviruses and WHV to HBV are mammalian hepadnaviruses. If RT-I binds to RNA, it would have to be on the surface of the reverse transcriptase domain to permit it to contact nucleic acid. While no crystal structures exist for a Hepatitis B Virus polymerase, a reliable model has been developed for the reverse transcriptase domain of HBV polymerase based on the human immunodeficiency virus (HIV) and Moloney murine leukemia virus reverse (MMLV) transcriptases (Das, K., J. Virol. 75: 4771-4779, 2001; Ding, J., et al, J. MoI. Biol. 284: 1095-1111, 1998; Hsiou, Y., Structure 4: 853-8601996; Huang, H., et al., Science 282: 1669-1675, 1998). As presented in FIG. 5, upper panels represent various views of the predicted structure of HBV reverse transcriptase domain; lower panels show RT-I rendered dark and overlaid on dimmed views of the predicted HBV reverse transcriptase domain. Mapping the HBV RT-I sequences onto the model revealed that most residues within RT-I were indeed predicted to be on the surface of the reverse transcriptase domain at one end of the DNA binding cleft.

[0089] Example 3

[0090] This example illustrates that mutations within RT-I inhibit priming, expose a mAb epitope near T3 and reduce binding to ε.

[0091] Mutating T3 inhibits priming, exposes buried mAb epitopes near T3 in the terminal protein domain and inhibits binding to ε (Cao, F., et al., J. Virol. 79: 10164- 10170, 2005, and Table 2). If RT-I binds to RNA in collaboration with T3, then mutations within RT-I should have similar effects. To test this prediction, we created ten sets of single or double mutations within RT-I. These mutants were translated in vitro and their ability to prime DNA synthesis, to expose the occluded mAb 6 epitope and to bind ε was tested (Table 2). All ten mutants were less active than wild-type polymerase in the priming assay and seven of the mutants retained less than 5% activity, indicating that RT-I is important for DNA priming. Exposure of the epitope for mAb 6 was increased by greater than two-fold for four of the mutants, and by more than 1.5 fold in three additional mutants. Therefore, altering RT-I had the predicted effects on the structure of the terminal protein domain, although the effects were substantially smaller than when T3 was mutated. Finally, seven of the mutants bound ε less efficiently than wild-type polymerase, although the magnitude of suppression was modest for all mutants except S410A/S413A.

[0092] Overall, seven of the ten RT-I mutants had the expected pattern of reduced priming, increased epitope exposure, and reduced ε binding (P383A/R385E, F391H/L392D, N399A/E402A, R404A/D408A, F409H/Q411R, S410A/S413A, N428A/L429A). The mutants with the predicted pattern all had lesions near the N- and C-terminal ends of RT-I (amino acids 383-392 and 404-429), whereas the three mutant polymerases that did not have the predicted pattern had substitutions near the middle of RT-I (residues 394-404). However, the alterations to epitope exposure and ε-binding were modest for the mutants with the expected phenotype, and the pattern of priming, epitope exposure and ε binding in the central set of mutations was complex. Therefore, these data are consistent with an interaction between T3 and the N- and C-termini of RT-I contributing to priming through modulation of RNA binding. However, these data do not exclude other possible roles for RT-I in DNA synthesis.

[0093] Example 4

[0094] This example illustrates that synthetic peptides containing T3 and RT-I sequences inhibit priming.

[0095] We have previously shown that a peptide containing T3 sequences specifically inhibits DNA priming in a dose-dependant manner (Cao, F., et al., J. Virol. 79: 10164- 10170, 2005). If RT-I binds to RNA in collaboration with T3, peptides containing RT- 1 sequences should also specifically block priming. Therefore we created 3 peptides containing RT-I sequences (Table 3). RT-IA contains the N-terminal half of RT-I (aa 385-399), RT-IB contains the C-terminal half (aa 399-415) and RT-IC contains the entire RT-I sequence (aa 385-415). We also created three larger T3 peptides in an attempt to improve the T3 peptide because the original T3 peptide was active only at high concentrations (>250 μM) (Table 3). Finally, we created T3-Scramble, a negative control peptide containing scrambled T3 sequences.

[0096] Synthetic peptides containing T3 sequences can inhibit purified polymerase in the absence of the cellular chaperones that are normally needed for P to function (FIG. 3). In these experiments, purified miniRT2 was incubated with ε and radiolabeled dGTP for 2 hours at 30°. Lane 1 shows Coomassie blue stained miniRT2 following purification. Lanes 2-6 show the priming signal following incubation of miniRT2 with increasing concentrations of T3 or an irrelevant peptide. Peptide concentrations are indicated in mM.

[0097] To determine the effects of these peptides on priming, increasing amounts of the peptides were added to the polymerase following termination of in vitro translation with cycloheximide, and the IC50 value for each peptide was calculated. The scrambled T3 peptide was essentially unable to inhibit priming (IC50=975 μM) and the original T3 peptide had a high IC50 (302 μM) (Table 3). The new T3 peptides all had IC50 values under 50 μM, with T3B and T3C having values near 10 μM. The RT-IA and RT-IB peptides were poor inhibitors of priming, with IC50 values greater than 500 μM. However, RT-IC was a much more effective inhibitor, with an IC50 of 46 μM (Table 3). The RT-I sequences needed to be present on a single molecule because mixing RT-IA and RT-IB together in the reaction did not inhibit priming any better than RT-IA and RT-IB individually (data not shown).

Table 3. Effects of T3 and RT-I peptides on priming and exposure of an occluded mAb epitope.

Values in μM.

[0098] Example 5

[0099] This example illustrates that synthetic peptides containing T3 or RT-I sequences bind to nucleic acids.

[00100] We predicted that inhibition of priming by the peptides could be due to disruption of an interaction of the T3 and RT-I motifs with the ε RNA. Therefore, we tested the ability of the T3 and RT-I peptides to bind RNA. In these experiments, the peptides (10 pMol) were bound to a nitrocellulose filter in a slot-blot apparatus and the filter was washed. Radiolabeled ε or its biologically inactive derivative ε-dlBulge were added with or without a 50-fold excess of yeast tRNA, the filter was washed, and retained RNA was detected by autoradiography As shown in FIG 6A. T3 and RT-I peptides bind ε. Top: The peptides were bound to a nitrocellulose filter in a slot-blot apparatus and then 32P-labeled ε RNA was passed through the filter and the filter was washed. T3 -scramble and T3B-scramble are negative controls in which the T3 motif sequences have been scrambled; UL13 and Pepl are irrelevant peptides. Bottom: T3B and RTlC peptides were loaded onto a filter in amounts ranging from 10 pMol to 0.3 pMol and RNA binding to 32P-labeled ε RNA was measured as in the top panel. [00101] FIG 6B illustrates that T3 and RT-I peptides bind nucleic acids non- specifically. Top: Filter binding assays were performed with T3 and RT-I peptides and either ε or the biologically inactive ε-dlBulge RNA in the presence or absence of a 50- fold excess of non-radioactive yeast tRNA. BSA was used as an irrelevant control protein. Bottom: Filter binding assays were performed with T3 and RT- 1 peptides and 32P-labeled DHBV core gene RNA or double-stranded DNA as irrelevant nucleic acid probes.

[00102] Experiments illustrated in FIG 6C disclose that high concentrations of

T3 and RT-I peptides precipitate RNA. In these experiments, T3 and RT-I peptides were incubated with 32P-labeled ε RNA in IPP 150 buffer, the mixture was briefly centrifuged and the supernatant and pellet fractions were resolved on a 15% polyacrylamide gel. The gel was stained with coomassie blue to detect peptides and radiolabeled RNA was detected by phosphorimage analysis.

[00103] These experiments demonstrate that there was no binding of RNA to two irrelevant peptides (UL13 and Pepl), nor to T3 or T3B peptides in which the T3 sequences were scrambled. However, robust binding to the T3, T3B, T3C, and RTlC peptides was detected. Furthermore, RNA binding was proportional to the amount of RNA loaded onto the filter, and the RTlC peptide appeared to bind RNA less well than the T3B peptide. This RNA binding was non-specific because the peptides bound well to both ε and ε-dlBulge and because binding was suppressed to nearly background by an excess of unlabeled tRNA (FIG. 6B). The T3B and RTlC peptides were also able to bind to an irrelevant unstructured RNA derived from the DHBV core gene and to a double-stranded DNA fragment.

[00104] Example 6

[00105] This example illustrates that miniRT2 binds to nucleic acids.

[00106] FIG. 7 illustrates that miniRT2 can bind RNA in vitro, and also illustrates that RNA binding by the polymerase is necessary but not sufficient to support DNA priming.

[00107] In this example, we extended the binding studies described in Example

5 from peptides to miniRT2 because miniRT2 retains ε-specific priming activity, is able to bind ε without the aid of the cellular chaperones, and can be purified easily from bacteria. Therefore, miniRT2 performs the authentic DNA priming reaction, but it is much more experimentally tractable than the full-length protein. [00108] In these experiments, miniRT2 was expressed in E. coli and purified under native conditions by nickel-affinity chromatography.

[00109] FIG. 7A illustrates miniRT2 purified under native conditions. Note that the bacterial chaperones co-purify with miniRT2 under native conditions but they are removed when the purification is performed under denaturing conditions (compare FIG. 7 and FIG. 3).

[00110] FIG 7B illustrates DNA priming by miniRT2, indicating that the enzyme is active. ε-dlBulge is a deletion variant of ε that cannot support the priming reaction by the polymerase. In these experiments, miniRT2 was incubated with ε and [(X³²PJdGTP, the reaction was resolved by SDS-PAGE, and priming activity was detected as ³²P labeling of miniRT2 due to the covalent linkage of [³²P] dGMP to the enzyme.

[00111] We next assessed RNA binding by miniRT2 employing the filter- binding assay we previously employed for the peptides. Wild-type miniRT2 bound both ε and its biologically inactive derivative ε-dlBulge, and this binding was efficiently competed by a 50-fold excess of unlabeled yeast tRNA (FIG. 7C). In these experiments, RNA binding by miniRT2 was tested in a filter-binding assay employing ³²P-labeled ε and ε-dlBulge RNAs as probes in the presence or absence of a 50-fold excess of unlabeled tRNA competitor; bovine serum albumen (BSA) was used as a negative control. Note that miniRT2 bound both ε and ε-dlBulge RNAs although only ε supported priming. Therefore, miniRT2 binds RNA nonspecifically, just as the T3 and RTl peptides do. This indicates that ε binding is not sufficient for miniRT2 to prime DNA synthesis; this is consistent with the lack of specificity in RNA binding by miniRT2 and the peptides.

[00112] Example 7

[00113] This example illustrates screening candidate anti-HBV compounds for anti-HBV activity using an ELISA-like format.

[00114] In this example, a series of candidate anti-HBV compounds are selected using a digital computer comprising a graphical user interface and coordinates of crystal structures of HIV-I RT (Das, K., et al, J. Virol. 75: 4771-4779, 2001; Ding, J., et al., J. MoI. Biol. 284: 1095-1111, 1998; Hsiou, Y., Structure 4: 853-8601996; Huang, H., et al., Science 282: 1669-1675, 1998) and MuLV RT (Georgiadis, M.M., et al., J. MoI. Biol. 284: 1095-1111, 1995). Models of the HBV polymerase domain are obtained using the amino acid sequence alignment-based three-dimensional structure- generating program MODELLER-4 (Sanchez, R., et al., Proteins 1997 (Suppl): 50-58, 1997) and crystal structures of HIV-I RT (PDB codes: IRTD, 2HMI, and IDLO) and of MULV RT (PDB code: IMML) as templates, the coordinates for which are incorporated by reference in their entirety and are available on the internet at the PDB website, http://www.rcsb.org/pdb/home/home.do, under their respective PDB codes. The protein conformation of the model obtained using the HIV-I RT-DNA-dNTP complex structure (IRTD) is used as an initial scaffold for the HBV polymerase model. The computer program O (Kleywegt, G. J., et al., Structure 3: 535-540, 1995) is used to construct models for less conserved regions, insertions, and side chains, with reference to databases of known main-chain conformations and preferred side-chain rotamers. Three models, derived from the HIV-I RT-DNA -Fab complex (2HMI), unliganded HIV-I RT (IDLO), and MULV RT (IMML), are used as additional guides in building a molecular model of HBV polymerase. Candidate compounds identified using these models are obtained from a commercial chemical source. A series of ELISA plates are prepared, comprising wells coated with a previously purified DHBV polymerase polypeptide. Following coating of the ELISA plates, non-specific binding is blocked with a blocking solution comprising 1% BSA and 0.1% NP40 detergent. Candidate anti-HBV compounds are applied to individual wells in concentrations varying from 1 μM to 100 μM, and an alkaline phosphatase-labeled nucleobase polymer is then added to the wells. Following incubation for 1 hr., the ELISA plates are washed, and a chemiluminescent substrate for alkaline phosphatase (CDP-Star®, Sigma-Aldrich Chemical, St. Louis, MO) is then added to the wells. Compounds in wells providing the least intense signals are thereby identified as candidate anti-HBV compounds.

[00115] Example 8

[00116] This example illustrates a screen for candidate anti-HBV compounds using a nucleic acid binding assay and HBV T3 and RT-I peptides.

[00117] In this example, a series of candidate anti-HBV compounds are obtained from a commercial provider. A series of ELISA plates are prepared as described in Example 7 except that purified HBV peptides containing T3 and RT- 1 sequences are employed. Candidate anti-HBV compounds are applied to individual wells in concentrations varying from 1 μM to 100 μM. A pUC18 plasmid, which does not

32 comprise an epsilon sequence, is labeled with P using a standard nick translation reaction. Radiolabeled DNA is added to the wells. After a 1 hr incubation, the wells are washed, and the ELISA plates are imaged on a phosphorimager. Compounds in wells providing the least intense signals are thereby identified as candidate anti-HBV compounds.

[00118] Example 9

[00119] This example illustrates a screen for candidate anti-HBV compounds using two polypeptides.

[00120] In this example, a series of compounds are obtained from a commercial provider. A series of ELISA plates are prepared as described in Example 8, except that the wells are coated with a previously purified T3 domain of a DHBV polymerase and a previously purified RT-I domain of a DHBV polymerase. After blocking of nonspecific binding, a fluorescein-labeled ε RNA is added. The wells are then washed, and candidate compounds are added to individual wells. After a 1 hour incubation, the wells are washed again and retained fluorescence is measured employing a plate reader. The samples yielding the lowest level of fluorescence identify wells containing candidate anti-HBV compounds.

[00121] All references cited herein are incorporated by reference in their entirety. Applicant reserves the right to challenge any conclusions or other statements made by the authors of cited references.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV), the method comprising: a) forming a mixture comprising i) a candidate compound, ii) a polypeptide comprising A) a T3 domain of a hepadnavirus polymerase and B) an RT-I domain of a hepadnavirus polymerase, iii) at least one nucleobase polymer; and b) detecting a decrease in binding compared to a mixture comprising the polypeptide and the nucleobase polymer in the absence of the candidate compound, whereby a decrease in the binding indicates anti-viral activity of the compound.

2. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus, and wherein the RT- 1 domain of a hepadnavirus polymerase is an RT-I domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus.

3. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus, and wherein the RT-I domain of a hepadnavirus polymerase is an RT-I domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus.

4. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the T3 domain and the RT-I domain are from the same species of hepadnavirus polymerase.

5. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 4, wherein the hepadnavirus polymerase is a human hepatitis B virus polymerase.

6. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 4, wherein the hepadnavirus polymerase is a duck hepatitis B virus polymerase.

7. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 1 , wherein the nucleobase polymer is a peptide- nucleic acid.

8. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 1, wherein the nucleobase polymer is a nucleic acid.

9. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is a nucleic acid other than a hepadnavirus epsilon RNA.

10. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is a hepadnavirus epsilon RNA.

11. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is a single stranded nucleic acid.

12. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is a double stranded nucleic acid.

13. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is an RNA.

14. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 8, wherein the nucleic acid is a DNA.

15. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 1, wherein the nucleobase polymer comprises a label.

16. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 15, wherein the label is selected from the group consisting of a radioisotope, a fluorophore, an enzyme, and a probe-binding target.

17. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 16, wherein the label is an enzyme, and wherein the method further comprises: adding to the mixture a substrate of the enzyme; and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate.

18. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 17, wherein the enzyme is selected from the group consisting of a peroxidase, a phosphatase, a galactosidase and a luciferase.

19. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 17, wherein the substrate is a chemiluminescent substrate, and the detecting comprises detecting light produced as a product of a reaction between the substrate and the enzyme.

20. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 16, wherein the label is a radioisotope selected from the

32 33 35 14 125 131 3 group consisting of a P, a P, an S, a C, an I, an I and a H.

21. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 16, wherein the label is a probe-binding target selected from the group consisting of a biotin, a digoxygenin, and a peptide.

22. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 21, wherein the probe-binding target and a probe which binds the probe-binding target are selected from the group consisting of a) a biotin and an avidin, b) a biotin and a streptavidin, c) a biotin and an anti-biotin antibody, d) a digoxygenin and an anti-digoxygenin antibody, and e) a peptide and an antibody directed against the peptide.

23. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide comprises a full length hepadnavirus polymerase.

24. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists essentially of A) a T3 domain of a hepadnavirus polymerase, and B) an RT- 1 domain of a hepadnavirus polymerase.

25. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists of A) a T3 domain of a hepadnavirus polymerase, and B) an RT-I domain of a hepadnavirus polymerase.

26. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide comprises a full length Duck Hepatitis B Virus polymerase.

27. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists essentially of A) a T3 domain of a Duck Hepatitis B Virus (DHBV) polymerase, and B) an RT- 1 domain of a DHBV polymerase.

28. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists of A) a T3 domain of Duck Hepatitis B Virus (DHBV) polymerase, and B) an RT-I domain of DHBV polymerase.

29. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide comprises a full length Hepatitis B Virus (HBV) polymerase.

30. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists essentially of A) a T3 domain of a Hepatitis B Virus (HBV) polymerase, and B) an RT-I domain of a HBV polymerase.

31. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the polypeptide consists of A) a T3 domain of Hepatitis B Virus (HBV) polymerase, and B) an RT-I domain of HBV polymerase.

32. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 1, further comprising immobilizing the polypeptide on a solid support.

33. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the method further comprises adding oligonucleotide primers to the mixture which hybridize to the at least one nucleobase polymer or its complement, and wherein the detecting binding comprises performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of a reaction product.

34. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the detecting a decrease in binding comprises a fluorescence resonance energy transfer (FRET) assay.

35. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 1, wherein the detecting a decrease in binding comprises a fluorescence anistropy polarization assay.

36. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV), the method comprising: a) forming a mixture comprising i) a candidate compound, ii) a first polypeptide comprising a T3 domain of a hepadnavirus polymerase iii) a second polypeptide comprising an RT-I domain of a hepadnavirus polymerase; b) adding to the mixture at least one nucleobase polymer; and c) detecting a decrease in binding between the nucleobase polymer and the polypeptides compared to a mixture comprising the polypeptides and the nucleobase polymer in the absence of the candidate compound, whereby a decrease in the binding indicates anti-viral activity of the compound.

37. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus, and wherein the RT-I domain of a hepadnavirus polymerase is an RT-I domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus.

38. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus, and wherein the RT-I domain of a hepadnavirus polymerase is an RT- 1 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus.

39. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the T3 domain and the RT-I domain are from the same species of hepadnavirus polymerase.

40. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 39, wherein the hepadnavirus polymerase is a human hepatitis B virus polymerase.

41. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 39, wherein the hepadnavirus polymerase is a duck hepatitis B virus polymerase.

42. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 36, wherein the nucleobase polymer is a peptide- nucleic acid.

43. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 36, wherein the nucleobase polymer is a nucleic acid.

44. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is a nucleic acid other than a hepadnavirus epsilon RNA.

45. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is a hepadnavirus epsilon RNA.

46. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is a single stranded nucleic acid.

47. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is a double stranded nucleic acid.

48. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is an RNA.

49. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 43, wherein the nucleic acid is a DNA.

50. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the nucleobase polymer comprises a label.

51. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 50, wherein the label is selected from the group consisting of a radioisotope, a fluorophore, an enzyme, and a probe-binding target.

52. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 51, wherein the label is an enzyme, and wherein the method further comprises: adding to the mixture a substrate of the enzyme; and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate.

53. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 51, wherein the enzyme is selected from the group consisting of a peroxidase, a phosphatase, a galactosidase and a luciferase.

54. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 52, wherein the substrate is a chemiluminescent substrate, and the detecting comprises detecting light produced as a product of a reaction between the substrate and the enzyme.

55. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 51, wherein the label is a radioisotope selected from the

56. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 51, wherein the label is a probe-binding target selected from the group consisting of a biotin, a digoxygenin, and a peptide.

57. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 56, wherein the probe-binding target and a probe which binds the probe-binding target are selected from the group consisting of a) a biotin and an avidin, b) a biotin and a streptavidin, c) a biotin and an anti-biotin antibody, d) a digoxygenin and an anti-digoxygenin antibody, and e) a peptide and an antibody directed against the peptide.

58. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the method further comprises adding oligonucleotide primers to the mixture which hybridize to the at least one nucleobase polymer or its complement, and wherein the detecting binding comprises performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of a reaction product.

59. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the detecting a decrease in binding comprises a fluorescence resonance energy transfer (FRET) assay.

60. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 36, wherein the detecting a decrease in binding comprises a fluorescence anistropy polarization assay.

61. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV), the method comprising: a) forming a mixture comprising i) a candidate compound, and ii) a polypeptide comprising a T3 domain of a hepadnavirus polymerase; b) adding to the mixture at least one nucleobase polymer; and c) detecting a decrease in binding between the nucleobase polymer and the polypeptides compared to a mixture comprising the polypeptides and the nucleobase polymer in the absence of the candidate compound, whereby a decrease in the binding indicates anti-viral activity of the compound.

62. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus.

63. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the T3 domain of a hepadnavirus polymerase is a T3 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus.

64. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 63, wherein the hepadnavirus polymerase is a human hepatitis B virus polymerase.

65. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 63, wherein the hepadnavirus polymerase is a duck hepatitis B virus polymerase.

66. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 61, wherein the nucleobase polymer is a peptide- nucleic acid.

67. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 61, wherein the nucleobase polymer is a nucleic acid.

68. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 65, wherein the nucleic acid is a nucleic acid other than a hepadnavirus epsilon RNA.

69. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 67, wherein the nucleic acid is a hepadnavirus epsilon RNA.

70. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 67, wherein the nucleic acid is a single stranded nucleic acid.

71. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 67, wherein the nucleic acid is a double stranded nucleic acid.

72. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 67, wherein the nucleic acid is an RNA.

73. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 67, wherein the nucleic acid is a DNA.

74. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the nucleobase polymer comprises a label.

75. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 74, wherein the label is selected from the group consisting of a radioisotope, a fluorophore, an enzyme, and a probe-binding target.

76. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 75, wherein the label is an enzyme, and wherein the method further comprises: adding to the mixture a substrate of the enzyme; and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate.

77. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 76, wherein the enzyme is selected from the group consisting of a peroxidase, a phosphatase, a galactosidase and a luciferase.

78. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 76, wherein the substrate is a chemiluminescent substrate, and the detecting comprises detecting light produced as a product of a reaction between the substrate and the enzyme.

79. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 75, wherein the label is a radioisotope selected from the

80. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 75, wherein the label is a probe-binding target selected from the group consisting of a biotin, a digoxygenin, and a peptide.

81. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 80, wherein the probe-binding target and a probe which binds the probe-binding target are selected from the group consisting of a) a biotin and an avidin, b) a biotin and a streptavidin, c) a biotin and an anti-biotin antibody, d) a digoxygenin and an anti-digoxygenin antibody, and e) a peptide and an antibody directed against the peptide.

82. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the method further comprises adding oligonucleotide primers to the mixture which hybridize to the at least one nucleobase polymer or its complement, and wherein the detecting binding comprises performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of a reaction product.

83. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the detecting a decrease in binding comprises a fluorescence resonance energy transfer (FRET) assay.

84. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 61, wherein the detecting a decrease in binding comprises a fluorescence anistropy polarization assay.

85. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV), the method comprising: a) forming a mixture comprising i) a candidate compound, ii) a polypeptide comprising a RT- 1 domain of a hepadnavirus polymerase; b) adding to the mixture at least one nucleobase polymer; and c) detecting a decrease in binding between the nucleobase polymer and the polypeptides compared to a mixture comprising the polypeptides and the nucleobase polymer in the absence of the candidate compound, whereby a decrease in the binding indicates anti-viral activity of the compound.

86. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the RT-I domain of a hepadnavirus polymerase is an RT- 1 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus, a human hepatitis B virus, a crane hepatitis B virus, a heron hepatitis B virus, a sheldgoose hepatitis B virus, a Ross's goose hepatitis B virus, a snow goose hepatitis B virus, a stork hepatitis B virus, a chimpanzee hepatitis B virus, a woolly monkey hepatitis B virus, an arctic ground squirrel hepatitis B virus, and a woodchuck hepatitis B virus.

87. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the RT-I domain of a hepadnavirus polymerase is an RT- 1 domain of a polymerase of a hepadnavirus selected from the group consisting of a duck hepatitis B virus and a human hepatitis B virus.

88. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 87, wherein the hepadnavirus polymerase is a human hepatitis B virus polymerase.

89. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 87, wherein the hepadnavirus polymerase is a duck hepatitis B virus polymerase.

90. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 85, wherein the nucleobase polymer is a peptide- nucleic acid.

91. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 85, wherein the nucleobase polymer is a nucleic acid.

92. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91 , wherein the nucleic acid is a nucleic acid other than a hepadnavirus epsilon RNA.

93. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91, wherein the nucleic acid is a hepadnavirus epsilon RNA.

94. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91 , wherein the nucleic acid is a single stranded nucleic acid.

95. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91, wherein the nucleic acid is a double stranded nucleic acid.

96. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91 , wherein the nucleic acid is an RNA.

97. A method of identifying an anti-viral compound against Hepatitis B Virus (HBV) in accordance with claim 91 , wherein the nucleic acid is a DNA.

98. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the nucleobase polymer comprises a label.

99. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 98, wherein the label is selected from the group consisting of a radioisotope, a fluorophore, an enzyme, and a probe-binding target.

100. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 99, wherein the label is an enzyme, and wherein the method further comprises: adding to the mixture a substrate of the enzyme; and detecting the presence and/or quantity of a product of a reaction between the enzyme and the substrate.

101. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 100, wherein the enzyme is selected from the group consisting of a peroxidase, a phosphatase, a galactosidase and a luciferase.

102. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 101, wherein the substrate is a chemiluminescent substrate, and the detecting comprises detecting light produced as a product of a reaction between the substrate and the enzyme.

103. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 99, wherein the label is a radioisotope selected from the

104. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 99, wherein the label is a probe-binding target selected from the group consisting of a biotin, a digoxygenin, and a peptide.

105. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 104, wherein the probe-binding target and a probe which binds the probe-binding target are selected from the group consisting of a) a biotin and an avidin, b) a biotin and a streptavidin, c) a biotin and an anti-biotin antibody, d) a digoxygenin and an anti-digoxygenin antibody, and e) a peptide and an antibody directed against the peptide.

106. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the method further comprises adding oligonucleotide primers to the mixture which hybridize to the at least one nucleobase polymer or its complement, and wherein the detecting binding comprises performing a polymerase chain reaction (PCR) amplification, and detecting the presence and/or quantity of a reaction product.

107. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the detecting a decrease in binding comprises a fluorescence resonance energy transfer (FRET) assay.

108. A method of identifying an anti-viral compound against Hepatitis B Virus in accordance with claim 85, wherein the detecting a decrease in binding comprises a fluorescence anistropy polarization assay.