WO1996010649A1 - Isolated human hepatitis b virus polymerase and uses thereof - Google Patents

Abstract

The invention is directed to an isolated active recombinant human HBV pol complex. The complex may comprise a human HBV pol fusion protein or an epitope useful for purification. The invention is also directed to an isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex. The nucleic acid may encode an HBV pol fusion protein. The invention is also directed to a replicable vector comprising the nucleic acids above. The invention is also directed to host cells containing the proteins or nucleic acids above. Methods are also described. In one embodiment the invention is a method of expressing and isolating active human HBV pol. In another embodiment the invention is a method of screening a compound as an inhibitor or stimulator of a recombinant human HBV pol activity. The invention is also directed to a kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol.

Description

ISOLATED HUMAN HEPATITIS B VIRUS POLYMERASE AND USES THEREOF

This application is a continuation in part of U.S. Serial No. 08/315,856 filed September 30, 1994 the contents of which are hereby incorporated by reference into the present application.

The invention was made in part with government funds from the National Institutes of Health. Therefore, the U.S. Government has certain rights in the invention.

Introduction

The members of Hepadnaviridae replicate their nucleic acid through a reverse transcription step (14,43,46). Human hepatitis B virus is a member of the Hepadnaviridae and is commonly designated HBV. The reverse transcriptase, designated pol, also has an RNaseH domain and an amino- terminal domain involved in the protein-priming of the synthesis of first strand DNA (4,35). The mechanism of genome replication has been elucidated by a variety of methods yielding the following presumptive replication scheme. The initial step appears to be the recognition of the pregenomic RNA by the polymerase. Recognition occurs best in cis, whereby pol binds to its own mRNA (2,16- 18,20,34) . More than one RNA sequence on pregenomic RNA may facilitate this recognition step (10,17) with the essential sequence being a stem loop structure termed epsilon that is present at both ends of pregenomic RNA (17,18,20,34). Although epsilon is present on both ends of pregenomic RNA, only the 5' copy appears to function in packaging (17,18). The epsilon sequence in itself is sufficient to induce the packaging of foreign RNA sequences by pol and the viral capsid protein (17,18) . The entire pol open reading frame is required for RNA packaging, even though no known pol enzymatic function is required (2,11,16) , and the packaging of pol is dependent upon an RNA molecule possessing a 5' copy of epsilon (5) . Thus, 'neither pol nor pregenomic RNA can be packaged in the absence of the other. A priming reaction ensues in which a nucleotide becomes covalently linked to pol (4,9A,31) via a phosphodiester bond with a tyrosine residue (4) . The addition of the first nucleotide appears to be templated by a sequence in a bulge in the 5' copy of the stem loop, and the reaction is then extended by three additional nucleotides also templated within the bulge (49,51) . Although it is generally believed that the priming reaction occurs after packaging, the possibility that this reaction occurs prior to packaging has not been formally tested. The primed pol complex is translocated to the 3' copy of DR1 where the synthesis of minus strand DNA is initiated (12,27,32,39,41,42,55) .

Synthesis of minus strand DNA terminates at the 5' end of pregenomic RNA yielding a molecule with an 8-9 base terminal redundancy (39,55) . The pregenomic RNA is degraded by the

RNaseH activity of pol with the exception of 12-18 nucleotides at the 5' end. This capped oligoribonucleotide is translocated, in the second translocation step, to DR2 on minus strand DNA and serves as the primer of plus strand DNA

(28,29,40,45) . Finally, a third strand transfer occurs once plus strand DNA synthesis has reached the 3 ' end of minus strand DNA, resulting in a noncovalently closed, partially double-stranded, circular DNA molecule. The synthesis of plus strand DNA is only partially completed in mature virions, yielding the gapped DNA substrate that is filled- in following infection of susceptible cells or during the endogenous polymerase assay (19,23,47) .

The events of hepadnavirus genome replication have been elucidated by studies on infected tissues, purified virions, virions expressed from cloned DNA in transfected cell lines and mutagenesis of these cloned genomes. Previous attempts to purify an active human HBV polymerase have been unsuccessful. Some studies have demonstrated that active pol can not be solubilized from cores nor can pol within cores switch to an exogenously supplied template (36) , while in other studies, polymerase released from virions has shown some function in activity gel assays (6,7,33) . However, even these studies have not been successfully reproduced by others and there are no examples of isolated fully functional HBC pol. Expression of pol in heterologous systems has met with limited success as well. Pol expressed in bacteria has been reported to retain specific RNA binding properties (21) , but no reverse transcriptase activity was observed. Again, these findings can not be reproduced by others. Reverse transcriptase activity in which the DNA products are bound to protein has been detected- in Xenopus oocyte lysates in association with the expression of HBV pol (44) , however the template and products of this reaction have not been fully characterized and are too large to represent faithful reverse transcription of HBV RNA. This activity is due to loss of selectivity for accurate priming and hence lack of fidelity in reverse transcriptase activity. Recently, two systems using the duck hepatitis B virus (DHBV) pol have demonstrated reverse transcriptase activity that is template-dependent and protein-primed. One system utilizes in vitro translation of DHBV pol to obtain a functional pol (51) , while the other packages a fusion protein of DHBV pol in a viral like particle from the yeast retrotransposon Tyl (49) . Both systems yield pol that possesses accurate protein-primed, reverse transcriptase activity that synthesizes minus strand DNA originating at DR1 (49,51) . The pol mRNA in both of these systems contains a 3' copy of the stem loop required for initiation of nucleotide priming, but no 5' copy of this sequence. The realization that nucleotide priming occurs at the stem loop prior to translocation to DRl was obtained in studies with these two systems (50,52) . The in vitro translation system has also been used to map the pol tyrosine residue at which nucleotide priming occurs (54,56). Surprisingly, the in vitro translation system for pol has not been successfully employed for the human counterpart, HBV, nor has the DHBV pol been expressed and purified in a functional form using a conventional expression system.

Summary of the Invention

The invention is directed to an isolated active recombinant human HBV pol complex. The complex may comprise a human HBV pol fusion protein and may further comprise an epitope useful for purification. The invention is also directed to an isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex. The nucleic acid may encode a HBV pol fusion protein. The invention is also directed to a replicable vector comprising the nucleic acids encoding a protein component of an active recombinant human HBV complex. The invention is also directed to host cells containing the nucleic acids.

Methods are also described. In one embodiment the invention is a method of expressing and isolating active human HBV pol. In another embodiment the invention is a method of screening a compound as an inhibitor or stimulator of a recombinant human HBV pol activity. The invention is also directed to a kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol. Brief Description of the Fiσures

Figure 1. Structure of FP -pol. The structure of the FP -pol transcript from the baculovirus construct is depicted. The HBV polymerase open reading frame is represented as a rectangular box. At the amino terminus, the hatched section represents the FLAG^* epitope with the amino acid sequence of the FLAG^* epitope shown below in single letter code. The terminal protein (TP) , reverse transcriptase (RT) and RNase H domains are indicated above the polymerase open reading frame. The 3' end of the transcript is enlarged below to detail the DR2, DRl and epsilon (e) regions. The four nucleotide homology between the bulge in epsilon and the first four nucleotides of DRl are shown in bold. Polymerase undergoes a nucleotide priming reaction templated by the bulge in epsilon and then the primed polymerase is translo¬ cated to homologous region in DRl.

Figure 2. Purification of FPL-pol. Sf9 cells were harvested 48 hr postinfection with the recombinant baculovirus FPL- pol. Cells were sonicated in extraction buffer, and a clarified extract was purified on an M2 monoclonal antibody immunoaffinity resin as described under Material and Methods. Samples from the extraction and purification were analyzed by SDS-PAGE and either Coo assie blue (CB) staining or Western blot (WB) with an anti-HBV pol antibody. The particulate (P) and soluble (S) fractions from the cell sonicate were analyzed by Coomassie blue (1/1000 of each) and Western blot (1/5000 of particulate and 1/1000 of soluble fractions) . The starting soluble fraction (ST) , the unbound fraction (UN) and the eluate (E) of the immunoaffinity purification were analyzed by Western blot

(1/250 each) . The purified pol was examined by Coomassie blue staining as well. Figure 3. in vitro polymerase assay and inhibition of HBV pol reverse transcriptase with an anti-viral drug. Immunoaffinity purified pol was examined in an in vitro nucleotide binding, polymerase assay as described in Materials and Methods. Purified pol was incubated in the presence of ³²P dGTP and cold TTP, dATP and dCTP for 30 min at 30C under various conditions. The products of the polymerase assay were analyzed by SDS-PAGE and autoradiography. A. Coomassie blue staining profile of partially purified pol in the polymerase assays. The position of pol is indicated by an arrow. B. Autoradiogram of polymerase assays. CN = control, no change in the polymerase assays; Ap = aphidicolin, ActD = actinomycin D; PFA = phosphonoformic acid; EDTA = ethylene diamine tetraacetic acid; RNase = RNase pretreatment; DNase = DNase posttreatment.

Figure 4A and 4B. Alkaline agarose analysis of polymerase products. Polymerase reactions were conducted with purified FPL-pol polymerase using ³²P TTP and unlabeled dATP, dGTP and dCTP as described in Materials and Methods. The DNA products were purified with (+) or without (-) prior treatment with proteinase K. The products were analyzed by denaturing alkaline gel electrophoresis and autoradiography. M denatured, ³²P-labeled Hindlll λ DNA markers. Figure 4B. Analysis of HBV DNA associated with further purified polymerase. DNA associated with purified FPL-pol was extracted with phenol with (+) or without (-) prior treatment with proteinase K. The DNA products were analyzed by alkaline agarose electrophoresis, were transferred to a nylon membrane and were hybridized with riboprobes complementary to minus strand DNA (-DNA) or positive strand DNA (+DNA) . M = labeled HindiII 1 DNA markers.

Figure 5. Nucleotide priming with single nucleotide triphos- phates. Polymerase reactions were conducted with purified FPL-pol either as described in the legend to Fig.4 with ³P- labeled TTP and the other three dNTPs unlabeled (+dNTPs/T) or with only the single, labeled dNTP present (- dNTPs/T,G,A,C) . The products were analyzed by SDS-PAGE and autoradiography.

Figure 6. Riboprobe analysis of minus and plus strand DNA. Polymerase assays were conducted with purified FPL-pol using all four unlabeled dNTPs as described under Materials and

Methods. The DNA products were purified with (+) or without

(-) prior treatment with proteinase K and analyzed by alkaline agarose electrophoresis. The samples were transferred to a nylon membrane and hybridized with riboprobes complementary to minus strand DNA (-DNA) or positive strand DNA (+DNA) . M = Denatured labeled HindiII λ DNA markers.

Figure 7. Primer extension analysis of minus strand DNA. Polymerase assays were conducted with purified FPL-pol using all four unlabeled dNTPs. The products were treated with proteinase K and RNase A prior to purification, purified DNA was annealed with an 5' end labeled oligonucleotide spanning nucleotides 1786-1805 and the primer was extended using AMV reverse transcriptase as described under Materials and Methods. The primer extension products (PE) were analyzed on an 8% polyacrylamide sequencing gel adjacent to a sequencing ladder (A,G,T,C) generated by dideoxysequencing reactions with HBV plasmid DNA and the same primer as used for the primer extension products. The 10 nucleotide sequence of DRl is bracketed and labeled with the sequence.

Figure 8. Phosphoamino acid analysis of in vitro labeled pol. Polymerase reactions were conducted with ³²P TTP in the absence of other unlabeled nucleotides. The labeled polymerase products were separated by SDS-PAGE and electrophoretically transferred to a PVDF blotting membrane, and the labeled pol band was excised. Phosphoamino acid analysis was conducted as ' described under Materials and Methods. Partial acid hydrolysis was conducted at HOC for lhr, the products were analyzed by 2 dimensional electrophoresis on TLC plates followed by autoradiography. The position of phosphotyrosine, phosphoserine and phosphothreonine were determined by ninhydrin staining of unlabeled amino acid standards.

Figure 9. Analysis of DNA synthesized in in vitro polymerase reactions. Polymerase reactions were conducted with purified FPL-pol using ³²P TTP and unlabeled dATP, dGTP and dCTP either in the absence (-PFA) or presence (+PFA) of PFA.. The DNA products were purified with (+) or without (-) prior treatment with proteinase K. The products were analyzed by denaturing urea-acrylamide gel electrophoresis and autoradiography. M = denatured, ³²P-labeled fX174 Hinfl DNA markers.

Detailed Description of the Invention

This is the first report of the expression of active human HBV pol in insect cells using the recombinant baculovirus system, i.e. having the reverse transcriptase activity and fidelity characteristic of the HBV virus polymerase. Pol was rapidly purified by immunoaffinity chromatography by virtue of the FLAG^* (IBI, New Haven, CT) epitope engineered at the amino terminus. The purified pol was active in in vitro assays for nucleotide priming and reverse transcription.

Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction that involves the covalent linkage of the first nucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerases in heterologous systems. We have expressed hepatitis B virus (HBV) polymerase (pol) in insect cells using the recombinant baculovirus system. The pol mRNA contained 3' noncoding sequences that include DRl and the epsilon stem loop that are required for in vitro activity by duck hepatitis B virus pol. A ten amino acid sequence was fused to the amino terminus of pol such that pol could be readily purified from insect cell lysates using immu¬ noaffinity chromatography. The purified pol was active in a nucleotide-priming assay in which incubation of pol with labeled deoxynucleotide triphosphates resulted in the labeling of the pol polypeptide. The reaction was insensitive to aphidicolin and actinomycin D, while the elongation, but not the nucleotide-priming reaction, was sensitive to phosphonoformic acid. Analysis of the DNA products on alkaline agarose gels revealed a smear from less than 100 nucleotides to approximately 2300 nucleotides. Southern hybridization demonstrated that the pol mRNA was the template for reverse transcription, and that both minus and plus strand DNA were synthesized. Primer extension analysis mapped the 5' end of the minus strand DNA to DRl. Thus, this system is suitable for in vitro analysis of polymerase function and for the screening of potential compounds for the treatment of HBV infected individuals.

This system allows purification of HBV pol by a rapid and simple method. The method can be conveniently used to provide unlimited quantities of isolated active recombinant HBV pol.

The invention is directed to an isolated active recombinant human HBV pol complex. The complex may comprise a human HBV pol fusion protein or an epitope useful for purification. Such epitopes useful for purification include but are not limited to the FLAG^® epitope, beta-galactosidase, protein A, chloramphenicol acetyl transferase, dihydrofolate reductase, agarase, protein G, lectins, arginine(s), cysteine(s), or histidine(s) , see Nilsson, B. et al. in Advances in Gene Technology edited Brew, K. et al. 1988, IRL Press, pp.122, 123.

The invention is also directed to an isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex. The nucleic acid may encode a HBV pol fusion protein. The invention is also directed to a replicable vector comprising the nucleic acids above. The vector may be an insect viral vector, a baculovirus vector or a baculovirus transfer vector. The invention is also directed to host cells containing the proteins or nucleic acids above. The host cells may be a eukaryotic cell, a bacterial cell, an insect cell, a Spodoptera frugiperda cell, or an sf9 cell. Methods are also described. In one embodiment the invention is a method of expressing and isolating active human HBV pol comprising (i) culturing a host cell containing a suitable recombinant vector encoding the active human HBV pol under conditions such that the active human HBV pol is expressed, and (ii) recovering the active human HBV pol.

A method of screening a compound as an inhibitor or stimulator of a recombinant human HBV pol activity which comprises (i) measuring the recombinant human HBV pol activity in the absence of the compound under conditions suitable for pol activity, (ii) measuring the recombinant human HBV pol activity in the presence of the compound under conditions suitable for pol activity, and comparing the activity in the absence of the compound with the activity in the presence of the compound so as to thereby determine whether the compound is a stimulator or inhibitor of the recombinant human HBV pol activity.

The invention is also directed to a kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol which comprises an isolated active recombinant human HBV complex above and a suitable reaction mixture. Alternatively, the kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol which comprises an isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex and a suitable host vector system.

Materials and Methods

Cells. The Sf9 cell line derived from Spodoptera frugipβrida was cultivated in spinner culture as previously described

(26) . The cultivation medium was TNMFH supplemented with 5% fetal bovine serum and 0.1% pluronic F68 prior to infection and was changed to Grace's medium supplemented with 2% fetal bovine serum and 0.1% pluronic F68 after infections. The methods for growth, isolation and assay of recombinant baculoviruses were as previously described (48) .

Plasmids. Pursuant to the provisions of the Budapest Treaty on the International Recognition of Deposit of Microorganisms For Purpose of Patent Procedure, the plasmids listed below have been deposited with the American Type Culture Collection ("ATCC"), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. :

1. plasmid designated FP-Pol, deposited under ATCC Accession No. 69692; and FPL-Poϊ.

2. plasmid designated FPL-Pol, deposited under ATCC

Accession No. 69693.

Viruses. HBV sequences of the ayw subtype are numbered as designated by Galibert and coworkers (13) . The FLAG^*-Pol- Stem Loop (FPL-pol) construct was cloned by the following procedure (ATCC No. 69693, deposited September 23, 1994) . The amino terminus of the pol open reading frame was first cloned into the Hindlll site of the bacterial expression vector pFLAG^*-2 (International Biotechnologies Inc., New Haven, CT) using PCR to generate the Hindlll site adjacent to the pol AUG such that the pol open reading frame was in- frame with the FLAG^* epitope. This construct encodes a fusion protein having the sequence Met Asp Tyr Lys Asp Asp Asp Asp Lys Leu (SEQ ID NO. 1) preceding the polymerase Met at nucleotide 2309 (Figure 1) . The amino terminus of the fusion protein was retrieved from pFLAG^*-2 and joined to the remainder of pol to create a construct terminating at the SSP1 site at nucleotide 1639, 6 nucleotides downstream of the polymerase TAG termination codon. This construct was designated FP-pol for FLAG^*-Pol (ATCC No. 69693, deposited September 23, 1994) . FPL-pol was created from FP-pol by the addition of 248 nucleotides to the Bglll site at nucleotide

1987 which results in the addition of DRl and the stem loop structure present in the 3' untranslated region of the pregenomic RNA. FPL-pol has a mutation changing nucleotide

1524 from a G to a C, and thus changing pol amino acid number 800 from a Gly to an Arg. This mutation introduces a protein kinase A recognition site into the carboxy-terminus of pol as previously described (3) . Both pol constructs were cloned into the baculovirus transfer vector pBacPAK9

(CLONTECH, Palo Alto, CA) and recombinant viruses were isolated following the supplier's procedure.

Baculovirus expression vectors and methods for their use are well known (see for example O'Reilly et al. "Baculovirus Expression Vectors, A Laboratory Manual. 1992. W.H. Freeman and Co., New York. The BacPAK9 vector is commercially available from Clontech Inc., Palo Alto, CA and the Flag^® epitope system is commercially available from International Biotechnologies Inc., New Haven CT.

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis. Insect cell lysates and purified pol were disrupted in electrophoresis sample buffer containing 2% SDS and 2% 2-mercaptoethanol and were heated to 100°C for 5 min. Proteins were separated by SDS-PAGE as previously described (22,24) . Gels from in vitro assays for pol function were stained with Coomassie blue, dried and autoradiographed. For immunoblot analysis, proteins were electrophoretically transferred to Genescreen™Plus membranes (New England

Nuclear, Boston, MA) , and membranes were processed as previously described (25) using a 1/4000 dilution of a rabbit antisera to full length pol followed by ¹⁵I-protein A (NEN) . The rabbit antibody was produced against gel purified full length pol derived from the insoluble pellet of baculovirus-infected insect cells. The antisera recognized epitopes in both the amino and carboxy terminus of pol and had an immunoblot titer in excess of 1/32,000.

Immunoaffinity purification of pol. Spinner cultures (250 ml) were harvested 48 hr post infection with recombinant baculoviruses. Cells were pelleted at 180 g for 10 min and washed two times in TNG (100 mM Tris, pH7.5, 30 mM NaCl, 10% glycerol) . The cell pellet was resuspended in 10 ml of TNG containing protease inhibitors (100 μM leupeptin, 1 mM prefablock, 10 μM aprotinin, 10 μg/ml pepstatin and 1 mM EDTA) and was sonicated three times for 20 sec with a miroprobe sonicator at full power. The sonicated extract was clarified at 30,000 g for 30 min at 4°C, and the clarified extract was passed three times over an affinity column containing the M2 monoclonal antibody (International Biotechnologies Inc.) at a flow rate of 0.5 ml/min. The M2 monoclonal antibody recognizes the sequence Asp-Tyr Lys Asp Asp Asp Asp Lys (SEQ ID NO. 2) within the FLAG^* epitope. The column was washed sequentially with 10 ml of TNG, TNG with 1 M NaCl and TNG at 0.5 ml/min. Bound polymerase was eluted with 0.1 M glycine, pH 3.0, 10% glycerol, collected in 1 ml fractions and neutralized with 25 μl of 1 M Tris base.

Polymerase assays. Purified polymerase was either exchanged into TNM (100 mM Tris, pH 7.5, 30 mM NaCl, 10 mM MgCl₂) by repeated dilution and concentration in a Centricon-30 microconcentrator (Amicon, Beverly, MA) or in latter experiments polymerase remained in the 25 mM Tris, 0.1 M glycine, 10% glycerol buffer from purification and MgCl₂ was added to 10 M. Polymerase (50-100 μl) was adjusted to contain 100 μM unlabeled deoxynucleotide triphosphates (dATP, dGTP, dCTP) and 5 μCi of [α-³²P] TTP (NEN; 3000 Ci/mmol) and was incubated at 30°C for 30 min. For Southern hybridization and 5' primer extension analyses, the reactions were performed with all four unlabeled dNTPs at 100 μM for 2 hr at 30°C.

DNA purification, gel analysis and Southern hybridization. The products from in vitro polymerase reactions were digested with proteinase K (1 mg/ml) in TNES (10 mM Tris, pH 7.4, 10 mM NaCl, 10 mM EDTA, 1% SDS) for 2 hr at 65°C. DNA was extracted with phenol/chloroform and then chloroform and ethanol precipitated as previously described (8) . DNA from labeled reactions was analyzed on alkaline agarose gels which were run in 30 mM NaOH, 1 mM EDTA for 4 hr at 50 volts as described (38) . DNA from unlabeled reactions was purified as described above except the DNA was first treated with lmg/ml RNase A for 30 min at 37°C. For Southern hybri- dization, DNA was electrophoresed in 1% alkaline agarose gels, was transferred to a Genescreen Plus ™ membrane (NEN) by capillary transfer in 0.4 M NaOH, and was hybridized with HBV riboprobes of both strand polarities. Hybridization was conducted in 50% formamide, 7% SDS, 0.25 M sodium phosphate, 0.25 M NaCl, and 1 mM EDTA at 42°C as previously described

(8) . The riboprobes were made by in vitro transcription using [α-³²P] UTP (NEN, 3000 Ci/mmol) and T7 and T3 riboprobe kits (Promega, Madison, WI) . The vector for riboprobes contained an HBV insert from Xbal nucleotide 1993 to BamHI nucleotide 1403 in pBluescript (-) (Stratagene, La Jolla, CA) . Transcription with T3 and T7 yielded probes complementary with minus and plus strand DNA, respectively.

Primer extension. Primer extension analysis was essentially as described (40) . The primer was complementary to minus strand DNA and the 3' end was 20 nucleotides upstream of DRl and spanned 1786-1805; 5' -TAGGCATAAATTGGTCTGCG-3 ' (SEQ ID NO. 3) . The primer was 5' end-labeled with [γ-³²P] ATP (NEN, 6000 Ci/mmol) and T4 polynucleotide kinase. DNA was purified as described above and annealed to 200 pg of primer in 10 μl by heating to 95°C for 5 min and cooling on ice. Primer extension was conducted in a 15 μl reaction (50 mM Tris, pH 8.0, 40 mM KC1, 6 mM MgCl₂, 1 mM DTT, 100 μM dNTPs and eight units of AMV RT) for 1 hr at 42°C. The product was purified by extraction with phenol/chloroform and chloroform, ethanol precipitated and analyzed on a sequencing gel with a sequencing ladder generated with the same oligonucleotide.

Phosphoamino acid analysis. Polymerase reactions were conducted in the presence of a single nucleotide (³²P TTP) for lhr at 30C, the labeled pol band was isolated by SDS-PAGE and electrophoretic transfer to a PVDF membrane. The pol band was localized by autoradiography and excised from the membrane, and partial acid hydrolysis was conducted in situ on the membrane. Phosphoamino acid analysis was conducted as previously described (10) . The membrane was treated with 6N HCl in a nitrogen atmosphere for lhr at HOC. The products were lyophilized and resuspended in pH 1.9 electrophoresis buffer (88% formic acid: glacial acetic acid: H₂0 in a ratio of 50:156:1794) . The products were mixed with unlabeled phosphoamino acid standards (Sigma) and spotted on thin layer cellulose plates (Eastman Kodak Co., Rochester, NY) . First dimension electrophoresis was at 1500 V for 20min in pH 1.9 buffer. The second dimension electrophoresis was at 1300 V for 16min in pH 3.5 buffer (pyridine: glacial acetic acid: H₂0 in a ratio of 10:100:1890) . The unlabeled amino acid standards were localized with ninhydrin (0.25% in acetone) and heating to 60C for 15min, and labeled phosphoamino acids were localized by autoradiography.

Results

Expression of HBV polymerase in insect cells. In attempts to develop an in vitro system for the analysis of HBV polymerase function, we have expressed pol in insect cells using the recombinant baculovirus expression system. In our initial studies, full length pol as well as individual pol domains were expressed from different recombinant viruses.

Analysis of posttranslational processing of pol demonstrated that at least two distinct sites on pol were phosphorylated

(1) . No definitive polymerase function was demonstrated using these original constructs despite efforts to biochemically purify a functional pol as well as attempts to refold pol using purified cellular chaperones (unpublished data) .

In the current studies, attempts to express a functional polymerase involved two changes in the pol expression vector. First, the 3' terminus of the construct was extended beyond the pol open reading frame to include DRl and the epsilon stem-loop structure (Figure 1) shown to be important for function with in vitro translated DHBV pol (51) . Second, an affinity tag was added to the amino terminus to facilitate rapid purification. A gene segment encoding the FLAG^® epitope was spliced to the 5' end of the

HBV pol gene. The ten amino acid FLAG^* sequence (Met Asp Tyr Lys Asp Asp Asp Asp Lys Leu) (SEQ ID NO 1) was fused to the amino terminus of pol such that pol could be purified on M2 monoclonal antibody columns that recognize an epitope in this sequence. The construct containing the 3' extension was designated FPL-pol to denote FLAG^*-Pol-Stem Loop.

Sf-9 cells were infected with the FPL-pol virus and were harvested at 48 hr post infection by sonication in TNG buffer as described in Materials and Methods. The sonicate was divided into soluble and particulate fractions by centrifugation. Analysis of these fractions by SDS-PAGE and Coomassie blue staining indicated that pol was expressed at high levels in insect cells and that the majority of the pol was detected in the particulate fraction despite the fact that five fold more soluble extract was analyzed in comparison to the particulate fraction (Figure 2) . A prominent pol band was observed in the particulate fraction by Coomassie blue staining of the gel. Although a band was observed at the same position in the soluble fraction, analysis of these fractions by Western blot revealed that insufficient pol was present in the soluble fraction to account for the band detected by Coomassie blue staining. The band detected by Coomassie blue staining in the soluble fraction is likely an insect cell or baculovirus protein.

Pol was affinity purified from the soluble fraction derived from a 250 ml spinner culture of Sf9 cells using an affinity resin containing the M2 monoclonal antibody. Immunoblot analysis of equivalent amounts of the starting material, and the unbound and bound fractions indicated that approximately 50% of the pol failed to bind to the column. The purified material was analyzed by SDS-PAGE and Coomassie blue staining and a prominent pol band was detected at approximately 85,000 molecular weight (Figure 2). Several other prominent polypeptides were present in the purified pol preparations. A band of approximately 70,000 molecular weight failed to immunoblot with either the rabbit antisera to pol or the M2 monoclonal antibody, as did other minor bands of lower molecular weight. A prominent band with a molecular weight of approximately 115,000 failed to react with the antisera to pol, but did react with the M2 antibody suggesting that this band was a cellular protein that contained an epitope cross reactive with M2 (data not shown) . Thus, despite the fact that most pol synthesized in insect cells is insoluble, the high level of expression in insect cells coupled with immunoaffinity purification rendered purification of significant quantities of pol feasible. Nucleotide binding -and reverse transcriptase activity. A nucleotide binding assay described for analysis of DHBV pol was utilized to examine insect cell expressed HBV pol. Initially, this assay was employed to examine the soluble extracts prior to purification, but no distinctive labeling of a band at the molecular weight of pol was observed (data not shown) . In contrast, when the purified material was examined, a heavily labeled smear appeared just above the position of the Coomassie blue stained pol band (Figure 3) and the labeling continued to a lesser degree to the top of the gel as well as some labeling of material below the pol band. Presumably, the labeling of the pol band represents covalent linkage of a few nucleotides to pol in the nucleotide priming reaction, and the labeling of upper molecular weight material represents extension of this product by reverse transcription.

As would be expected for a hepadnavirus polymerase (46,51) , the reaction was not inhibited by aphidicolin, an inhibitor of cellular DNA polymerases, or actinomycin D, an inhibitor of DNA templated DNA synthesis. Inclusion of phosphonoformic acid in the reaction buffer resulted in the labeling of the pol band without labeling of higher molecular weight species indicating that PFA does not inhibit the nucleotide priming reaction but does inhibit elongation (30,51) . Inclusion of EDTA in the buffer blocked all labeling confirming that both polymerase activity (19) and the priming reaction (51) require Mg^2*. Treatment of purified pol with RNase A prior to the polymerase reaction abolished all activity indicating that the reaction was dependent on an RNA template. Treatment with DNase after the polymerase reaction removed the higher molecular weight material, but the labeled material at the position of pol was protected from DNase digestion (Figure 3) . This demonstrates the utility of purified FPL-pol for the screening of anti-viral drugs. The RT activity of FPL-pol could also be inhibited by dideoxyanalogues of all four deoxyribonucleotide triphosphates.

To examine whether the DNA products of the polymerase reac¬ tion were covalently bound to protein, polymerase reactions were extracted with phenol with or without prior treatment with protease to remove covalently bound protein (15) , and the DNA was examined on an alkaline agarose gel. In the absence of treatment with proteinase K, the labeled DNA products were diminished but not completely lost to the phenol phase in comparison to the sample treated with proteinase K prior to phenol extraction. Both samples yielded an intensely labeled smear on the denaturing DNA gel (Figure 4A) . The lack of treatment with proteinase K resulted primarily in the loss of the smallest of the DNA products. The size of the single stranded product was estimated by comparison to denatured Hindlll lambda DNA markers. The products ranged from less than 100 to approximately 1500 nucleotides. Longer exposure of the same gel revealed products of up to 2300 nucleotides which approaches the expected size for the full length cDNA of the pol mRNA if initiation occurs at DRl. Analysis of the same material on a urea-acrylamide gel demonstrated that much of the product was less than 24 nucleotides (data not shown) .

Detection of HBV minus strand DNA covalently linked to purified pol. Further purified preparations of pol were examined for the presence of HBV DNA to determine whether nucleotide priming and minus strand DNA synthesis had occurred in the baculovirus-infected insect cells. Purified pol was extracted with phenol with or without prior treatment with proteinase K to determine whether the minus strand DNA was covalently-linked to pol. The DNA products were analyzed by electrophoresis on denaturing, alkaline agarose gels and Southern hybridization using strand specific riboprobes. The results demonstrated that the pol mRNA had been reverse transcribed in the insect cells, since a DNA product was detected with a minus strand specific probe. No product was detected with a plus strand specific probe (Figure 4B) . The minus strand DNA ranged from approximately 100 to 500 nucleotides. The lack of proteinase K treatment resulted in the complete loss of the DNA products suggesting that the minus strand DNA was covalently linked to pol.

The appropriate synthesis of minus strand DNA would not only involve covalent linkage of the DNA to pol, but would also result in a product with 5' ends mapping to DRl. The 5' end of virion associated minus strand DNA for HBV maps to either the third or fourth nucleotide of DRl, a G residue at 1828 or a T residue at 1829 (37,55) . These residues are within the sequence TGAA, the complement of which is found on pregenomic RNA within DRl and in the bulge within the epsilon stem loop. In vitro reactions with DHBV pol have demonstrated that the priming reaction is initiated on the stem loop by the addition of four nucleotides to pol and this initiation complex is transferred to DRl for elongation of minus strand DNA (50,52). The first nucleotide of minus strand HBV DNA is either the third or fourth nucleotide of the DRl sequence, a G or T residue, respectively (37,55) . Recent studies with DHBV pol have demonstrated that the first nucleotide of minus strand DNA (the third nucleotide of DRl, a G residue) is templated by a nucleotide sequence in epsilon (50,52) and that the priming reaction has a high degree of specificity for a G residue unless the templated residue is changed by mutagenesis. To determine whether the in vitro priming reaction for HBV pol displayed specificity for a T residue or whether other residues could be covalently linked to pol, the pol reactions were conducted with a single labeled nucleotide in the absence of unlabeled nucleotides.

Incorporation of label in the TTP labeled reaction in the presence of unlabeled dATP, dCTP and dGTP was much greater than incorporation in the absence of unlabeled nucleotides

(Figure 5) . Although incorporation was the greatest with

TTP, other deoxynucleotides were capable of labeling pol when present as the only nucleotide. The order of preference based on labeling intensity was T> G> A> C (Figure 5) . This is the same order of appearance of nucleotides at the 5' end of minus strand DNA if minus strand initiates with the fourth nucleotide of DRl.

Detection of HBV minus and plus strand DNA. Southern hybri- dization experiments were conducted to demonstrate that the template for the polymerase reaction was the pol mRNA and to determine whether both minus and plus strand DNA were produced. The reaction was conducted with all four unlabeled dNTPs, the DNA was purified from the reaction with and without prior proteinase K treatment, and the products were analyzed by alkaline agarose gel electrophoresis and Southern hybridization with riboprobes specific for either minus or plus strand DNA. The results demonstrated that the template for reverse transcription was the pol mRNA since the product was detected with a minus strand specific probe, however a plus strand specific probe also hybridized with the polymerase reaction products (Figure 6) . These results suggest that at least some second strand synthesis was occurring via DNA-templated, DNA synthesis. The minus strand products ranged from 100 to 1500 nucleotides. Again the lack of proteinase K treatment primarily resulted in the loss of the smallest of the products. The plus strand products were similar in size to the minus strand products except that less smaller products were detected. Since the majority of minus strand DNA was much less than the size of the pol mRNA, it is unclear as to what triggered the switch to second strand synthesis.

Primer extension analysis. In order to determine whether reverse transcription was initiating at DRl of the RNA template, primer extension analysis was performed. The 5' end of virion associated minus strand DNA for HBV maps to either the third or fourth nucleotide of DRl, a G residue at 1828 or a T residue at 1829 (37,55). The 5' ends of both GSHV and WHV have been mapped to the T residue (39,41) . This T residue is within the sequence TGAA, the complement of which is found on pregenomic RNA within DRl and in the bulge within the epsilon stem loop. JEn vitro reactions with DHBV pol have demonstrated that the priming reaction is initiated on the stem loop by the addition of four nu¬ cleotides to pol and this initiation complex is transferred to DRl for elongation of minus strand DNA (50,52) . The 5' terminus of minus strand DNA from these in vitro reactions maps to both DRl and the 3' stem loop. Presumably, minus strand DNA mapping to the stem loop reflects a failure to translocate the initiated complex to DRl. To map the 5' end of minus strand DNA from the in vitro reactions with HBV pol, the polymerase reactions were conducted with all four unlabeled dNTPs, and the DNA products were purified by RNase digestion, proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. The DNA was annealed with a ³²P-labeled oligonucleotide complementary to a sequence 24 nucleotides from the 5' end of viral minus strand DNA, and the oligo was extended with AMV reverse transcriptase. Analysis of the primer extension products on a sequencing gel along with a sequencing ladder generated by sequencing cloned HBV DNA with the primer used in the extension analysis revealed that the most abundant product mapped to the A residue at 1827, while longer exposure of the gel revealed two additional bands and suggested that the 5' end mapped to the T residue at nucleotide 1829 (Figure 7) . The same 5' extension product was obtained from viral DNA purified from an infectious plasma (data not shown) .

Discussion

The expression of a functional human HBV polymerase with accurate and faithful reverse transcriptase activity has been especially problematic. Although pol is highly active in an endogenous polymerase assay using secreted mature virions or immature cytoplasmic cores, attempts to purify pol in an active soluble form have for the most part yielded unsatisfactory results. The inability of encapsidated pol to switch to an exogenously provided template was especially discouraging. Limited success has been reported for the refolding of denatured pol in activity gel assays and binding to an rAdT homopolymer in a northwestern blot assay was claimed, but an accurate faithful reverse transcriptase activity was neither demonstrated, nor isolated. These approaches have limited utility for the in vitro analysis of genome replication. The failure to express functional pol led to speculation that encapsidation or some accessory function of core may be required for pol function. Reports of two in vitro models for analysis of DHBV pol dispelled these hypotheses. In the first DHBV system, the in vitro translation of DHBV pol demonstrated activity in the absence of any other viral protein. The second utilized encapsidation in a yeast Tyl viral like particle. Although both systems negated an absolute requirement for core protein for function, an accessory role of core in completion of genomic replication is not unlikely. Despite the success with DHBV pol, an active form of HBV pol has not been described, nor has the expression and purification of DHBV pol.

Our initial studies with pol focused on the purification of pol expressed from a mRNA lacking 5' or 3' copies of DRl and epsilon. Although limited activity was observed with an occasional preparation, these studies were mostly negative. Additional efforts focused on the denaturation of pol and refolding with cellular chaperones in the presence and absence of synthetic pregenomic RNA (unpublished data) . In the current studies, we chose to include a 3' copy of DRl and epsilon on the pol mRNA due to the success of such constructs with DHBV pol. No activity was detected in cellular extracts of insect cells expressing these altered pol constructs. Inhibitors of pol activity may be present in cellular extracts as have been reported for adenovirus DNA polymerase (53) . The inclusion of an affinity tag at the amino terminus permitted the rapid purification of large quantities of pol active in nucleotide priming and reverse transcriptase assays. The lack of activity in cellular extracts and the high level of activity of purified pol suggest that in HBV-infected cells pol may not be functional prior to encapsidation due to specific inhibitors rather than activation by core following encapsidation.

Our initial studies with FPL-pol detected both minus and positive strand HBV DNA following in vitro pol reactions, and surprisingly, most of the minus strand DNA was not cova- lently linked to pol (unpublished observations) . Further analyses suggested that low levels of contaminating HBV DNA from the baculovirus vector was serving as the template in these reactions. Modification of the purification procedure reduced contaminating baculovirus DNA to undetectable levels and resulted in the detection of only minus strand DNA, all of which was covalently linked to pol. These data suggested that pol can utilize templates other than the pol mRNA. Recent studies with DHBV pol indicate that pol possesses polymerase activity in the absence of nucleotide priming (56) . This observation would in part explain the high level of activity we observed for pol using a baculovirus DNA template, as well as the lack of covalent linkage of pol to minus strand DNA in these reactions. Northern hybridization analyses of pol-associated RNA suggest that significant levels of pol mRNA copurify with pol, albeit in a highly degraded state (unpublished observation) . The copurification of pol mRNA with pol is consistent with the cis preference of pol in the packaging of pregenomic RNA (2,17-19,21,36) and may rely on the 3' copy of epsilon in the FPL-pol construct. Taken together the above data indicate that template may be limiting in the pol reactions, and in support of this assumption, we have observed that pol activity and the size of the DNA products are increased in the presence of exogenously supplied synthetic RNA templates (unpublished data) .

Our analyses suggest that a least two populations of pol are present in the purified preparations. One population consists of molecules possessing covalently linked minus strand DNA with the 5' end mapping to DRl. These molecules were presumably active in the insect cells but did not appear to be active following purification. The lack of in vitro activity for pol primed in vivo is assumed, since labeling with a single nucleotide did not label higher molecular weight material to detectable levels, which would be consistent with the addition of a nucleotide to the preexisting minus strand DNA. Presumably, a second pol population was active in vitro and underwent nucleotide priming in vitro based on the appearance of ³²P-labeled phosphotyrosine. A third population likely exists as well which was not active either in the insect cells or in vitro, since much of the pol band did not shift in mobility following in vitro polymerase reactions. At this time it is not clear why some but not all pol is active in vivo. The fraction of pol that is inactive following purification could be due to inactivation during purification. Preliminary experiments suggest that only a small fraction of pol is active in the in vitro priming reaction. When priming reactions were conducted in the presence of PFA to prevent elongation, less than one molecule of TTP was incorporated per 1000 molecules of pol; however, the concentration of labeled substrate was not sufficient for maximum activity in these studies. Whether high levels of PFA partially suppress the priming activity of pol is not known. Attempts are underway to increase the fraction of pol active in in vitro assays by modification of the purification scheme as well as by alteration of the conditions for the in vitro assay, including the addition of exogenous templates.

The in vitro polymerase reaction of HBV pol purified from insect cells differs in several properties from the DHBV in vitro pol systems previously described. DHBV pol appears to display an absolute specificity for the first nucleotide in the priming reaction, whereas HBV pol could be labeled with all four dNTPs. For DHBV pol, the priming reaction requires dGTP which is templated by the bulge in epsilon. When the C residue in the bulge is mutated to G, the priming reaction requires dCTP (52) . However, when the DHBV pol reaction is conducted in the presence of a single labeled nucleotide and the other three nucleotides are present as dideoxynucleotide triphosphates (ddNTP) , ddGTP reduced but did not completely block incorporation of other nucleotides, suggesting that DHBV pol could incorporate other nucleotides in the priming reaction at a reduced efficiency.

In vitro, HBV pol was capable of covalent linkage with each of the four deoxynucleotide triphosphates, although a preference for a T residue was apparent in reactions with a single nucleotide present. HBV pol was also capable of labeling with each of the four dNTPs when the other three nucleotides were present as ddNTPs (data not shown) . Mutations in the epsilon bulge as well as studies to ascertain the nucleotide actually linked to pol will be required to determine the specificity of the priming reaction. Incorporation of label into a band comigrating with the pol polypeptide was much greater when all four nucleotides were present in comparison to any single labeled nucleotide, suggesting that significant extension has occurred even when the migration of the pol band was not appreciably retarded. Alternatively, the presence of cold nucleotides enhances the priming reaction.

A second difference was observed in the 5' extension products from minus strand DNA. i vitro reactions from both DHBV and HBV map to the correct site for minus strand DNA within DRl, but a second product in the DHBV reactions maps to the epsilon bulge. No 5' extension product of minus strand DNA mapped to epsilon for the HBV in vitro reactions, perhaps indicating a more efficient in vitro strand transfer to DRl.

The third difference was the synthesis of minus strand DNA that was not covalently linked to pol. This might be explained by the presence of partially degraded templates. The fact that an RNA template has survived the purification is surprising in itself and may suggest that the RNA is partially protected by pol. Upon reaching the 5' end of a short template, pol may fall off and seek a second template. Since nucleotide priming has already occurred pol may be unconstrained and may initiate reverse transcription with priming due to a hairpin loop at the 3' end of the RNA or a template primed with short degraded oligonucleotides. Such a mechanism would also explain why the smaller DNA products were lost to the phenol phase in the absence of proteinase K treatment. A more detailed analysis of the various minus strand DNA products will be required to determine the nature and origin of minus strand DNA not bound to pol.

The fourth difference in the products from the in vitro polymerase reactions of HBV and DHBV was the synthesis of plus strand DNA by HBV pol. The switch to plus strand DNA synthesis could occur by several mechanisms. As in the above hypothesis, the template may be of variable lengths due to degradation of the pol mRNA during purification. Upon reaching the 5' end of the template, pol may either fall off or use the short undegraded oligoribonucleotide as a primer to begin synthesizing plus strand DNA. This would be similar to what is observed during in vivo replication of mutants in which the 5' copy of DRl has been altered, and thus the undegraded oligoribonucleotide at the 5' end of pregenomic RNA is no longer suitable as a primer at DR2, either due to the lack of sufficient homology for strand transfer or mismatch at the 3' end of the primer and DR2. The product of such mutants is predominantly linear DNA (12,29,45). Thus, for in vitro reactions, a degraded template would result in short linear DNA products. Even with an undegraded template, the lack of a 5' copy of DRl may result in the synthesis of a short plus strand DNA product. Studies with WHV have demonstrated that a purine-rich sequence of pregenomic RNA, 3' of DR2 on minus strand DNA, can serve as a primer for plus strand DNA (40) . Presumably, the purine- rich sequence is not degraded by RNase H and thus remains on minus strand DNA to serve as an alternate site for priming of plus strand DNA synthesis. A purine-rich sequence is located between nucleotides 1727 and 1754 of HBV and may serve as a primer of plus strand synthesis in the in vitro reactions. Inefficient RNase H activity during in vitro pol reactions could result in numerous primers being present for the priming of plus strand DNA synthesis. Detailed analysis of the plus strand DNA product will be required to determine which if any of these mechanisms is employed.

ADDITIONAL INFORMATION ON THE PROPERTIES OF PURIFIED POL

In addition to the utility of FPL-Pol, we have found that FP-Pol is also active in nucleotide priming and reverse transcription. The nature of this reaction at this time is not entirely clear, since this construct lacks the RNA sequences at the 3' end of the pol mRNA that are thought to be essential for templating the nucleotide priming reaction. It appears that FP-Pol binds to an RNA sequence that closely mimics the epsilon RNA stem loop that is at the 3 ' end of the FPL-Pol mRNA. This sequence that closely resembles epsilon may be on the FP-Pol mRNA or may be present on an insect cellular RNA or an mRNA encoded by baculovirus. Nonetheless, since FP-Pol also exhibits nucleotide priming and reverse transcriptase activity, it is suitable for screening antiviral inhibitors of HBV replication.

One of our other observations is that the addition of a synthetic HBV RNA to the purified pol greatly increases the activity of pol in the RT assay. The probable explanation is that during purification most of the template RNA is lost. It is assumed that pol carries its own mRNA through the purification and uses it as the template for reverse transcription. This is demonstrated in part by the fact that pretreatment of pol with RNase abolishes the RT activity. Thus, addition of more template increases the RT activity. This can be used in the drug screening assay to increase the strength of signal in the RT assay. Previous screens using HBV producer cell lines (See for example Furman et al. (1992) Antimicrobial Agents and Chemother. 36 (12) :2686-2692) have employed unpurified cultures for screening. The present invention allows cell free in vitro screening specifically for antiviral drug activities which act at the level of the HBV pol.

Another observation is the pol can also be expressed using other affinity tags for purification. A series of 6 histidines (His tag) allows proteins to be purified on metal columns such as a nickel column (Hochuili et al. (1987) J. Chromatog. 411:177-184; Hochuli (1991) In "Genetic Engineering: Principles and Methods" Ed. J.K. Setlow, Plenum Press, NY) Cloning, expression and purification kits with detailed protocols for this system are available from several suppliers (e.g. OTAGEN, Chatsworth,' CA) . Recently, we have expressed a functional pol in insect cells using this affinity purification method.

We have also used other eucaryotic expression systems to express FPL-Pol. We have made a series of vaccinia virus constructs that express pol at high levels in mammalian cells (HeLa) , but the levels are not as high as is seen in insect cells with baculovirus. The pol has not been tested for activity but is expected to be active.

It is also possible that with Flag^® affinity purification, bacterially expressed pol may be functional. The original commercial Flag^® vectors are bacterial vectors. We have cloned Pol in this vector. In order to make the baculovirus Flag^® vector we obtained the Flag^® sequence from this commercial vector.

To conclusively demonstrate that nucleotide priming occurs in vitro requires the detection of labeled phosphotyrosine on pol following the in vitro reaction. If the first nucleotide is added to pol in vitro, the ³² P-labeled phosphate in the alpha position of the labeled dNTP will become covalently attached to tyrosine. In vitro pol reactions were conducted in the presence of a single nucleotide (³²P TTP) , and the pol band was isolated by SDS-PAGE and transfer to a PVDF membrane. Phosphoamino acid analysis was conducted by 2-dimensional electrophoresis as described under Materials and Methods. A labeled spot was detected at the position of phosphotyrosine demonstrating that the purified pol was competent for in vitro nucleotide priming (Figure 8) . Consistent with the above data that the nucleotide priming reaction could occur with different nucleotides, phosphotyrosine was also detected when the reaction was conducted with ³P dGTP in the absence of other nucleotides (data not shown) .

Analysis of in vitro synthesized HBV DNA. To examine the size of the in vitro synthesized DNA products and to determine whether the DNA products were covalently bound to protein, polymerase reactions were extracted with phenol with or without prior treatment with proteinase K to remove covalently bound protein (15) , and the DNA products were examined by electrophoresis in urea acrylamide gels followed by autoradiography. In the absence of treatment with proteinase K, the labeled DNA products were almost completely lost to the phenol phase, while the sample treated with proteinase K prior to phenol extraction yielded an intensely labeled smear on the denaturing DNA gel (Figure 9) . The size of the single stranded DNA product was estimated by comparison to denatured fX174 Hinfl DNA markers. The products ranged from less than 25 to greater than 60 nucleotides. The small size of the in vitro synthe- sized DNA products were in part due to the limiting size of the RNA template (see Discussion) . No DNA products were detected from reactions conducted in the presence of PFA.

POTENT TARGETS FOR ANTI-HBV COMPOUNDS Anyone knowledgeable in the field of the invention would readily understand that the purified, functional HBV pol is extremely valuable for use in assays to screen for antiviral compounds to treat HBV infections. Several functions are expressed by the functional pol and assays can be designed to screen for inhibitors of each of these functions. First, pol must bind to specific HBV RNA sequences in order to initiate nucleotide priming and reverse transcription. An assay to measure this RNA binding or the inhibition of binding is useful to screen for inhibitors of priming or of binding of RNA for priming. Second, the first nucleotide of minus strand DNA becomes covalently linked to pol in a nucleotide priming reaction. An assay which measures this is useful in screening. Third, purified pol following the nucleotide priming event functions as a reverse transcriptase. This is one of the most common targets of antiviral screening. Phosphonoformic acid was capable of blocking this activity in the absence of inhibition of nucleotide priming. Finally, purified HBV pol exhibited DNA dependent DNA polymerase activity and assays to specifically measure this activity are useful for screening. Some compounds may be found which only inhibit this activity without inhibiting reverse transcriptase activity or vice versa.

POTENTIAL MASS SCREENING ASSAY FORMATS

The format of the assay would be 96 well plates and the assay would measure inhibition of deoxynucleotide incorporation in a reverse transcriptase reaction. One possible assay format would use unpurified cell lysates from insect cells infected with the recombinant baculovirus expressing FPL-pol and the pol would be captured and purified directly on the plate by coating the plates with the monoclonal antibody to the Flag sequence. Following incubation of the plates with unpurified cell lysates unbound proteins would be removed by washing. Assay plates with captured pol would be incubated with a potential pol inhibitor and indicator deoxynucleotide triphosphates. Following an incubation for the RT reaction, plates would be washed to remove unbound nucleotides and incorporation of the indicator nucleotide would be measured.

Alternatively, assay plates could be coated with pol previously purified from cell lysates by affinity chromatography. The remainder of the assay would be the same as above starting with the addition of potential pol inhibitors and indicator deoxynucleotide triphosphates.

Another alternative would be to use purified pol in solution in assay plates for the RT reactions and then to capture the RT product after the assay for measurement of incorporation of indicator deoxynucleotide triphosphates. This is possible since the RT product is covalently linked to the pol itself due to the nucleotide priming activity. This complex can be captured on nitrocellulose filters, while unbound nucleotides are washed through the filter. Assay plates are available with nitrocellulose filters as the bottom of the well for use in such an assay.

The indicator nucleotide could be a fluorescent nucleotide that can be directly measured by a fluorometer. The nucleotide could be radioactive (³²P or ³⁵S) such that it could be directly measured in a device that measures radioactivity, i.e. a gamma counter or scintillation counter. In perhaps the most sensitive assay, the nucleotide could be a biotinylated nucleotide that could then be detected by the binding of streptavidin alkaline phosphatase. Unbound streptavidin alkaline phosphatase would be removed by washing, and measured by the addition of a substrate for alkaline phosphatase. Such substrates could either undergo a colorimetric change upon cleavage and be measured by a conventional ELISA plate reader or substrates could emit light in a chemiluminescent reaction upon cleavage by alkaline phosphatase and be measured by photometer ELISA plate reader.

To one knowledgeable in the field it will be immediately apparent that many other potential assay designs are possible.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Lanford, Robert E. Novtall, Lena M. Beames, Burton D.

(ii) TITLE OF INVENTION: ISOLATED HUMAN HEPATITIS B VIRUS

POLYMERASE AND USES THEREOF

(iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Cooper & Dunham LLP

(B) STREET: 1185 Avenue of the Americas

(C) CITY: New York (D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10036

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 29-SEP-1995

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/315,856

(B) FILING DATE: 30-SEP-1994

(C) CLASSIFICATION: 435 (viii) ATTORNE /AGENT INFORMATION:

(A) NAME: White, John P.

(B) REGISTRATION NUMBER: 28,678

(C) REFERENCE/DOCKET NUMBER: 46406-A-PCT (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 212 278 0400

(B) TELEFAX: 212 391 0526

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Asp Tyr Lys Asp Asp Asp Asp Lys Leu 1 5 10 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

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

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Asp Tyr Lys Asp Asp Asp Asp Lys

1 5

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 : TAGGCATAAA TTGGTCTGCG 20

Claims

CLAIMS :

1. An isolated active recombinant human HBV pol complex.

2. The isolated active recombinant human HBV pol complex of claim 1 comprising a human HBV pol fusion protein.

3. The isolated active human HBV pol fusion protein of claim 2 comprising an epitope useful for purification.

4. An isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex.

5. The nucleic acid of claim 4, wherein the protein component is a human HBV pol fusion protein.

6. A replicable vector comprising the nucleic acid of claim 4.

7. A replicable vector comprising the nucleic acid of claim 5.

8. A host cell containing the vector of claim 6.

9. A host cell containing the vector of claim 7.

10. A method of expressing and isolating active human HBV pol comprising (i) culturing a host cell containing a suitable recombinant vector encoding the active human

HBV pol under conditions such that the active human HBV pol is expressed, and (ii) recovering the active human HBV pol.

11. A method according to claim 10 in which the host cell is a eukaryotic cell .

12. A method according to claim 10 in which the host cell is a bacterial cell .

13. A method according to claim 11 in which the eukaryotic cell is an insect cell.

14. A method according to claim 13 in which the insect cell is a Spodoptera frugiperda cell.

15. A method according to claim 14 in which the Spodoptera frugiperda cell is an sf9 cell.

16. A method according to claim 15 in which the recombinant vector is an insect viral vector.

17. A method of claim 16 in which the insect viral vector is a baculovirus vector.

18. A method of claim 17 in which the recombinant vector is a baculovirus transfer vector.

19. A method of screening a compound as an inhibitor or stimulator of a recombinant human HBV pol activity which comprises : (i) measuring the recombinant human HBV pol activity in the absence of the compound under conditions suitable for pol activity, (ii) measuring the recombinant human HBV pol activity in the presence of the compound under conditions suitable for pol activity, and comparing the activity in the absence of the compound with the activity in the presence of the compound so as to thereby determine whether the compound as a stimulator or inhibitor of the recombinant human HBV pol activity.

20. A kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol which comprises an isolated active recombinant human HBV complex of claim 1 and a suitable reaction mixture.

21. A kit useful for screening a compound for activity as an inhibitor or stimulator of a human HBV pol which comprises an isolated nucleic acid encoding a protein component of an active recombinant human HBV pol complex of claim 4 and a suitable host vector system.