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WO1996010649A1 - Polymerase isolee du virus de l'hepatite b chez l'homme et ses utilisations - Google Patents

Polymerase isolee du virus de l'hepatite b chez l'homme et ses utilisations Download PDF

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Publication number
WO1996010649A1
WO1996010649A1 PCT/US1995/012960 US9512960W WO9610649A1 WO 1996010649 A1 WO1996010649 A1 WO 1996010649A1 US 9512960 W US9512960 W US 9512960W WO 9610649 A1 WO9610649 A1 WO 9610649A1
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pol
human hbv
activity
hbv pol
dna
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PCT/US1995/012960
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English (en)
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Robert E. Lanford
Lena M. Novtall
Burton D. Beames
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Southwest Foundation For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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) .
  • 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) .
  • 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) .
  • '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) .
  • 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) .
  • 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
  • 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.
  • 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.
  • the invention is a method of expressing and isolating active human HBV pol.
  • 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.
  • FIG. 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
  • FIG. 1 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.
  • CB Coo assie blue
  • WB Western blot
  • 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
  • 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.
  • FIG 4A and 4B Alkaline agarose analysis of polymerase products. Polymerase reactions were conducted with purified FPL-pol polymerase using 32 P 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, 32 P-labeled Hindlll ⁇ 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.
  • FIG. 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 3 P- 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 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.
  • PE primer extension products
  • FIG. 8 Phosphoamino acid analysis of in vitro labeled pol. Polymerase reactions were conducted with 32 P 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.
  • 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.
  • HBV hepatitis B virus
  • pol hepatitis B virus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Sf9 cell line derived from Spodoptera frugip ⁇ rida was cultivated in spinner culture as previously described
  • 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. :
  • plasmid designated FP-Pol deposited under ATCC Accession No. 69692; and FPL-Po ⁇ .
  • 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
  • FPL-pol has a mutation changing nucleotide
  • 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.
  • 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 ⁇ l 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 MgCl 2 ) 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 MgCl 2 was added to 10 M.
  • TNM 100 mM Tris, pH 7.5, 30 mM NaCl, 10 mM MgCl 2
  • Polymerase 50-100 ⁇ l was adjusted to contain 100 ⁇ M unlabeled deoxynucleotide triphosphates (dATP, dGTP, dCTP) and 5 ⁇ Ci of [ ⁇ - 32 P] TTP (NEN; 3000 Ci/mmol) and was incubated at 30°C for 30 min.
  • dATP deoxynucleotide triphosphate
  • dGTP dGTP
  • dCTP 5 ⁇ Ci of [ ⁇ - 32 P] TTP
  • NPN 3000 Ci/mmol
  • 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.
  • DNA was electrophoresed in 1% alkaline agarose gels, was transferred to a Genescreen Plus TM 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
  • the riboprobes were made by in vitro transcription using [ ⁇ - 32 P] 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 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 [ ⁇ - 32 P] ATP (NEN, 6000 Ci/mmol) and T4 polynucleotide kinase.
  • DNA was purified as described above and annealed to 200 pg of primer in 10 ⁇ l by heating to 95°C for 5 min and cooling on ice.
  • Primer extension was conducted in a 15 ⁇ l reaction (50 mM Tris, pH 8.0, 40 mM KC1, 6 mM MgCl 2 , 1 mM DTT, 100 ⁇ M 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 ( 32 P 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: H 2 0 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: H 2 0 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.
  • HBV polymerase in insect cells.
  • pol in insect cells using the recombinant baculovirus expression system.
  • full length pol as well as individual pol domains were expressed from different recombinant viruses.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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) .
  • HBV minus strand DNA covalently linked to purified pol 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.
  • 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.
  • 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) .
  • DHBV pol 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.
  • 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.
  • 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.
  • 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.
  • 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 32 P-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.
  • 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.
  • 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.
  • the priming reaction requires dGTP which is templated by the bulge in epsilon.
  • the priming reaction requires dCTP (52) .
  • ddNTP dideoxynucleotide triphosphates
  • 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.
  • 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.
  • 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.
  • a purine-rich sequence of pregenomic RNA, 3' of DR2 on minus strand DNA can serve as a primer for plus strand DNA (40) .
  • 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.
  • FP-Pol is also active in nucleotide priming and reverse transcription.
  • 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.
  • 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.
  • pol can also be expressed using other affinity tags for purification.
  • His tag His tag
  • metal columns such as a nickel column
  • Cloning, expression and purification kits with detailed protocols for this system are available from several suppliers (e.g. OTAGEN, Chatsworth,' CA) .
  • OTAGEN e.g. OTAGEN, Chatsworth,' CA
  • 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.
  • 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 ( 32 P 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.
  • 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.
  • 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.
  • 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.
  • the indicator nucleotide could be a fluorescent nucleotide that can be directly measured by a fluorometer.
  • the nucleotide could be radioactive ( 32 P or 35 S) such that it could be directly measured in a device that measures radioactivity, i.e. a gamma counter or scintillation counter.
  • 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.
  • Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell 21:801-809.
  • RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation. J. Virol. 67:3254-3263.
  • Hepadnavirus P protein utilizes a tyrosine residue in the TP domain to prime reverse transcription. J. Virol. 68:2994-2999.
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention concerne un complexe isolé, actif et recombinant de la transcriptase inverse, désignée pol, du virus de l'hépatite B (HBV) chez l'homme. Ce complexe peut comprendre une protéine de fusion de pol de HBV ou un déterminant antigénique utile à la purification. L'invention concerne également un acide nucléique isolé codant un constituant protéique d'un complexe actif recombinant de pol de HBV. L'acide nucléique peut coder une protéine de fusion de pol de HBV. L'invention concerne également un vecteur réplicable comprenant lesdits acides nucléiques. Elle concerne également des cellules hôtes contenant lesdites protéines ou lesdits acides nucléiques. Des procédés sont également décrits. Dans un mode de réalisation, l'invention concerne un procédé d'expression et d'isolation de pol actif de HBV chez l'homme. Dans un autre mode de réalisation, l'invention concerne un procédé de criblage d'un composé, afin de déterminer s'il inhibe ou stimule l'activité recombinante de pol de HBV chez l'homme. L'invention concerne enfin un kit permettant de cribler un composé, afin de déterminer s'il inhibe ou stimule l'activité de pol de HBV.
PCT/US1995/012960 1994-09-30 1995-09-29 Polymerase isolee du virus de l'hepatite b chez l'homme et ses utilisations WO1996010649A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007869A1 (fr) * 1996-08-16 1998-02-26 Dong Wha Pharm. Ind. Co., Ltd. Polymerase du virus de l'hepatite b, enzyme rnase h derive de la polymerase du virus de l'hepatite b, procedes de preparation et utilisation dans le criblage d'agents antiviraux
WO2000018968A1 (fr) * 1998-09-29 2000-04-06 Government Of The Republic Of Singapore Essai in vitro de dosage de l'activite de l'adn-polymerase du virus de l'hepatite b (vhb) humain et son utilisation pour cribler des inhibiteurs de l'adn-polymerase du vhb
WO2001057244A1 (fr) * 2000-02-03 2001-08-09 Melbourne Health Test de detection de virus d'hepatite b variants montrant une reponse deficiente aux agents
KR20030075638A (ko) * 2002-03-20 2003-09-26 (주) 비엔씨바이오팜 Ｂ형 간염바이러스의 프라이밍 저해제의 스크리닝 방법
US7037682B2 (en) 1999-03-13 2006-05-02 Government Of The Republic Of Singapore In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening for inhibitors of HBV DNA polymerase

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4851341A (en) * 1986-12-19 1989-07-25 Immunex Corporation Immunoaffinity purification system
US4937074A (en) * 1988-03-29 1990-06-26 Fox Chase Cancer Center Method of treating retrovirus infection
US5194376A (en) * 1989-02-28 1993-03-16 University Of Ottawa Baculovirus expression system capable of producing foreign gene proteins at high levels
US5360714A (en) * 1992-08-28 1994-11-01 Fox Chase Cancer Center Hepadnavirus polymerase gene product having RNA-dependent DNA priming and reverse transcriptase activities and methods of measuring the activities thereof
US5432165A (en) * 1992-04-06 1995-07-11 Oclassen Pharmaceuticals, Inc. Methods for the treatment of infection caused by Hepatitis B virus (HBV)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4851341A (en) * 1986-12-19 1989-07-25 Immunex Corporation Immunoaffinity purification system
US4937074A (en) * 1988-03-29 1990-06-26 Fox Chase Cancer Center Method of treating retrovirus infection
US5194376A (en) * 1989-02-28 1993-03-16 University Of Ottawa Baculovirus expression system capable of producing foreign gene proteins at high levels
US5432165A (en) * 1992-04-06 1995-07-11 Oclassen Pharmaceuticals, Inc. Methods for the treatment of infection caused by Hepatitis B virus (HBV)
US5360714A (en) * 1992-08-28 1994-11-01 Fox Chase Cancer Center Hepadnavirus polymerase gene product having RNA-dependent DNA priming and reverse transcriptase activities and methods of measuring the activities thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007869A1 (fr) * 1996-08-16 1998-02-26 Dong Wha Pharm. Ind. Co., Ltd. Polymerase du virus de l'hepatite b, enzyme rnase h derive de la polymerase du virus de l'hepatite b, procedes de preparation et utilisation dans le criblage d'agents antiviraux
AU721695B2 (en) * 1996-08-16 2000-07-13 Dong Wha Pharmaceutical Industrial Co., Ltd. HBV polymerase, RNase H enzyme derived from HBV polymerase, processes for preparation and uses for screening antiviral agents thereof
WO2000018968A1 (fr) * 1998-09-29 2000-04-06 Government Of The Republic Of Singapore Essai in vitro de dosage de l'activite de l'adn-polymerase du virus de l'hepatite b (vhb) humain et son utilisation pour cribler des inhibiteurs de l'adn-polymerase du vhb
US6593082B1 (en) 1998-09-29 2003-07-15 Government Of The Republic Of Singapore In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening for inhibitors of HBV DNA polymerase
US7037682B2 (en) 1999-03-13 2006-05-02 Government Of The Republic Of Singapore In vitro activity assay for human hepatitis B virus (HBV) DNA polymerase, and its use for screening for inhibitors of HBV DNA polymerase
WO2001057244A1 (fr) * 2000-02-03 2001-08-09 Melbourne Health Test de detection de virus d'hepatite b variants montrant une reponse deficiente aux agents
KR20030075638A (ko) * 2002-03-20 2003-09-26 (주) 비엔씨바이오팜 Ｂ형 간염바이러스의 프라이밍 저해제의 스크리닝 방법

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