title: Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid or the viral replication origin, DR1.
abstract: The double-stranded DNA genome of hepatitis B virus (HBV) is reverse transcribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elongation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it from viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT proteins and expressed them in Xenopus oocytes via a synthetic RT mRNA which does not include the viral capsid protein or the known initiation site for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in vitro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosphate polymerization reaction by using an as-yet-unidentified RNA template and (ii) is sensitive to the RT inhibitors actinomycin D and phosphonoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reaction. Electrophoretic analysis reveals a heterogeneous array of RT reaction products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalently bound protein, consistent with the notion that the RT protein may have primed their synthesis. We conclude that HBV RT activity can be uncoupled from both the nucleocapsid and the replication origin, DR1. Our results raise the possibility that unless HBV employs novel mechanisms to regulate its constitutively active RT, cellular RNAs may be reverse transcribed during HBV infection, with potential implications for the development of HBV-related liver cancer. The use of the oocyte system should facilitate studies of HBV RT, including the development of HBV RT inhibitors for antiviral therapy.
authors.affiliation: Hormone Research Institute, University of California, San Francisco, California 94143-0534.
authors.forename: M
authors.initials: M
authors.lastname: Seifer
authors.forename: D N
authors.initials: DN
authors.lastname: Standring
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis; D005260:Female; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication; D014981:Xenopus
chemical_list: D004279:DNA, Viral; D000939:Epitopes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase