RECOMBINANT HUMAN HEPATITIS-B VIRUS REVERSE-TRANSCRIPTASE IS ACTIVE IN THE ABSENCE OF THE NUCLEOCAPSID OR THE VIRAL REPLICATION ORIGIN, DR1

The double-stranded DNA genome of hepatitis B virus (HBV) is reverse t ranscribed from the viral pregenome RNA template by a virally encoded reverse transcriptase enzyme (RT) that possesses both priming and elon gation activities. Prior efforts have failed to express an active form of HBV RT outside the nucleocapsid in animal cells or to release it f rom viral nucleocapsids, thus restricting the characterization of this important enzyme. Here, we have engineered epitope-tagged HBV RT prot eins and expressed them in Xenopus oocytes via a synthetic RT mRNA whi ch does not include the viral capsid protein or the known initiation s ite for viral DNA synthesis, DR1. We demonstrate the production of an immunoprecipitable 96-kDa HBV RT protein and show, using a simple in v itro RT assay, that oocyte lysates containing this protein possess an activity that (i) catalyzes an RNA-dependent deoxynucleotide triphosph ate polymerization reaction by using an as-vet-unidentified RNA templa te and (ii) is sensitive to the RT inhibitors actinomycin D and phosph onoformate. Experiments with the chain terminator ddATP suggest that a significant amount of chain elongation occurs in our in vitro reactio n. Electrophoretic analysis reveals a heterogeneous array of RT reacti on products with sizes ranging from about 100 bases to far larger than that of the input RT mRNA. These products appear to contain covalentl y bound protein, consistent with the notion that the RT protein may ha ve primed their synthesis. We conclude that HBV RT activity can be unc oupled from both the nucleocapsid and the replication origin, DRI. Our results raise the possibility that unless HBV employs novel mechanism s to regulate its constitutively active RT, cellular RNAs may be rever se transcribed during HBV infection, with potential implications for t he development of HBV-related liver cancer. The use of the oocyte syst em should facilitate studies of HBV RT, including the development of H BV RT inhibitors for antiviral therapy.