Expression of an enzymatically active polymerase of human hepatitis B virus in a coupled transcription-translation system

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possess es protein-priming, reverse transcriptase, DNA polymerase, and RNase H acti vities. Characterization of this enzyme has been hampered by failure to pur ify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hep atitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-transla tion system. In vitro site-directed mutagenesis confirmed that the recombin ant polymerase cDNA produced three products: a full-length protein (similar to 94 kDa), an internally initiated protein (similar to 81 kDa), and an N- terminal protein (similar to 40 kDa). The in vitro expressed polymerase pos sessed protein priming activity, as demonstrated by P-32-dGTP-labeling of t he full size polymerase and the N-terminal portion of the molecule in an in vitro priming assay. The polymerase also exhibited polymerization activity , as detected in an in vitro polymerase assay by incorporation of radionucl eotides into acid-precipitable polynucleotides and by synthesis of human he patitis B virus (HBV) specific DNA with product lengths between 100 and 500 nucleotides. In addition, the polymerase was found to use an RNA sequence bearing HBV DR1/epsilon stem-loop motif as a template for DNA synthesis. Bo th the protein-priming and the reverse transcription activities of this rec ombinant polymerase are dependent on the RNA fragment containing the HBV DR 1/epsilon stem-loop sequence known to be required for the polymerase activi ties. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.