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

Abstract

Genome replication of hepadnavirus proceeds by reverse transcription from a viral pregenomic RNA template by a virally encoded polymerase that possesses protein-priming, reverse transcriptase, DNA polymerase, and RNase H activities. Characterization of this enzyme has been hampered by failure to purify an active enzyme from virions and difficulties in expressing an active polymerase in heterologous systems. In this study, we constructed human hepatitis B virus polymerase cDNA under the control of a phage T7 promoter and expressed it in a rabbit reticulocyte lysate-coupled transcription-translation system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produced three products: a full-length protein (~94 kDa), an internally initiated protein (~81 kDa), and an N-terminal protein (~40 kDa). The in vitro expressed polymerase possessed protein priming activity, as demonstrated by32P-dGTP-labeling of the 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 radionucleotides into acid-precipitable polynucleotides and by synthesis of human hepatitis 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. Both the protein-priming and the reverse transcription activities of this recombinant polymerase are dependent on the RNA fragment containing the HBV DR1/epsilon stem-loop sequence known to be required for the polymerase activities. The in vitro systems used in this study will be applicable to further functional and biochemical studies of this enzyme.Key words: hepadnavirus, HBV polymerase, protein priming, reverse transcription, rabbit reticulocyte lysate.