The Catalytic Properties of Human Hepatitis B Virus Polymerase

Abstract

The DNA-dependent DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities of hepadnavirus polymerases are both essential for viral replication. Human hepatitis B virus (HBV) polymerase has been successfully expressed inEscherichia colias a fusion protein in frame with maltose-binding protein. The present study was undertaken to characterize these two activities and introduce anin vitroassay system.In situactivity gel assays show that the polymerase has both types of activities. One hundred thirty-four kilodaltons of active full-length product was proteolytically cleaved into ∼73 kDa of active fragment by proteinase K preincubation. Mutation of conserved YMDD motif also confirms that the activities were due to the recombinant polymerase and that this motif is essential to polymerase activity. Two activities of the polymerase show their optima under conditions of 1 mM (DDDP) or 0.25 mM (RDDP) of MnCl2, 400 mM KCl, 37°C (DDDP) or 24°C (RDDP), and pH 7.0–7.7. Substitution of Mg2+for Mn2+results in reduction of processivity, which may explain why Mn2+supports nucleotide incorporation to a higher level than Mg2+. The polymerase is resistant to aphidicolin. Actinomycin D acts selectively on DDDP activity, whereas phosphonoformic acid inhibits both activities. Thein vitroHBV polymerase assay system demonstrated herein will be useful for screening potential HBV polymerase inhibitors for the development of anti-HBV drugs.