RNASE-H ACTIVITY OF HUMAN HEPATITIS-B VIRUS POLYMERASE EXPRESSED IN ESCHERICHIA-COLI

Human Hepatitis B Virus (HBV) replication is accomplished by its own p olymerase. The HBV RNase H domain of HBV polymerase has been expressed in Escherichia coil and purified by affinity column chromatography. T he MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H doma in) was proved to be RNase H by in vitro activity assay, inhibitor stu dies, and mutagenesis, The HBV RNase H domain represented the optimal RNase H activity in the presence of either 8 mM MgCl2 or 16 mM MnCl2. In Tris-Cl buffer, the optimum pH for MBP-RNase H fusion protein is be tween 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monov alent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM, Ribonucleoside Vanadyl c omplex (RAV) and S'-deoxyadenosine B'-monophosphate (dAMP) inhibited t he RNase H activity. Moreover, the mutation of highly conserved amino acids in the HBV RNase H domain diminished the RNase H activity. These results clearly suggest that the RNase H activity is separable from v iral HBV polymerase enzymatic activities. (C) 1997 Academic Press.