RNase H activity of human hepatitis B virus polymerase expressed in Escherichia coli.
Human Hepatitis B Virus (HBV) replication is accomplished by its own polymerase. The HBV RNase H domain of HBV polymerase has been expressed in Escherichia coli and purified by affinity column chromatography. The MBP-RNase H fusion protein (43 kDa MBP plus 17 kDa HBV RNase H domain) was proved to be RNase H by in vitro activity assay, inhibitor studies, 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 between 7.7 and 8.2. The MBP-RNase H fusion protein required 40 mM monovalent cation for its enzyme activity, whereas it showed lower activity at a salt concentration of more than 100 mM. Ribonucleoside Vanadyl complex (RAV) and 2'-deoxyadenosine 5'-monophosphate (dAMP) inhibited the 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 viral HBV polymerase enzymatic activities.
Lee
Y I
YI
Department of Biology Education, Seoul National University, Korea.
Hong
Y B
YB
Kim
Y
Y
Rho
H M
HM
Jung
G
G
D015153:Blotting, Western; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli; D006515:Hepatitis B virus / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012263:Ribonucleosides / Q000378:metabolism; D014638:Vanadates / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology
D003838:Deoxyadenine Nucleotides; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D012263:Ribonucleosides; C057724:vanadyl ribonucleoside complex; C028191:2'-deoxy-3'-adenosine monophosphate; D014638:Vanadates; D016914:Ribonuclease H