title: Residues Arg703, Asp777, and Arg781 of the RNase H domain of hepatitis B virus polymerase are critical for viral DNA synthesis.
abstract: Hepatitis B virus (HBV) synthesizes its DNA genome through reverse transcription, which is catalyzed by viral polymerase (Pol). Previous studies suggested that the RNase H domain of hepadnaviral Pol may contribute to multiple steps of the viral genome replication, such as RNA encapsidation and viral DNA synthesis. However, specific residues of the RNase H domain that contribute to viral reverse transcription have not been determined. Therefore, we employed charged-to-alanine scanning mutagenesis to generate a set of single-substitution mutants of the RNase H domain and then analyzed their ability to support viral reverse transcription. Southern blot analysis showed that three mutants (R703A, D777A, and R781A mutants) yielded significantly reduced amounts of viral DNAs. However, none of these mutants were defective in RNA encapsidation. The data indicated that in the R703A and D777A mutants, minus-strand DNA synthesis was incomplete due to loss of catalytic activity of RNase H. In contrast, in the R781A mutant, the minus-strand DNA synthesis was near complete to some extent, while the plus-strand DNA synthesis (i.e., relaxed circular DNA) was severely impaired due to the defect in RNase H activity. Overall, our analysis revealed that three charged residues of the HBV Pol RNase H domain contribute to the catalysis of RNase H in removing the RNA template, but not in the RNA encapsidation.
authors.affiliation: Department of Biochemistry, Yonsei University, Seoul, Republic of Korea.
authors.forename: Chunkyu
authors.initials: C
authors.lastname: Ko
authors.forename: Youn-Chul
authors.initials: YC
authors.lastname: Shin
authors.forename: Woo-Jin
authors.initials: WJ
authors.lastname: Park
authors.forename: Seungtaek
authors.initials: S
authors.lastname: Kim
authors.forename: Jonghwa
authors.initials: J
authors.lastname: Kim
authors.forename: Wang-Shick
authors.initials: WS
authors.lastname: Ryu
mesh_terms: D001120:Arginine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis*; D056945:Hep G2 Cells; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D009154:Mutation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D001224:Aspartic Acid; D001120:Arginine; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H