Hepatitis B virus (HBV) synthesizes itsDNAgenome through reverse transcription, which is catalyzed by viral polymerase (Pol). Previous studies suggested that the RNaseHdomain of hepadnaviral Pol may contribute to multiple steps of the viral genome replication, such asRNAencapsidation and viralDNAsynthesis. However, specific residues of the RNaseHdomain that contribute to viral reverse transcription have not been determined. Therefore, we employed charged-to-alanine scanning mutagenesis to generate a set of singlesubstitution mutants of the RNaseHdomain 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 inRNAencapsidation. The data indicated that in the R703A and D777A mutants, minus-strand DNAsynthesis was incomplete due to loss of catalytic activity of RNase H. In contrast, in the R781A mutant, the minus-strandDNA synthesis was near complete to some extent, while the plus-strandDNAsynthesis (i.e., relaxed circular DNA) was severely impaired due to the defect in RNaseHactivity. Overall, our analysis revealed that three charged residues of theHBVPol RNaseHdomain contribute to the catalysis of RNaseHin removing theRNAtemplate, but not in theRNAencapsidation.
All Science Journal Classification (ASJC) codes
- Insect Science