Residues Arg703, Asp777, and Arg781 of the rnase H domain of hepatitis B virus polymerase are critical for viral DNA synthesis

Chunkyu Ko, Youn Chul Shin, Woo Jin Park, Seungtaek Kim, Jonghwa Kim, Wang-Shick Ryu

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)154-163
Number of pages10
JournalJournal of Virology
Volume88
Issue number1
DOIs
Publication statusPublished - 2014 Jan 1

Fingerprint

Ribonuclease H
Hepatitis B virus
Viral DNA
Reverse Transcription
mutants
synthesis
reverse transcription
DNA
Circular DNA
Viral Genome
Southern Blotting
Catalysis
catalytic activity
Mutagenesis
Alanine
circular DNA
mutagenesis
alanine
Southern blotting
genome

All Science Journal Classification (ASJC) codes

  • Immunology
  • Virology

Cite this

Ko, Chunkyu ; Shin, Youn Chul ; Park, Woo Jin ; Kim, Seungtaek ; Kim, Jonghwa ; Ryu, Wang-Shick. / Residues Arg703, Asp777, and Arg781 of the rnase H domain of hepatitis B virus polymerase are critical for viral DNA synthesis. In: Journal of Virology. 2014 ; Vol. 88, No. 1. pp. 154-163.
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Residues Arg703, Asp777, and Arg781 of the rnase H domain of hepatitis B virus polymerase are critical for viral DNA synthesis. / Ko, Chunkyu; Shin, Youn Chul; Park, Woo Jin; Kim, Seungtaek; Kim, Jonghwa; Ryu, Wang-Shick.

In: Journal of Virology, Vol. 88, No. 1, 01.01.2014, p. 154-163.

Research output: Contribution to journalArticle

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