Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis
Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis
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DOI：
10.1128/jvi.01916-13
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发表时间：
2014-01-01
影响因子：
5.4
通讯作者：
Ryu, Wang-Shick
中科院分区：
文献类型：
--
作者：
Ko, Chunkyu;Shin, Youn-Chul;Ryu, Wang-Shick
B型肝炎病毒（HBV）通过逆转录合成其DNA基因组，逆转录由病毒聚合酶（Pol）催化。以往的研究表明，嗜肝DNA病毒Pol的RNase H结构域可能有助于病毒基因组复制的多个步骤，如RNA合成和病毒DNA合成。然而，RNA酶H结构域的特定残基，有助于病毒逆转录尚未确定。因此，我们采用电荷-丙氨酸扫描诱变产生一组RNase H结构域的单取代突变体，然后分析它们支持病毒逆转录的能力。Southern印迹分析表明，三个突变体（R703 A，D 777 A和R781 A突变体）产生的病毒DNA的量显着减少。然而，这些突变体中没有一个是缺陷的RNA折叠。数据表明，在R703 A和D 777 A突变体中，由于RNase H的催化活性丧失，负链DNA合成不完全。相比之下，在R781 A突变体中，负链DNA合成在一定程度上接近完成，而正链DNA合成（即，松弛的环状DNA）由于RNase H活性的缺陷而严重受损。总的来说，我们的分析表明，HBV Pol RNA酶H结构域的三个带电残基有助于RNA酶H在去除RNA模板中的催化作用，但不参与RNA水解。
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 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.