Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: Effects of metal ions
Functional and structural dynamics of hepadnavirus reverse transcriptase during protein-primed initiation of reverse transcription: Effects of metal ions
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DOI：
10.1128/jvi.02760-07
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发表时间：
2008-06-01
影响因子：
5.4
通讯作者：
Ha, Jianming
中科院分区：
文献类型：
--
作者：
Lin, Li;Wan, Fen;Ha, Jianming
肝病毒的逆转录是由病毒逆转录酶(RT)(蛋白质启动)启动的，需要RT和称为epsilon的特定病毒RNA模板之间的相互作用。蛋白质启动对一些逆转录酶抑制物具有抵抗力，可以阻止随后的病毒DNA延伸，并且可能需要一个独特的“启动”构象。此外，蛋白质启动可能包括两个不同的阶段，即第一个脱氧核苷酸连接到RT(起始)和随后添加2个或3个脱氧核苷酸(聚合)。特别是，截短型鸭乙型肝炎病毒RT(MiniRT2)在镁离子存在下进行测试时，能够启动，但在聚合方面存在缺陷。鉴于金属离子对各种DNA和RNA聚合酶活性的已知影响，我们测试了金属离子是否会影响庚型肝炎病毒RT启动。我们报道，与镁离子相比，Mn2+对MiniRT2的启动活性和全长RT都有显著的影响。首先，也是最重要的是，MiniRT2在Mn2+存在下表现出完全的聚合活性，这表明MiniRT2包含了聚合所需的所有序列，但不能与Mg2+从引发向聚合过渡。第二，在Mn2+的作用下，MiniRT2和全长RT的启动活性更强。第三，在Mn2+存在的情况下，蛋白质启动过程中核苷酸和模板的特异性降低。第四，在Mn2+存在下，聚合反应对焦磷酸类似物的抑制很敏感，而在Mg2+存在下，聚合抑制作用不敏感。最后，有限的蛋白质分解提供了直接的证据，表明启动活性的MiniRT2根据Mn2+和Mg2+的存在而采用不同的构象，并且从引发到聚合的转变伴随着RT构象的变化。
Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the interaction between the RT and a specific viral RNA template termed epsilon. Protein priming is resistant to a number of RT inhibitors that can block subsequent viral DNA elongation and likely requires a distinct "priming" conformation. Furthermore, protein priming may consist of two distinct stages, i.e., the attachment of the first deoxynucleotide to RT (initiation) and the subsequent addition of 2 or 3 deoxynucleotides (polymerization). In particular, a truncated duck hepatitis B virus RT (MiniRT2) is competent in initiation but defective in polymerization when tested in the presence of Mg2+. Given the known effects of metal ions on the activities of various DNA and RNA polymerases, we tested if metal ions could affect hepadnavirus RT priming. We report here that Mn2+, in comparison with Mg2+, showed dramatic effects on the priming activity of MiniRT2 as well as the full-length, RT. First and foremost, MiniRT2 exhibited full polymerization activity in the presence of Mn2+, indicating that MiniRT2 contains all sequences essential for polymerization but is unable to transition from initiation to polymerization with Mg2+. Second, the initiation activities of MiniRT2 and the full-length RT were much stronger with Mn2+. Third, the nucleotide and template specificities during protein priming were decreased in the presence of Mn2+. Fourth, polymerization was sensitive to inhibition by a pyrophosphate analog in the presence of Mn2+ but not in the presence of Mg2+. Finally, limited proteolysis provided direct evidence that the priming active MiniRT2 adopted distinct conformations depending on the presence of Mn2+ versus that of Mg2+ and that the transition from initiation to polymerization was accompanied by RT conformational change.