A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein
A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein
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DOI：
10.1128/jvi.00482-11
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发表时间：
2011-08-01
影响因子：
5.4
通讯作者：
Nassal, Michael
中科院分区：
文献类型：
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作者：
Beck, Juergen;Nassal, Michael
嗜肝DNA病毒是唯一已知的通过蛋白质引发的逆转录复制的病毒。除了保守的逆转录酶（RT）和RNase H结构域，它们的聚合酶（P蛋白）携带一个独特的末端蛋白（TP）结构域，该结构域提供了鸭乙型肝炎病毒（DHBV）中的特异性Tyr残基Tyr96，负链DNA的第一个核苷酸自催化连接并延伸三个核苷酸。用DHBV P蛋白和细胞伴侣进行的这种引发反应的体外重建揭示了对作为模板的D β RNA茎环和RT结构域的催化活性加上TP结构域的RNA结合能力的严格要求。通过使用截短的P蛋白（miniP）可以避免伴侣依赖性。在这里，我们发现在TP和RT之间具有烟草蚀纹病毒（TEV）蛋白酶切割位点的miniP（miniP（TEV））在提供α-（32）P-标记的脱氧核苷三磷酸时显示真实的引发活性;然而，蛋白酶切割令人惊讶地揭示，RT结构域也被标记。RT标记具有与在Tyr96处引发相同的要求，并且起源于dNMP转移到推定的RT引物夹持基序中鉴定为Tyr561的独特Tyr残基。突变的Tyr561并不影响Tyr96引发在体外，只有适度降低复制能力的完整的DHBV基因组，因此，脱氧核苷酸化的Tyr561是不是一个专性的中间在TP引发。然而，作为精致复杂的蛋白质引发反应的第一替代底物，dNMP转移到Tyr561是进一步阐明嗜肝DNA病毒复制起始机制的新工具，并表明可以找到特异性引发抑制剂。
Hepadnaviruses are the only known viruses that replicate by protein-primed reverse transcription. Beyond the conserved reverse transcriptase (RT) and RNase H domains, their polymerases (P proteins) carry a unique terminal protein (TP) domain that provides a specific Tyr residue, Tyr96 in duck hepatitis B virus (DHBV), to which the first nucleotide of minus-strand DNA is autocatalytically attached and extended by three more nucleotides. In vitro reconstitution of this priming reaction with DHBV P protein and cellular chaperones had revealed strict requirements for the D epsilon RNA stem-loop as a template and for catalytic activity of the RT domain plus RNA-binding competence of the TP domain. Chaperone dependence can be obviated by using a truncated P protein (miniP). Here, we found that miniP with a tobacco etch virus (TEV) protease cleavage site between TP and RT (miniP(TEV)) displayed authentic priming activity when supplied with alpha-(32)P-labeled deoxynucleoside triphosphates; however, protease cleavage revealed, surprisingly, that the RT domain was also labeled. RT labeling had identical requirements as priming at Tyr96 and originated from dNMP transfer to a unique Tyr residue identified as Tyr561 in the presumed RT primer grip motif. Mutating Tyr561 did not affect Tyr96 priming in vitro and only modestly reduced replication competence of an intact DHBV genome; hence, deoxynucleotidylated Tyr561 is not an obligate intermediate in TP priming. However, as a first alternative substrate for the exquisitely complex protein-priming reaction, dNMP transfer to Tyr561 is a novel tool to further clarify the mechanism of hepadnaviral replication initiation and suggests that specific priming inhibitors can be found.