The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase
The two-component NS2B-NS3 proteinase represses DNA unwinding activity of the West Nile virus NS3 helicase
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DOI：
10.1074/jbc.m801719200
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发表时间：
2008-06-20
影响因子：
4.8
通讯作者：
Strongin, Alex Y.
中科院分区：
文献类型：
--
作者：
Chernov, Andrei V.;Shiryaev, Sergey A.;Strongin, Alex Y.
与包括登革热和黄热病病毒在内的许多黄病毒类型类似，西尼罗河病毒（WNV）的非结构NS 3多功能蛋白具有N-末端丝氨酸蛋白酶结构域和RNA三磷酸酶、NTR结构域和C-末端结构域中的RNA解旋酶，其涉及多蛋白加工和RNA复制，因此是有希望的药物靶标。为了显示其蛋白水解活性，NS 3蛋白酶需要存在由上游NS 2B序列编码的辅因子。在我们详细研究WNV解旋酶的生物学过程中，我们表征了全长NS 2B-NS 3蛋白酶-解旋酶蛋白以及缺乏NS 2B辅因子和NS 3蛋白酶序列两者的单个NS 3解旋酶结构域和仅缺乏NS 2B辅因子的单个NS 3蛋白酶-解旋酶的ATP酶和RNA/DNA解旋活性。我们确定NS 3解旋酶和NS 3蛋白酶-解旋酶构建体都能够解旋DNA和RNA模板。相反，全长NS 2B-NS 3蛋白酶解旋酶仅解旋RNA模板，而其DNA解旋活性受到严重抑制。我们的数据表明，生产性的，有催化能力的折叠NS 2B-NS 3蛋白酶部分代表了一个重要组成部分的RNA-DNA底物的选择性机制在西尼罗河病毒，并可能在其他黄病毒。根据我们的数据，我们假设，我们已经确定的机制发挥了作用，但尚未确定在病毒诱导的膜结合复制复合物在宿主细胞质和感染细胞的细胞核内发生的WNV复制。
Similar to many flavivirus types including Dengue and yellow fever viruses, the nonstructural NS3 multifunctional protein of West Nile virus (WNV) with an N-terminal serine proteinase domain and an RNA triphosphatase, an NTPase domain, and an RNA helicase in the C-terminal domain is implicated in both polyprotein processing and RNA replication and is therefore a promising drug target. To exhibit its proteolytic activity, NS3 proteinase requires the presence of the cofactor encoded by the upstream NS2B sequence. During our detailed investigation of the biology of the WNV helicase, we characterized the ATPase and RNA/DNA unwinding activities of the full-length NS2B-NS3 proteinase-helicase protein as well as the individual NS3 helicase domain lacking both the NS2B cofactor and the NS3 proteinase sequence and the individual NS3 proteinase-helicase lacking only the NS2B cofactor. We determined that both the NS3 helicase and NS3 proteinase-helicase constructs are capable of unwinding both the DNA and the RNA templates. In contrast, the full-length NS2B-NS3 proteinase-helicase unwinds only the RNA templates, whereas its DNA unwinding activity is severely repressed. Our data suggest that the productive, catalytically competent fold of the NS2B-NS3 proteinase moiety represents an essential component of the RNA-DNA substrate selectivity mechanism in WNV and, possibly, in other flaviviruses. Based on our data, we hypothesize that the mechanism we have identified plays a role yet to be determined in WNV replication occurring both within the virus-induced membrane-bound replication complexes in the host cytoplasm and in the nuclei of infected cells.