Chernov Andrei V, Shiryaev Sergey A, Aleshin Alexander E, Ratnikov Boris I, Smith Jeffrey W, Liddington Robert C, Strongin Alex Y
Burnham Institute for Medical Research, La Jolla, California 92037, USA.
J Biol Chem. 2008 Jun 20;283(25):17270-8. doi: 10.1074/jbc.M801719200. Epub 2008 Apr 28.
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.
与包括登革热病毒和黄热病病毒在内的许多黄病毒类型相似,西尼罗河病毒(WNV)的非结构NS3多功能蛋白在N端具有丝氨酸蛋白酶结构域,在C端具有RNA三磷酸酶、NTPase结构域和RNA解旋酶,参与多蛋白加工和RNA复制,因此是一个有前景的药物靶点。为了展现其蛋白水解活性,NS3蛋白酶需要上游NS2B序列编码的辅因子存在。在我们对WNV解旋酶生物学特性的详细研究中,我们表征了全长NS2B-NS3蛋白酶-解旋酶蛋白以及缺少NS2B辅因子和NS3蛋白酶序列的单个NS3解旋酶结构域以及仅缺少NS2B辅因子的单个NS3蛋白酶-解旋酶的ATP酶活性和RNA/DNA解旋活性。我们确定NS3解旋酶和NS3蛋白酶-解旋酶构建体都能够解开DNA和RNA模板。相比之下,全长NS2B-NS3蛋白酶-解旋酶只能解开RNA模板,而其DNA解旋活性则受到严重抑制。我们的数据表明,NS2B-NS3蛋白酶部分的有效、具有催化活性的折叠是WNV以及可能其他黄病毒中RNA-DNA底物选择性机制的重要组成部分。基于我们的数据,我们推测我们所确定的机制在宿主细胞质中病毒诱导的膜结合复制复合物内以及感染细胞核内发生的WNV复制中发挥着尚未确定的作用。