A complex zinc finger controls the enzymatic activities of nidovirus helicases
A complex zinc finger controls the enzymatic activities of nidovirus helicases
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DOI：
10.1128/jvi.79.2.696-704.2005
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发表时间：
2005-01-01
影响因子：
5.4
通讯作者：
Ziebuhr, J
中科院分区：
文献类型：
--
作者：
Seybert, A;Posthuma, CC;Ziebuhr, J
巢状病毒（冠状病毒科、动脉炎病毒科和罗尼病毒科）编码一种非结构蛋白，在动脉炎病毒中称为nsp 10，在冠状病毒中称为nsp 13，其由C-末端超家族1解旋酶结构域和N-末端推定的锌结合结构域（ZBD）组成。以前，马动脉炎病毒（EAV）nsp 10 ZBD的突变被证明可以通过破坏RNA合成和可能的病毒粒子生物合成来阻断动脉炎病毒的繁殖。在这里，我们的特点的ATP酶和解旋酶的活性细菌表达的突变形式的nsp 10和它的人冠状病毒229 E直系同源物，nsp 13，并与这些在体外活动与特定的病毒表型。用Ala替换保守的Cys或His残基被证明比Cys替换His或His替换Cys更有害。此外，变性-复性实验表明，在蛋白质的复性过程中，Zn 2+是必不可少的救援的酶活性的巢病毒解旋酶。两者合计，数据强烈支持锌结合功能的N-末端结构域的巢病毒解旋酶。由于ZBD中的单残基取代或整个结构域的缺失导致的nsp 10 ATP酶/解旋酶缺陷不能被野生型ZBD反式互补，这表明ZBD顺式的关键功能。一致地，在将编码ATP酶/解旋酶缺陷型nsp 10的EAV全长RNA转染到易感细胞中后，未检测到病毒RNA合成。与此相反，不同的表型与酶活性的nsp 10，这在许多情况下与在体外测得的活动的突变体进行了观察。总的来说，我们的数据表明，ZBD是至关重要的巢病毒复制和转录通过调节解旋酶结构域的酶活性和其他，但未知的机制。
Nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn2+ is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in trans by wild-type ZBD, suggesting a critical function of the ZBD in cis. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.