The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
The SARS-coronavirus nsp7+nsp8 complex is a unique multimeric RNA polymerase capable of both de novo initiation and primer extension.
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DOI：
10.1093/nar/gkr893
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发表时间：
2012-02
影响因子：
14.9
通讯作者：
Snijder EJ
中科院分区：
文献类型：
--
作者：
te Velthuis AJ;van den Worm SH;Snijder EJ
在RNA病毒中独一无二的是，据信1030-kb SARS冠状病毒基因组的复制涉及两种RNA依赖性RNA聚合酶（RdRp）活性。第一种是引物依赖性的，与106 kDa的非结构蛋白12（nsp 12）相关，而第二种是由22 kDa的nsp 8催化的。后一种酶能够从头起始，并已被提议作为引发酶操作。有趣的是，该蛋白质仅与10-kDa nsp 7一起结晶，形成十六聚体的dsRNA环绕环结构[即nsp（7+8），由8个拷贝的两种nsp组成]。为了更好地理解这些结构特征对nsp 8驱动的RNA合成的影响，我们研究了nsp（7+8）复合物形成的先决条件及其聚合酶活性。我们发现，特别是nsp 8的天然N-末端残基的暴露对于蛋白质与nsp 7缔合的能力和增强其RdRp活性都是至关重要的。此外，这种“改进的”重组nsp 8能够延伸引物RNA模板，这是迄今为止未被注意到的特性。然而，后者的活性比相同单体浓度下的引物依赖性nsp 12-RdRp弱20倍。最后，采用保守D/ExD/E基序的定点突变来鉴定对nsp（7+8）RdRp活性至关重要的残基。
Uniquely among RNA viruses, replication of the ∼30-kb SARS-coronavirus genome is believed to involve two RNA-dependent RNA polymerase (RdRp) activities. The first is primer-dependent and associated with the 106-kDa non-structural protein 12 (nsp12), whereas the second is catalysed by the 22-kDa nsp8. This latter enzyme is capable of de novo initiation and has been proposed to operate as a primase. Interestingly, this protein has only been crystallized together with the 10-kDa nsp7, forming a hexadecameric, dsRNA-encircling ring structure [i.e. nsp(7+8), consisting of 8 copies of both nsps]. To better understand the implications of these structural characteristics for nsp8-driven RNA synthesis, we studied the prerequisites for the formation of the nsp(7+8) complex and its polymerase activity. We found that in particular the exposure of nsp8's natural N-terminal residue was paramount for both the protein's ability to associate with nsp7 and for boosting its RdRp activity. Moreover, this ‘improved’ recombinant nsp8 was capable of extending primed RNA templates, a property that had gone unnoticed thus far. The latter activity is, however, ∼20-fold weaker than that of the primer-dependent nsp12-RdRp at equal monomer concentrations. Finally, site-directed mutagenesis of conserved D/ExD/E motifs was employed to identify residues crucial for nsp(7+8) RdRp activity.
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DOI：
10.1038/nrmicro2147
发表时间：
2009-06
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
通讯作者：
--
影响因子：
14.9
作者：
Gorbalenya AE;Koonin EV;Donchenko AP;Blinov VM
通讯作者：
Blinov VM
SARS-Coronavirus复制/转录复合物受到膜保护，需要在体外活动的宿主因子。
影响因子：
6.7
作者：
van Hemert, Martijn J.;van den Worm, Sjoerd H. E.;Knoops, Kevin;Mommaas, A. Mieke;Gorbalenya, Alexander E.;Snijder, Eric J.
通讯作者：
Snijder, Eric J.
影响因子：
5
作者：
Imbert I;Snijder EJ;Dimitrova M;Guillemot JC;Lécine P;Canard B
通讯作者：
Canard B
DOI：
10.1073/pnas.0608952104
发表时间：
2007-03-13
影响因子：
11.1
作者：
Castro, Christian;Smidansky, Eric;Cameron, Craig E.
通讯作者：
Cameron, Craig E.