One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities
One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities
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DOI：
10.1073/pnas.1323705111
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发表时间：
2014-09-16
影响因子：
11.1
通讯作者：
Imbert, Isabelle
中科院分区：
文献类型：
--
作者：
Subissi, Lorenzo;Posthuma, Clara C.;Imbert, Isabelle
冠状病毒科除了引起较轻微的人类感染的成员外，还包括引起严重急性呼吸道综合征（SARS）和最近出现的中东呼吸道综合征的潜在致命的人畜共患病原体。冠状病毒的30-kb正链RNA基因组编码一种复制/转录机制，该机制异常复杂，由16种非结构蛋白（nsps）组成。SARS-CoV nsp 12是典型的RNA依赖性RNA聚合酶（RdRp），在体外表现出较差的RNA合成过程，与体内非常大的RNA基因组的有效复制不一致。在这里，我们报告说，SARS-CoV nsp 7和nsp 8激活并赋予nsp 12的RNA合成活性的持续合成能力。使用生物化学测定和反向遗传学，保守的nsp 7和nsp 8残基的重要性进行了探讨。尽管几个nsp 7突变在有限程度上影响病毒复制，但对与nsp 12相互作用至关重要的两个nsp 8残基（P183和R190）和对聚合酶复合物与RNA相互作用至关重要的第三个残基（K58）的替换对病毒都是致命的。在不损失持续合成能力的情况下，nsp 7/nsp 8/nsp 12复合物可以与nsp 14缔合，nsp 14是一种双功能酶，具有分别涉及复制保真度和5 '-RNA加帽的3'-5 '核糖核酸外切酶和RNA帽N7-鸟嘌呤甲基转移酶活性。这种三重聚合酶复合物的鉴定反过来又与nsp 14校正酶相关联，这揭示了冠状病毒如何组装RNA合成机器以复制最大的已知RNA基因组。这种蛋白质复合物是RNA聚合酶、加帽和校对活动功能整合的一个迷人的例子。
In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The similar to 30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3'-5'exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5'-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.