The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication.
The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication.
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DOI：
10.1128/jvi.00671-22
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发表时间：
2022-08-24
影响因子：
5.4
通讯作者：
中科院分区：
文献类型：
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作者：
正链RNA病毒使用病毒编码的RNA依赖性RNA聚合酶（RdRP）复制其基因组，所述RNA依赖性RNA聚合酶具有共同的活性位点结构和闭合机制，复制速度和保真度可以在此基础上进化以优化病毒适应性。冠状病毒（CoV）形成大的多组分RNA复制-转录复合物，其包含由nsp 12 RdRP蛋白制成的核心RNA合成机器，其中一个nsp 7和两个nsp 8蛋白是活性所需的必需亚基。我们表明，该复合物的组装可以加速5倍的预孵育的nsp 12与nsp 8和进一步优化与使用一种新的nsp 8L 7异源二聚体融合蛋白的构建。使用快速动力学方法，我们测量的核心复制酶的延伸率高达260个核苷酸（nt）/s，这是一个速度非常快的病毒聚合酶。为了解决这种快速的起源，我们研究了RdRP活性位点中两个CoV特异性残基的作用：Ala 547，其取代了结合NTP上方的保守谷氨酸，和Ser 759，其将棕榈结构域GDD序列突变为SDD。我们的数据显示，Ala 547允许复制率加倍，但这是以保真度为代价的，通过在棕榈域中使用SDD序列可以减轻保真度。我们的生物化学数据表明，固定在聚合酶基序F和C的突变发挥了关键作用，在巢病毒的进化调整复制速率和保真度，以适应他们的大基因组。复制大基因组对RNA病毒来说是一个挑战，因为需要快速RNA合成来逃避先天免疫防御，但更快的聚合酶本质上是低保真度的酶。尽管如此，冠状病毒使用所有正链RNA病毒共有的核心聚合酶结构和机制复制它们的30-kb基因组。对它们成功的经典解释是，大基因组巢病毒获得了一种基于核酸外切酶的修复系统，以补偿高聚合酶突变率。在这项工作中，我们确定了巢病毒聚合酶本身也通过两个关键活性位点残基的突变在维持基因组完整性方面发挥关键作用，这两个关键活性位点残基能够实现非常快的复制率，同时保持典型的突变率。我们的研究结果进一步证明了核心聚合酶平台的进化可塑性，显示了它是如何在从短基因组小核糖核酸病毒扩展到长基因组巢状病毒的过程中适应的。
Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes. IMPORTANCE Replicating large genomes represents a challenge for RNA viruses because fast RNA synthesis is needed to escape innate immunity defenses, but faster polymerases are inherently low-fidelity enzymes. Nonetheless, the coronaviruses replicate their ≈30-kb genomes using the core polymerase structure and mechanism common to all positive-strand RNA viruses. The classic explanation for their success is that the large-genome nidoviruses have acquired an exonuclease-based repair system that compensates for the high polymerase mutation rate. In this work, we establish that the nidoviral polymerases themselves also play a key role in maintaining genome integrity via mutations at two key active-site residues that enable very fast replication rates while maintaining typical mutation rates. Our findings further demonstrate the evolutionary plasticity of the core polymerase platform by showing how it has adapted during the expansion from short-genome picornaviruses to long-genome nidoviruses.
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影响因子：
5
作者：
Gorbalenya AE;Enjuanes L;Ziebuhr J;Snijder EJ
通讯作者：
Snijder EJ
影响因子：
16
作者：
Bravo JPK;Dangerfield TL;Taylor DW;Johnson KA
通讯作者：
Johnson KA
影响因子：
4.8
作者：
Moustafa, Ibrahim M.;Korboukh, Victoria K.;Cameron, Craig E.
通讯作者：
Cameron, Craig E.
影响因子：
2.9
作者：
Arnold, JJ;Cameron, CE
通讯作者：
Cameron, CE
DOI：
10.1016/bs.enz.2021.06.002
发表时间：
2021-01-01
期刊：
VIRAL REPLICATION ENZYMES AND THEIR INHIBITORS, PT A
影响因子：
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作者：
Gong, Peng
通讯作者：
Gong, Peng