Department of Biochemistry & Molecular Biology, Colorado State Universitygrid.47894.36, Fort Collins, Colorado, USA.
Centre National de la Recherche Scientifique, Aix-Marseille Université CNRS UMR 7257, AFMB, Marseille, France.
J Virol. 2022 Aug 24;96(16):e0067122. doi: 10.1128/jvi.00671-22. Epub 2022 Aug 4.
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. 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.
正链 RNA 病毒利用病毒编码的 RNA 依赖性 RNA 聚合酶（RdRP）复制其基因组，该酶具有常见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）形成大型多组分 RNA 复制-转录复合物，其中包含由 nsp12 RdRP 蛋白组成的核心 RNA 合成机器，该蛋白与一个 nsp7 和两个 nsp8 蛋白作为必需亚基结合在一起。我们发现，通过预先孵育 nsp12 和 nsp8，可以将该复合物的组装速度提高 5 倍，并且使用新型 nsp8L7 异二聚体融合蛋白构建体可以进一步优化。使用快速动力学方法，我们测量了核心复制酶的延伸速率高达 260 个核苷酸（nt）/s，对于病毒聚合酶来说，这是一个异常快的速率。为了解决这个快速速率的起源，我们检查了 RdRP 活性位点中的两个 CoV 特异性残基的作用：取代结合的 NTP 上方保守的谷氨酸的 Ala547，以及将 palm 结构域 GDD 序列突变为 SDD 的 Ser759。我们的数据表明，Ala547 可以使复制速率提高一倍，但代价是保真度降低，而 palm 结构域中的 SDD 序列可以减轻这种影响。我们的生化数据表明，聚合酶基序 F 和 C 中的突变固定在调节复制速率和保真度以适应其大型基因组方面发挥了关键作用。复制大型基因组对 RNA 病毒来说是一个挑战，因为需要快速的 RNA 合成来逃避先天免疫防御，但更快的聚合酶固有地是低保真度酶。尽管如此，冠状病毒还是使用所有正链 RNA 病毒共有的核心聚合酶结构和机制复制其约 30kb 的基因组。它们成功的经典解释是，大型基因组的 nidoviruses 已经获得了基于外切酶的修复系统，该系统补偿了聚合酶的高突变率。在这项工作中，我们通过两个关键活性位点残基的突变证实了 nidoviral 聚合酶本身在维持基因组完整性方面也发挥了关键作用，这些突变允许非常高的复制速率，同时保持典型的突变率。我们的发现进一步证明了核心聚合酶平台的进化灵活性，表明它如何在从小基因组的肠病毒扩展到长基因组的 nidoviruses 的过程中进行了适应。
