Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens
Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens
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DOI：
10.1128/jvi.00080-16
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发表时间：
2016-08-01
影响因子：
5.4
通讯作者：
Denison, Mark R.
中科院分区：
文献类型：
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作者：
Sexton, Nicole R.;Smith, Everett Clinton;Denison, Mark R.
正义 RNA 病毒编码基因组复制必需的 RNA 依赖性 RNA 聚合酶 (RdRps)。除了冠状病毒 (CoV) 等大型巢状病毒外，RNA 病毒缺乏校对，因此依赖 RdRps 来控制核苷酸选择性和保真度。冠状病毒在非结构蛋白 14 (nsp14-ExoN) 中编码一种校对核酸外切酶，与其他 RNA 病毒相比，其保真度提高了 10 倍以上。目前尚不清楚 CoV 聚合酶 (nsp12-RdRp) 在多大程度上参与复制保真度。我们试图确定同源模型是否可以识别 CoV RdRps 中核苷酸选择性和保真度的假定决定因素。我们对 CoV 鼠肝炎病毒 (MHV) nsp12-RdRp 结构进行了建模，并将其叠加在已解决的小核糖核酸病毒 RdRp 结构上。先前在柯萨奇病毒 B3 (CVB3) 中发现的保真度改变突变被映射到 nsp12-RdRp 模型结构上，然后工程化到具有 [nsp14-ExoN(+)] 或不具有 [nsp14-ExoN(-)] ExoN 活性的 MHV 基因组中。使用这种方法，我们鉴定了两种赋予对诱变剂 5-氟尿嘧啶 (5-FU) 抗性的突变：nsp12-M611F 和 nsp12-V553I。对于 nsp12-V553I，我们还证明了对诱变剂 5-氮杂胞苷 (5-AZC) 的抗性并减少了突变的积累。 nsp14-ExoN 校对活性有效掩盖了对 5-FU 的耐药性以及基因组突变数量的减少。这些结果表明，nsp12-RdRp 可能在保真度调节中发挥作用，并且尽管序列保守性较低，但 RdRp 核苷酸选择性的一些决定因素在 RNA 病毒中是保守的。结果还表明，就核苷酸选择性而言，nsp14-ExoN 相对于 nsp12-RdRp 具有上位性，这与其在多蛋白复制酶校对复合物中的拟议作用一致。 重要性 RNA 病毒具有进化微调的复制保真度，以平衡遗传稳定性和多样性的要求。 RNA 病毒复制保真度的责任归因于 RNA 依赖性 RNA 聚合酶，多种 RNA 病毒的 RdRps 突变显示可改变保真度并减弱病毒复制和毒力。冠状病毒 (CoV) 是唯一已知的编码校对核酸外切酶 (nsp14-ExoN) 以及参与保真度调节的其他复制酶蛋白的 RNA 病毒。该报告表明，冠状病毒 RdRp (nsp12) 可能在复制保真度方面发挥作用；冠状病毒 RdRp 核苷酸选择性的残基决定簇映射到其他不相关的 RNA 病毒聚合酶的相似结构区域；对于 CoV，nsp14-ExoN 的校对活性与 RdRp 的功能保真度是上位的。
Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(-)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex.IMPORTANCERNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.