Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
Vanderbilt Institute for Infection, Immunology, and Inflammation (VI4), Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.
PLoS Pathog. 2021 Jan 19;17(1):e1009226. doi: 10.1371/journal.ppat.1009226. eCollection 2021 Jan.
Recombination is proposed to be critical for coronavirus (CoV) diversity and emergence of SARS-CoV-2 and other zoonotic CoVs. While RNA recombination is required during normal CoV replication, the mechanisms and determinants of CoV recombination are not known. CoVs encode an RNA proofreading exoribonuclease (nsp14-ExoN) that is distinct from the CoV polymerase and is responsible for high-fidelity RNA synthesis, resistance to nucleoside analogues, immune evasion, and virulence. Here, we demonstrate that CoVs, including SARS-CoV-2, MERS-CoV, and the model CoV murine hepatitis virus (MHV), generate extensive and diverse recombination products during replication in culture. We show that the MHV nsp14-ExoN is required for native recombination, and that inactivation of ExoN results in decreased recombination frequency and altered recombination products. These results add yet another critical function to nsp14-ExoN, highlight the uniqueness of the evolved coronavirus replicase, and further emphasize nsp14-ExoN as a central, completely conserved, and vulnerable target for inhibitors and attenuation of SARS-CoV-2 and future emerging zoonotic CoVs.
重组被认为对冠状病毒（CoV）的多样性以及 SARS-CoV-2 和其他人畜共患 CoV 的出现至关重要。虽然 RNA 重组是 CoV 复制过程中的必需步骤，但 CoV 重组的机制和决定因素尚不清楚。CoV 编码一种 RNA 校对外切核酸酶（nsp14-ExoN），它与 CoV 聚合酶不同，负责高保真 RNA 合成、对核苷类似物的抗性、免疫逃避和毒力。在这里，我们证明包括 SARS-CoV-2、MERS-CoV 和模式 CoV 鼠肝炎病毒（MHV）在内的 CoV 在培养中复制时会产生广泛而多样的重组产物。我们表明，MHV nsp14-ExoN 是天然重组所必需的，而 ExoN 的失活会导致重组频率降低和重组产物改变。这些结果为 nsp14-ExoN 增加了另一个关键功能，突出了进化的冠状病毒复制酶的独特性，并进一步强调 nsp14-ExoN 作为 SARS-CoV-2 和未来新兴人畜共患 CoV 的抑制剂和减毒的核心、完全保守和脆弱的靶标。
