Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing

Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.

多数RNA病毒缺乏识别并校正基因组复制过程中产生突变的机制，由此形成的准种（quasispecies）多样性是病毒致病与适应宿主的必要条件。然而，对于冠状病毒科（Coronaviridae，基因组长度26至32 kb）这类携带大型RNA基因组的病毒，其如何平衡基因组稳定性与准种多样性的需求，目前尚不明确。此外，大型RNA基因组复制过程中的复制保真性极限，以及保真性降低如何随时间影响病毒适配性，同样尚无定论。我们此前的研究显示，在鼠肝炎病毒的非结构蛋白14（nsp14）中，冠状病毒核糖核酸外切酶（ExoN）的遗传失活会导致复制保真性下降15倍。然而，目前尚不清楚nsp14-ExoN是否为所有冠状病毒的复制保真性所必需，也不清楚保真性降低对病毒复制与传代过程中的基因组多样性及适配性的影响。本研究报道了严重急性呼吸综合征冠状病毒（SARS-CoV）的nsp14-ExoN突变病毒的构建与拯救，该突变病毒存在稳定的生长缺陷，且在体外培养复制过程中的突变频率升高21倍。对SARS-ExoN突变病毒克隆的全基因组序列进行分析后发现，同一轮复制产生的所有被测基因组均带有独特的突变集合，且全基因组范围内共存在100种独特突变。通过使用新型生物信息学工具，并在体外进行10次群体传代后对全基因组进行深度测序，我们证实ExoN突变得以保留，且与野生型SARS-CoV相比，突变病毒的多样性与突变负荷仍持续升高。本研究结果构建了一套全新的遗传学与生物信息学模型，可在具有复制能力的病毒中引入并鉴定多等位基因突变，该模型将成为研究保真性降低与准种多样性升高对病毒复制、致病机制及进化的影响的有力工具。