Dissecting the role of the coronavirus proofreading exoribonuclease in RNA recombination

项目摘要 冠状病毒（Coronaviruses，CoV）是一种引起人类呼吸道疾病的正义RNA病毒家族 从普通感冒到严重致命的疾病。严重急性呼吸道综合征的流行 2002-2004年SARS冠状病毒和中东呼吸综合征冠状病毒的出现 自2012年以来，MERS-CoV强调了CoV通过菌株重组出现并引起新的 具有大流行潜力的人畜共患传染病。尽管这些感染的死亡率很高， 或针对任何冠状病毒的疫苗是目前可用的。冠状病毒，其基因组大几倍 与其他正链RNA病毒不同，它在非结构蛋白14中编码一种校正核糖核酸外切酶 RNA依赖性RNA聚合酶（nsp 12-RdRp）与nsp 14-ExoN不同。我们已经证明 nsp 14-ExoN（ExoN（-））的催化失活降低了复制保真度，导致突变增加， 频率，对RNA诱变剂的敏感性增加，复制减少，适应性降低，稳定 体内衰减。最近在其他RNA病毒中的发现将复制保真度和重组联系起来， 我们的初步研究支持了这样一个科学前提，即复制保真度有助于重组， 这表明，模型冠状病毒，小鼠肝炎病毒（MHV）的ExoN（-）突变体已经损害了 重组该提案的目标是确定nsp 14-ExoN在CoV重组中的作用，并测试 ExoN保真度调节的结构和氨基酸决定因素是否与重组有关。在 具体目标1：我们将比较WT和ExoN（-）MHV在下一代细胞中RNA重组的变化 测序和体外感染测定。在具体目标2中，我们将使用结构定向诱变， 确定nsp 14-ExoN结构元件和氨基酸残基对MHV重组的影响， 复制保真度和适应性。总之，这些研究将定义CoV的nsp 14-ExoN决定因素 重组，并探索nsp 14-ExoN在新菌株出现中的潜在作用。这项研究也将 为下一代测序方法的开发提供信息，以研究CoV重组和RNA 合成.最后，这些研究将利用重组RNA作为工具，以更好地了解冠状病毒复制 忠诚度，适应性，病毒的出现和进化

PROJECT SUMMARY Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause respiratory illnesses in humans ranging from the common cold to severe and lethal disease. The epidemic of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002-2004 and emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) since 2012 emphasize the capacity of CoVs to emerge by strain recombination and cause new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. Coronaviruses, whose genomes are several times larger than other positive-strand RNA viruses, encode a proofreading exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is distinct from the RNA-dependent RNA polymerase (nsp12-RdRp). We have demonstrated that catalytic inactivation of nsp14-ExoN (ExoN(-)) decreases replication fidelity, resulting in increased mutation frequency, increased sensitivity to RNA mutagens, decreased replication, decreased fitness, and stable attenuation in vivo. Recent findings in other RNA viruses have linked replication fidelity and recombination, and our preliminary studies support scientific premise that replication fidelity contributes to recombination by demonstrating that ExoN(-) mutants of the model coronavirus, murine hepatitis virus (MHV) have impaired recombination. The goals of this proposal are to define the role of nsp14-ExoN in CoV recombination and test whether the structural and amino acid determinants of ExoN fidelity regulation are linked to recombination. In Specific Aim 1 we will compare WT and ExoN(-) MHV for changes in RNA recombination by next-generation sequencing and in vitro infection assays. In Specific Aim 2 we will use structure-directed mutagenesis to determine the impact of nsp14-ExoN structural elements and amino acid residues on MHV recombination, replication fidelity, and fitness. Together, these studies will define the nsp14-ExoN determinants of CoV recombination and explore the potential role of nsp14-ExoN in new strain emergence. This research also will inform the development of next-generation sequencing approaches to investigate CoV recombination and RNA synthesis. Finally, these studies will utilize recombinant RNA as tools to better understand CoV replication fidelity, fitness, and viral emergence and evolution.