Coronavirus RNA synthesis by multicomponent protein machines

摘要 新冠肺炎的病原体SARS-CoV-2已经成为一种席卷全球的人类病原体 社区。制定预防或治疗新冠肺炎的策略对于减轻疾病和限制 进一步的病毒传播。抗病毒治疗的一个关键靶点是病毒RNA复制复合体。SARS-CoV-2 复制复合体是一个由多种辅因子、酶和宿主调节蛋白组成的大型多亚基机器。 这些蛋白质亚基如何组装和协作进行病毒RNA复制和转录仍不清楚。 这项工作的主要主题是用一种 重点描述现有抗病毒疗法的效果，以及发现新的靶点和 化合物。我们感兴趣的是病毒nsp14核酸外切酶如何有助于病毒RNA的校对，减少 核苷酸错误结合，并对核苷类似物产生天然耐药性。我们还将探索 神秘的nsp12核苷酸转移酶(Niran)的功能并将病毒蛋白网络拼接在一起 负责组装RNA合成复合体的相互作用。为此，我们将使用多种方法，包括 生物化学、生物物理学、细胞生物学、化学生物学和低温电子显微镜。这些研究将提供新的 深入了解这台复杂机器的工作原理，为现有疗法提供新的作用机制 发现新的抗病毒化合物。此外，复制机械部件的高度保守性 允许这些对SARS-CoV-2的研究适用于未来出现的冠状病毒 在未来的病毒大流行成为全球危机之前阻止它们。

SUMMARY SARS-CoV-2, the causative agent of COVID-19, has emerged as a global human pathogen sweeping through our communities. The development of strategies to prevent or treat COVID-19 is essential for mitigating disease and limiting further viral spread. A key target of antiviral therapeutics is the virus RNA replication complex. The SARS-CoV-2 replication complex is a large multi-subunit machine with multiple co-factors, enzymes and host modulating proteins. How these protein subunits assemble and cooperate to carryout viral RNA replication and transcription remains unclear. The overarching theme of this work is to examine understudied aspects of the coronavirus replication complex with an emphasis on characterizing the effects of existing antiviral therapeutics as well as the discovery of novel targets and compounds. We are interested in how the virus nsp14 exonuclease contributes to viral RNA proofreading, reducing nucleotide misincorporations and providing natural resistance to nucleoside analogue drugs. We will also explore the function of the enigmatic nsp12 nucleotidyltransferase (NiRAN) and will piece together the network of viral protein interactions responsible for the assembly of the RNA synthesis complex. To this we will use diverse methods including biochemistry, biophysics, cell biology, chemical biology and cryo-electron microscopy. These studies will provide new insight into the workings of this complicated machine, provide new mechanisms of action for existing therapeutics and discover novel antiviral compounds. In addition, the high conservation of components of the replication machinery across the coronavirus family allows these studies of SARS-CoV-2 to be applicable to future emerging coronaviruses to head off future viral pandemics before they become global crises.