Mechanistic insights into multifaceted roles of coronavirus exoribonuclease complex

随着SARS-CoV-2继续在地球仪肆虐，了解其机制并 病毒基因组复制和转录的调控，这是冠状病毒生命的基本过程 循环并代表治疗干预的重要靶点。冠状病毒基因组复制和 转录是通过动态复制-转录复合物（RTC）进行的，该复合物由一系列 病毒非结构蛋白（NSP）。在RTC内，一种独特的校对核糖核酸外切酶（ExoN）复合物， nsp 14-nsp 10通过切除错误掺入的核苷酸和许多抗病毒核苷酸来提高复制保真度 类似物除了其在校正病毒RNA合成中的作用之外，ExoN复合物还参与病毒RNA合成。 RNA 5′加帽，这对冠状病毒的免疫逃避至关重要。尽管在研究中付出了非凡的努力 冠状病毒生物学和复制，主要差距仍然存在于我们的理解的关键作用exoN复合体 在冠状病毒生命周期的各个基本方面发挥作用。首先，目前尚不清楚ExoN复合物如何协调 用低保真病毒聚合酶来校对RNA合成。第二，人们对ExoN是如何运作的了解甚少。 复合物由病毒辅因子调节。第三，这是未知的两种不同的酶功能，RNA ExoN复合物的切割和加帽活性在病毒生命周期中相互耦合。的中心目标 我们提出的实验是通过系统地解剖结构来填补这些理解上的空白。 ExoN复合物及其与病毒辅因子在病毒RNA合成中的作用 和处理我们将使用SARS-CoV-2作为模型系统，并采用低温电子 显微镜，单分子生物物理学，蛋白质-RNA生物化学和细胞病毒学来实现这一中心目标 通过以下几个方面：项目1，错配过程中聚合酶和核糖核酸外切酶的协调 纠正一下我们将定义RNA错配从聚合酶转移到ExoN的机制 并确定其功能相互作用的分子决定因素。项目2，ExoN复合物的调节， 病毒辅因子我们将阐明ExoN复合物与关键RTC之间相互作用的分子细节 亚基，nsp 8，并确定这种相互作用如何调节ExoN复合物的校对活性， 失配校正项目3，RNA外切核酸酶切和ExoN的5′加帽活性的偶联 复杂.基于我们新发现的ExoN复合物二聚体形式的冷冻电镜结构， 消化和封盖活动耦合，我们将确定分子和生物化学基础 并建立ExoN两种酶活性之间功能联系的生理意义 复杂.通过这项研究计划，我们将揭示的原则和分子细节， 这种独特的病毒RNA校对器的多方面作用，并提供了新的见解的机制， 冠状病毒基因组复制和转录的调控。更广泛地说，现有的工具和 实验平台很容易适用于研究其他RNA病毒。

As SARS-CoV-2 continues to wreak havoc across the globe, it is imperative to understand the mechanism and regulation of the viral genome replication and transcription, which are essential processes in coronavirus life cycle and represent important targets for therapeutic interventions. Coronavirus genome replication and transcription are carried out by a dynamic replication-transcription complex (RTC), assembled from an array of viral non-structural proteins (nsps). Within the RTC, a unique proofreading exoribonuclease (ExoN) complex, nsp14-nsp10, boosts replication fidelity by excising mis-incorporated nucleotides and many antiviral nucleotide analogs. In addition to its role in proofreading viral RNA synthesis, the ExoN complex is also involved in viral RNA 5′ capping, which is critical for immune evasion by coronaviruses. Despite extraordinary efforts in studying coronavirus biology and replication, major gaps remain in our understanding of the key roles ExoN complex plays in various fundamental aspects of coronavirus life cycle. First, it is unclear how ExoN complex coordinates with the low-fidelity viral polymerase to proofread RNA synthesis. Second, it is poorly understood how ExoN complex is modulated by viral cofactors. Third, it is unknown how the two different enzymatic functions, RNA cleavage and capping activities, of ExoN complex are coupled in the virus life cycle. The central objective of our proposed experiments is to fill these gaps in understanding through a systematic dissection of the structural basis and functional roles of ExoN complex and its dynamic interlay with viral cofactors in viral RNA synthesis and processing. We will use SARS-CoV-2 as a model system and employ a combination of cryo-electron microscopy, single-molecule biophysics, protein-RNA biochemistry, and cell virology to achieve this central goal through the following aspects: Project 1, coordination of polymerase and exoribonuclease during mismatch correction. We will define the mechanism by which RNA mismatches are transferred from polymerase to ExoN and identify the molecular determinants for their functional interplay. Project 2, modulation of ExoN complex by viral cofactors. We will elucidate the molecular details of the interaction between ExoN complex and a key RTC subunit, nsp8, and determine how this interaction modulates the proofreading activity of ExoN complex during mismatch correction. Project 3, coupling of RNA exonucleolytic digestion and 5′ capping activities of ExoN complex. Building on our newfound cryo-EM structure of a dimeric form of the ExoN complex in which its RNA digestion and capping activities are coupled, we will determine the molecular and biochemical underpinnings and establish the physiological significance of the functional link between the two enzymatic activities of ExoN complex. Through this research program, we will reveal the principles and molecular details governing the multifaceted roles of this unique viral RNA proofreader and provide new insights into the mechanisms and regulation of coronavirus genome replication and transcription. More broadly, the established tools and experimental platforms are readily applicable to studying other RNA viruses.