Emerging mechanisms of viral gene regulation from battles between host and SARS-CoV-2

项目摘要/摘要 在过去的二十年里，包括SARS-CoV-2在内的三种高致病性人类冠状病毒 出现，认为未来新的或重新出现的冠状病毒和其他RNA病毒的致命大流行是 几乎是不可避免的。治疗这些致命传染病的迫切需要使这一发现和 开发新型有效的抗病毒药物。然而，尽管做出了前所未有的努力来应对新冠肺炎 在危机中，由于缺乏机械性的理解，高效的新冠肺炎疗法受到严重限制 冠状病毒复制和致病机制的研究。目前的大多数研究和治疗开发努力 关注SARS-CoV-2 RNA聚合酶(RdRp)或Spike蛋白，但它们的成功可能会严重破坏 通过一种独特的SARS-CoV-2校对机制，该机制切除了掺入的RdRp抑制剂或通过快速 出现抗药性刺突蛋白突变体。因此，开发创新的抗病毒药物势在必行 针对病毒生命周期中不同的、在某些情况下尚未确定的基本组成部分的战略。 错综复杂的病毒-宿主相互作用构成了一个复杂的调控网络，决定了病毒的结果 感染，为探索创新的抗病毒疗法提供了很有希望的机会。但是，当前 冠状病毒-宿主相互作用的知识大多局限于病毒-宿主在激活或阻断过程中的军备竞赛。 干扰素依赖的信号通路或在竞争宿主翻译机制。相比之下，病毒- 宿主在冠状病毒基因表达和调控中的相互作用在很大程度上是未知的领域。这一重大差距很大 阻碍了针对这些被忽视的冠状病毒-宿主相互作用的新型抗病毒药物的设计 对病毒存活和宿主抗病毒反应至关重要。这项提案的中心目标是界定 多个新近发现的SARS-CoV-2-宿主相互作用的新分子机制和功能作用 在调节病毒复制和转录过程以及确定新的SARS-CoV-2与宿主的相互作用方面 与病毒基因调控的新机制有关。结合结构生物学，蛋白质-RNA 生物化学、单分子生物物理学、细胞病毒学和计算方法，我们将完成这些 通过三个紧密交织的目标完成任务。在目标1中，我们将确定分子和结构基础 这些新发现的宿主因子和病毒复制-转录机制之间的相互作用。在目标2中，我们 将描述这些关键宿主因子在调节病毒复制和转录方面的生物学功能。 在目标3中，我们将开发一个创新的捕获-识别-可视化实验流程，以发现新的 宿主因素和新的病毒复制-转录调节机制。总而言之，这些研究将 建立一个创新的概念框架来研究冠状病毒与宿主的相互作用并揭示新的靶点 用于开发新的抗冠状病毒疗法。

PROJECT SUMMARY/ABSTRACT Over the last two decades, three highly pathogenic human coronaviruses, including SARS-CoV-2, have emerged, arguing that future deadly pandemics of new or re-emerging coronaviruses and other RNA viruses are almost inevitable. The urgent need to treat these fatal infectious diseases has prioritized the discovery and development of novel effective antivirals. However, despite the unprecedented efforts to address the COVID-19 crisis, highly effective COVID-19 therapeutics are severely limited due to the lack of a mechanistic understanding of coronavirus replication and pathogenesis. Most of the current research and therapeutic development efforts focus on SARS-CoV-2 RNA polymerase (RdRp) or spike protein, but their success can be severely undermined by a unique SARS-CoV-2 proofreading mechanism that excises incorporated RdRp inhibitors or by the rapid emergence of drug-resistant spike protein mutants. Therefore, it is imperative to develop innovative antiviral strategies targeting distinct, and in some cases yet to be identified, essential components of the viral life cycle. The intricate virus-host interplay constitutes a sophisticated regulatory network that dictates the outcome of virus infection, affording promising opportunities to be explored for innovative antiviral therapeutics. However, current knowledge of coronavirus-host interactions is mostly limited to the virus-host arms race in activating or blocking the interferon-dependent signaling pathways or in competing for host translation machinery. By contrast, virus- host interplay in coronavirus gene expression and regulation is largely uncharted territory. This major gap greatly hinders the design of novel antiviral agents targeting these much-overlooked coronavirus-host interactions that are critical for viral survival and host antiviral responses. The central objectives of this proposal are to define novel molecular mechanisms and functional roles of multiple recently discovered SARS-CoV-2-host interactions in modulating the viral replication and transcription processes and to identify new SARS-CoV-2-host interactions linked to novel mechanisms of viral gene regulation. With a combination of structural biology, protein-RNA biochemistry, single-molecule biophysics, cell virology, and computational approaches, we will accomplish these tasks through three tightly interwoven aims. In Aim 1, we will determine the molecular and structural basis of these newfound interactions between host factors and the viral replication-transcription machinery. In Aim 2, we will delineate the biological functions of these crucial host factors in modulating viral replication and transcription. In Aim 3, we will develop an innovative capture-identification-visualization experimental pipeline to discover new host factors and novel viral replication-transcription-regulating mechanisms. Together, these studies will establish an innovative conceptual framework to study coronavirus-host interactions and reveal new targets for the development of novel anti-coronavirus therapeutics.