Constructing the nest - understanding the mechanisms of nidoviridae RNA genomes transcription and recombination

项目总结 冠状病毒属于新城疫病毒科的高级目，有包膜阳性，单链 感染脊椎动物和无脊椎动物的RNA病毒。NidoVirus来源于拉丁语“Nidus”，意思是 这种病毒在感染过程中产生多个不同的具有共同末端的基因组转录本。 这些转录本表达所有的病毒结构蛋白，比全长基因组短， 被称为亚基因组RNA。 亚基因组RNA是通过一种称为不连续转录的过程产生的，在这种过程中，病毒RNA 聚合酶在转录过程中从模板上解离，并在不同的 模板。尽管对“嵌套”病毒的研究已有近半个世纪，但我们对宿主的了解有限 蛋白质，RNA模板特征，以及参与不连续转录的机制。此外， 管理亚基因组RNA生产的相同原则和参与者被认为参与了 冠状病毒的重组，其中两种不同的亲本病毒共感染同一宿主细胞并产生 双亲都有基因的病毒后代。重组是病毒进化的核心，也是至关重要的 病毒适应新宿主的能力。对冠状病毒间断传播过程的认识 转录和重组将为治疗干预开辟新的途径。 我的总体目标是使用高吞吐量和不偏不倚的方法来识别RNA特性，包括 序列和结构，以及亚基因组RNA生产所必需的蛋白质因子和 SARS-CoV-2的病毒重组。我将在我之前的工作基础上开发下一代 用测序方法研究SARS-CoV-2等病毒RNA在活动性感染过程中的顺式作用特征。我 将建立新的检测方法来研究病毒重组，并使用CRISPR筛查来识别宿主反式 这些过程所需的因素。 拟议的项目将解决冠状病毒生物学的基本问题。这些问题包括 SARS-CoV-2重组频率，促进重组的因素，以及 重组“热点”。这些问题的答案将揭示病毒漏洞，并帮助我们的 了解冠状病毒的演变，所有这些都将有助于预防未来的大流行。

PROJECT SUMMARY Coronaviruses belong to the higher order of Nidovirales, which are enveloped positive sense, single stranded RNA viruses that infect vertebrates and invertebrates. Nidoviruses are named from the Latin “nidus”, meaning nest as viruses in this order produce multiple distinct genomic transcripts with common ends during infection. These transcripts, which express all of the viral structural proteins, are shorter than the full-length genome and are termed subgenomic RNAs. Subgenomic RNAs are generated through a process called discontinuous transcription, in which the viral RNA polymerase dissociates from its template midway through transcription and finishes transcribing on a different template. Despite nearly half a century of research on “nested” viruses, we have limited knowledge of the host proteins, the RNA template features, and the mechanisms involved in discontinuous transcription. Moreover, the same principles and players governing production of subgenomic RNAs are thought to be involved in recombination of coronaviruses, in which two distinct parental viruses coinfect the same host cell and generate virus progeny that have genes from both parents. Recombination is at the heart of viral evolution and is critical for the ability of a virus to adapt to new hosts. Understanding the process of coronavirus discontinuous transcription and recombination will open new avenues for therapeutic intervention. My overall goal is to use high throughput and unbiased approaches to identify the RNA features, including sequence and structure, as well as the protein factors that are necessary for subgenomic RNA production and viral recombination for SARS-CoV-2. I will build upon my previous work on developing next generation sequencing approaches to study the cis features of viral RNAs such as SARS-CoV-2 during active infection. I will establish new assays to study viral recombination and use CRISPR screens to identify the host trans factors required for these processes. The proposed project will address fundamental questions of coronavirus biology. Amongst these questions are the SARS-CoV-2 recombination frequency, factors that promote recombination, and the existence of recombination “hot spots”. The answers to these questions will uncover viral vulnerabilities and aid our understanding of coronavirus evolution, all which will help prevent future pandemics.