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

项目摘要 冠状病毒属于巢状病毒目的高级目，是有包膜的正义单链病毒， 感染脊椎动物和无脊椎动物的RNA病毒。Nidoviruses的名称来自拉丁语“nidus”，意思是 在感染过程中，这种顺序的病毒产生具有共同末端的多个不同的基因组转录物。 这些表达所有病毒结构蛋白的转录物比全长基因组短， 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，其中病毒RNA 聚合酶在转录中途从其模板解离，并在不同的 template.尽管对“巢状”病毒的研究已经进行了将近半个世纪，但我们对宿主的了解仍然有限 蛋白质、RNA模板特征以及不连续转录中涉及的机制。此外，委员会认为， 控制亚基因组RNA产生的相同原理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 具有双亲基因的病毒后代。病毒进化的核心是进化， 病毒适应新宿主的能力了解冠状病毒不连续的过程 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通量和无偏见的方法来识别RNA特征，包括 序列和结构，以及亚基因组RNA产生所必需的蛋白质因子， 病毒重组的研究我将在我以前的工作基础上发展下一代 测序方法来研究病毒RNA的顺式特征，如SARS-CoV-2在活动性感染期间。我 将建立新的检测方法来研究病毒重组，并使用CRISPR筛选来识别宿主的反式 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的漏洞，并帮助我们 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.