Determinants of Coronavirus Fidelity in Replication and Pathogenesis

冠状病毒复制和发病机制保真度的决定因素

基本信息

  • 批准号:
    8597862
  • 负责人:
  • 金额:
    $ 56万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2013
  • 资助国家:
    美国
  • 起止时间:
    2013-08-01 至 2017-07-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Emerging human and zoonotic RNA viruses including SARS-CoV cause significant global morbidity, mortality, and social disruption. The current model for RNA virus host-species movement, adaptation, evolution, disease, and resistance to vaccines and antivirals is that they generate vast diversity of related mutants around the central consensus genome, also referred to as quasispecies diversity or "mutant swarms". This allows for rapid selection of adaptive change, and until recently was thought to result solely from the action of RNA- dependent RNA polymerases with low fidelity (high basal mutation rate) and no error repair - proofreading - during RNA synthesis. Coronaviruses (CoVs) are broadly represented in humans, bats, and other mammals and avian species, and are adept at host-species movement and adaptation, as demonstrated by SARS-CoV and the novel HCoV-EMC/2012. CoVs contain the largest positive-strand RNA genomes, up to 32 kb, posing unique challenges to models of intrinsic low-fidelity replication. CoVs encode a DEDDh family 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN), and inactivating mutations in ExoN (ExoN-) of murine hepatitis virus (MHV) and SARS-CoV result in viable mutator phenotypes (20-fold decreased fidelity) in vitro and in vivo. CoV ExoN- mutator viruses manifest decreased fitness compared to ExoN+ (WT) viruses, are sensitive to RNA mutagens, and are stably attenuated in mouse models of lethal SARS-CoV infection. Thus, nsp14-ExoN is likely the first known RNA virus proofreading exonuclease, and is critically involved in virus replication, fitness, and pathogenesis. However, how it regulates fidelity is unknown. Nsp14-ExoN is a distinct protein from the RNA dependent RNA polymerase (nsp12) and also has been shown to have enhanced exoribonuclease activity in vitro by interactions with nsp10. The contributions of these and other CoV proteins to fidelity regulation also is undetermined. Experiments in this proposal will test the hypothesis that CoVs encode multiple proteins, including nsp14-ExoN, nsp12, and nsp10, that together regulate replication fidelity. The specific aims of the proposal are: 1) To define fidelity determinants in nsp14 and the impact on viral fitness and RNA synthesis; 2) To identify proteins and determinants within the CoV fidelity complex; and 3) To determine the effect of altered fidelity on in vivo replication and pathogenesis. The proposed aims build on the complementary strengths and collaborations of the Denison and Baric labs in CoV (MHV and SARS-CoV) reverse genetics, replicase protein mutagenesis and functions, pathogenesis, immune response, and synthetic genomics. The results of the proposed experiments will identify fidelity determinants critical for replication, pathogenesis and virulence, define the range of tolerated fidelity, and identify novel approaches for CoV attenuation and inhibition.
描述(由申请方提供):包括SARS-CoV在内的新出现的人类和人畜共患RNA病毒导致全球显著的发病率、死亡率和社会混乱。RNA病毒宿主种运动、适应、进化、疾病和对疫苗和抗病毒药的抗性的当前模型是它们在中央共有基因组周围产生大量多样性的相关突变体,也称为准种多样性或“突变体群”。这允许适应性变化的快速选择,并且直到最近才被认为仅仅是由具有低保真度(高基础突变率)并且在RNA合成期间没有错误修复-校对的RNA依赖性RNA聚合酶的作用引起的。冠状病毒(CoV)广泛存在于人类,蝙蝠和其他哺乳动物和鸟类物种中,并且擅长宿主物种的运动和适应,如SARS-CoV和新型HCoV-EMC/2012所证明的。冠状病毒包含最大的正链RNA基因组,高达32 kb,对内在低保真度复制模型提出了独特的挑战。CoV编码DEDDh家族非结构蛋白14(nsp 14-ExoN)中的3 '-to-5'核糖核酸外切酶,并且鼠肝炎病毒(MHV)和SARS-CoV的ExoN(ExoN-)中的失活突变在体外和体内导致可行的突变体表型(保真度降低20倍)。与ExoN+(WT)病毒相比,CoV ExoN-突变体病毒表现出降低的适应性,对RNA诱变剂敏感,并且在致死SARS-CoV感染的小鼠模型中稳定减毒。因此,nsp 14-ExoN可能是第一个已知的RNA病毒校正核酸外切酶,并且关键地参与病毒复制、适应性, 和发病机制。然而,它如何调节忠诚度是未知的。Nsp 14-ExoN是与RNA依赖性RNA聚合酶(nsp 12)不同的蛋白质,并且也已显示通过与nsp 10相互作用而在体外具有增强的核糖核酸外切酶活性。这些和其他CoV蛋白对保真度调节的贡献也未确定。该提案中的实验将测试CoV编码多种蛋白质的假设,包括nsp 14-ExoN,nsp 12和nsp 10,这些蛋白质共同调节复制保真度。该提案的具体目标是:1)定义nsp 14中的保真度决定因素以及对病毒适应性和RNA合成的影响; 2)鉴定CoV保真度复合物中的蛋白质和决定因素; 3)确定保真度改变对体内复制和发病机制的影响。拟议的目标建立在丹尼森和Baric实验室在CoV(MHV和SARS-CoV)反向遗传学,复制酶蛋白诱变和功能,发病机制,免疫反应和合成基因组学方面的互补优势和合作基础上。拟议实验的结果将确定复制,发病机制和毒力的关键保真度决定因素,定义耐受保真度的范围,并确定新的方法CoV减毒和抑制。

项目成果

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Ralph S Baric其他文献

Ralph S Baric的其他文献

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{{ truncateString('Ralph S Baric', 18)}}的其他基金

Core A: Administrative Core
核心A:行政核心
  • 批准号:
    10513680
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
Development of direct-acting flavivirus inhibitors
直接作用黄病毒抑制剂的开发
  • 批准号:
    10513687
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
Core B: Virology Core
核心 B:病毒学核心
  • 批准号:
    10425027
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
Research Project 1: Coronavirus antiviral lead development and combination testing
研究项目1:冠状病毒抗病毒先导药物开发和组合测试
  • 批准号:
    10513684
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
RAPIDLY EMERGING ANTIVIRAL DRUG DEVELOPMENT INITIATIVE- AViDD CENTER (READDI-AC)
迅速崛起的抗病毒药物开发计划 - AViDD 中心 (READDI-AC)
  • 批准号:
    10513679
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
Development of Antivirals against Filovirus Replication
抗丝状病毒复制的抗病毒药物的开发
  • 批准号:
    10513686
  • 财政年份:
    2022
  • 资助金额:
    $ 56万
  • 项目类别:
Systems Immunogenetics of Emerging Coronavirus Infections in the Collaborative Cross
协作交叉中新出现的冠状病毒感染的系统免疫遗传学
  • 批准号:
    10180497
  • 财政年份:
    2020
  • 资助金额:
    $ 56万
  • 项目类别:
North Carolina Seronet Center for Excellence
北卡罗来纳州 Seronet 卓越中心
  • 批准号:
    10855051
  • 财政年份:
    2020
  • 资助金额:
    $ 56万
  • 项目类别:
Project 1: Serological Correlates of SARS CoV2 Immunity and Disease
项目 1:SARS CoV2 免疫与疾病的血清学相关性
  • 批准号:
    10688377
  • 财政年份:
    2020
  • 资助金额:
    $ 56万
  • 项目类别:
Human antibody-based countermeasures against the Coronavirus SARS-CoV-2
基于人类抗体的冠状病毒 SARS-CoV-2 对策
  • 批准号:
    10264078
  • 财政年份:
    2020
  • 资助金额:
    $ 56万
  • 项目类别:

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Mechanistic insights into multifaceted roles of coronavirus exoribonuclease complex
冠状病毒外核糖核酸酶复合物多方面作用的机制见解
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探索冠状病毒外核糖核酸酶作为抗病毒靶点
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利用 SARS-CoV-2 nsp14 3-5-核糖核酸外切酶作为抗病毒化疗的靶点
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