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：
10265802
负责人：
Ralph S Baric
金额：
$ 44.73万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-06 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10265802
关键词：
5&apos -exoribonuclease Active Sites Address Animal Model Animals Antiviral Agents Attenuated Cell Culture Techniques Cells Chiroptera Complex Coronavirus Crystallization Data Engineering Evolution Exonuclease Exoribonucleases Family Funding Future Genetic Genetic Transcription Genetic Variation Genome Human Immune Immune Evasion Immune response Immunologic Surveillance In Vitro Infection Innate Immune Response Interferon-beta Interferons Maintenance Maps Mediating Middle East Respiratory Syndrome Coronavirus Movement Murine hepatitis virus Mutagenesis Mutagens Mutation Natural Immunity Nonstructural Protein Pathogenesis Phenotype Play Positioning Attribute Proteins Public Health Publishing RNA RNA Viruses RNA chemical synthesis RNA-Directed RNA Polymerase Regulation Resistance Resolution Respiratory Tract Infections Risk Role SARS coronavirus Sequence Alignment Severe Acute Respiratory Syndrome Structure System Testing Therapeutic Intervention Vaccines Viral Pathogenesis Virulence Virus Virus Replication Zinc Fingers Zoonoses airway epithelium attenuation betacoronavirus cost experimental study fitness human coronavirus human disease human model immunoregulation in vivo mortality mouse model mutant new therapeutic target novel novel coronavirus pandemic disease pressure programs protein function replicase reverse genetics sensor targeted treatment therapy development vaccine development viral RNA viral fitness zoonotic coronavirus

项目摘要

PROJECT SUMMARY Viruses in the Coronaviridae family (CoVs) have emerged as zoonoses with pandemic potential twice in the 21st century, causing severe human disease. Middle East respiratory syndrome (MERS)-CoV continues to cause new cases of lethal respiratory infections with 35% mortality. Further, severe acute respiratory syndrome (SARS)-like bat CoVs currently circulating are capable of infecting human cells, establishing the risk for future emergence of zoonotic CoVs. There are no approved vaccines or antivirals for any human or zoonotic CoV, emphasizing the importance of identifying vulnerable and broadly conserved CoV targets for therapeutic intervention and vaccine development. Most RNA viruses generate genetic diversity required for interspecies movement and adaptation via error-prone RNA-dependent RNA polymerases (RdRps) that lack proofreading. In contrast, all CoVs encode a 3'-to-5' exoribonuclease (ExoN) in nonstructural protein 14 (nsp14-ExoN) that is a key driver of CoV evolution and adaptation via RNA-dependent RNA proofreading. During the four years of funding for this program, we have shown that CoV nsp14-ExoN mediates high-fidelity replication and that CoVs lacking ExoN activity (ExoN(-)) are less fit during infection in cell culture, more sensitive to RNA mutagens, and attenuated in a murine model of SARS-CoV infection. Our findings suggest that divergent β-CoVs - MERS-CoV, SARS-CoV, and murine hepatitis virus (MHV) - have differential requirements for ExoN to sustain viability and overall fitness. Finally, ExoN may play important and previously unpredicted functions in CoV resistance to host innate immune surveillance. Thus, our published and preliminary studies support the scientific premise that nsp14-ExoN is a master regulator of CoV fitness, evolution, and pathogenesis via functions in viral replication, fidelity, and evasion of host innate immune responses. Specific aims of this proposal will define: 1) Sequence and structural determinants of nsp14-ExoN-mediated functions in CoV replication, fidelity, and interferon sensitivity; 2) Adaptations in nsp14, nsp12-RdRp, and elsewhere in the CoV replicase that compensate for loss of ExoN-mediated fidelity; and 3) Mechanisms of ExoN regulation of the innate antiviral immune response in vitro and in vivo. The availability of a high-resolution structure of nsp14; facile reverse genetics systems for MHV, SARS-CoV, and MERS-CoV; and robust, relevant animal models for SARS-CoV and MERS-CoV will allow us to address these questions, resulting in a comprehensive understanding of ExoN roles and mechanisms in CoV replication, adaptation, and pathogenesis. These studies will catalyze approaches targeting ExoN as a basis for stably attenuated CoV vaccines and novel antiviral drugs.

项目摘要冠状病毒家族（COV）中的病毒已成为人畜共患病，两次是大流行潜力 21世纪，导致严重的人类疾病。中东呼吸综合征（MERS）-COV继续引起新的致命呼吸道感染病例，死亡率为35％。此外，严重的急性呼吸综合症（SARS） - 类似于蝙蝠COV当前循环的能够感染人类细胞，确立了未来的风险人畜共患病的出现。任何人类或人畜共患疫苗或抗病毒药都没有批准的疫苗或抗病毒药。强调确定脆弱和广泛构成治疗的COV目标的重要性干预和疫苗开发。大多数RNA病毒产生种间所需的遗传多样性通过缺乏校对的依赖RNA的RNA聚合酶（RDRP）的运动和适应。相比之下，所有COV在非结构蛋白14（NSP14-EXON）中编码3'至5'Exoriboneclease（外显子）通过RNA依赖性RNA校对的COV进化和适应的关键驱动力。在四年为该计划的资金，我们已经证明COV NSP14- exon介导了高保真复制，并且COVS 缺乏外显子活性（外显子（ - ）在细胞培养中感染期间的拟合度较小，对RNA诱变剂和更敏感在SARS-COV感染的鼠模型中减弱。我们的发现表明β-covs-mers-cov， SARS -COV和鼠肝炎病毒（MHV） - 对外显子具有不同的要求，以维持生存能力和整体健身。最后，外显子可能在COV抗性中起重要且以前没有预测的功能主持先天免疫监视。这，我们已发表和初步研究支持科学前提 NSP14-EXON是通过病毒中功能的COV适应性，进化和发病机理的主要调节剂宿主先天免疫反应的复制，忠诚和演变。该提议的具体目的将定义：1） NSP14-外年介导的功能的序列和结构确定剂在COV复制，保真度和干扰素灵敏度； 2）在NSP14，NSP12-RDRP和COV复制酶的其他位置进行改编补偿外显子介导的保真度的损失； 3）先天抗病毒的外显子调节机制体外和体内免疫反应。 NSP14的高分辨率结构的可用性；便利的反向 MHV，SARS-COV和MERS-COV的遗传系统； SARS-COV的强大，相关的动物模型 MERS-COV将使我们能够解决这些问题，从而对外显子有全面的了解 COV复制，适应和发病机理中的作用和机制。这些研究将催化接近靶向外显子作为稳定减弱的COV疫苗和新型抗病毒药的基础。