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.

项目概要冠状病毒科 (CoV) 中的病毒曾两次作为人畜共患病出现，具有大流行的潜力进入 21 世纪，中东呼吸综合征 (MERS)-CoV 仍在继续造成严重的人类疾病。导致新的致命呼吸道感染病例，死亡率高达 35%，此外，还导致严重急性呼吸道综合症。目前正在传播的类（SARS）蝙蝠冠状病毒能够感染人类细胞，从而确定了未来的风险人畜共患冠状病毒的出现没有针对任何人类或人畜共患冠状病毒的批准疫苗或抗病毒药物，强调识别脆弱且广泛保守的 CoV 治疗靶点的重要性大多数RNA病毒产生种间所需的遗传多样性。通过缺乏校对的容易出错的 RNA 依赖性 RNA 聚合酶 (RdRps) 进行运动和适应。相比之下，所有 CoV 都在非结构蛋白 14 (nsp14-ExoN) 中编码 3' 至 5' 核糖核酸外切酶 (ExoN)，即四年来，通过依赖RNA的RNA校对，冠状病毒进化和适应的关键驱动力。为该计划提供资金，我们已经证明 CoV nsp14-ExoN 介导高保真复制，并且 CoV 缺乏 ExoN 活性 (ExoN(-)) 在细胞培养物感染期间不太适合，对 RNA 诱变剂更敏感，并且我们的研究结果表明，不同的 β-CoV（中东呼吸综合征冠状病毒）在小鼠模型中减弱。 SARS-CoV 和鼠肝炎病毒 (MHV) - 对 ExoN 维持活力和维持活性有不同的要求最后，ExoN 可能在 CoV 抗性中发挥重要且之前未预测到的功能。因此，我们发表的初步研究支持了这一科学前提。 nsp14-ExoN 是通过病毒中的功能调节 CoV 适应性、进化和发病机制的主要调节因子该提案的具体目标将定义：1) nsp14-ExoN 介导的 CoV 复制、保真度和功能的序列和结构决定因素干扰素敏感性；2) nsp14、nsp12-RdRp 和 CoV 复制酶其他部位的适应补偿 ExoN 介导的保真度损失；3) ExoN 调节先天抗病毒的机制体外和体内免疫反应 nsp14 的高分辨率结构的可用性； MHV、SARS-CoV 和 MERS-CoV 的遗传学系统以及 SARS-CoV 的稳健相关动物模型； MERS-CoV 将使我们能够解决这些问题，从而全面了解 ExoN 这些研究将促进冠状病毒复制、适应和发病机制中的作用和机制。以 ExoN 为目标的方法作为稳定减毒 CoV 疫苗和新型抗病毒药物的基础。