Innate immune factors governing restriction of human coronaviruses

控制人类冠状病毒限制的先天免疫因素

• 批准号: 10506519
• 负责人: Caitlin Stoddard
• 金额: $ 12.86万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10506519
• 关键词: 2019-nCoV; Address; Antiviral Agents; Attention; Biochemical; COVID-19; COVID-19 pandemic; CRISPR screen; CRISPR/Cas technology; Cell Death; Cell Line; Cell model; Cells; Cessation of life; Co-Immunoprecipitations; Communities; Comparative Study; Complex; Coronavirus; Coronavirus Infections; Custom; Disease; Disease Outbreaks; Disease Outcome; Disease Progression; Equilibrium; Evaluation; Future; Gene Targeting; Genes; H1299; Human; Immune; Immune response; Immunologic Factors; Infection; Infection Control; Infectious Diseases Research; Innate Immune Response; Integration Host Factors; Interferon Type I; Interferon-beta; Interferons; Knock-out; Libraries; Link; Literature; Mass Spectrum Analysis; Mediating; Mentors; Middle East Respiratory Syndrome Coronavirus; Natural Immunity; Outcome; Pathogenesis; Pathogenicity; Phase; Play; Pneumonia; Production; Proteins; Reporting; Risk; Role; SARS coronavirus; SARS-CoV-2 infection; Series; Signal Transduction; Syndrome; Testing; Therapeutic; Time; Up-Regulation; Viral; Viral Proteins; Virus; Work; base; comparative; coronavirus disease; experimental study; gene interaction; genome-wide analysis; high throughput screening; human coronavirus; knockout gene; lung Carcinoma; novel therapeutics; protective effect; respiratory; response; screening; transcriptome sequencing; zoonotic coronavirus

PROJECT SUMMARY/ABSTRACT The recent SARS-CoV-2 pandemic has caused global catastrophe and needs little introduction. One of seven coronaviruses known to infect humans (HCoVs), SARS-CoV-2 infection leads to a range of pathogenic outcomes ranging from asymptomatic infection to severe pneumonia and death. Conversely, four HCoVs are endemic, circulate globally and typically cause only mild illness. The differences in pathogenesis between more lethal HCoVs and endemic HCoVs raises the possibility that there are differences in the innate immune response upon infection with the different HCoV species. The Type I interferon (IFN) response is the first line of innate immune defense against viruses and involves activation of a suite of IFN-stimulated genes (ISGs) that maintain IFN production and generate a cellular antiviral state. Several high-throughput screening studies have identified ISGs that contribute to the protective IFN response against SARS-CoV-2 infection, several of which did not overlap, suggesting much more remains to be learned. A smaller subset of ISGs is known to participate in IFN-mediated signaling in cells infected with endemic HCoVs such as HCoV-OC43, but the studies have not been comprehensive, and the role for IFN remains complex and understudied. Therefore, comprehensive approaches are needed to identify ISGs that are restrictive against HCoVs, particularly endemic HCoVs. Identifying ISGs that are active against endemic HCoVs will pave the way for comparative studies of ISG activity between HCoVs that are more pathogenic. Identifying the mechanisms that govern differential ISG activity against broad HCoV species may inform broad antiviral therapeutic strategies targeting current HCoVs and those that are likely to emerge in the future. In preliminary studies, we identified H1299 cells as permissible to infection with HCoV-OC43, and found a 92-fold protective effect against HCoV-OC43 infection in the presence of IFNβ. Here, we propose to identify IFN-mediated restriction factors against HCoV-OC43. To address the hypothesis that HCoVs with different pathogenic features are regulated by different features of the innate immune response, we will do a comparative study of ISG activity between SARS-CoV-2 and HCOV-OC43 and will seek to identify mechanistic explanations for differences we identify. During the mentored K99 phase: we will (1) develop a customized CRISPR-Cas9 knockout library for use with HCoV-OC43 in H1299 cells, (2) screen for ISGs responsible for the 92-fold protective IFNβ effect, (3) validate hits with single gene knockout cell lines and test for activity against SARS-CoV-2 and (4) initiate experiments to determine if these ISGs reflect direct viral protein-host protein interactions, if time allows. During the independent R00 phase: we will (1) comparatively test viral protein-host ISG interactions identified by co-immunoprecipitation and mass spectrometry, (2) complete expansion of CRISPR screening to other respiratory cell lines, and (3) initiate preliminary structural studies to characterize ISG-viral protein interfaces.

项目摘要/摘要 最近的SARS-CoV-2大流行已经造成了全球灾难，不需要介绍什么。七个中的一个 已知可感染人类的冠状病毒(HCoV)，SARS-CoV-2感染会导致一系列致病后果 从无症状感染到严重肺炎和死亡。相反，四种HCoV是地方性的， 在全球范围内传播，通常只会导致轻微的疾病。致命性较强的两种疾病的发病机制不同 HCoV和地方性HCoV增加了在先天免疫反应中存在差异的可能性 感染不同种类的HCoV。 I型干扰素(干扰素)反应是针对病毒和 包括激活一套干扰素刺激基因(ISG)，维持干扰素的产生并产生细胞 抗病毒状态。几项高通量筛查研究已经确定了有助于保护 对SARS-CoV-2感染的干扰素反应，其中几种不重叠，表明还有更多剩余 要被学习。已知有较小的ISG亚群参与感染的细胞中干扰素介导的信号转导 地方性HCoV如HCoV-OC43，但研究还不全面，而干扰素的作用 仍然很复杂，研究还不够深入。因此，需要综合的方法来确定符合以下条件的ISG 限制HCov，特别是地方性HCov。识别对地方性HCoV有积极作用的ISG 将为更具致病性的HCoV之间的ISG活性的比较研究铺平道路。识别 控制ISG对广泛的HCoV物种的不同活性的机制可能为广泛的抗病毒提供信息 针对目前和未来可能出现的HCoV的治疗策略。 在初步研究中，我们确定H1299细胞允许感染HCoV-OC43，并发现 干扰素β对人类冠状病毒OC43感染的92倍保护作用在这里，我们建议确定 干扰素介导的对HCoV-OC43的限制因子。为了解决HCov与不同的 致病特性是由不同特性调节的先天免疫反应，我们将做一个比较 SARS-CoV-2和hCoV-OC43间ISG活性的研究及其机制解释 对于我们确定的差异。 在指导的K99阶段：我们将(1)开发一个定制的CRISPR-Cas9基因敲除库，用于 HCoV-OC43在H1299细胞中的表达，(2)筛选具有92倍保护性干扰素β效应的ISGs，(3)验证 用单基因敲除细胞系命中并检测抗SARS-CoV-2的活性和(4)启动实验以 如果时间允许，确定这些ISG是否反映了病毒蛋白-宿主蛋白的直接相互作用。 在独立的R00阶段：我们将(1)比较测试病毒蛋白与宿主ISG的相互作用 免疫共沉淀和质谱学，(2)CRISPR筛查完全扩展到其他 (3)启动初步结构研究，以确定ISG-病毒蛋白界面的特征。