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: 10673163
• 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/10673163
• 关键词: 2019-nCoV; Address; Antiviral Agents; Attention; Biochemical; COVID-19; COVID-19 pandemic; CRISPR screen; CRISPR/Cas technology; Cell Death Induction; Cell Line; Cell model; Cells; Cessation of life; Co-Immunoprecipitations; Communities; Comparative Study; Complex; Coronavirus; Coronavirus Infections; Disease; 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; Learning; Libraries; Link; Literature; Mass Spectrum Analysis; Mediating; Mentors; Middle East Respiratory Syndrome Coronavirus; Natural Immunity; Nucleic Acids; 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 Physiology; Viral Proteins; Virus; Work; comparative; coronavirus disease; experimental study; future outbreak; gene interaction; genome-wide analysis; high throughput screening; human coronavirus; knockout gene; lung Carcinoma; novel therapeutics; permissiveness; 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大流行造成了全球灾难，几乎不需要引入。七个 已知感染人类的​​冠状病毒（HCOV），SARS-COV-2感染导致一系列致病结果 从不对称感染到严重的肺炎和死亡。相反，四个HCOV是内在的， 全球循环，通常仅引起轻度疾病。更致命的发病机理的差异 HCOV和内在HCOVS增加了一种可能性，即先天免疫反应有所不同 感染不同的HCOV物种。 I类干扰素（IFN）反应是针对病毒和 涉及激活一组IFN刺激的基因（ISG） 抗病毒状态。几项高通量筛查研究已经确定了有助于受保护的ISG IFN对SARS-COV-2感染的反应，其中几种没有重叠，这表明远不止保留 要学习。已知ISG的较小子集参与感染的细胞中IFN介导的信号传导 诸如HCOV-OC43之类的内粒HCOV，但研究并不全面，而IFN的作用 保持复杂和理解。因此，需要全面的方法来识别 对HCOV的限制，尤其是内在HCOV。识别具有活性的ISG针对内在HCOV 将为更具致病性的HCOV之间的ISG活性进行比较研究铺平道路。识别 针对广泛的HCOV物种的差异性ISG活性的机制可能告知广泛的抗病毒 针对当前HCOV的治疗策略以及将来可能出现的治疗策略。 在初步研究中，我们识别出H1299细胞被允许感染HCOV-OC43，并发现 在IFNβ存在下，针对HCOV-OC43感染的92倍受保护作用。在这里，我们建议确定 IFN介导的限制因子针对HCOV-OC43。解决以下假设： 致病特征由先天免疫反应的不同特征调节，我们将进行比较 研究SARS-COV-2和HCOV-OC43之间的ISG活性，并将寻求识别机械解释 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淘汰库供与 H1299细胞中的HCOV-OC43，（2）ISG的筛选，负责92倍受保护的IFNβ效应，（3）验证 用单基因基因敲除细胞系进行命中，并测试针对SARS-COV-2的活性，（4）启动实验 确定这些ISG是否反映了直接的病毒蛋白宿主蛋白相互作用，如果允许的话。 在独立的R00阶段：我们将（1）相对测试的病毒蛋白宿主ISG ISG相互作用 共免疫沉淀和质谱法，（2）将CRISPR筛选到其他 呼吸细胞系，（3）启动初步结构研究，以表征ISG病毒蛋白界面。