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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要最近的 SARS-CoV-2 大流行造成了全球灾难，几乎不需要介绍七大灾难之一。已知感染人类的冠状病毒 (HCoV)、SARS-CoV-2 感染会导致一系列致病结果从无症状感染到严重肺炎和线下死亡，四种 HCoV 呈地方性流行。在全球范围内被破坏，通常只引起轻微的疾病。更致命的疾病之间的发病机制存在差异。 HCoV 和地方性 HCoV 提出了先天免疫反应存在差异的可能性。不同 HCoV 物种的感染。 I 型干扰素 (IFN) 反应是针对病毒和病毒的先天免疫防御的第一道防线。涉及激活一组 IFN 刺激基因 (ISG)，这些基因维持 IFN 产生并产生细胞一些高通量筛选研究已经确定了有助于保护性的 ISG。针对 SARS-CoV-2 感染的 IFN 反应，其中几个没有重叠，表明还有更多的反应已知一小部分 ISG 参与感染细胞中 IFN 介导的信号传导。地方性 HCoV，如 HCoV-OC43，但研究尚不全面，IFN 的作用仍然复杂且尚未得到充分研究，因此需要采取全面的方法来识别 ISG。限制 HCoV，特别是地方性 HCoV 识别对地方性 HCoV 有活性的 ISG。将为鉴定更具致病性的 HCoV 之间的 ISG 活性的比较研究铺平道路。控制针对广泛 HCoV 物种的不同 ISG 活性的机制可能为广泛的抗病毒药物提供信息针对当前 HCoV 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细胞可以感染HCoV-OC43，并发现在 IFNβ 存在的情况下，对 HCoV-OC43 感染具有 92 倍的保护作用。 IFN 介导的针对 HCoV-OC43 的限制因子旨在解决 HCoV 具有不同的假设。致病特征是由先天免疫反应的不同特征调节的，我们将做一个比较研究 SARS-CoV-2 和 HCOV-OC43 之间的 ISG 活性，并将寻求确定机制解释对于我们识别的差异。在指导的 K99 阶段：我们将 (1) 开发定制的 CRISPR-Cas9 敲除文库，用于 H1299 细胞中的 HCoV-OC43，(2) 筛选负责 92 倍保护性 IFNβ 效应的 ISG，(3) 验证用单基因敲除细胞系进行攻击并测试针对 SARS-CoV-2 的活性，以及 (4) 启动实验如果时间允许，确定这些 ISG 是否反映直接的病毒蛋白-宿主蛋白相互作用。在独立R00阶段：我们将（1）比较测试由以下方法确定的病毒蛋白-宿主ISG相互作用：免疫共沉淀和质谱分析，(2) 将 CRISPR 筛选完全扩展到其他领域呼吸道细胞系，(3) 启动初步结构研究以表征 ISG-病毒蛋白界面。