Innate immune factors governing restriction of human coronaviruses

项目总结/摘要 最近的SARS-CoV-2大流行造成了全球性的灾难，几乎不需要介绍。兄弟姐妹共有七 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 从无症状感染者到重症肺炎和死亡。相反，四种HCoV是地方性的， 在全球范围内传播，通常只引起轻微疾病。在发病机制上的差异，更致命的 HCoV和地方性HCoV提出了先天免疫反应存在差异的可能性 感染不同种类的HCoV。 I型干扰素（IFN）应答是针对病毒的先天免疫防御的第一线， 涉及一组IFN刺激基因（ISG）的激活，这些基因维持IFN的产生并产生细胞免疫应答。 抗病毒状态几项高通量筛选研究已经确定了有助于保护性的ISG IFN对SARS-CoV-2感染的反应，其中几个没有重叠，表明还有更多的剩余 学习。已知ISG的一个较小子集参与感染了IFN-γ的细胞中IFN-介导的信号传导。 地方性HCoV，如HCoV-OC 43，但研究还不全面， 仍然很复杂而且研究不足。因此，需要采取综合办法来确定 限制性抗HCoV，特别是地方性HCoV。鉴定对地方性HCoV有活性的ISG 将为致病性更强的HCoV之间ISG活性的比较研究铺平道路。识别 控制ISG对广泛HCoV物种的差异活性的机制可能为广泛的抗病毒药物提供信息， 治疗策略针对目前的HCoV和那些可能出现在未来。 在初步研究中，我们鉴定了H1299细胞可被HCoV-OC 43感染，并发现 在IFNβ存在下，对HCoV-OC 43感染的92倍保护作用。在这里，我们建议确定 IFN介导的针对HCoV-OC 43的限制性因子。为了解决这一假设，HCoV与不同的 致病特征受先天免疫反应的不同特征调节，我们将做一个比较 研究SARS-CoV-2和HCOV-OC 43之间ISG活性，并将寻求确定机制解释 我们发现的差异。 在指导K99阶段：我们将（1）开发定制的CRISPR-Cas9敲除文库，用于 HCoV-OC 43在H1299细胞中的表达，（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命中，并测试抗SARS-CoV-2的活性，以及（4）启动实验， 如果时间允许，确定这些ISG是否反映了直接的病毒蛋白-宿主蛋白相互作用。 在独立R 00阶段：我们将（1）比较性检测通过以下方法鉴定的病毒蛋白-宿主ISG相互作用： 免疫共沉淀和质谱，（2）将CRISPR筛选完全扩展到其他 呼吸道细胞系，和（3）启动初步的结构研究，以表征ISG-病毒蛋白质界面。

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.