Innate immune factors governing restriction of human coronaviruses

点击翻译按钮获取中文摘要

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.