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 IFNß. 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 IFNß 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.