Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

Interferons establish an antiviral state in responding cells through the induction of hundreds of interferon-stimulated genes (ISGs). ISGs antagonize viral pathogens directly through diverse mechanisms acting at different stages of viral life cycles, and indirectly by modulating cell cycle and promoting programmed cell death. The mechanisms of action and viral specificities for most ISGs remain incompletely understood. To enable the high throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating the potentially confounding effects of endogenous IFN and potential antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG induction in isogenic cell lines with and without the capacity to respond to IFN. Engineered CRISPRa cell lines demonstrated inducible, robust, and specific gRNA-directed expression of ISGs, which are functional in restricting viral infection. Using this platform, we screened for ISGs that restrict SARS-CoV-2, the causative agent of the COVID-19 pandemic. Results included ISGs previously described to restrict SARS-CoV-2 as well as multiple novel candidate antiviral factors. We validated a subset of candidate hits by complementary targeted CRISPRa and ectopic cDNA expression infection experiments, which, among other hits, confirmed OAS1 as a SARS-CoV-2 restriction factor. OAS1 exhibited strong antiviral effects against SARS-CoV-2, and these effects required OAS1 catalytic activity. These studies demonstrate a robust, high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by the identification of multiple novel SARS-CoV-2 restriction factors.

A diverse collection of viral pathogens target airway epithelial cells for infection, with effects ranging from mild upper respiratory tract symptoms to death of the infected individual. Among these pathogens are recently discovered and/or emergent viruses that sometimes fail to infect commonly used, immortalized cell lines and for which infection phenotypes in the respiratory tract remain unknown. Human airway epithelial cultures have been developed over the past several decades and have proven to be a useful model system in culturing hard-to-grow viruses and assaying various features of infection in a physiologically relevant setting. This article includes methods for the generation of well-differentiated human airway epithelial cell cultures at air-liquid interface that recapitulate the mucosal epithelium of the trachea/bronchus in vivo. We further detail inoculation of these cultures with respiratory viruses-specifically rhinovirus, influenza virus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-and provide a protocol for the detection of double-stranded RNA or viral antigen-positive cells by immunofluorescence microscopy. These techniques, together with a post-imaging analysis, can be applied to characterize the efficiency of infection and kinetics of spread within the airway epithelium. Furthermore, these methods can be utilized in conjunction with antibodies against cellular targets to determine cell tropism and colocalization with specific host factors during infection.

Lymph nodes are the primary site of replication for measles virus (MeV). Here, we modeled MeV infection in human tonsil explants, utilizing a clinical strain of MeV that expresses GFP. We show that MeV replicates efficiently in this tissue as measured by increasing infectious virus production and GFP+ cells (>10% of tonsillar cells by day 8). Using scRNA-Seq, we identified 29 cell populations, all of which were susceptible to MeV. While T cells were the most abundant cell type in the lymphoid explants, B cells were the dominant infected population. Flow cytometry analysis revealed that the preferential infection of B cells was associated with higher CD150 expression. We found that while germinal center B cells were the largest population of infected B cells, there were no differences in susceptibility to MeV among individual B cell subsets. Among CD3+ T cells, infection in both the CD4+ and CD8+ compartments displayed bias towards antigen experienced subsets and away from naive cells, consistent with relative CD150 expression. Differential gene expression analysis revealed that the host response to MeV was dominated by the potent induction of interferon stimulated genes within both T and B cells. These data provide new insights into how MeV infection progresses in lymph nodes, a critical launching point for pathogenesis.