Fluorescence-Activated Cell Sorting-Based Analysis Reveals an Asymmetric Induction of Interferon-Stimulated Genes in Response to Seasonal Influenza A Virus

Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and IFN-stimulated genes (ISGs) in infected cell cultures. However, the spatiotemporal dynamics of the IFN reaction are incom-pletely understood, as previous studies investigated mainly the population responses of virus-infected cultures, although sub-stantial cell-to-cell variability has been documented. We devised a fluorescence-activated cell sorting-based assay to simultane-ously quantify expression of viral antigens and ISGs, such as ISG15, MxA, and IFIT1, in IAV-infected cell cultures at the single-cell level. This approach revealed that seasonal IAV triggers an unexpected asymmetric response, as the major cell populations expressed either viral antigen or ISG, but rarely both. Further investigations identified a role of the viral NS1 protein in blocking ISG expression in infected cells, which surprisingly did not reduce paracrine IFN signaling to noninfected cells. Interestingly, viral ISG control was impaired in cultures infected with avian-origin IAV, including the H7N9 virus from eastern China. This phenotype was traced back to polymorphic NS1 amino acids known to be important for stable binding of the polyadenylation factor CPSF30 and concomitant suppression of host cell gene expression. Most significantly, mutation of two amino acids within the CPSF30 attachment site of NS1 from seasonal IAV diminished the strict control of ISG expression in infected cells and sub-stantially attenuated virus replication. In conclusion, our approach revealed an asymmetric, NS1-dependent ISG induction in cultures infected with seasonal IAV, which appears to be essential for efficient virus propagation