Modulation of Influenza A virus NS1 expression reveals prioritization of host response antagonism at single-cell resolution

Influenza A virus (IAV) is an important human respiratory pathogen that causes significant seasonal epidemics and potential devastating pandemics. As part of its life cycle, IAV encodes the multifunctional protein NS1, that, among many roles, prevents immune detection and limits interferon (IFN) production. As different host immune pathways exert different selective pressures against IAV as replication progresses, we expect a prioritization in the host immune antagonism by NS1. In this work, we profiled bulk transcriptomic differences in a primary bronchial epithelial cell model facing IAV infections at distinct NS1 levels. We further demonstrated that the intracellular NS1 levels in-part shapes the host response heterogeneity at single cell level. We found that modulation of NS1 levels reveal a ranking in its inhibitory roles: modest NS1 expression is sufficient to inhibit immune detection, and thus the expression of pro-inflammatory cytokines (including IFNs), but higher levels are required to inhibit IFN signaling and ISG expression. Lastly, inhibition of chaperones related to the unfolded protein response requires the highest amount of NS1, often associated with later stages of viral replication. This work demystifies some of the multiple functions ascribed to IAV NS1, highlighting the prioritization of NS1 in antagonizing the different pathways involved in the host response to IAV infection. Overall design: To better capture the relationship between IAV NS1 expression and the host antiviral response, we characterized the transcriptional landscape of primary bronchial epithelial cells upon infection with wild type and recombinant viruses with varying levels of NS1. We utilized a previously described recombinant virus (lab adapted A/Puerto Rico/8/34 (H1N1)), in which the viral NS1 and NEP open reading frames are separated to enable the insertion of two miRNA targets. We hence compared the NS1 expression levels during active infections via RNA-seq, contrasting IAV-NS1-T with wildtype IAV (IAV-WT) and IAV-?NS1, a recombinant virus lacking NS1-specific open reading frame while expressing NEP. In addition, we sought to investigate whether the host antagonism prioritization can also be demonstrated by cell-to-cell NS1 expression heterogeneity during wild-type IAV infection. We employed single cell 3' RNA-seq technology to characterize both viral transcript abundance and host response in NHBE infected with IAV-WT.