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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 distinct 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, at single cell level, the intracellular amount of NS1 in-part shapes the heterogeneity of the host response. 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.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38β is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38β substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38β and supports exploring p38β inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.

Summary: Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence. : Muñoz-Moreno et al. report that influenza A virus NS1 undergoes diverse and unpredictable evolutionary pathways based on its different phylogenetic lineages. A high-throughput approach using a barcoded library is used to test the interactions between NS1-recombinant viruses and to study their preference for specific or multiple hosts. Keywords: influenza virus, orthomyxovirus, NS1 evolution, IFN response, innate immunity, barcoded library

Responding to an influenza A virus (IAV) infection demands an effective intrinsic cellular defense strategy to slow replication. To identify contributing host factors to this defense, we exploited the host microRNA pathway to perform an in vivo RNAi screen. To this end, IAV, lacking a functional NS1 antagonist, was engineered to encode individual siRNAs against antiviral host genes in an effort to rescue attenuation. This screening platform resulted in the enrichment of strains targeting virus-activated transcription factors, specific antiviral effectors, and intracellular pattern recognition receptors (PRRs). Interestingly, in addition to RIG-I, the PRR for IAV, a virus with the capacity to silence MDA5 also emerged as a dominant strain in wild-type, but not in MDA5-deficient mice. Transcriptional profiling of infected knockout cells confirmed RIG-I to be the primary PRR for IAV but implicated MDA5 as a significant contributor to the cellular defense against influenza A virus.