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ABSTRACT Influenza A viruses (IAVs) can cause zoonotic infections with pandemic potential when most of the human population is immunologically naive. After a pandemic, IAVs evolve to become seasonal in the human host by acquiring adaptive mutations. We have previously reported that the interferon (IFN)-inducible tripartite motif 22 (TRIM22) protein restricts the replication of seasonal IAVs by direct interaction with the viral nucleoprotein (NP), leading to its polyubiquitination and proteasomal degradation. Here we show that, in contrast to seasonal H1N1 IAVs, the 2009 pandemic H1N1 strain as well as H1N1 strains from the 1930s are resistant to TRIM22 restriction. We demonstrate that arginine-to-lysine substitutions conferring an increased sensitivity to TRIM22-dependent ubiquitination accumulated progressively in the NP of seasonal influenza A (H1N1) viruses between 1918 and 2009. Our findings suggest that during long-term circulation and evolution of IAVs in humans, adaptive mutations are favored at the expense of an increased sensitivity to some components of the innate immune response. IMPORTANCE We have uncovered that long-term circulation of seasonal influenza A viruses (IAV) in the human population resulted in the progressive acquisition of increased sensitivity to a component of the innate immune response: the type I interferon-inducible TRIM22 protein, which acts as a restriction factor by inducing the polyubiquitination of the IAV nucleoprotein (NP). We show that four arginine residues present in the NP of the 1918 H1N1 pandemic strain and early postpandemic strains were progressively substituted for by lysines between 1918 and 2009, rendering NP more susceptible to TRIM22-mediated ubiquitination. Our observations suggest that during long-term evolution of IAVs in humans, variants endowed with increased susceptibility to TRIM22 restriction emerge, highlighting the complexity of selection pressures acting on the NP.

ABSTRACT The optimized exploitation of cell resources is one cornerstone of a successful infection. Differential mapping of host-pathogen protein-protein interactions (PPIs) on the basis of comparative interactomics of multiple strains is an effective strategy to highlight correlations between host proteome hijacking and biological or pathogenic traits. Here, we developed an interactomic pipeline to deliver high-confidence comparative maps of PPIs between a given pathogen and the human ubiquitin proteasome system (UPS). This subarray of the human proteome represents a range of essential cellular functions and promiscuous targets for many viruses. The screening pipeline was applied to the influenza A virus (IAV) PB2 polymerase proteins of five strains representing different levels of virulence in humans. An extensive PB2-UPS interplay has been detected that recapitulates the evolution of IAVs in humans. Functional validation with several IAV strains, including the seasonal H1N1pdm09 and H3N2 viruses, confirmed the biological relevance of most identified UPS factors and revealed strain-independent and strain-specific effects of UPS factor invalidation on IAV infection. This strategy is applicable to proteins from any other virus or pathogen, providing a valuable resource with which to explore the UPS-pathogen interplay and its relationship with pathogenicity. IMPORTANCE Influenza A viruses (IAVs) are responsible for mild-to-severe seasonal respiratory illness of public health concern worldwide, and the risk of avian strain outbreaks in humans is a constant threat. Elucidating the requisites of IAV adaptation to humans is thus of prime importance. In this study, we explored how PB2 replication proteins of IAV strains with different levels of virulence in humans hijack a major protein modification pathway of the human host cell, the ubiquitin proteasome system (UPS). We found that the PB2 protein engages in an extended interplay with the UPS that evolved along with the virus’s adaptation to humans. This suggests that UPS hijacking underlies the efficient infection of humans and can be used as an indicator for evaluation of the potential of avian IAVs to infect humans. Several UPS factors were found to be necessary for infection with circulating IAV strains, pointing to potential targets for therapeutic approaches.