RESUMEN
Homotypic co-infections with influenza viruses are described to increase genetic population diversity, to drive viral evolution and to allow genetic complementation. Less is known about heterotypic co-infections between influenza A (IAV) and influenza B (IBV) viruses. Previous publications showed that IAV replication was suppressed upon co-infection with IBV. However, the effect of heterotypic co-infections on IBV replication was not investigated. To do so, we produced by reverse genetics a pair of replication-competent recombinant IAV (A/WSN/33) and IBV (B/Brisbane/60/2008) expressing a GFP and mCherry fluorescent reporter, respectively. A549 cells were infected simultaneously or 1 h apart at a high MOI with IAV-GFP or IBV-mCherry and the fluorescence was measured at 6 h post-infection by flow cytometry. Unexpectedly, we observed that IBV-mCherry infection was enhanced upon co-infection with IAV-GFP, and more strongly so when IAV was added 1 h prior to IBV. The same effect was observed with wild-type viruses and with various strains of IAV. Using UV-inactivated IAV or type-specific antiviral compounds, we showed that the enhancing effect of IAV infection on IBV infection was dependent on transcription/replication of the IAV genome. Our results, taken with available data in the literature, support the hypothesis that the presence of IAV proteins can enhance IBV genome expression and/or complement IBV defective particles.
RESUMEN
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.