Role of deubiquitylases in influenza A virus infection and immunity

Abstract

Ubiquitylation is a cellular post-translational modification (PTM), routinely targeted by pathogens to deregulate and hijack host intracellular pathways. During Influenza A virus (IAV) infection, the host ubiquitylation machinery is significantly altered and exploited for RIG-I mediated viral sensing and immune response. To comprehensively characterize the ubiquitylation machinery hijacked by IAV in mammalian cells, we designed and employed a chemoenzymatic strategy to identify specific deubiquitylases (DUBs) that are induced upon IAV infections. Using a C-terminal vinyl methyl ester modified HA-tagged ubiquitin (HA-Ub-vme) combined with large-scale immunoprecipitation and mass spectrometry we identified DUBs that are upregulated in IAV infected cells.

Among others, OTUB1 was identified as a deubiquitylase that is upregulated upon IAV infection. Interferon β treatment alone could also induce OTUB1 expression in A549 cells, suggesting it is an IFN-I induced host factor. To functionally characterize OTUB1 in IAV infection, CRISPR/Cas9 genome editing was utilized to generate OTUB1-deleted A549 cells. OTUB1-deficient (OTUB1-/- ) cells produced significantly less viral progeny, which were rescued by introduction of OTUB1 in OTUB1-/- cells. Further investigation showed severely defective polymerase activity in OTUB1-/- cells compared to WT. Influenza A viral protein PB2 which is essential for viral polymerase activity interacted directly with OTUB1 as measured by co-immunoprecipitation and co-localization score. Lack of OTUB1 was shown to be associated with a higher K-48 linked polyubiquitylation of PB2, suggesting a vital role of OTUB1 in regulating polymerase activity through deubiquitylation of PB2.

Additionally, OTUB1-/- cells were defective in production of both IFN-α as well as pro-inflammatory cytokines compared to the WT when subjected to IAV infection. Further investigation of OTUB1-/- cells upon infection showed a significant redution in IRF and NFB activation, upstream of IFN and cytokines respectively. Through the use of proximity-based labeling, components of the RIG-I signaling pathway were found to be highly enriched in the OTUB1 interactome. Furthermore, OTUB1 was shown to directly interact with IAV viral protein NS1 known for its RIG-I antagonism and was shown to be degraded without proteasome inhibitor treatment upn IAV infection. Immunoprecipitated NS1 samples were shown to be enriched in K-48 and K-63 linked polyubiquitylation and formed complexes with TRAF3 and RIG-I, which have been previously reported as NS1 interactors. In OTUB1-/- cells, neither the ubiquitylated material, nor the RIG-I complex was detectable upon NS1 pull-down. The NS1-RIG-I interaction is known to be integral in the function of NS1 to limit IRF and NFB activation, eventually leading to dampened antiviral response. We find OTUB1 to be an essential determinant in the formation of NS1-RIG-I complex and mediation of the downstream antiviral responses.

Apart from innate immune responses associated with IAV, ubiquitylation also plays a significant role in the adaptive immune responses, such as T cell receptor (TCR) signaling. We identified Ubiquitin-specific peptidase (Usp) 12 as a positive regulator highly enriched T lymphocytes and a crucial component of TCR expression at the cell surface. These findings provided novel insights in fine-tuning host signaling cascades both in IAV infection and immunity, identifying new avenues for the development of potential therapeutic targets.