Deubiquitylase OTUB1 Plays Critical Role in Influenza A Virus Infection and Immune Response Akhee Sabiha Jahan, Dr. Sumana Sanyal1, Prof. Roberto Bruzzone1 1HKU-Pasteur Research Pole, School of public health, HKU
Ubiquitylation is a cellular post-translational modification (PTM), routinely targeted by pathogens to deregulate and hijack host intracellular pathways. Most viruses utilize ubiquitylation to target central signaling pathways such as NFB and MAPK that are crucial for their replication, propagation and host immune evasion. During Influenza A virus (IAV) infection, the host ubiquitylation machinery is significantly altered and exploited for RIG-I mediated viral sensing and immune response. In an effort 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 vinylmethylester 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, we identified OTUB1, a deubiquitylase highly upregulated upon IAV infection. Interferon β treatment alone could also induce OTUB1 expression in A549 cells, suggesting it is an IFN-type 1 induced host factor. To functionally characterize OTUB1 in IAV infection, we utilized CRISPR/Cas9 genome editing to generate knock-outs of OTUB1 in A549 cells. OTUB1-deficient A549 cells produced significantly less viral progeny at a multi-replication cycle and were defective in production of both IFN type 1 as well as pro-inflammatory cytokines compared to the wildtype when subjected to IAV infection. Further investigation showed a lack of IRF and NFB activation upon infection in OTUB1 deficient cells. Through the use of proximity based labeling, we found components of the RIG-I signaling pathway to be highly enriched in the OTUB1 interactome. Furthermore, IAV viral proteins PB2 and NS1, directly interacted with host factor OTUB1. Our results suggest that induction of OTUB1 achieved upon IAV infection is critical for both IAV viral progeny production as well as IRF and NFB activation leading to prodution of interferon and cytokines in A549 cells. Our findings provide novel insights into the mechanism of IAV infection and indicate new avenues for the development of potential therapeutic targets.
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