Role of Deubiquitylating Enzymes in Innate Immune Signaling Pathways and During Virus Infection Qiwen Teo1, Jingshu Zhang1, Akhee Sabiha Jahan1, Sumana Sanyal1, 1HKU-Pasteur Research Pole, School of Public Health, The University of Hong Kong, Hong Kong, China
Ubiquitylation is a reversible post-translational modification implemented in a cascade of three enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). These enzymatic activities work together to conjugate the conserved 76-residue polypeptide ubiquitin to protein substrates, which can be hydrolased by deubiquitylating enzymes (DUBs). The ubiquitylation machinery is vitally important in regulating Influenza A virus (IAV) life cycle. In contrast to some other viruses, IAVs do not express their own DUBs. However, IAVs exploit the host cellular machinery to upregulate or downregulate DUBs to support its life cycle. For example, IAV viral RNA replication can be inhibited by cellular DUBs via deubiquitylating IAV nucleoprotein. Using a DUB specific activity-based probe, we identified and validated an upregulation of USP25 and UCHL3 activities in the cytosol upon IAV infection through a proteomics screen. USP25 and UCHL3 are cysteine proteases belonging to the Ubiquitin Specific Protease(USP) and Ubiquitin C-terminal hydrolases (UCH) family, respectively. We generated cells with the corresponding gene deletions using CRISPR/Cas9 strategy. Our results displayed a significant increase in virus production in USP25 knock-out cells compared to in wild-type cells during IAV infection, suggesting an antiviral role of USP25. Additionally, given the timeframe of enhanced virus production in USP25 knock-out cells, it is likely that USP25 functions in the interferon response pathway to establish an antiviral state. Furthermore, UCHL3 redistributed from the nucleus to the cytosol upon IAV infection as determined by confocal imaging, suggesting that UCHL3 was hijacked by IAV for its replicative advantage. However, the details of the mechanism are yet to be elucidated. Therefore, we will be studying the mechanism by employing different biochemical approaches, including proximity-dependent labeling to decipher the biochemical pathway of USP25 and UCHL3 function. Our data implicates that these DUBs are crucial for host defense against IAV infection, and can be manipulated for antiviral therapeutic purpose.