Characterization of USP25 and SMURF1 function in virus-induced autophagy and innate immune response

Abstract

Ubiquitination 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 hydrolysed by deubiquitinating enzymes (DUBs). The ubiquitination machinery is critical for regulation of the Influenza A virus (IAV) life cycle as well as innate immune responses to infection. However, the interplay between ubiquitination and influenza remains poorly defined.

Using a DUB specific activity-based probe, we identified and validated several that are activated upon IAV infection. Among others we identified USP25. To determine its functional implications in IAV infection, we generated cells with the gene deletion of USP25 using CRISPR/Cas9 strategy. Our results show a significant increase in virus production in USP25 knock-out cells compared to those in wild-type cells during IAV infection, suggesting that USP25 functions as a restriction factor. The increase of virus production in the knock-out cells was not a result of increased entry or replication but correlated with increased autophagy as reflected by the appearance of LC3-II. Our results demonstrated that USP25 restricts autophagy dependent transport and release of IAV by interacting with M2 to hydrolyse K6-, K29- and K63-polyubiquitination chains of M2. USP25 is required to maintain integral lysosome functions as deletion of USP25 or reconstitution with a catalytically deficient variant of USP25 resulted in hyperglycosylation of LAMP1. This caused lysosomal dysfunction and accumulation of LC3+ autophagosome-like compartments in mock-infected cells, a phenotype that was significantly exacerbated in influenza-infected cells.

Additionally, deletion of USP25 also dampened RIG-I-mediated immunity as reflected by decreased RIG-I and downstream effector protein expression in the USP25 knock-out cells. USP25 knock-out cells displayed attenuated interferon and elicited cytokine production, making the cells more susceptible to virus infection. On the other hand, depleting SMURF1, an E3 ligase that functions to regulate USP25 expression, protected cells from infection by a range of viruses including SARS-CoV-2 and the highly pathogenic H5N1 and H7N9 by inducing robust Type I interferon response.

Taken together, our data implicate USP25 as a critical restriction factor for IAV. Deletion of USP25 not only inhibited RIG-I dependent immune signalling but also induced autophagy to enhance viral release. Furthermore, we identified SMURF1 as a pan-RNA virus host factor required for establishing successful viral infection. The well-defined E3 ligase catalytic cleft of SMURF1 offers an attractive target for developing potential antiviral drugs for treating RNA virus infections.