STING (also known as MITA) mediates the innate antiviral signaling and ubiquitination of STING is key to its function. However, the deubiquitination process of STING is unclear. Here we report that USP18 recruits USP20 to deconjugate K48-linked ubiquitination chains from STING and promotes the stability of STING and the expression of type I IFNs and proinflammatory cytokines after DNA virus infection. USP18 deficiency or knockdown of USP20 resulted in enhanced K48-linked ubiquitination and accelerated degradation of STING, and impaired activation of IRF3 and NF-κB as well as induction of downstream genes after infection with DNA virus HSV-1 or transfection of various DNA ligands. In addition, Usp18 mice were more susceptible to HSV-1 infection compared with the wild-type littermates. USP18 did not deubiquitinate STING in vitro but facilitated USP20 to catalyze deubiquitination of STING in a manner independent of the enzymatic activity of USP18. In addition, reconstitution of STING into Usp18 MEFs restored HSV-1-induced expression of downstream genes and cellular antiviral responses. Our findings thus uncover previously uncharacterized roles of USP18 and USP20 in mediating virus-triggered signaling and contribute to the understanding of the complicated regulatory system of the innate antiviral responses.

Host pathogen-recognition receptors detect nucleic acid from invading viruses and initiate a series of signaling pathways that lead to the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we found that a viral infection-induced deubiquitinase (DUB), ubiquitin-specific protease 25 (USP25) was required for host defense against RNA and DNA viruses. The activation of transcription factors IRF3 and NF-κB was impaired and the production of type I IFNs and proinflammatory cytokines was inhibited in Usp25−/− cells compared with the wild-type counterparts after RNA or DNA viruses infection. Consistently, USP25 deficient mice were more susceptible to H5N1 or HSV-1 infection compared with the wild-type mice. USP25 was associated with TRAF3 and TRAF6 after infection by RNA or DNA viruses and protected virus-induced proteasome-dependent or independent degradation of TRAF3 and TRAF6, respectively. Moreover, reconstitution of TRAF3 and TRAF6 into Usp25−/− MEFs restored virus-triggered production of type I IFNs and proinflammatory cytokines. Our findings thus reveal a previously uncovered positive feedback regulation of innate immune responses against RNA and DNA viruses by USP25.

USP22 is a cytoplasmic and nuclear deubiquitinating enzyme, and the functions of cytoplasmic USP22 are unclear. Here, we discovered that cytoplasmic USP22 promoted nuclear translocation of IRF3 by deubiquitianting and stabilizing KPNA2 after viral infection. Viral infection induced USP22-IRF3 association in the cytoplasm in a KPNA2-depedent manner, and knockdown or knockout of USP22 or KPNA2 impaired IRF3 nuclear translocation and expression of downstream genes after viral infection. Consistently, Cre-ER Usp22fl/fl or Lyz2-Cre Usp22fl/fl mice produced decreased levels of type I IFNs after viral infection and exhibited increased susceptibility to lethal viral infection compared with the respective control littermates. Mechanistically, USP22 deubiquitinated and stabilized KPNA2 after viral infection to facilitate efficient nuclear translocation of IRF3. Reconstitution of KPNA2 into USP22 knockout cells restored virus-triggered nuclear translocation of IRF3 and cellular antiviral responses. These findings define a previously unknown function of cytoplasmic USP22 and establish a mechanistic link between USP22 and IRF3 nuclear translocation that expands potential therapeutic strategies for infectious diseases.

Mediator of IRF3 activation (MITA, also known as STING and ERIS) is an essential adaptor protein for cytoplasmic DNA-triggered signaling and involved in innate immune responses, autoimmunity and tumorigenesis. The activity of MITA is critically regulated by ubiquitination and deubiquitination. Here, we report that USP49 interacts with and deubiquitinates MITA after HSV-1 infection, thereby turning down cellular antiviral responses. Knockdown or knockout of USP49 potentiated HSV-1-, cytoplasmic DNA- or cGAMP-induced production of type I interferons (IFNs) and proinflammatory cytokines and impairs HSV-1 replication. Consistently, Usp49 -/- mice exhibit resistance to lethal HSV-1 infection and attenuated HSV-1 replication compared to Usp49 +/+ mice. Mechanistically, USP49 removes K63-linked ubiquitin chains from MITA after HSV-1 infection which inhibits the aggregation of MITA and the subsequent recruitment of TBK1 to the signaling complex. These findings suggest a critical role of USP49 in terminating innate antiviral responses and provide insights into the complex regulatory mechanisms of MITA activation.