STING (also known as MITA) is critical for host defence against viruses and the activity of STING is regulated by ubiquitination. However, the deubiquitination of STING is not fully understood. Here, we show that ubiquitin-specific protease 13 (USP13) is a STING-interacting protein that catalyses deubiquitination of STING. Knockdown or knockout of USP13 potentiates activation of IRF3 and NF-κB and expression of downstream genes after HSV-1 infection or transfection of DNA ligands. USP13 deficiency results in impaired replication of HSV-1. Consistently, USP13 deficient mice are more resistant than wild-type littermates to lethal HSV-1 infection. Mechanistically, USP13 deconjugates polyubiquitin chains from STING and prevents the recruitment of TBK1 to the signalling complex, thereby negatively regulating cellular antiviral responses. Our study thus uncovers a function of USP13 in innate antiviral immunity and provides insight into the regulation of innate immunity.

The activity and stability of the adapter protein MAVS (also known as VISA, Cardif and IPS-1), which critically mediates cellular antiviral responses, are extensively regulated by ubiquitination. However, the process whereby MAVS is deubiquitinated is unclear.Here, we report that the ovarian tumor family deubiquitinase 4 (OTUD4) targets MAVS for deubiquitination. Viral infection leads to the IRF3/7-dependent upregulation of OTUD4 which interacts with MAVS to remove K48-linked polyubiquitin chains, thereby maintaining MAVS stability and promoting innate antiviral signaling. Knockout or knockdown of OTUD4 impairs RNA virus-triggered activation of IRF3 and NF-κB, expression of their downstream target genes, and potentiates VSV replication in vitro and in vivo. Consistently, Cre-ER Otud4 fl/fl or Lyz2-Cre Otud4 fl/fl mice produce decreased levels of type I interferons and proinflammatory cytokines and exhibit increased sensitivity to VSV infection compared to their control littermates. In addition, reconstitution of MAVS into OTUD4-deficient cells restores virus-induced expression of downstream genes and cellular antiviral responses. Together, our findings uncover an essential role of OTUD4 in virus-triggered signaling and contribute to the understanding of deubiquitination-mediated regulation of innate antiviral responses.

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.

MAVS is essential for antiviral immunity, but the molecular mechanisms responsible for its tight regulation remain poorly understood. Here, we show that NLK inhibits the antiviral immune response during viral infection by targeting MAVS for degradation. NLK depletion promotes virus-induced antiviral cytokine production and decreases viral replication, which is potently rescued by the reintroduction of NLK. Moreover, the depletion of NLK promotes antiviral effects and increases the survival times of mice after infection with VSV. NLK interacts with and phosphorylates MAVS at multiple sites on mitochondria or peroxisomes, thereby inducing the degradation of MAVS and subsequent inactivation of IRF3. Most importantly, a peptide derived from MAVS promotes viral-induced IFN-β production and antagonizes viral replication in vitro and in vivo. These findings provide direct insights into the molecular mechanisms by which phosphorylation of MAVS regulates its degradation and influences its activation and identify an important peptide target for propagating antiviral responses.

The subcellular localization and intracellular trafficking of Toll-like receptors (TLRs) critically regulate TLRs-mediated antimicrobial immunity and autoimmunity. Here, it is demonstrated that the E3 ubiquitin ligase RNF115 inhibits the post-endoplasmic reticulum (ER) trafficking of TLRs and TLRs-mediated immune responses by catalyzing ubiquitination of the small GTPases RAB1A and RAB13. It is shown that the 14-3-3 chaperones bind to AKT1-phosphorylated RNF115 and facilitate RNF115 localizing on the ER and the Golgi apparatus. RNF115 interacts with RAB1A and RAB13 and catalyzes K11-linked ubiquitination on the Lys49 and Lys61 residues of RAB1A and on the Lys46 and Lys58 residues of RAB13, respectively. Such a modification impairs the recruitment of guanosine diphosphate (GDP) dissociation inhibitor 1 (GDI1) to RAB1A and RAB13, a prerequisite for the reactivation of RAB proteins. Consistently, knockdown of RAB1A and RAB13 in Rnf115 +/+ and Rnf115 −/− cells markedly inhibits the post-ER and the post-Golgi trafficking of TLRs, respectively. In addition, reconstitution of RAB1A K49/61R or RAB13 K46/58R into Rnf115 +/+ cells but not Rnf115 −/− cells promotes the trafficking of TLRs from the ER to the Golgi apparatus and from the Golgi apparatus to the cell surface, respectively. These findings uncover a common and step-wise regulatory mechanism for the post-ER trafficking of TLRs.