USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

Abstract

Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity.

The nucleolar protein GLTSCR2 is required for efficient viral replication

“…USP21 and USP3 have also been shown to remove K63-linked polyubiquitin chains from RIG-I, resulting in deactivation of RIG-I244142. Here, we ectopically co-expressed Flag-tagged GLTSCR2 and Myc-tagged TRIM25 (Fig.…”

“…The amino-terminal caspase recruitment domains (CARDs) of RIG-I (RIG-N) undergo robust ubiquitination induced by TRIM2514 and TRIM415, which effectively deliver the Lys 63-linked polyubiquitin chains to the RIG-N, to increase the activity of RIG-I. However, an ubiquitin-specific proteases USP1524, USP2141, or USP342, removes the 63-linked polyubiquitin chains from the RIG-N to attenuate the activity of RIG-I. K63-linked ubiquitination of RIG-I results in activation of RIG-I, leading to induction of type I IFN14, whereas K48-linked ubiquitination results in inactivation of RIG-I26.…”

Ubiquitination in the antiviral immune response

“…This interaction led to RIG-I deubiquitination and a decrease in type-I IFN induction. More recently, USP3 has been shown to deubiquitinate RIG-I, leading to a decrease in IFN-β induction [78]. Upon virus infection, USP3 interacted with RIG-I, likely acting as a negative feedback regulator.…”

Activation and regulation of pathogen sensor RIG-I

“…While TRIM25, Riplet and free K63-linked ubiquitin chains promote signaling by enhancing association of RIG-I-RNA complexes with MAVS on mitochondria, several deubiquitinases have been discovered that reverse this process by removing the polyubiquitin chains from RIG-I. CYLD and ubiquitin-specific protease (USP) 3 associate with RIG-I CARDs and remove K63-linked polyubiquitin chains [109,110]. USP21 is another deubiquitinase that has been shown to reverse the polyubiquitination catalyzed by both TRIM25 and Riplet [111].…”