Ubiquitination in the antiviral immune response

Abstract

Ubiquitination has long been known to regulate fundamental cellular processes through the induction of proteasomal degradation of target proteins. More recently, 'atypical' non-degradative types of polyubiquitin chains have been appreciated as important regulatory moieties by modulating the activity or subcellular localization of key signaling proteins. Intriguingly, many of these non-degradative types of ubiquitination regulate the innate sensing pathways initiated by pattern recognition receptors (PRRs), ultimately coordinating an effective antiviral immune response. Here we discuss recent advances in understanding the functional roles of degradative and atypical types of ubiquitination in innate immunity to viral infections, with a specific focus on the signaling pathways triggered by RIG-I-like receptors, Toll-like receptors, and the intracellular viral DNA sensor cGAS.

Keywords: Antiviral immunity; E3 ligases; RIG-I-like receptors; STING; TRIM proteins; TRIM25; Toll-like receptors; Type-I interferon; Ubiquitin; cGAS.

Figure 1. Functional roles of the different linkage types of polyubiquitination

The 8 different linkage types of polyubiquitination are illustrated. Known fates of modified substrates as well as key pathways regulated by specific polyubiquitins are shown. The specific details of how different ubiquitin polymers regulate substrate proteins are described in the text.

Figure 2. Regulation of RLRs by ubiquitination

RIG-I and MDA5, members of the RIG-I-like receptor (RLR) family, recognize cytoplasmic viral RNA species and subsequently signal through the adaptor protein MAVS (also called Cardif, IPS-1, or VISA) on mitochondria. Through various steps (not illustrated), MAVS activates downstream signaling, leading to gene expression of type-I IFNs (IFN-α/β). The stability and signaling activities of RIG-I, MDA5 and MAVS are tightly regulated by K48- and K63-linked polyubiquitination, respectively. RIG-I is activated by K63-linked ubiquitination mediated by TRIM25, an IFN-inducible E3 ubiquitin ligase belonging to the large family of TRIM proteins. TRIM25 ubiquitinates several lysines in the N-terminal caspase activation and recruitment domains (CARDs) of RIG-I (not illustrated). TRIM25-mediated ubiquitination specifically at K172 in RIG-I is critical for RIG-I signaling. In addition, TRIM4 and MEX3C were shown to induce K63-linked ubiquitination of the RIG-I CARDs. Furthermore, RIPLET induces K63-linked ubiquitination of K788 (and also other residues) in the C-terminal domain (not illustrated). Both RIG-I and MDA5 have been reported to non-covalently bind unanchored K63-linked ubiquitin chains in vitro; however, the role of MDA5 activation by K63-linked ubiquitin chains remains unclear. TRIM25 itself undergoes K48-linked ubiquitination catalyzed by LUBAC, leading to TRIM25 degradation. Inversely, USP15 antagonizes the LUBAC-induced K48-linked ubiquitination of TRIM25, thereby stabilizing TRIM25 during viral infection, which leads to a sustained IFN response. MAVS is ubiquitinated with K63-linked polyubiquitin chains; however, the E3 ligase for MAVS K63-ubiquitination is unknown. Multiple different E3 ubiquitin ligases have been reported to induce the K48-linked ubiquitination of RLRs and MAVS, triggering their degradation by the proteasome: RNF125 for RIG-I; TRIM13 and RNF125 for MDA5; and AIP4/Itch, Smurf1/2, RNF5 and RNF125 for MAVS. The K48-linked ubiquitination of RIG-I can be actively removed by USP4. Furthermore, TRIM44, an atypical TRIM protein that lacks the RING E3 ligase domain, inhibits the K48-linked ubiquitination of MAVS through an unidentified mechanism.

Figure 3. Regulation of TLRs by ubiquitination

Toll-like receptors (TLRs), found on the cell surface or on endosomal membranes, survey the extracellular milieu for viral nucleic acid or proteins. After binding to their respective viral ligands, TLRs signal through one of two critical adaptor proteins, MyD88/IRAK or TRIF. Signaling by IRAK1 is perpetuated through modification with K63-linked ubiquitination by Pellinos or TRAF6. The activation of TLRs and their adaptor proteins is regulated by degradative K48-linked ubiquitination mediated by the E3 ligases indicated. The specific details of how K48-polyubiquitin regulates TLR signaling are described in the text.

Figure 4. Regulation of the cGAS-STING pathway by ubiquitination

cGAS recognizes viral DNA in the cytoplasm and synthesizes cyclic GMP-AMP (cGAMP). cGAMP then activates STING on the ER, inducing downstream signaling for type-I IFN induction. STING is regulated by three types of polyubiquitination: K11-linked and K63-linked ubiquitination of K150 by RNF26 and TRIM56 or TRIM32, respectively, facilitating STING activation and type-I IFN gene expression. Besides K500, TRIM32 ubiquitinates three other residues in STING (K20, K224 and K236). Furthermore, K500 in STING is covalently modified by K48-linked ubiquitination mediated by RNF5. RNF5-induced STING ubiquitination leads to STING degradation.

Figure 5. Ubiquitin-dependent regulation of common downstream molecules of PRRs

After recognition of viral nucleic acids, RLRs and TLRs signal through their downstream adaptors MAVS and MyD88/TRIF, respectively. These adaptors then propagate this signal to TRAF6 for NF-κB activation and TRAF3 for IRF3/7 activation. TRAF6 and TRAF3 both induce autoubiquitination, creating a scaffold for downstream signaling partners to interact. K63-linked polyubiquitin on TRAF6 leads to the recruitment of the TAK1/TAB2/3 complex, which in turn recruits NEMO and the IKK complex to phosphorylate IκBα. Phosphorylation of IκBα then leads to its K48-polyubiquitin-dependent degradation. Degradation of IκBα releases the NF-κB subunits p50 and p65, allowing for their translocation into the nucleus to activate transcription of target genes. On the other hand, K63-linked ubiquitination of TRAF3 recruits NEMO specifically complexed TBK1/IKKε. sTING also directly binds to and activates TBK1. TBK1/IKKε then phosphorylate IRF3 and IRF7, leading to their dimerization and translocation to the nucleus to induce transcription of type-I IFN and antiviral genes. Many proteins in these signaling cascades are targets for degradative K48-linked ubiquitination and non-degradative types of polyubiquitination. The E3 ligases involved in these ubiquitination events, as well as the DUBs responsible for removal of polyubiquitin, are indicated. The details of how specific ubiquitin marks regulate the activities of the illustrated signaling molecules are described in the text.