To mount an effective antiviral response, the immune system must trigger multiple signaling pathways to promote the production of cytokines and other antiviral factors that collectively suppress viral replication and assembly 1
. The most robust antiviral response can be attributed to the induction of type I interferons (IFN) – IFNα and IFNβ. On immunological assault, cellular pattern recognition receptors are rapidly stimulated following the recognition of specific molecular signatures of pathogens, which are often referred to as pathogen-associated molecular patterns 2, 3
. In the case of an infection by RNA viruses and some DNA viruses, both the membrane-bound Toll-like receptors (TLRs) and cytosolic sensors, such as retinoic acid-inducible gene-I (RIG-I
)-like receptors (RLRs), have been clearly shown to detect 5′ triphosphate-containing viral RNA structures, generated as a consequence of early virus transcription and/or replication 3, 4
. Consequently, detection of viral RNA triggers the rapid activation of various transcription factors, such as NF-κB, AP-1 and members of the IFN regulatory factor (IRF) family (IRF1/3/7), that specifically bind to the IFNβ promoter to stimulate gene expression. Following production, IFNβ engages its cognate receptor and induces a complex intracellular signaling process to specifically activate the expression of multiple IFN-stimulated genes (ISGs), all of which contain a variation of the DNA sequence referred to as IFN-stimulated response element 5
RLRs including RIG-I, melanoma differentiation factor 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) reside in the cellular cytoplasm, whereas TLRs are located on the plasma membrane or at the endosomal surface 2, 6, 7, 8, 9
. Structurally, all three RLRs contain a DExD-box RNA helicase domain for RNA binding and with the exception of LGP2, also possess a caspase recruitment domain (CARD) that mediates downstream protein-protein interactions. Upon viral RNA recognition, activation of RIG-I promotes self-dimerization and other structural modifications that permit CARD-CARD interaction with the downstream adapter molecule mitochondrial antiviral signaling protein (MAVS; also known as IPS-1/Cardif/VISA) 10, 11, 12, 13
. MAVS is composed of multiple motifs including: a C-terminal transmembrane domain (TM), which is essential for targeting to the outer mitochondrial membrane, three TRAF-interacting motifs (TIM), two included in the N-terminal proline-rich region (Pro), and an N-terminal CARD domain. Functionally, the CARD and TM domains appear to be critical for MAVS dimerization and for relaying the signal from RIG-I to downstream adapter molecules 12, 14, 15, 16, 17
. The Pro region harbors two distinct TIMs, one located at aa 143-PVQET-147 that binds TRAF2 and TRAF3, and a second TIM located at aa 153-PGENSE-158 that exclusively binds TRAF6 13, 18
. An alternate TRAF6-binding site is located in the C-terminus of MAVS at aa 455-PEENEY-460 13
, and both N- and C-terminal sites are required for TRAF6-mediated activation of the NF-κB pathway 13
. Additionally, MAVS interacts with multiple proteins, including LGP2, TRADD, FADD, RIP1, TRAF5, IKKε, caspase 8/9 and MITA/STING/MYPS/ERIS, TOM70, NLRX1, PCBP2/AIP4, OPTN, PLK1, deubiquitination enzyme A (DUBA), A20, 9, 10, 11, 15, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32
, all indicating that MAVS functions as a molecular scaffold to which key adapter proteins bind to orchestrate the RIG-I dependent antiviral response.
TRAF family members are primarily involved in the regulation of inflammation, antiviral responses and apoptosis, and function downstream of the TNF, CD40 and LTβR receptors, to name a few. TRAF proteins play non-overlapping roles in signaling; e.g., TRAF3 uniquely regulates the type I IFN response 33, 34, 35
, while both TRAF2 and TRAF6 activate the NF-κB pathway downstream of RIG-I 13
. Thus, the RIG-I pathway bifurcates at the level of the TRAFs into two distinct pathways. TRAF2 and TRAF6 activate the classical IKKα/β kinases, and induce phosphorylation of IκBα inhibitor, resulting its proteasomal degradation. Active p65/p50 NF-κB dimers are then released and translocate into the nucleus to activate NF-κB-dependent target genes 36, 37, 38, 39
. TRAF3 stimulates the non-canonical IKK-related kinases, TBK1 and IKKε, which induce C-terminal phosphorylation of IRF3/IRF7, leading to IRF3 dimerization, nuclear translocation, DNA binding and activation of IRF-dependent antiviral genes 26, 40, 41, 42, 43
Cytokine signal transduction is a transient process that is tightly regulated to prevent inappropriate inflammatory or autoimmune responses. Dynamic control of the antiviral response is coordinated at transcriptional levels, as well as by post-translational modifications, such as phosphorylation and ubiquitination. Multiple proteins are involved in the coordination of the RIG-I pathway including A20, DUBA, CYLD, NLRX1, OPTN, PCBP2-AIP4, PSMA7, miR146a, LGP2, NLRC5, gC1qR, FLN29, ISG15, RNF125, Pin1, SIKE and TOM70, to name a few 19, 27, 28, 30, 32, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58
. Previously, we demonstrated that IKKε, but not TBK1, was recruited to the MAVS adapter via a Lys63-linked polyubiquitination on Lys500, thus revealing an unexpected function of IKKε in the negative regulation of the inflammatory and antiviral response 15
. We also identified Triad3A as an E3 ubiquitin ligase involved in the termination of the IFN response following viral infection, by targeting TRAF3 for K48-linked ubiquitination and proteasomal degradation 59
. Recently, Tseng et al.
demonstrated that TRAF3 can either positively or negatively control the expression of type I IFN and pro-inflammatory cytokines through distinct types of ubiquitination.
Elucidating the spatiotemporal events involved in the recruitment of adapters, kinases and transcription factors to MAVS is essential for an understanding of the inflammatory and antiviral response. Although TRAF3 is essential for the IFN response, Seth et al. previously demonstrated that removal of the Pro domain from MAVS had no effect on IFN signaling, suggesting the presence of an alternate functional TRAF3-binding site. In this study, we identify a novel TRAF3-binding site in the C-terminus of MAVS (455-PEENEY-460) that is essential for MAVS-mediated antiviral responses. Furthermore, IKKε, recruited to MAVS via K63-mediated ubiquitination of Lys500, decreases the binding and protein stability of TRAF3, thus contributing to negative regulation of the antiviral response.