UBIQUITINATION-MEDIATED REGULATION OF RLR-TRIGGERED ANTIVIRAL SIGNALING IN FISH AND MAMMALS

In mammals, type I interferon (IFN) response is the first line of defense against viral invasion. In the case of viral infection, host cells recognize the corresponding pathogen-associated molecular pattern (PAMPs) by pathogen recognition receptor (PRRs) and recruit different downstream linker molecules to initiate the corresponding signal transduction pathway, eventually resulting in the expression of IFN, which in turn activates the transcription of numerous downstream IFN-stimulated gene (ISG) expression for establishment of host antiviral immune state. RLR retinoic acid-inducible gene I (RIG-I)-like Receptors represents a family of cytosolic pattern recognition receptors, composed of RIG-I, MDA5 (Melanoma differentiation-associated gene 5) and LGP2 (Laboratory of genetics and physiology 2). RLR receptors are structurally highly conserved and contain a DEX (D/H)-box RNA helicase domain and a C-terminal RNA binding domain and regulatory domain. Compared to RIG-I and MDA5, LGP2 shares the helicase domain and RD but lacks the two N-terminal CARDs that are required for RLR-triggered IFN signaling. 　　Extensive studies have suggested a canonical paradigm of RIG-I and MDA5 signaling. Upon binding to 5’ppp- or 5’pp-dsRNA through RD and helicase domain, RIG-I undergoes a conformational change, thus releasing N-terminal CARDs from an auto-inhibitory state in an ATP-dependent manner and initiating downstream signaling cascade after interaction with adaptor mitochondrial antiviral signaling protein (MAVS). MDA5 preferentially binds to long dsRNAs to form protein-coated filaments, therefore leading to tandem CARDs oligomerization to activate MAVS. The resultant MAVS activation finally facilitates protein kinases TANK-binding kinase 1 (TBK1) to phosphorylate and activate IFN regulatory factors 3/7 (IRF3/7). The phosphorylated IRF3/7 translocate from cytoplasm to nucleus, bind to the promoters of IFN genes, and turn on their expression. The secreted IFNs combine to the cognate receptors in an autocrine or a paracrine manner, to activate the JAK-STAT pathway inducing the expression of ISGs. Upon some RNA virus infection, mediator of IRF3 activation (MITA) also functions as a scaffold protein to link TBK1 and IRF3 to MAVS complex for the expression of IFN and ISGs, although it is well-known that MITA primarily participates in virus DNA-directed IFN signaling. As the third member of RLR family, LGP2 is initially considered to be dysfunctional. Recent studies have shown that LGP2 is a key factor for regulation of a switch between positive and negative roles in RLR signal transduction: at lower levels, LGP2 synergies with MDA5 but not RIG-I to augment IFN signaling; at higher levels, LGP2 instead acts as an inhibitor of RIG-I and MDA5 signaling. 　　Many ISGs encode antiviral effector proteins that are essential for eradication of virus invasion. Which beneficial for host cells to rapidly turn on the expression of IFN and ISGs upon viral infection, overproduction of IFNs leads to the development of immunopathological conditions partially due to continuous expression of those IFN-induced antiviral proteins. Therefore, it is important for host cells to immediately terminate the IFN-mediated antiviral response after virus has been cleared. That is, RLR-triggered IFN antiviral response should be fine tune controlled at an appropriate extent as relative to that of viral infection in given cells. Consistently, host cells develop multiple mechanisms to precisely modulate IFN signaling for appropriate production of IFNs.　　Actually, some factors display abilities to appropriately regulate RLR-triggered IFN signaling. They include E3-ubiquintin ligases and deubiquitinases (DUBs), which mediate ubiquitination or deubiquitination of targeted signaling components of RLR pathway to cooperatively regulate their biological activity. A best example is the family of tripartite motif (TRIM) proteins, which play pivotal roles in the innate antiviral response. Generally, TRIMs function as a major class of E3 ubiquitin ligase enzymes, and work together with ubiquitin-activating enzymes (E1s) and ubiquitin-conjugating enzymes (E2s) to participate in an ubiquitination cascade process. Such process results in diverse ubiquitination modifications of RLR signaling factors, to activate them from an inactive state, or target them to degrade by proteasome or lysosomal pathways, thus regulating RLR-triggered IFN antiviral response. Notably, ubiquitination modification process is inducible and also is reversible. In the case, many deubiquitinases, including ubiquitin-specific proteases (USP) and ovarian tumor protein (OUT), are capable to remove the related ubiquitination-mediated modification from the targeted proteins, finally convert the E3 ligase-mediated ubiquitination to shape IFN antiviral response at a given infection time. 　　Since the first fish IFN gene was identified in zebrafish in 2003, accumulating evidence has shown that fish exist the conserved RLR-mediated IFN signaling pathway. Although fish IFNs are not classified into IFNα/β but instead group I and group II IFNs based on cysteine numbers, fish RIG-I and MDA-5 direct IFN expression through a conserved RIG-I/MDA5-MAVS/MITA-TBK1-IRF3/7 signaling pathway. In addition, fish LGP2 confers protection on fish cells against SVCV infection through a similar signaling pathway and at limited expression levels, LGP2 exerts more significant protection than either RIG-I or MDA5. Importantly, RLR-triggered fish IFN antiviral response is also regulated appropriately by similar mechanisms, in which ubiquitination- and deubiquitination-mediated regulation is involved. In this review, we summarize the recent progress on ubiquitination-mediated regulation of RLR-triggered IFN antiviral response in mammals and fish.