The E3 Ubiquitin Ligase TRIM40 Attenuates Antiviral Immune Responses By Targeting MDA5 And RIG-I

Retinoic acid-inducible gene-?(RIG-?)-like receptors(RLRs)including melanoma differentiation-associated gene 5(MDA5)and RIG-? are crucial for host recognizing non-self RNAs,especially viral RNA.Optimal expression and activation of RIG-? and MDA5 are crucial for the secretion of IFN-? and induction of antiviral innate immune responses.However,uncontrolled expression of RIG-? and MDA5,and excessive activation of RLR-induced signaling,play crucial causative or contributing roles in the initial of autoinflammatory and autoimmune diseases.Thus,the expression and activation of RLRs play fundamental roles in eliminating the invading RNA viruses and maintaining immune homeostasis.However,how RLRs expression is tightly regulated remains to be further investigated.Ubiquitination is one of the most important posttranslational modifications modulating RIG-? activity.Several E3 ubiquitin ligases,such as tripartite interaction motif 25(TRIM25),TRIM4,RING finger protein 135(RNF135)and RNA-binding E3 ubiquitin-protein ligase MEX3C(also called RNF194),mediate lysine 63(K63)-linked ubiquitination of RIG-? and facilitates its activation.On the other hand,E3 ubiquitin ligases specifically target K48-linked ubiquitination of RIG-?,such as RNF125,RNF122,casitas B-lineage lymphoma proto-oncogene(c-Cb1,also known as RNF55)and carboxyl terminus of HSC70-interacting protein(CHIP)promote its degradation through a proteasome pathway and thus terminate the downstream signal transduction.However,the potential regulatory effect of ubiquitination modification on MDA5 activity remains largely unknown.Moreover,new E3 ligases regulating RLR-triggered antiviral responses need to be further identified.Tripartite interaction motif 40(TRIM40)is a member of the TRIM protein family,which is encoded within the major histocompatibility complex(MHC)class? region.It has been reported that several TRIM family members organized in a tight cluster within the MHC class ? region possess vital regulatory effects in innate immunity,such as TRIM26,TRIM 15,TRIM31 and TRIM39.However,the biological function of TRIM40 in immunity remains unknown.In this study,we identified theTRIM40 as a suppressor of RLR signaling by directly targeting MDA5 and RIG-?.TRIM40 selectively inhibits RLR(MDA5/RIG-?)signal pathways,with no effects on TLR3/4 signal pathways.As a physiological feedback regulator of antiviral immune responses,TRIM40 eliminates RLR-induced IFN-P production and subsequent antiviral immune responses in vitro and in vivo.TRIM40 deficiency greatly increases the resistance of Sev or VSV infected mice with enhanced antiviral immune responses and decreased viral replication.TRIM40 interacts with the highly conserved caspase-recruitment domains(CARD)domain of MDA5 and RIG-I through its own coiled-coil(CC)domain.As an E3 ubiquitin ligase,TRIM40 promotes their K27-and K48-linked polyubiquitination via its E3 ligase activity,leading to their proteasomal degradation.Our results also revealed that the lysine 23,lysine 43 and lysine 68 of human MDA5 are the main ubiquitination sites modified by TRIM40.Consequently,we demonstrate that TRIM40 limits RLR-triggered innate activation.For the first time our research identified the ubiquitination sites in human MDA5 and uncovered the association between the K27 ubiquitination of RIG-I/MDA5 and their proteasomal degradation.These results expand the research of the regulation network on RLR signal pathway by TRIM family memebers,suggesting TRIM40 as a novel therapeutic target for the control of viral infection.