Regulation Of RL Rs-mediated Innate Antiviral Responses By GPATCH3

The innate immune system is an evolutionally conserved host defense mechanism against pathogens and acts as the first line of defense against viral infection.Upon viral infection,germline-encoded pathogen recognition receptors(PRRs)recognize pathogen-associated molecular patterns(PAMPs)derived from viruses and trigger a series of signaling events,leading to the induction of type I interferons and proinflammatory cytokines.Subquently,type I interferons activate JAK-STAT signaling and lead to the production of hundreds of interferon stimulated genes(ISGs).ISGs and proinflammatory cytokines cooperate to inhibit viral infection and activate adaptive immunity.It has been demonstrated that cytosolic RNAs derived from viral genome or its replication intermediates are mainly recognized by the RLR family members RIG-I(retinoic acid–inducible gene-1)and MDA5(melanoma differentiation-associated gene 5).RIG-I and MDA5 undergo conformational changes and recruite the downstream adaptor protein VISA(also known as MAVS,IPS-1,Cardif).VISA then forms large prion-like polymers and serves as a platform for the assembly of VISA signalosome which contain multiple components,including TRAF proteins(TRAF2/3/5/6),TBK1 and IKKs kinases.TBK1 and IKKs then phosphorylate IRF3 and NF-?B,respectively,leading to the induction of type I IFNs and proinflammatory cytokines.Since abnormal production of type I IFNs could cause serious diseases,the process of type I interferons(IFNs)induction is stringently controlled by host immune system.In this study,to further investigate the regulatory mechanisms of virus-induced innate imunne signaling,we screened ~10,000 human c DNA clones by luciferase assays and identified GPATCH3(G-patch domain-containing protein 3)as a negative regulator of RLR-mediated signaling.GPATCH3 is ubiquitously expressed in mammalian tissues.However,the function of GPATCH3 has not yet been characterized.We found that overexpression of GPATCH3 reduced Se V-triggered activation of IFN-? promoter,ISRE and NF-?B in a dose-dependent manner.In contrast,knockdown of GPATCH3 significantly enhanced Se V-induced transcription of IFNB1,ISGs and TNFA.Interestingly,knockdown of GPATCH3 had no effects on DNA virus HCMV-or double strand DNA HSV120-triggered transcription of IFNB1.Consistently,knockdown of GPATCH3 markedly impaired replications of both Se V and VSV.To further confirm these results,we generated GPATCH3-deficient 293 T cells.Compared with the control cells,GPATCH3-deficiency significantly enhanced transcription of IFNB1 induced by RNA virus infection.Mechanistically,we found that GPATCH3 interacted with VISA and the binding of GPATCH3 with VISA was increased upon viral infection.Intriguingly,interactions of VISA-TBK1 and VISA-TRAF6 were markedly reduced by GPATCH3.These data indicate that GPATCH3 interferes with the assembly of VISA signalosomes,leading to negative regulation of the RLR-mediated signaling.The biological function of GPATCH3 has been elusive.In this study,we identified GPATCH3 as a negative regulator of the RLR-mediated signaling by disrupting the assembly of VISA signalosome.As a central adaptor of RLR-mediated signaling,the assembly of VISA signalosome plays pivotal roles in the activation of RLRs-mediated signaling.Thus,these findings not only expand our understanding on the regulation of RLRs-mediated antivairal responses,but also shed the first light on the biological function of GPATCH3.