Regulation Mechanisms Of Grass Carp(Ctenopharyngodon Idella) LGP2 And HMGB1b In Response To GCRV Infection

Retinoic acid-inducible gene I(RIG-I)like receptors(RLRs)play an improtant role in sensing of RNA virus infection and initiate antiviral immune response.RIG-I and melanoma differentiation-associated gene 5(MDA5)are two essential RLRs members that detect viral double strand RNA(ds RNA)and induce downstream signaling activation via interaction with IFN-? promoter stimulator 1(IPS-1)and post-translational modifications.But for laboratory of genetics and physiology 2(LGP2),the third RLR member,lacking caspase-recruitment domain(CARD)results in it failing to recruit signaling proteins to activate a cascade of downstream signaling events.So far,studies have uncovered the controversial functional performance of LGP2 as a negative or positive regulator in immune regulation.However,it is currently unclear why LGP2 mediates opposing roles.Especially,no sufficient evidence uncovers the functional mechanisms of LGP2 in fish RLR signaling pathway.High-mobility group box proteins(HMGBs)are conserved chromatin-associated nuclear proteins,which are also a novel family of nucleic acid sensors.Previous studies have demonstrated the essential role of grass carp(Ctenopharyngodon idella)HMGBs in response to grass carp reovirus(GCRV)infection,and the dynamic subcellular localization induced by virus infection and pathogen-asociated molecular patterns(PAMPs)stimulation was regulated by intramolecular interaction between different domains of HMGB.However,the regulation mechanism by which HMGBs mediate antivial immunity is still unknown.In present study,we investigated the regulation mechanism of grass carp LGP2 in RIGI-and MDA5-mediated antiviral responses against GCRV infection.We also identified heat shock protein 70(HSP70)as an interaction partner of HMGB1 b,and demonstratd the positive role of HSP70 in HMGB1b-mediated antiviral autophagy.1.LGP2 plays negative role in RIG-I-and MDA5-mediated antiviral signaling in resting state and early stage of GCRV infection.In present study,we demonstrated that LGP2 overexpression inhibited synthesis and phosphorylation of interferon regulatory factor(IRF)3 and IRF7,and m RNA levels and promoter activities of all the family members of interferons(IFNs)(IFN1,IFN2,IFN3,IFN4,IFN?1,IFN?2,)and nuclear factor ?B(NF-?Bs)(NF-?B1,NF-?B2)in resting state and early phase of GCRV infection.Knockdown of LGP2 obtained opposite effects.Dual luciferase report assay indicated that LGP2 worked at the upstream of RIG-I and MDA5.Meanwhile,LGP2 interacted with RIG-I and MDA5,and which was independent of GCRV infection.In domain interaction assay,we demonstrated that LGP2 interacted with all the three domains of MDA5,but failed to interact with RIG-I-CARD domain.Furthermore,LGP2 remarkably restrained K63-linked ubiquitination of RIG-I and MDA5.But for the CARD domain,LGP2 overexpression just inhibited the K63-linked ubiquitination of RIGI-CARD domain,and had no influence on MDA5-CARD domain.These differences resulted in disparate repressive mechanisms of LGP2 to RIG-I and MDA5-mediated signal activations of PS-1 and mediator of IRF3 activation(MITA).Interestingly,LGP2 also inhibited K48-linked ubiquitination of RIG-I and MDA5,which guaranteed the basal protein levels of RIG-I and MDA5 for subsequently rapid signal activation.All these results reveal a mechanism that LGP2 plays a negative role in RLR signaling pathways to maintain cellular homeostasis in resting state and early phase during GCRV infection.2.HSP70 promotes HMGB1b-mediated antiviral autophagy against GCRV infection by interacting with HMGB1 b,inducing HMGB1 b cytoplasmic translocation,and enhancing HMGB1b-Beclin 1 association in C.idella kidney(CIK)cellsAutophagy plays a wide variety of physiological and pathophysiological roles.However,the roles and the regulatory mechanisms of autophagy in response to viral infections are poorly defined in teleost.In the present study,we found that GCRV infection and hydrogen peroxide(H2O2)treatment induced the accumulation of reactive oxygen species(ROS)in CIK cells,and subsequently intensive autophagy.Whereas ROS-induced autophagy in turn restricted GCRV proliferation.Further investigations demonstrated that grass carp HMGB1 b served as a HSP70-dependent pro-autophagic protein.Upon H2O2 treatment,cytoplasmic HSP70 translocated to the nucleus where it interacted with HMGB1 b,and promoted cytoplasmic translocation of HMGB1 b.In cytoplasm,HSP70 and HMGB1 b synergistically enhanced ROS-induced autophagic activation.Moreover,HSP70 reinforced the HMGB1b-Beclin 1 association and formation of autophagosome clusters via directly interacting with Beclin 1.This study delineates the inducible process of antiviral autophagy,and demonstrates the essential role of HSP70 in HMGB1b-mediated autophagy initiation in teleost.These findings will instruct us of a new means to develop an effective therapeutic strategy for grass carp hemorrhagic disease through activating autophagy initiation and autophagic signal pathway.