Structural and biochemical studies of RIG-I antiviral signaling

Retinoic acid-inducible gene I(RIG-I)is an important pattern recognition receptor that detects viral RNA and triggers the production of type-I interferons through the downstream adaptor MAVS(also called IPS-1,CARDIF,or VISA).A series of structural studies have elaborated some of the mechanisms of dsRNA recognition and activation of RIG-I.Recent studies have proposed that K63-linked ubiquitination of,or unanchored K63-linked polyubiquitin binding to RIG-I positively regulates MAVS-mediated antiviral signaling.Conversely phos-phorylation of RIG-I appears to play an inhibitory role in controlling RIG-I antiviral signal transduction.Here we performed a combined structural and biochemical study to further define the regulatory features of RIG-I signaling.ATP and dsRNA binding triggered dimeriza-tion of RIG-I with conformational rearrangements of the tandem CARD domains.Full length RIG-I appeared to form a complex with dsRNA in a 2:2 molar ratio.Com-pared with the previously reported crystal structures of RIG-I in inactive state,our electron microscopic struc-ture of full length RIG-I in complex with blunt-ended dsRNA,for the first time,revealed an exposed active conformation of the CARD domains.Moreover,we found that purified recombinant RIG-I proteins could bind to the CARD domain of MAVS independently of dsRNA,while S8E and T170E phosphorylation-mimick-ing mutants of RIG-I were defective in binding E3 ligase TRIM25,unanchored K63-linked polyubiquitin,and MAVS regardless of dsRNA.These findings suggested that phosphorylation of RIG inhibited downstream signaling by impairing RIG-I binding with polyubiquitin and its interaction with MAVS.

Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model

AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP),which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence.Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy.Cellular localization and protein levels were examined by Western blotting.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom mitochondria to block the activation of interferon (IFN)-β promoter,thus resulting in downregulation of SEAP activity.The decrease in SEAP activity was proportional to the dose of active NS3/4A protease.Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.CONCLUSION:Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors.This system will constitute a new tool to allow the efficient screening of HCV inhibitors.

Retinoic acid inducible gene-I,more than a virus sensor

Retinoic acid inducible gene-I(RIG-I)is a caspase recruitment domain(CARD)containing protein that acts as an intracellular RNA receptor and senses virus infection.After binding to double stranded RNA(dsRNA)or 5′-triphosphate single stranded RNA(ssRNA),RIG-I transforms into an open conformation,translocates onto mitochondria,and interacts with the downstream adaptor mitochondrial antiviral signaling(MAVS)to induce the production of type Ⅰ interferon and inflammatory factors via IRF3/7 and NF-κB pathways,respectively.Recently,accumulating evidence suggests that RIG-I could function in non-viral systems and participate in a series of biological events,such as inflammation and inflammation related diseases,cell proliferation,apoptosis and even senescence.Here we review recent advances in antiviral study of RIG-I as well as the functions of RIG-I in other fields.

TAX1BP1 protects against myocardial infarction-associated cardiac anomalies through inhibition of inflammasomes in a RNF34/MAVS/NLRP3-dependent manner

Acute myocardial infarction(MI),one of the most common cardiovascular emergencies,is a leading cause of morbidity and mortality.Ample evidence has revealed an essential role for inflammasome activation and autophagy in the pathogenesis of acute MI.Tax1-binding protein 1(TAX1BP1),an adaptor molecule involved in termination of proinflammatory signaling,serves as an important selective autophagy adaptor,but its role in cardiac ischemia remains elusive.This study examined the role of TAX1BP1 in myocardial ischemic stress and the underlying mechanisms involved.Levels of TAX1BP1 were significantly downregulated in heart tissues of patients with ischemic heart disease and in a left anterior descending(LAD)ligation-induced model of acute MI.Adenovirus carrying TAX1BP1 was delivered into the myocardium.The acute MI induced procedure elicited an infarct and cardiac dysfunction,the effect of which was mitigated by TAX1BP1 overexpression with little effect from viral vector alone.TAX1BP1 nullified acute MI-induced activation of the NLRP3 inflammasome and associated mitochondrial dysfunction.TAX1BP1 overexpression suppressed NLRP3 mitochondrial localization by inhibiting the interaction of NLRP3 with mitochondrial antiviral signaling protein(MAVS).Further investigation revealed that ring finger protein 34(RNF34)was recruited to interact with TAX1BP1 thereby facilitating autophagic degradation of MAVS through K27-linked polyubiquitination of MAVS.Knockdown of RNF34 using siRNA nullified TAX1BP1 yielded protection against hypoxia-induced MAVS mitochondrial accumulation,NLRP3 inflammasome activation and associated loss of mitochondrial membrane potential.Taken together,our results favor a cardioprotective role for TAX1BP1 in acute MI through repression of inflammasome activation in a RNF34/MAVS-dependent manner.