Study On Molecular Mechanism Of Interaction Of RIG-Ⅰand Several Viruses

Retinoic acid inducible-gene I (RIG-I) is a cytosolic pattern-recognition receptor that senses5’-triphosphate single-stranded RNA (5’ppp-ssRNA) or double-stranded RNA (dsRNA) from viruses and triggers an antiviral immune response by activating type-I interferons. In this study, Enterovirus71(EV71), coxsackievirus A16(CA16) and Human Immunodeficiency Virus (HIV) were used as models. To investigate the effect of RIG-I activation on EV71replication, whether the UTR of EV71, CA16and HIV were recognized by RIG-I as dsRNA, the effect of CA16or HIV on RIG-I mediated signal transduction. To elaborate the mechanism of RIG-I mediated antiviral immunity and inhibition of RIG-I by viruse.1. RD cells were transfected with Poly(I:C) or plasmid expressing RIG-I N-terminal, after24h, cells were infected with EV71. At24h post-infection, cells were harvested for RNA and protein analysis and cell culture supernatants were collected for determination of viral titer. The transcription of,IFN-β and RIG-I was detected by Real-Time PCR. The protein expression of RIG-I and EV71was detected by immunoblotting. The results showed that the expression of RIG-I and IFN-P mRNAs were upregulated, RIG-I protein expression could be detected, EV71RNA and protein expression in cells was reduced, Viral titers exhibited a reduction after RIG-I activation.2.293T cells were transfected with an IFN-β promoter-luc construct as well as plasmid expressing RIG-I and24h later were transfected with in vitro-transcribed RNA representing the UTR of EV71, CA16and HIV. IFN-β promoter activity was measured by reporter gene assay after24h. RD cells were transfected with indicated UTR and24h later native PAGE and immunoblotting were performed to detect dimerization of IRF-3. RD cells were transfected with GFP-IRF3.24h later, cells were transfected with UTR.12h later, cells were visualized with fluorescence microscopy. UTR and RIG-I binding assay and co-immunoprecipitation experiement were performed. The results showed that the UTR of EV71, CA16and HIV induced IFN-β promoter and activation of IRF3. Moreover, the UTR of enterovirus co-precipitated with RIG-I in the co-immunoprecipitation experiement.3. Cells were transfected with the plasmids pIFN-P-luc and pRL-CMV and then infected with CA16.24h later, IFN-β promoter activity was measured by reporter gene assay. Cells were infected with CA16, the transcription of IFN-β was detected by Real-Time PCR after6h,12h,24h and48h. Cells were transfected with the plasmids pIFN-β-luc, pRL-CMV, RIG-I and3C protein of CA16or protease of HIV.24h later, IFN-β promoter activity was measured by reporter gene assay. Cells were transfected with the plasmids RIG-I N-terminal, GFP-IRF3and3C protein of CA16or protease of HIV.24h later, cells were visualized with fluorescence microscopy. Cells were transfected with the plasmids RIG-I, IPS-1and3C protein of CA16or protease of HIV.24h later, immunoprecipitation was used to detect the interaction of RIG-I and the3C protein of CA16or protease of HIV. The results showed that CA16infection does not induce IFN-P production, the3C protein of CA16or protease of HIV inhibits RIG-I mediated induction of IFN-β promoter activation and IRF3nuclear translocation. The3C protein of CA16or protease of HIV interacts with RIG-I and disrupts the interaction of RIG-I and IPS-1.These findings indicated that RIG-I activation and subsequent induction of DFN-β have inhibitory effects on EV71replication. The UTR of EV71, CA16and HIV were recognized by RIG-I, leading to the induction of type I IFNs. The3C protein of CA16or protease of HIV inhibits RIG-I mediated activation of induction of IFN-β by interacting with RIG-I.