Mechanism Of Rotavirus NSP3 Protein Inhibiting Type I Interferon Signaling

Rotavirus(RV)belongs to the family revirus family.The genome consists of 11Double-stranded RNA(ds RNA)and encodes 12 proteins,including 6 structural proteins(VP1?VP4,VP6 and VP7)and 6 non-structural proteins(NSP1?NSP6).Rotavirus is one of the important pathogens causing viral diarrhea in young children and young animals.Previous studies found that NSP1 protein functions to inhibit IFN-signaling,but whether other proteins affect type I interferon signaling function needs further investigation.The purpose of this study was to explore the mechanism of action of RV-encoded proteins in regulating type I interferon signaling.In this paper,the RV genomic DNA of SA11 strain was obtained by RT-PCR,constructed with eukaryotic expression plasmid.By using dual luciferase reporter system,protein degradation and phosphorylation test,we elucidated the regulatory mechanism of rotavirus-encoded protein in the IFN-signaling pathway.It mainly includes two parts: firstly,constructed the eukaryotic expression vector of RV SA11 strain encoding protein to screen the rotavirus protein of SA11 strain that regulates IFN-signaling pathway.Secondly,clarified the mechanism of SA11 NSP3 protein on the regulation of type I interferon signaling pathway.The research contents are summarized as follows:1.In this study,the RV of SA11 strain was used as the research material,and the target genes VP4,VP6,VP7,NSP1,NSP2,NSP3,NSP4,NSP5 and NSP6 were obtained by RT-PCR method to construct the eukaryotic expression plasmid p RK-Flag-VP4,PRK-Flag/HA-VP6,p RK-Flag/HA-VP7,p RK-Flag/HA-NSP1,p RK-Flag-NSP2,p RK-Flag/HA-NSP3,p RK-Flag/HA-NSP4,p RK-HA-NSP5,p RK-HA-NSP6,Western blotting proved that the above plasmids were expressed successfully in HEK293 T.The above recombinant plasmids and IFN-?-Luc/ISRE-Luc plasmids were co-transfected into HEK239 T cells.After stimulation by RIG-IN(the activation domain of RIG-I),the dual luciferase reporter gene system was used to determine the protein's influence on IFN-?/ISRE reporter gene activities.In summary,the eukaryotic expression plasmids p RK-Flag-NSP1 and p RK-Flag-NSP3 could significantly reduce the dual luciferase activity induced by RIG-IN which proved that NSP3 protein had the same inhibition function of IFN-I signaling pathway.2.In this study,the RV of the SA11 strain was used as the research material to clarify the mechanistic effects of the eukaryotic expression plasmid p RK-Flag /HA-NSP3 in regulating the type I interferon signaling pathway.We based on the ISRE and IFN-dual-luciferase reporter systems,detecting the dose-dependent effect of signal-molecule-mediated reporter experiments,possibly upstream of MAVS.subsequently,We overexpressed of NSP3 protein in HEK293 T cells,measuring the key molecules IRF3 and I?B?,showed that NSP3 protein inhibited Se V-mediated phosphorylation of IRF3 and I?B?.The m RNA levels of IFN-?RLRs and its downstream ISGs(ISG56,CXCL10,OASL)were measured by quantitative PCR in the presence of NSP3 protein,and showed that NSP3 protein significantly inhibited the expression of IFNB1,IFIT2,and CXCL10 stimulated by Se V,RIG-IN,MDA5-N.Finally,through cellular degradation experiments,the NSP3 protein and RIG-I actions did not interact with other signaling molecules.Overall,it suggests that NSP3 protein functions to downregulate RNA virus-mediated interferon production.