Antagonism Of Selected Key Factors Of RLRs Signal Transduction Pathway In Human-original Cells By Bovine Rotavirus UK Strain

Rotavirus is one of the leading pathogens,resulting in gastroenteritis in young animals and infants worldwide annually,with 500 to 600 thousand deaths in infants and young children worldwide annually.Rotavirus is a zoonotic pathogen,however,specific drugs for treatment of diseases associated with rotavirus infection are currently unavailable and there is the vaccine administration is the most useful regimes to prevent the diseases due to rotaviruses.The clinical efficacy of oral,live rotavirus reassortant human vaccine based on UK strain through the modified “Jenner principle” is less than 50% in clinical trials in Africa and other places.To date,it is demonstrated that RVs enables to antagonize the innate immune response of the host by various route,which may be related to immunosuppression of innate immune response targeting such kind of rotavirus vaccines.RV NSP1 is the key protein that antagonizes host innate immunity by varied route.RIG-I like receptors(RLRs)are the cytoplasmic pattern recognition receptors,which enables to recognize viral double-stranded RNAs,induce downstream interferon regulatory factor,promote the secretion of type I IFN and activate the host innate immune response.In this study,RV UK strain as the stereotype was selected to explore the mechanism of rotavirus on the selected key factors of RLRs signal transduction pathway in human colon adenocarcinoma cell line Caco-2 at the level of RNA transcription and protein,and as well as the effect of UK NSP1 protein on the selected key factors of RLRs signal transduction pathway in human embryonic kidney cell 293 T in protein level.The Caco-2 cells infected by UK RV were harvested 6 h,12 h and 24 h post infection,respectively.The transcription level of RIG-I,MDA5,IRF3(IFN regulation factor 3),IRF7 and RV VP6 mRNA was quantified by real-time quantitative PCR.The results showed that VP6 mRNA increased gradually over the transfection time,and IRF3,IRF7,RIG-I and MDA5 mRNA increased slightly over the time of infection,and the expression of RIG-I and MDA5 in the early stage of infection increased significantly.The results demonstrated that RV UK strain infection activated RLRs pathway of human-original innate immune response.In addition,the Caco-2 cells infected by UK RV were harvested 6 h,12 h and 24 h post infection,respectively and the kinetics of IRF3,RIG-I and MDA5 were confirmed by western blot.The IRF3 protein decreased,and the RIG-I and MDA5 increased slightly over the time of infection.The results validated that RV UK strain mediated degradation of IRF3 protein.In order to investigate the effect of UK NSP1 on the key factors of congenital immune RLRs signal transduction pathway,a pCMV-3×FLAG-NSP1 eukaryotic expression plasmid was constructed and transfected into 293 T cells.The cells were harvested at 4 h,8 h and 12 h post transfection,respectively and the results showed that IRF3 protein content decreased over transfection time,in contrast,RIG-I and MDA5 protein did not change significantly.Meanwhile,the deletion mutation of the IRF3 binding domain of pCMV-3 × FLAG-NSP1 △ IRF3 eukaryotic expression plasmid was transfected into 293 T cells.The cells were harvested at 4 h,8 h and 12 h post transfection,respectively.The contents of IRF3,RIG-I and MDA5 were detected by western blot.The results showed that the IRF3,RIG-I and MDA5 did not change significantly over transfection time.The pEGFP-C2-RIG-I,pEGFP-C2-MDA5 and pEGFP-N3-IRF3 eukaryotic expression plasmids were transfected into 293 T cells with pCMV-3×FLAG-NSP1 eukaryotic expression plasmids respectively.The cells were harvested at 4 h,8 h and 12 h post transfection,respectively.The contents of IRF3,RIG-I and MDA5 were detected by western blot.The results showed that IRF3 protein content decreased,RIG-I and MDA5 increased over co-transfection time.Prior to transfection,293 T cells were pretreated by proteasome inhibitor MG132 for 1 h and the content of IRF3 protein did not change significantly in pCMV-3×FLAG-NSP1 transfected group over transfection time.The contents of NSP1 and IRF3 protein increased in the pCMV-3 ×FLAG-NSP1 and pEGFP-N3-IRF3 co-transfected group.The data showed that the expression of RIG-I and MDA5 in the RLRs signal transduction pathway increased and activated the host innate immune response after Caco-2 cells was infected with RV UK strain,however,the RV NSP1 protein mediated the degradation of the downstream signal molecule IRF3 to antagonize the innate immune response.NSP1 mediated the degradation of IRF3 protein,which depended on integrity of IRF3 binding domain of NSP1 and the proteasome-dependent pathway.Elucidation of the antagonism of selected key factors of RLRs signal transduction pathway in human-original cells by bovine rotavirus UK strain may help understand the cause that the efficacy of reassortant vaccines was unsatisfied and facilitate the development of novel recombinant vaccines,as well as drugs,by which degradation of IRF3 by NSP1 in proteasome-dependent manner is suppressed.In addition,it may contribute to understanding of rotavirus molecular biology.