Inhibitory Effect Of The Foot-and-Mouth Disease Virus Proteins 2B,3B On The RLRs-Mediated Type ? Interferon Response

Foot-and-mouth disease virus?FMDV?is the causative agent of Foot-and-mouth disease?FMD?,which is an acute and highly contagious disease affecting pigs,cattles,sheep and other cloven-hoofed animals.Due to the serious economic consequences it causes to the livestock production,FMD is listed as a notifiable animal disease by the Office International des Epizooties?OIE?.Also it is enlisted as ClassI animal disease in China.Previous studies have shown that FMDV encoded proteins,including the non-structural protein L^pro,3A,3C^pro,and structural protein VP1,play important roles in host innate immune evasion.Yet the function of FMDV non-structural proteins 2B and 3B in inhibiting the host's innate immune response is largely unknown.Therefore,in the current study we mainly carry out research on FMDV 2B and 3B,to clarify whether they inhibit the RIG-?-like receptor-mediated type ? interferon response and its related mechanisms.In this study,we found that over-expression of FMDV 3B in HEK293T cells markedly inhibited the expression of IFN-?,IL-6,ISG15 at the mRNA level,and significantly inhibited Poly?I:C?-induced or SeV-triggered activation of the IFN-?,NF-?B and ISRE promoter were examined by reverse transcription real-time quantitative PCR?RT-qPCR?and dual-luciferase reporter assays,it is speculated that FMDV 2B inhibited the transcriptional activation of type ? interferons and pro-inflammatory cytokines.To explore the mechanism of FMDV 2B inhibits RLR-mediated type ? interferon response in HEK293T cells.Phosphorylation assay showed that the phosphorylation levels of TBK1 and IRF3 in HEK293T cells were decreased after overexpression of FMDV 2B.Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-? and MDA5.Moreover,our data showed that FMDV 2B inhibited the transcriptional abundance of RIG-? and MDA5,and inhibited the expression of both exogenous and endogenous RIG-? and MDA5,but not VISA,TRAF3,TRAF6,TBK1 and IRF3.During another study,we found that over-expression of FMDV 3B in HEK293T cells markedly inhibited the expression of IFN-?,IL-6,ISG15 at the mRNA level,and significantly inhibited Poly?I:C?-induced or SeV-triggered activation of the IFN-?,NF-?B and ISRE promoter.The results of dual-luciferase reporter assays demonstrated that FMDV 3B protein targeted at the RIG-? and MDA5 level to regulate the RLR-mediated type ? interferon response.RT-qPCR analysis showed that over-expression of FMDV 3B down-regulated the transcription level of the RIG-? and MDA5.Meanwhile,we also verified that all three proteins 3B1/3B2/3B3 had inhibitory effects on the activation of type ? interferon-related promoters in HEK293T cells.In summary,we identified that FMDV 2B could interact with RIG-? and MDA5,and inhibit the transcription and expression of the RIG-? and MDA5.Furthermore,we also revealed that FMDV 2B could inhibit the phosphorylation of TBK1 and IRF3 in the RLR signaling pathway,thus inhibiting the expression of type ? IFNs.In addition,FMDV 3B presents inhibitory effects on type ? interferon response in HEK293T cells,and all proteins 3B1/3B2/3B3 inhibit the activation of type ? interferon-related promoters.These findings could improve our understandings of the mechanisms of FMDV's evasion of the host immune system,and provide reference for developing novel FMD vaccines and formulating more effective prevention and control strategies against FMD.