Study On The Antagonism Mechanism Of Host RIP2-mediated Anti-foot-and-mouth Disease Virus Infection

Foot-and-mouth disease?FMD?is an acute,febrile and highly contagious disease that caused by foot-and-mouth disease virus?FMDV?.The host innate immune response and vaccination are effective measures to prevent FMD.There are many serotypes of FMDV,but lack of the cross-protective efficacy among these serotypes of FMDV,which increases the difficulty of preventing and controlling FMD.Therefore,the host innate immune response plays important roles in inhibiting FMDV replication.FMDV has evolved several strategies during long-term proliferation to inhibit the host innate immune response,promoting viral replication.For example,the mechanisms that FMDV antagonizes TLRs-and RLRs-induced innate immune signaling pathways have been reported.In 2009,an article reported that nucleotide-binding oligomerization domain 2?NOD2?induced innate immune signaling pathway plays important roles in anti-viral infection.NOD2 and its downstream molecule RIP2 are specific proteins in the NOD2-induced innate immune signaling pathway.Our laboratory had reported that FMDV can inhibit NOD2-induced innate immune signaling pathway by inhibiting the expression of NOD2 protein.However,the impact of RIP2 on FMDV replication remains unknown.Therefore,in the present study,the innate immune signaling pathways regulated by RIP2 and FMDV were used as the entry point to study the antagonistic mechanism of FMDV.The specific contents are as follows:?1?Luciferase experiment is used to determine that RIP2 affected FMDV-induced IFN-?and NF-?B pathways activation.Further study indicated that the downregulation of NOD2 significantly impairs FMDV-induced the phosphorylation of endogenous IRF3 and P65 and decreases the expression of IFN-?,ISG15,IL1?,and CCL3L1 mRNA.?2?PK-15 cells that transfected with the different dose of RIP2 plasmid and RIP2siRNA were infected with FMDV,the viral VP1 protein,RNA,and titers were determined.The result indicated that RIP2 can inhibit FMDV replication.?3?PK-15 cells were infected with FMDV,the mRNA levels and abundance of RIP2 were determined.The results showed that the mRNA levels of RIP2 were found to be significantly upregulated as the infection progressed,but the abundance of RIP2were found to be significantly decreased as the infection progressed.Further study indicated that FMDV also induced the reduction of RIP and RIP3.?4?PK-15 cells were transfected with plasmids expressing various FLAG-tagged viral proteins,the expression of RIP2 was detected.The results showed that 2B,2C,L^pro,and 3C^pro protein significantly decreased abundance of RIP2 protein in a dose-dependent manner.Further study indicated that 2B,2C,L^pro,and 3C^pro protein induced the reduction of RIP2 were independent of the lysosomes,proteasomes,and caspases pathways.PK-15 cells were transfected with plasmids expressing 2B,2C,L^pro,and 3C^pro mutants,the expression of RIP2 was detected.The results showed that the protease activity of L^pro and 3C^pro is essential for the reduction of RIP2,the carboxyl terminal 105-114 and 135-144 regions of 2B are essential for the reduction of RIP2,the nterminal 1-61 region of 2C is essential for the reduction of RIP2.Co-immunoprecipitation assays showed that 2C interacted with RIP2,and the nterminal 1-61 region of 2C is essential for the interaction between 2C and RIP2.?5?To explore the mechanism that 2C inhibited the expression of RIP2,PK-15cells were transfected with plasmids expressing 2C,the expression of PABPC1 was detected.The results showed that FMDV 2C decreased the abundance of PABPC1protein,but did not affected the mRNA levels of PABPC1.The nterminal 1-61 region of 2C is essential for the reduction of PABPC1.Further study indicated that the reduction of PABPC1 protein induced the reduction of RIP2,revealing a novel mechanism of 2C to reduce the expression of RIP2 protein.In conclusion,this study confirmed that RIP2 affected FMDV-induced IFN-?and NF-?B pathways activation,and RIP2 can inhibit FMDV replication.In addition,FMDV 2B,2C,L^pro,and 3C^pro play antagonistic roles in RIP2-induced antiviral effect,and we explore the mechanism of 2B,2C,L^pro,and 3C^pro to decrease the expression of RIP2 protein.This study revealed a new mechanism of FMDV immune escape,and can help better control of FMD and also provide new theoretical basis for the development of vaccines.