Foot-and-mouth Disease Virus Antagonizes NOD2-mediated Host Antiviral Effects

Foot-and-mouth disease?FMD?,an acute,febrile and highly contagious disease that caused by foot-and-mouth disease virus?FMDV?,is defined the first class animal epidemic disease in China.Vaccination is an effective measure to prevent FMD in developing countries.There are seven serotypes and many subtypes of FMDV,but lack of the cross-protective efficacy among these serotypes of FMDV,which increases the difficulty of preventing and controlling FMD.Innate immunity is the first line of host defense against invading pathogens.Proteins expressed by FMDV can counteract the host innate immune response to promote their own replication and spreading,resulting in persistent infection.To date,how FMDV antagonizes TLRs-and RLRs-induced innate immune signaling pathways were reported.Nucleotide-binding oligomerization domain 2?NOD2?,a member of NLRs,plays important roles in anti-bacterial,anti-parasitic,and anti-viral infection.However,the role of NOD2-induced signaling pathways in FMDV-infected cells remains unknown.The molecules in the NOD2-induced signaling pathways consist of NOD2,RIP2,MAVS,IRF3,and IKK.NOD2 and RIP2 are specific in NOD2-induced signaling pathways.Therefore,in this study,the innate immune signaling pathways regulated by FMDV and NOD2 were used as the entry point to study the antagonistic mechanism of FMDV on NOD2-induced antiviral effect.The specific contents are as follows:?1?To study the involvement of NOD2-mediated innate immune signal pathways in FMDV infection,we constructed porcine IFN-?and NF-?B promoter luciferase reporter plasmids?IFN-?-Luc and NF-?B-Luc?using PGL3-Basic vector.Our results indicated that FMDV infection triggers NOD2-mediated IFN-?and NF-?B pathways activation,and downregulation of NOD2 significantly impairs IFN-?and NF-?B pathways activation during FMDV infection.A further study indicated that the phosphorylation of endogenous RIP2 and IRF3 was impaired and the expression of IFN-?,OAS1,ISG15,IL1?,and CCL3L1 mRNA were significantly decreased in the NOD2 siRNA cells compared with that in the NC siRNA cells after FMDV infection.?2?PK-15 cells were infected with FMDV?MOI 0.5?,the transcription and translation levels of NLRs were determined.The results showed that transcription of NOD2,NLRP3,and CIITA were found to be significantly upregulated as the infection progressed,but FMDV infection reduced the expression of NOD2,NLRP3,and CIITA proteins.In addition,FMDV infection had no impact on the transcription and translation levels of NLRX1,NLRC5,and NOD1.Replication of FMDV in NOD2-,NLRP3-,and CIITA-down-regulated cells was also assessed.Expression of NOD2mRNA and viral RNA was determined by qPCR assay.Expression of the NOD2 and viral VP1 protein was detected by western blotting,and viral titers were determined by TCID50 assay.The results demonstrate an important antiviral role of NOD2,NLRP3,and CIITA in FMDV replication.?3?To investigate the viral proteins that may be responsible for reduction of NOD2,PK-15 cells were transfected with plasmids expressing various FLAG-tagged viral proteins.It was observed that the expression of 2B,2C,and 3C^pro protein significantly decreased NOD2 protein abundance in a dose-dependent manner.In addition,the regulation of 2B,2C,and 3C^pro on NOD2 transcription was evaluated by qPCR assay,which indicated that 2B,2C,and 3C^pro did not disrupt the transcription of NOD2.A further study indicated that 2B,2C,and 3C^proro induced decrease of NOD2 were independent of the proteasomes,lysosomes,and caspases pathways.In addition,both2B and 2C induced reduction of NOD2 were also independent of the cleavage of host eukaryotic translation initiation factor 4 gamma?eIF4G?and induction of cellular apoptosis.FMDV 2C also significantly induced reduction of RIG-I,TRAF3,VISA,and IRF3,whereas 2C did not induce reduction of TBK1.Over-expression of 2C significantly promoted FMDV replication.PK-15 cells were transfected with plasmids expressing 2B,2C,and 3C^pro mutants,which showed that the mutant that included the carboxyl terminal 101-154 amino acid region of 2B was observed to induce the reduction of NOD2,and the carboxyl terminal 105-114 and 135-144 regions are essential for inducing the reduction of NOD2;all the 2C truncated mutants did not induce the reduction of NOD2;the protease activity of 3C^pro is essential for induction of decrease of NOD2.?4?Software was used to analyze the antigenicity of 2B,2C,3C^pro proteins,and the highly antigenic polypeptides were synthesized to prepare the freund's adjuvant vaccine.Polyclonal antibodies against FMDV 2B,2C,3C^pro were prepared after repeated immunization with rabbits.To investigate the interaction of 2B,2C,3C^pro and NOD2,PK-15 cells were transfected with FLAG–2B-,2C-,3C^pro-expressing plasmid or empty FLAG vector.At 24 hpt,the lysates were immunoprecipitated with anti-NOD2antibody or anti-FLAG antibody and analyzed by western blotting.The results indicated that 2B and 2C interacted with NOD2;3C^proro did not interact with NOD2.The interaction of 2B and 2C with NOD2 in the context of viral infection was further confirmed.PK-15 cells were mock-infected or infected with FMDV for 12 h.The cell lysates were immunoprecipitated with anti-NOD2,2B,or 2C antibody and subjected to immunoblotting analysis.The results confirmed the 2B-and 2C-NOD2 interaction during FMDV infection.In addition,the possible interaction regions of 2B and 2C with NOD2 were also determined.PK-15 cells were transfected with FLAG–2B mutants-expressing plasmid,FLAG–2C mutants plasmids,or empty FLAG vector.Cells lysates were immunoprecipitated with anti-FLAG antibody and analyzed by western blotting.The results indicated that the 2B mutants with the deletion of the carboxyl terminal 105-114 region failed to interact with NOD2;the 2C mutants with the deletion of the carboxyl terminal 116-260 regions failed to interact with NOD2.In conclusion,this study confirmed that FMDV was involved in NOD2-mediated signal pathways,and it was found that NOD2 can inhibit the replication of FMDV.In addition,FMDV 2B,2C,and 3C^pro play an antagonistic role in NOD2-induced antiviral effect,and we preliminarily explore the mechanism of 2B,2C,and 3C^pro to reduce the expression of NOD2 protein.These results have broadened the molecular mechanism of host innate immune system to resist FMDV infection,and revealed new mechanism of FMDV immune escape,which can help better control the prevalence and spread of FMD and also provide new strategies and theoretical basis for the development of new vaccines.