Mechanism Of Non-structural Protein 3B Of Foot-and-mouth Disease Virus Type O Affecting Host Innate Immunity

Foot-and-mouth disease(FMD)is a severe infectious disease of artiodactyls caused by foot-and-mouth disease virus(FMDV),which is extremely harmful to the animal husbandry worldwide and China.The innate immune response is the first line of defense against pathogen invasion and plays an important role in the prevention of pathogen transmission and disease development.The retinoic acid-induced gene protein I-mediated signaling pathway could be activated after FMDV invades the host cells,thereby inducing the synthesis and secretion of type I interferon and inflammatory factors to inhibit the proliferation of FMDV.The 3B protein(VPg)of FMDV has a low molecular structure and is therefore relatively stable.Only when the structure and activity of the 3B protein are normal,infective virus cells could be produced in the host cells.Early studies on host anti-FMDV infection found that FMDV could regulate the host's natural immunity and induce type I interferon production after infection.FMDV's multiple structural and non-structural proteins,as well as genomic RNA,are involved in this regulation.At present,there are few studies on FMDV 3B protein,its function during FMDV replication,and its interaction with host proteins are unclear.In this study,FMDV 3B protein was used as the bait protein,and the host proteins interacting with FMDV 3B protein were screened in host cells by immunoprecipitation-mass spectrometry.The important related proteins in the natural immune system were selected.Gene overexpression experiments and siRNA interference experiments were conducted to study the mechanism of FMDV non-structural protein 3B affecting host congenital immunity.The results obtained are as follows:1.Host proteins that interact with the FMDV 3B protein were screened in PK-15 cells.The nucleotide sequence of 3B was amplified by using FMDV China/1/99 strain genome as template.The recombinant vector Flag-3B and Flag-GFP were constructed and transfected into PK-15 cells for detection by Western blot.The expression of GFP was observed by fluorescence microscopy and the expression of GFP was examined by fluorescence microscopy.The optimal expression time of the target protein was 36 h after transfection,so the cells were harvested for co-immunoprecipitation experiments at 36 h.Then using the 3B protein of FMDV as bait protein,the co-immunoprecipitation experiment against Flag antibody was carried out,and the host protein interacting with 3B protein was precipitated,and the precipitated product was sent to Shanghai Shengke Biotechnology Co.,Ltd.for massspectrometry analysis.Finally the central junction protein VISA in the natural immune system was selected from the mass spectrometry results as the next research object.2.FMDV 3B protein interacts with VISA protein both inside and outside the cell.The host cell protein VISA interacting with the FMDV 3B protein was previously screened by co-immunoprecipitation-mass spectrometry.In order to avoid the existence of false positive results,we verified the interaction between 3B protein and VISA protein in vivo and in vitro by immunoprecipitation and GST pull-down.Firstly,the VISA nucleotide sequence was amplified from PK-15 cells and the eukaryotic expression vector VISA-Myc was constructed.The plasmid VISA-Myc was transfected into 293 T cells for expression,and the VISA protein was successfully detected in 293 T cells by Western blot.VISA-Myc and Flag-3B were co-transfected into PK-15 cells,and anti-Flag agarose beads were used for co-immunoprecipitation experiments.The presence of VISA-Myc protein was detected in the precipitate using rabbit anti-Myc polyclonal antibody,proving that 3B protein can interact with VISA protein in cells.Then,the fusion protein GST-3B was expressed in E.coli.Western blot experiment showed that the optimal expression time was 6 h after induction.At the same time,fusion protein VISA-Myc was expressed into 293 T cells.Fusion protein was extracted for GST pull-down experiment.The presence of VISA-Myc protein was detected in the pellet using the rabbit anti-Myc polyclonal antibody,demonstrating that the 3B protein can also interact with the VISA protein in vitro.To identify the interaction domain of the 3B protein,a series of 3B truncated plasmids Flag-3B1,Flag-3B2 and Flag-3B3 were constructed,and it was identified by immunoprecipitation experiments that it is the 3B3 protein interacted with the VISA protein.3.Effect of VISA protein on the proliferation of FMDV.After infection of PK-15 cells by FMDV,After FMDV infected PK-15 cells,the transcription and protein expression of VISA gene were detected at 0h,2h,4h,6h,8h,12 h and 24 h after infection.Quantitative real-time PCR showed that both the FMDV and VISA mRNA levels increased with the duration of FMDV infection,suggesting that FMDV infection can up-regulate the transcription of VISA gene in PK-15 cells.The amount of endogenous VISA protein detected by Western blot gradually decreased with the extension of FMDV infection time.PK-15 cells were transfected with VISA-Myc plasmids of different concentrations and inoculated with1 MOI FMDV 24 h after transfection.Quantitative real-time PCR showed that with the increase of VISA-Myc plasmid transfection,the amount of VISA mRNA increased significantly,while the amount of FMDV mRNA decreased significantly.TCID50 test showed that the virus titer of FMDV in the cell culture supernatant was also significantly reduced,indicating that overexpression of VISA in PK-15 cells can significantly inhibit theproliferation of FMDV.The siRNA was used to interfere with the transcription of VISA gene in PK-15 cells,then 0.5 MOI of FMDV was inoculated,and FMDV mRNA transcription levels of FMDV were detected at 0,4,8 and 12 h after inoculation.Fluorescence quantitative PCR showed that compared with the control group siVISA-nc,the FMDV genomic RNA in PK-15 cells transfected with siVISA-1 was significantly increased,indicating that interference with VISA could promote the replication of FMDV.4.The effect of FMDV 3B and VISA on the type I interferon pathway.The VISA-Myc plasmid was transfected into PK-15 cells.Real-time quantitative PCR showed that overexpression of VISA in PK-15 cells could up-regulate the mRNA levels of VISA-mediated antiviral factors TRAF2?TRAF3?TRAF6?TBK1?IFN-??IFN-??IL-6?IL-8?ISG15and OAS1,indicating that VISA can induce the production of host antiviral factors.IFN-?and IL-6 ELISA kits were used to detect the expression of IFN-? and IL-6 in the supernatant of PK-15 cells.The results showed that overexpression of VISA in PK-15 cells could increase the secretion of IFN-? and IL-6.FMDV infection can reduce VISA-mediated mRNA expression of IFN-? and IL-6.This results shows that FMDV infection could inhibit the production of natural immune factors mediated by VISA.In summary,this study demonstrates that FMDV 3B protein interacts with host cell VISA protein both intracellularly and extracellularly.The level of endogenous VISA transcription increased in FMDV-infected PK-15 cells,and the amount of VISA protein gradually decreased with the extension of FMDV infection time.Overexpression of VISA in PK-15 cells inhibited the replication of FMDV,while down-regulation of VISA expression in PK-15 cells promoted the replication of FMDV,indicating that VISA plays an important role in the host anti-FMDV infection.Further studies have shown that overexpression of VISA could up-regulate VISA-mediated mRNA levels of innate immune factors,while overexpression of VISA could enhance the secretion of IFN-? and IL-6.FMDV infection could inhibit the secretion of IFN-? and IL-6,and overexpression of 3B protein could also inhibit the secretion of IFN-? and IL-6.These experimental results indicate that FMDV 3B interacts with VISA protein through its 3B3 protein to inhibit VISA signaling pathway and reduce the expression of IFN-? and IL-6,thereby evading the host's innate immune system and promoting FMDV replication.