Leader Protein Of Foot-and-Mouth Disease Virus Inhibits NF-?B-Mediated Expression Of Type ? Interferon

Foot-and-mouth disease virus(FMDV)is the pathogen of foot-and-mouth disease(FMD)in livestock,which mainly infects cloven-hoofed animals,such as cattle,sheep,and pigs,and causes highly contagious epidemics.The characteristic symptoms of animals infected with FMD is the occurrence of vesiculation in the mouth,nose,hoof,and breasts,indicating that FMDV primarily infects epithelial tissue of the host.IFN-?3,discovered in recent years,has been identified as a distinct member of type III interferon(IFN)and displays specific antiviral effects targeting epithelial tissues.At present,little is known about the relationship of porcine-derived IFN-?3 and FMDV infection.Therefore,the study of porcine-derived IFN-?3 will provide new insights for effective FMD prevention and control.The leader(L)protease(Lpro)plays important roles in antagonizing the host innate immune responses during FMDV infection.It can significantly regulate expression of IFN through multiple pathways.In order to obtain anti-Lpro antibody,prokaryotic expression method was employed to produce recombinant Lpro.Subsequently,high-titer rabbit polyclonal antibody serum against Lpro was prepared using recombinant Lpro,and it could specifically recognize Lpro synthesized during FMDV infection.For the purpose of preparing porcine-derived IFN-?3 protein,the IFN-?3 gene was cloned into the prokaryotic expression plasmid p ET-28a(+),and the target protein was induced and expressed using E.coli BL21.Finally,high concentration of recombinant porcine-derived IFN-?3 was generated after protein purification and refolding.The effect of porcine IFN-?3 on FMDV replication was tested.The results showed that IFN-?3 treatment significantly inhibited FMDV proliferation in a dose-dependent manner,and knockdown of IFN-?3 markedly increased the titer of FMDV,all demonstrating that porcine-derived IFN-?3 suppressed FMDV replication.The influence of FMDV infection on IFN-?3 expression was also assessed and we found that FMDV downregulated poly(I:C)-induced IFN-?3 production.Lpro encoded by FMDV was identified as a viral antagonist of IFN-?3 and the inhibition was related to the cleavage function of Lpro.The predicted result using Jaspar software showed that there was a number of NF-?B binding sites on IFN-?3 promoter sequence.It has been reported that p65/Rel A,the subunit of NF-?B,was involved in the regulation of IFN transcription.Therefore,we hypothesized that p65/Rel A might act as a regulator of IFN-?3.Our experimental data proved that overexpression of p65/Rel A significantly increased the m RNA level of IFN-?3 and inhibited FMDV replication,which was consistent with our hypothesis above.Subsequently,we investigated whether Lpro regulated the production of IFN-?3 by dependence on p65/Rel A.The results indicated that Lpro could not only degrade endogenous p65/Rel A,but also cleave exogenous p65/Rel A,and the catalytic activity of Lpro was required for p65/Rel A cleavage.In addition,Lpro and p65/Rel A were examined to be co-localized in the nucleus.These findings suggested that Lpro could directly cleave p65/Rel A.The interaction between Lpro and p65/Rel A led to the downregulation of p65/Rel A-mediated IFN-?3 expression and this phenomenon was also related to the catalytic function of Lpro.In summary,the rabbit polyclonal serum against Lpro was prepared,and porcine-derived IFN-?3 protein was obtained.We found that the Lpro could antagonize IFN-?3 antiviral response through cleaving p65/Rel A,which helped to understand the pathogenic mechanism of FMDV and develop new strategy to control FMD in swine.