The Molecular Mechanism Underlying Foot-and-Mouth Disease Virus Nonstructural Protein2C Affects Host Innate Immunity

Foot and mouth disease is a highly contagious zoonotic disease caused byFoot-and-Mouth Disease Virus, a single positive stranded RNA virus, which causes blisters in the mouth and feet of bovids and other cloven-hoofed animals. FMDV is the most contagious OIE listed disease which has a great potential for causing economic loss in susceptible animals. FMDV can replicate in the host rapidly and infect other animals via aerosol or through contact. FMDV has seven serotypes, namely A, O, C, Asial, SAT1, SAT2and SAT3and each serotype can be further differentiated as several subtypes. Infection with one serotype does not confer immunity to another, thus increasing difficulties to control FMD.The non structural protein2C is the largest membrane binding protein involved in FMDV replication. The2C protein is a multi-functional proteinlocalized in the cytosol, whichcan cause the rearrangement of the intracellular membrane system, bind to the viral RNA cellular protein and other viral proteins.2C is expected to be capable of binding the organelles and act as an ATPase.2C plays a vital role in viral replication complex as it can interact with many host cellular components. However its role in host cells need to be furthered.We performed the Yeast Two-hybrid assay using FMDV protein2C as the bait and the PK15cDNA library as the prey to screen the2C-interacting proteins. We found that N-Myc and STAT interactor (Nmi) intract with2C after the online sequence blast at NCBI.The interaction between2C and Nmi in293T, BHK-21and PK15cells was confirmed by Co-IP, endogenous Nmi pull-down and Confocal assay using monoclonal antibodies.2C protein shows the membrane binding activity, distributes in the vesicular structures in cytosol, and binds the Mitochondria and ER.After binding2C, Nmi is taken to the vesicular structure in the cytosol, while under natural conditions Nmi is homogeneously distributed in the cytosol. These findings may incicate the role of2C in cellular membrane system rearrangement.IFP35is a member of IFP family. This35KD protein is one of the Nmi binding partners and can inhibit the viral genes expression. Like2C, IFP35localizes Nmi to the vesicular structures. We also found that2C can interact with Nmi-IFP35complex as it co localize with this complex on the vesicular structures. Expression of2C has no effects on the protein levels of endogenous Nmi and IFP35, although2C changes the distribution of them in the host cells.At the level of promoter、mRNA and protien, We used Dual-luciferase、Real-time PCR and plaque formation assay showed that transfection of HEK293cells with pEGFP-2C or pEGFP-Nmi induced expression of type I interferon and NF-κB promoters but not AP-1promoters. The plaque formation assay using VSV confirmed the results. Knockdown of IFP35expression by siRNA abolished pEGFP-2C or pEGFP-Nmi induced activation of type I interferon and NF-κB promoters, suggesting that IFP35may play a critical role in FMDV2C-induced type I interferon expression acting as Nmi downstream adaptor.These data further our understandings and provide new insights into the molecular mechanisms of the persistent infections and immune escapes of FMDV, and also help to elucidate the host-virus interaction for the development of better strategies to control FMD.