Research On The Interaction Mechanism Between Vimentin And 3A Protein Of Foot-and-mouth Desease And Viruse

Foot-and-mouth disease?FMD?,a highly contagiouse and economically devastating viral disease of cloven-hoofed animals,is caused by foot-and-mouth disease viruse?FMDV?.FMD is topped the list of highly contagious viral diseases reportable to the World Organization for Animal Health?OIE?,and is a first class animal infectious diseases in China.China as one of the largest animal husbandry countries in the word,the sum of pigs,cattles,and sheeps was more than 1.8 billion annually in China.The outbreak and epidemic of FMD disease have caused serious damage to the breeding industry in China.However,the pathogenesis of FMDV infection has not yet been fully revealed.FMDV is a specific intracellular parasitic organism.Viral protein interacts with host proteins extensively during the formation of FMDV replication complexes.In the earlier period,we screened host vimentin protein which may interacts with non-structural protein 3A of FMDV interact with the vimentin of the host by co-immunoprecipitation technology,but its mechanism of action is still unclear.1.To evaluate the improtance of the binding between 3A and vimentin to FMDV replication,we tried to identify amino acid residues within 3A that directly mediate the interaction with vimentin.We used an alanine scanning mutagenesis approach and GST pull-down to determine the binding site?s?for vimentin present in 3A.The result revealed that 3A proteins containing mutations in areas 3 was unable to bind vimentin.Therefore,the 15^th-21^thh amino acids in FMDV 3A protein are the key amino acids which interact with vimentin.2.Immunofluorescence was performed to identify the localization of the cellular vimentin,and the results suggested that infection of FMDV to PK-15 cells lead to rearrangement of vimentin.,Mock-infected cells have the regular pattern of vimentin immunostaining with filaments distributed throughout the cell and extending from the perinuclear region to the plasma membrane.In contrast,this pattern was altered in cells infected with FMDV for 3 h,yielding a vimentin network that has withdrawn from the cellular periphery and accumulated at the nuclear periphery.In addition,both the protein expression levels and mRNA transcription levels of cellular vimentin were a clear transitory downturn at first time and then upturn during FMDV infecton.However,FMDV infecton did not lead to cleavage of vimentin.These results demonstrated that FMDV infection results in vimentin rearrangement,and the rearrangement is not a result of proteolytic cleavage of vimentin.3.PRRSV infection of Marc-145 cells was blocked partially by vimentin which was purified and renatured from E.coli expression systems.The result showed that vimentin was biological activity.However,FMDV infection of PK-15 cells was not blocked by vimentin.Moreover,flow cytomertry indicated that the vimentin distributed in cytoplasm instead of the surface of epicyte.Therefore,vimentin did not play role in FMDV adsorption and penetration.In summary,these results revealed that vimentin had no effect on the process of FMDV adsorption and penetration to host cells.However,FMDV 3A interacted with vimentin during FMDV infection.These studies provided important informations for further study the pathogenesis of FMDV infection,and new ideas for prevent and control FMDV infection.