Effects Of CD44 On Foot-and-mouth Disease Infection

Foot-and-mouth disease(FMD),caused by infection with Foot-and-mouth disease virus(FMDV),is a highly contagious disease of cloven-hoofed animals.FMD leads to significant economic losses due to an epidemic of FMDV all over the world.Our early research using SILAC quantitative proteomic methods found that the expression of CD44 significantly increased in the early stage of FMDV infection.In order to further explore the role of CD44 in BHK-21 cells during FMDV infection,we monitored endogenous CD44 expression in BHK-21 cells using Western blot and RT-PCR assay,the results showed that CD44 level is increased after FMDV infection.Moreover,immunofluorescence assay indicated that cellular CD44 protein colocalized with FMDV capsid proteins under confocal microscopy.In order to further understand the relationship between CD44 and FMDV,we preformed CD44 interference using specific siRNA and the molecular inhibitor,M?CD.FMDV replication was evaluated by Western blot,TCID50 and RT-PCR assay.The results showed that interference of CD44 suppressed FMDV proliferation.In addition,CD44 overexpression was performed using eukaryotic expression plasmid.FMDV replication assay showed that overexpression of CD44 facilitated FMDV replication.Previous studies reported that FMDV entry into host cells via macropinocytosis-dependent manner.The binding of FMDV to RTKs may activate cellular actin modulators and other factors,including p-?PAK,FGF,FLT3,Rac1,which trigger actin rearrangement and plasma membrane ruffling.To elucidate the mechanism of CD44 in BHK-21 cells with FMDV infection,We detected the expression of p-?PAK,FGF,FLT3,P-Rac1,Rac1 after siRNA interference or overexpression of CD44 by Western blot.The result indicated that expression of p-?PAK,P-Rac1,Rac1,FGF,FLT3 were synergistic with CD44,indicating that CD44 participate in macropinocytosis during FMDV infection.To further research whether CD44 is involved in RTKs signaling-mediated macropinocytosis pathway,RTKs specific inhibitor genistein(Gen)was used to block RTKs-mediated macropinocytosis signal in BHK-21 cell.Expression of CD44 was detected by western blot with or without FMDV infection.The result suggested that CD44 was involved in RTKs-mediated micropinocytosis during FMDV infection.In conclusion,this study preliminarily revealed the role of CD44 during FMDV infection and attempted to elucidate the mechanism of the FMDV infection,which will help to provide new target for development of novel anti-FMDV strategy in the future.