Studies On The Role Of Caveolin-1 In EMCV Infected Host Cells By Endocytosis Pathway

To confirm whether EMCV infected cells by endocytosis,endocytic pathway inhibitors were added before EMCV incubated with BHK-21 cells.The EMCV replication was detected by virus titres,virus copies and viral structural protein VP1.The results suggested that endocytic pathway took part into replication of EMCV in BHK-21 cells.In addition,during EMCV infect cells,co-localization of endocytic pathway marker-EEA1 with EMCV VP1 was observed,this result suggested that EMCV could infect BHK-21 cells by endocytic pathway.To confirm which way does EMCV pass through,different endocytic pathway inhibitors was added into BHK-21 cells before EMCV infected,the results indicated that EMCV infected BHK-21 cells through Caveolin-dependent endocytosis but not Clathrin-dependent endocytosis pathway and Macropinocytosis.Then we examined the function of Dynamin and Actin in EMCV replication in BHK-21 cells,which were related with Caveolin-dependent endocytosis.The results suggested that Actin and Dynamin also affected EMCV infection in BHK-21 cells.Finally,we do the experiments that added specific inhibitors after the EMCV incubated with BHK-21 cells,to detect whether those endocytic pathway played a role in the late stage of virus infection cycle.The results suggested that the endocytic pathway only played a role in the early stage of EMCV replication in BHK-21 cells in vitro.To detected the function of the main structural proteins-Caveolin-1 in EMCV replication in BHK-21 cells,EMCV was inoculated to cells that Caveolin-1was overexpressed and silenced,quantitative polymerase chain reaction,virus titres and western blot analyses showed that Caveolin-1 can promote EMCV infected BHK-21 cells.In addition,immunofluorescent confocal microscopy analysis suggested that Caveolin-1 was temporally colocalized with EMCV VP1 in infection.These results suggested EMCV could infect BHK-21 cells by Caveolin-1.Because the endocytosis pathway always closely linked with virus attachment and internalization.Therefore,we explored whether Caveolin-1 related with EMCV attachment andinternalization.The results of qRT-PCR and virus titres suggested that Caveolin-1affected virus internalization process but not attachment.These results suggest that:(1)EMCV can infect BHK-21 cells by endocytosis.(2)EMCV infect BHK-21 cells through Caveolin-dependent endocytosis,don’t through Clathrin-dependent endocytosis and Macropinocytosis.(3)Related proteins of Caveolin-dependent endocytosis-Actin and Dynamin is involved in the process of EMCV infected BHK-21 cells.(4)Caveolin-1 plays an important role in EMCV infected BHK-21 cells,and mainly plays a role in virus internalization process.