Research On The Function Of Three Kinds Of Transmembrane Proteins In Encephalomyocarditis Virus On The Proliferation In Vitro

Encephalomyocarditis virus(EMCV)is a zoonotic pathogen,which can cause encephalitis,myocarditis,neurological diseases,dysgenesis and diabetes.EMCV outbreaks have led to great economic losses in public health and breeding industry.There are more and more researches on EMCV epidemic investigation,diagnosis and monitoring,however,the molecular mechanism of infection,replication and pathogenesis of EMCV is still unclear.We screened host proteins interacting with EMCV structural proteins VP1,VP2,and VP3 respectively by yeast two hybrid,and identified CD63,TMEM39A,and VCAM-1 preliminarily.Many previous studies have shown that these three kinds of membrane proteins are important host proteins involved in the regulation of multiple viral proliferation.The results of yeast two hybrid assay indicated that CD63,TMEM39A and VCAM-1 might take part in the process of EMCV infecting host cells.And this study verified this inference.The techniques of transient overpression and gene knockdown were used to regulate this three proteins expressions in host cells,then this host cells were infected with virus to test the effects of different expression levels of CD63,TMEM39A and VCAM-1 on the proliferation of EMCV.The full-length cDNA of CD63/TMEM39A/VCAM-1 was amplified by RT-PCR and cloned into integrated expression vector pCDNA3.1.CD63/TMEM39A/VCAM-]knockdown cells and cells transfected with recombinant construct pCDNA3.1-CD63/TMEM39A/VCAM-1 or empty vector pCDNA3.1 were infected with EMCV and harvested after 24h and 48h,then EMCV replication and pathogenicity were measured by qRT-PCR and TCID50.The results of EMCV replication suggested that the change of EMCV proliferation had the similar trend with the expressions of CD63/TMEM39A,while VCAM-1 showed the opposite effect on the proliferation of EMCV.Both the statistical differences were significant.These findings indicated that expressions of CD63/TMEM39A/VCAM-1 could change the proliferation of EMCV in host cells,suggesting that CD63/TMEM39A/VCAM-1 were related to proliferation of EMCV in host cells.All the results laid foundations for the further research of EMCV infection mechanism.