The Mechanism Of Localization Of Enterovirus71Nonstructural Protein3A

EV71epidemics and pandemics have been periodically reported worldwide. Different clinical features such as neurological diseases are caused by EV71infection. Enterovirus infection could affect the integrity of membrane structure and induce the membrane vesicel as virus replication factory.The mechanism underlying the membrane reconstitution and vesicle formation is not clear, but it has been speculated that the nostructural proteins may play a role. Enterovirus3A protein is found in vesicle and is a membrane associated protein, but how the3A protein localized to the vesicle membrane and the function in vesicle genesis need futher investigation.This research consisted of three parts:The first part was the construction of full length infectious cDNA clone of EV71. Genome cDNA of EV71was obtained by reverse transcription. Three overlapping genome fragments were amplified and cloned into pBluescript Ⅱ vector, respectively. Then three genome fragments were orderly cloned into pBluescriptⅡ to construct full length infectious cDNA clone of EV71under T7promoter control, which was the foundation for research of the virus.The second part include the prokaryotic expression and antibody development of EV713A protein.The3A gene was amplified by PCR and cloned into vector PET28a to make PET28a-3a for the prokaryotic expression of3A protein.The recombinant3A protein was expressed in E. coli BL21and subsequently used to immunize mouse. Western blot analysis showed the resultant mouse anti-3A sera reacted with eukaryotic expressed EGFP-3A fusion protein. Moreover, the antisera recognized EV71infected cells by immunofluorescence staining, demonstrating that anti-3A antibody was successfully prepared and may provide a useful tool for function study of3a gene.The third part is the mechanism research of localization of3A protein.Different domains of3a were amplified by PCR and cloned into vector pEGFP-C1to express GFP and3A mutant fusion proteins. The localization of fusion protein was observed in the RD cells.The results showed that3A protein was localized the endoplasmic reticulum, and the TMD domian is the localization signal. The results of Co-IP demonstrated that3A protein interacted with host ASNA1protein, a membrane of membrane localization appratus. Virus replication was reduced after ASNA1was interferenced by RNAi,, demonstrated that ASNA1protein may be involved in the virus replication.