The2A Protease Of Enterovirus71Cleaves Nup62to Inhibit Nuclear Transport

OBJECTIVE: To investigate the interaction of human enterovirus71(EV71) and its2A protease with nuclear pore protein p62(Nup62) and the resulting influence on thenuclear pore complex (NPC) and its nuclear transport function. METHODS: A greenfluorescent protein carrier with nuclear localization signal (pGFP-NLS) wastransfected into RD cells, and24hours later, the RD cells were separately infected with1TCID50,10TCID50,100TCID50of EV71virus, then the cells were observed for thelocalization of green fluorescent protein under fluorescence microscope after infectedfor8hours; EV712A protease constructed in eukaryotic expression vector andpGFP-NLS were transfected into RD cells together for observing the localization ofgreen fluorescent protein under fluorescence microscope after transfected for48h;RD cells were infected by1TCID50,10TCID50and100TCID50of EV71virus.8hourslater, cells were collected and lysis for detection of Nup62by Western blotting.Meanwhile, proliferation of EV71was detected by RT-PCR of2A gene;2A proteaseconstructed in eukaryotic expression vector was transfected into RD cells, the cells werecollected respectively24h,36h and48h after transfection for detection of Nup62byWestern blotting. At the same time, we extracted the cell RNA and detected theamplification of EV71-2A by RT-PCR. RESULTS: After transfection of pGFP-NLSplasmid for24h and infection with virus for8h, we can see under the microscope thatthe expression of green fluorescent protein appeared in cytoplasm, while in uninfectedcells, the expression of green fluorescent protein in the nucleus because the EV71virusstopped the nuclear transfer of green fluorescent protein(GFP-NLS); After transfection of2A protease conducted in eukaryotic expression vector and pGFP-NLS together for48h, we observed under microscope that green fluorescent protein expressed in thecytoplasm, while in the control cells untransfected with2A protease, the expression ofgreen fluorescent protein in the nucleus;The western blotting results of Nup62showedthat expression of Nup62in cells infection with different titers of EV71virus decreasewith the increase of the virus virulence, and Nup62was significantly cut in cellsinfected with EV71virus of100TCID50. RT-PCR of gene2A results also showed thatthe virus can replicate in cells;The western blotting results of RD cells transfected fordifferent time indicated that the Nup62decreased, which was most in the48th hours,RT-PCR results showed that expression of2A protease has time dependence. With thetransfection time became longer, the expression of mRNA of2A increased, reflectingthat the expression of2A protease also increased. CONCLUSION: This studydemonstrates that the EV71virus damages the structure of the nuclear pore complex bycleaving nuclear pore protein p62with2A protease, thus affecting its nuclear transportfunction.