Effects of N-linked glycosylation on the infection and proliferation of Japanese encephalitis virus (JEV) | N-糖基化对乙型脑炎病毒感染与增殖的影响

Chino. N-糖基化对于病毒的感染与增殖中均具有重要作用，切除病毒表面糖链或抑制病毒糖基化修饰的方法已成为登革热病毒等的潜在治疗方法。为阐明日本乙型脑炎病毒(Japanese encephalitis virus，JEV)表面的糖链在其感染和增殖过程中的作用，本研究用肽N-糖苷酶F(peptide N-glycosidase F, PNGase F)和内切糖苷酶H(endoglycosidase H, Endo H)处理乙型脑炎病毒，将酶切后的病毒用Western blot验证糖基化酶切的效果，将酶切的JEV接种BHK-21细胞进行蚀斑试验，观察蚀斑的大小及病毒滴度的变化情况。同时用衣霉素处理BHK-21细胞并同步接种JEV，进行蚀斑试验，观察蚀斑大小的变化与病毒增殖情况。结果显示，用Endo H和PNGase F处理JEV后，病毒形成的蚀斑均显著小于未处理的病毒对照，且病毒增殖滴度显著降低，说明病毒粒子表面的糖基化修饰对于病毒的侵染具有重要作用。衣霉素处理细胞后，病毒产生的蚀斑大小没有明显差异，但增殖滴度却显著降低，说明糖基化在JEV增殖过程中也具有重要的功能。本研究说明JEV的糖基化对于其感染和增殖均具有重要的作用，为开发抑制乙型脑炎病毒药物研究提供一个新的思路。

Inglés. N-linked glycosylation site is critical for the infection and proliferation of most virus, elimination or inhibition of glycosylation is becoming a potent therapy of some virus such as Dengue virus. To elucidated roles of N-linked glycosylation in the infectivity and duplication of Japanese encephalitis virus (JEV), in this study, endo-glycosidase enzymes peptide N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) were used to digest JEV to remove the the N-glycosylation on the virus particles surface. Western blotting analysis showed that the glycan chains of JEV particles were successfully removed. To observe the changes in JEV infectivity and proliferation, plaque assay was performed using treated JEV on BHK-21 cells. In another exprement, BHK-21 cells were treated with tunicamycin and infected with JEV. The results showed that JEV digested by PNGase F or Endo H formed smaller plaques than that of control group, while JEV titer was reduced significantly. The plaques formed by JEV in cells treated with tunicamycin were not observed significant differences in size, but JEV titer was also reduced significantly. From these results we conformed that N-glycosylation plays an important role in the process of JEV infection and proliferation, without N-glycosylation, the infectivity and proliferative ability of JEV will be reduced remarkably.This study arises a new potential strategy on JEV therapy.