Effects Of CVB3 Infection On Both Golgi Apparatus And The Cell Cycle Of Hela Cells

Objective:This study investigated the effects of CVB3 infection on Golgi apparatus and the cell cycle of Hela cells with an aim to further research on the pathogenesis of CVB3 infection.Methods:HeLa cells were used as sensitive host cells to do the research as follows which were infected with CVB3 at MOI of 5:(1) To detect the location of the viral protein VP1, cellular proteins GM130(Golgi matrix protein130)and GRASP65(Golgi reassembly stacking Protein 65), these proteins were double labelled immunofluorescently in Hela cells at 0 h,1.5 h,3 h,4.5 h and 6 h post infection.(2) The total cellular proteins were collected for quantitative analysis at 0 h,1.5 h,3 h,4.5 h,6 h,7.5 h,9 h,10.5 h,12 h,13.5 h,15 h,16.5 h,18 h,21 h and 24 h post infection. And then Western blot was used to observe the expression of VP1,GM130 and GRASP65.(3) G1/S phase synchronized or G2/M phase synchronized cells were selected to investigated the effects of CVB3 infection on the cell cycle.After released for 0 h,3 h,6 h and 9 h,the cell cycles of the CVB3 infected group and the mock infected group were detected with flow cytometric assay.Results:1. Double labeling immunofluorescent analysis show:(1) The results of double immunolabelled GM130 and GRASP65 showed that GM130 and GRASP65 colocalized obviously. The Golgi apparatus located perinuclear in the normal Hela cells and the Golgi ribbon was succession.(2) The results of double immunolabelled GM130 and VP1 showed that the immunofluorescence of GM130 was ribbon-like in the normal Hela cells and then become disperse in the infected cells at 3 h post infection.(3) The results of double immunolabelled GRASP65 and VP1 showed that the immunofluorescence of GRASP65 become diffuse obviously in the infected cells at 3h post infection.it was far different from ribbon-like fluorescence in the normal cells.2. Western blot analysis revealed that the expression of GM130 and GRASP65 decreased gradually over time post infection. The expression of GM130 was hardly detected at 18 h post infection ,and also can not detect the expression of GRASP65 at 12 h post infection. The high expression of VP1 had lasted since Hela cells were infected with CVB3 for 6 h.3. Flow cytometry analysis revealed :(1) The Hela was synchronized to G1/S phase by Thymidine treated sequentially for two times.After treated by Thymidine,the cells were released for 0 h,3 h,6 h and 9 h.The cell proportion of G1 phase in the mock infected group is about 89.14%,65.77%,14.14% and 13.11%; The cell proportion of G1 phase in the CVB3 infected group is about 91.08%,84.41%,81.81%,and 81.87%.(2) The hela cells was synchronized to G2/M phase by Nocodazole treated with 1μg/ml for 18 hours.The cell proportion of G2 phase was increased heavily from 4.62% to 40% above. After treated by Nocodazole ,the synchronized cells were released for 0 h,3 h,6 h and 9 h. The cell proportion of G2 phase in the mock infected group is about 42.99%,51.93%,37.97% and 21.35%; but the cell proportion of G2 phase is about 61.25%,43.34%,29.37% and 27.62%,and the G1 phase is about 8.48%,28.01%,28.10% and 33.46% in the CVB3 infected group.Conclusions:1. The Golgi ribbon was brokendown and the structure of Golgi apparatus was destroyed 3h post infection,which was a possible mechanism of causing CPE(cytopathic effect)in CVB3 infected cells.2 . The infection of CVB3 influence the expression of GM130 and GRASP65.The expression of both proteins decreased gradually over time after CVB3 infection. It would be a possible mechanism of causing Golgi apparatus destruction in the CVB3 infected cells.3.After infection with CVB3 ,the Hela cell cycle should be mainly arrest in G1,but could not caused G2 phase cell cycle block .