The Influence Of ATF5 Expression On HCMV Replication In Glioma Cells

Objective:1. To detect the expression of human cytomegalovirus(human cytomegalovirus, HCMV) genes, proteins and ATF5 in U87, SY5 Y and A172 glioma cells infected with HCMV.2. To investigate the effects of down-regulation of ATF5 gene on the virus replication in the glioma cells.Methods:1. The U87, SY5 Y and A172 cell were infected with HCMV virus at a multiplicity of infection(MOI) of 5 then the changes of the cell morphology were observed.2. The U87、SY5Y and A172 cell were infected with HCMV virus at a multiplicity of infection(MOI) of 5 then the samples of culture supernatants were removed for virus titer determination at 24, 48, 72, 96,120 h post-infection(hpi). Plaque assay was applied for quantitative determination of virus titer.3. Real-time PCR and WB(WB) were used to test the Immediate-Early gene(IE2), early gene(UL44), late gene(UL99)and ATF5 genes and protein expression.4. Use lentivirus expressing ATF5 samll interfering RNA to knockdown the ATF5 gene expression in U87 and SY5 Y cells, fluorescence microscope observation of the efficiency of infection, WB testing of ATF5 protein expression levels, then choosing the best conditions and parameters.5. Down-regulation of the expression level of ATF5 in the U87 and SY5Y glioma cells to detect the level of virus replication.Results:1. After infected with HCMV the U87 and SY5 Y were found to be having shorter cell processes and becoming thicker and more aggregated, compared with the uninfected cells. In contrast, the A172 cells have no significant difference.2. Plaque assay was applied to quantitative determination of virus titer the results show that the U87 and SY5 Y culture supernatants have a significant difference. In contrast, the A172 cells have no significant difference.3. Real-time PCR and WB results show that the HCMV genes and proteins were expressed in U87 and SY5 Y were obviously higher compared with the corresponding hours in the A172 group(P<0.05), and the ATF5 gene and protein were also higher than the A172 group(P<0.05).4. Pearson analyse show that after the U87 and SY5 Y cells were infected with HCMV the expression of IE protein has a positive correlation with ATF5 protein,U87(r=0.955,P <0.05)and SY5Y(r=0.956,P <0.05).5. In the condition of Complete Medium+5μg/ml Polybrene, U87( MOI=1)、SY5Y(MOI=15) and red fluorescent protein can be detected by fluorescence microscopy, at 72 h the transfection efficiency excees 95% after the cells were infected with lentivirus. The WB was used to test the ATF5 protein expression. The relative expression levels of normal group, negative control group CON053 and LV-ATF5-RNAi group were 0.31±0.02,0.29±0.02 and 0.07±0.03 in U87 cells,in the SY5 Y cells The relative expression were 0.29±0.01,0.28±0.04 and 0.06±0.03.6. A recombinant lentivirus expressing ATF5 samll interfering RNA was constructed and used to infect U87 and SY5 Y cells to knockdown ATF5 gene expression, which resulted in decreased levels of viral replication.Conclusions:1. The level of HCMV replication is higher in U87 and SY5 Y cells than A172 cells.2. The level of HCMV replication is higher in U87 and SY5 Y cells than A172 cells can be correlated with ATF5 expression level.3. Down regulation of ATF5 expression in glioma cells can inhibit the replication of HCMV.