Human Cytomegalovirus Ie86 Protein Research In Arpe-19 Cell Cycle

Objective:To study the effects of HCMV infection on cell cycles of ARPE-19, observe the relationship between the effects and HCMV IE86 genes, and explore the pathogenesis of HCMV infection.Methods:Synchronized ARPE-19 were infected with HCMV, the mock-infected group was set up as control group simultaneously. Infected ARPE-19 were harvested at 1,2,3, 4 and 5 days of post-infection. HCMV IE mRNA was detected by RT-PCR, HCMV IE proteins were detected by immunofluorescence assay, HCMV IE proteins and pp65 were detected by western blot assays. Cell proliferation of ARPE-19 after HCMV infection was detected by MTT assay. Cell cycles of ARPE-19 was detected by flow cytometry analysis. Cyclin D1 and Cyclin E were detected by western blot assays. Eukaryotic expression plasmid pEGFP-N3-IE86 was constructed and transfected into ARPE-19 cells with Lipofectamine 2000. The expression of IE86 was tested in ARPE-19 cells by RT-PCR and immunofluorescence assay. Cell cycles of transfected ARPE-19 was detected by flow cytometry analysis.Results:The HCMV IE72 and IE86 mRNA were expressed respectively at 1 and 3 days of post infection. The HCMV IE72, IE86 and pp65 were expressed respectively at 2,3 and 5 days of post infection. MTT show that the OD ratio of HCMV were respectively (0.646±0.0558),(0.664±0.0765),(0.723±0.1474) and (0.803±0.1073) at 3,4,5 and 6 days of post infection. The FCM results show that the S phase ratio in the cell cycles were respectively 21.78%±2.65%,26.65%±0.71% and 31.80%±3.97% at 3,4 and 5 days of post infection. There were significant difference(P<0.05)in the number of cells in S phase between the mock-infected group and HCMV-infected group at 3,4 and 5 days of post infection. The amount of cyclin E protein significantly increased in HCMV-infected cells at 3 dpi, while cyclin D1 levels dramaticly decreased at 4 dpi. The recombinant plasmid pEGFP-N3-IE86 had been constructed correctly and the expression of IE86 gene could be detected in APRE-19 cells. The FCM analytic results showed that cell cycles had the same changes after cells were transient transfected by pEGFP-N3-IE86.Conclusions:HCMV can replicate and propagate in ARPE-19, but HCMV genes had a delayed kinetics. HCMV infection influences the progression of cell cycle of ARPE-19 and arrests cell cycle at S phase. This may be induced by HCMV IE86.