Effect Of Human Cytomegalovirus Infection On Cell Cycle And Pre-replication Complex Of Human Embryonic Lung Fibroblast

Objective To study the effect of human cytomegalovirus(HCMV) infection on cell cycle and pre-replication complex(pre-RC)of human embryonic lung fibroblast(HELF).Methods1.To synchronize the HELF in G₀/G₁ phase by serum starvation and measure cell cycle of HELF by flow cytometry analysis.Synchronized HELF were infected with HCMV as HCMV-infected group(n=1×10⁶), the simulant-infection group was set up as control group(n=1×10⁶) simultaneously.Observation of the pathomorphological change of infected HELF was performed until 7 days of post infection by inverted phase contrast microscope.Infected HELF were harvested at 12,24,48, 96 hours of post infection.2.Cell cycle of HELF was detected by flow cytometry analysis.3.HCMV IE₁72 protein and Cdt1 protein of HELF was detected by immunocytochemistry method.4.To extract the total RNA.The expression levels of Cdt1 mRNA, Cdc6 mRNA,Mcm2 mRNA,Mcm8 mRNA were detected by semiquantitative reverse transcription-polymerase chain reaction(RTPCR).5.The expression level of Geminin mRNA was detected by RT-PCR.Results1.The change of morphology of HELF:The change of morphology was not observed in the simulant-infected cells.In HCMV-infected group, the partial cells became round,bulgy,and their cytoplasm and nucleus became large at 72 hours of post-infection,cytopathic effect was observed in all infected cells at 7 days of post-infection.2.The expression of positive products ofHCMV IE₁72 protein:The positive products of HCMV IE₁72 protein were not detected in the simulant-infected cells.In HCMV-infected group,the positive products of HCMV IE₁72 protein were detected at 12 hours of post-infection.3.The cell cycle of HELF:At 12 hours of post-infection,70.4%of HELF were in G₁ phase in the HCMV-infected group;but 65.7%of HELF were in G₁ phase in the simulant-infected group.At 24 hours of post-infection,69.9%of the HCMV-infected cells were in G₁ phase but 43.8%of the simulant-infected cells were in G₁ phase.The statistical analysis suggested that there were significant difference(P＜0.01)in the number of cells in G₁ phase between the simulant-infected group and HCMV-infected group both at 12 and 24 hours of post-infection.4.The expression level of Cdt1 mRNA:The expression level of Cdt1 mRNA reached a peak at 12 hours of post-infection,then decreased gradually,and reached a nadir at 72 hours,but then returned a little at 96 hours.In the simulant-infection group;while the expression level of Cdt1 mRNA was low at 12 hours of post simulant-infection,then increased slightly at 24 hours,and reached a peak at 48 hours,but then began to decrease at 72 hours,and at 96 hours,maintained the same level as at 72 hours.The statistical analysis suggested that the expression levels of Cdt1 mRNA in HCMV-infected group were significantly lower(P＜0.05)than the ones in the simulant-infection group at 12,24 hours of post-infection, but the expression levels of Cdt1 mRNA in HCMV-infected group were significantly higher(P＜0.05)than the ones in the simulant-infection group at 48,72hours of post-infection.The graphs of the expression level of Cdt1 mRNA suggested that the expression level reached a peak at 12 hours of post simulant-infection,but the expression level reached a peak at 48 hours of post infection,which markedly lagged behind the one in the simulant-infection group.5.The expression level of Cdt1 protein:There were significantly difference in expression levels of Cdt1 protein in two groups(P＜0.05)at 12,24,96 hours of post infection,but there were not significantly difference in expression levels of Cdt1 protein in two groups(P＞0.05)at 48,72 hours of post infection.The expression levels of Cdt1 protein in HCMV-infected group were significantly lower than the ones in the simulant-infection group at 12,96 hours of post infection,but the expression levels of Cdt1 protein in HCMV-infected group were significantly higher than the ones in the simulant-infection group at 24 hours of post infection.The expression level of Cdt1 protein in simulant- group reached at a peak at 12 hours of simulant-infection,reached a nadir at 24 hours,but then returned a little at 96 hours.In the HCMV-infection group;while the expression levels of Cdt1 protein were lower at 12 hours of post infection,reached a peak at 24 hours,and then decreased sharply at 48 hours,and maintained constantly lower stable level.6.The expression level of Cdc6 mRNA:There were significantly difference in expression levels of Cdc6 mRNA in two groups(P＜0.05)at 12,24,48,72,96 hours of post infection,the expression levels of Cdc6 mRNA in HCMV-infected group were significantly higher than the ones in the simulant-infection group at 12,24,48 hours of post infection.,but the expression levels of Cdc6 mRNA in HCMV-infected group were lower than the ones in the simulant-infection group at 72,96 hours of post infection.In the HCMV-infected group the expression level of Cdc6 mRNA reached at a peak at 12 hours of infection,then decreased gradually,and reached a nadir at 72h,but then returned a little at 96h, while in the simulant-infection group the expression level of Cdc6 mRNA reached at a peak at 12 hours of post simulant-infection,then decreased gradually,and reached a nadir at 48h,but then returned a little at 72h,and continued increasing at 96hours.7.The expression levels of Mcm2 mRNA,Mcm8 mRNA:The statistical analysis suggested that the expression levels of Mcm2 mRNA and Mcm8 mRNA in HCMV-infected group were significantly lower (P＜0.05)than the ones in the simulant-infection group at 12,24 hours of post infection,but the expression levels of Mcm2 mRNA and Mcm8 mRNA in HCMV-infected group were significantly higher(P＜0.05)than the ones in the simulant-infection group at 48,72,96 hours of post infection.The graphs of the expression levels of Mcm2 mRNA and Mcm8 mRNA suggested that the expression levels reached a peak at 24 hours of post simulant-infection in the simulant-infection group,but in HCMV-infected group the expression levels reached a peak at 48 hours of post infection,which markedly lagged behind the ones in the simulant-infection group.8.The expression levels of Geminin mRNA:The expression levels of Geminin mRNA in HCMV-infected group were significantly higher (P＜0.05)than the ones in the simulant-infected group at 12,24 hours of post infection,but the expression levels of Geminin mRNA in HCMV-infected group were significantly lower(P＜0.05)than the ones in the simulant-infected group at 48,72 hours of post infection. The graphs of the expression levels of Geminin mRNA suggested that the expression levels of Geminin mRNA reached at a peak at 12 hours of post infection in the HCMV-infected group,but the expression levels of Geminin mRNA reached at a peak at 48 hours of post simulant infection in simulant-infection group.Conclusion1.HCMV can replicate and propagate in HCMV-infected HELF.2.HCMV infection influences the progression of cell cycle of HELF and arrests cell cycle at G₁ phase.3.The expression level of HCMV IE₁72 protein suggested the early infection of HCMV.4.HCMV infection has an effect on the expression level of replication licensing factor as Cdt1mRNA,Mcm2 mRNA,Mcm8 mRNA, and makes the expression of them slag behind.HCMV can induce the expressions delaying of Cdt1 protein.HCMV can induce the over expressions of Cdc6 mRNA and degradation pathway of Cdc6 by the HCMV infection,accumulation of Cdc6.5.HCMV infection can induce the abnormal expression of Geminin mRNA,and resulting in the Geminin/pre-replication complex imbalance, which could be responsible for the blockage in cell cycle in G₁ phase by HCMV infection.