The Effect And Mechanism Of Human Herpesvirus 6 Infection On HSB2 Cell Cycle And Apoptosis

Human herpesvirus-6 belongs to the beta-herpes virus subfamily and can be further classified into two variants, A and B. It is identified as the causative agent of exanthem subitum, and also related with lymphoproliferative disease, multiple sclerosis, encephalitis, chronic fatigue syndrome, viral myocarditis and reactivation or reinfection in immunocompromised hosts. HHV-6 infection can influence cell cycle progression and induce cell apoptosis, however, the mechanism underlying its actions remain unclear. In this study, we try to elucidate some mechanisms of influencing cell cycle and apoptosis of HSB2 cells infected by HHV-6.Firstly, The GS strain of HHV-6 was propagated in CBMCs stimulated with PHA. HHV-6 infected and uninfected cells were collected and subjected to the freeze-thaw cycle. HHV-6 supernatant was concentrated by high speed centrifugation (80,000rpm 2h). The standard plasmid of pMD19T-U22 was constructed, then the virus titer was determined by real-time PCR.Secondly, we observed negative effect of HHV-6 on the proliferation of HSB2 cells in time-dependent manner through MTT assay and cell counting; Cell cycle profiles by flow cytometry showed that G2/M phase arrest was induced by HHV-6. The further study showed that G2 phase arrest was induced; Using immunoblotting analysis and real-time RT-PCR, we found mRNA and protein levels of cyclinA2,cyclinB1,cyclinE1 were increased in HHV-6 infected cell. But there was no change in cyclinD1. Moreover, it is suggested G2/M arrest promotes HHV-6 replication and protein expression.Thirdly, we further studied the mechanism of HHV-6 induced G2 arrest. In vitro kinase assay showed HHV-6 infection inhibited cyclin B1-Cdk1 kinase activity; After HHV-6 infection, total cdc2 and phospho-cdc2 (Tyr15) levels were increased in a time-dependent manner; The high level of phospho-cdc2 (Tyr15) was due to increase Wee1 protein level, Myt1 kinase activity and decrease Cdc25C activity; We also found the mRNA and protein levels of p53, p21 and p27 were increased markedly in HHV-6 infected cell, furthermore, the phospho-p53 (Ser15) were also increased, these results indicate that HHV-6 activates p53 and then induces p21 expression. Phospho-Chk2 (Thr68) and Phospho-Cdc25C(Ser216) was also induced after HHV-6 infection. These results suggest HHV-6 induced G2 arrest is associated with change of many kinds checkpoint regulatory protein.Lastly,we study the mechanism of HHV-6-induced apoptosis. Apoptosis was determined by staining cells with Annexin V- FITC/PI in flow cytometry; Chromatin condensation and nuclear fragmentation along with dramatic changes of mitochondria were observed by electron microscopy; The cell death was associated with activation of caspase 3, caspase 9, and cleavage of PARP, which is known to be an important substrate for activated caspase 3, but no change in caspase-8 activity; Moreover, HHV-6 infection increased Bax, decreased Bcl-2 protein levels. HHV-6 infection also induced disruption of mitochondrial transmembrane potential. Taken together, our present results suggest that HHV-6 infection induces apoptosis of the host cell through mitochondrion-mediated, caspase 3-dependent pathway.In summary, our observations highlight the presence of multiple distinct but interrelated mechanism to induce G2 arrest and apoptosis after infection with HHV-6. Understanding interactions between viruses and host cells provides new targets for treatment HHV-6 related diseases.