人巨细胞病毒感染对宿主细胞周期素依赖性蛋白激酶2的影响

目的 观察HCMV感染细胞周期素依赖性蛋白激酶2（Cdk2）的亚细胞定位，研究HCMV感染对Cdk2蛋白水平及对细胞周期蛋白E（CyclinE）／Cdk2激酶活性的影响。方法通过密度抑制使细胞同步化于G0／G1期，用HCMV AD169毒株感染人胚肺成纤维细胞（HEL），用免疫细胞化学技术分别测定HCMV感染前及感染后24 h Cdk2亚细胞定位；Western Blot法测定HCMV Cdk2蛋白丰度；用免疫沉淀，激酶活性分析法检测HCMV感染细胞内Cdk2的活性。结果 接触抑制阻止在Go期细胞Cdk2游离在细胞质，HCMV感染24h内导致Cdk2从细胞质移位到细胞核。同时HCMV感染可引起cvclinE／Cdk2激酶的强烈激活，但HCMV感染并不诱导Cdk2蛋白丰度增加。结论HCMV感染G0／G1细胞，在24h内导致Cdk2从细胞质移位到细胞核，使之与细胞核内的调节亚单位CyclinE结合，激活CyclinE／Cdk2激酶，使细胞周期越过G1，S限制点，进展至晚G1期。

Influence of human cytomegalovirus infection on CDK2 in host cell

To investigate the subcellular location of Cdk2 in HCMV-infected cells and the effect of HCMV infection on Cdk2 protein level and activity of cyclin E/Cdk2. Methods The subcellular location of Cdk2 in HCMV-infected cell was detected by immunohistochemical method, the abundance of Cdk2 protein determined by Western Blot, the activity of Cdk2 assayed by immunoprecipitation and kinase assay. Results The expression of Cdk2 was sequestered in the cytoplasm of cells arrested in GO by contact inhibition, HCMV infection resulted in translocation of Cdk2 expression from the cytoplasm into the nucleus within 24h after infection in contact-inhibited cells. Meanwhile HCMV infection resulted in robust activation of cyclinE/Cdk2 kinase. However HCMV infection can not induce the increase of the abundance of Cdk2 protein. Conclusion The GO/G1 cells infected by HCMV lead to the transloeation of Cdk2 from eytolymph to nucleus within 24h PI, which results in the combination of Cdk2 and CyelinE.All the above leads to skip GI/S restriction points and cell cycle progresses to later G1.