摘要
人类疱疹病毒6型(human herpesvirus 6, HHV-6)属于疱疹病毒β亚科,分为HHV-6A和B两个亚型。HHV-6是引起婴幼儿急疹(exanthem subitum, ES)的病原。文献报道HHV-6与淋巴系统增生性疾病、多发性硬化症(multiple sclerosis, MS)、急性脑炎、慢性疲劳综合症、病毒性心肌炎、器官移植后感染等多种疾病有关。HHV-6感染能影响宿主细胞周期进程甚至能诱导细胞凋亡,但其具体的分子机制目前还未进行过详细的研究。本课题以人T淋巴细胞系HSB2为研究对象,探讨HHV-6感染对HSB2细胞周期进程及细胞凋亡的影响及其分子机制。
首先,我们大量收集HHV-6 GS标准株感染的CBMC细胞,反复冻融后,高速离心对病毒浓缩纯化。然后构建标准质粒,制作标准曲线,用实时荧光定量PCR的方法测定病毒滴度。
其次,分别用细胞计数法和MTT法测定HHV-6感染对HSB2细胞增殖的影响,结果显示HHV-6感染能显著抑制细胞增殖,其作用具有时间依赖性;流式细胞术结果显示,感染细胞发生了G2/M期阻滞,进一步检测M期的标志分子磷酸化H3(Ser10),证明细胞周期停滞于G2期;实时荧光定量RT-PCR和Western blot结果显示,HHV-6感染使cyclinA2、cyclinB1、cyclinE1的蛋白水平和mRNA水平提高,cyclinD1的蛋白水平和mRNA水平均无明显变化;此外,Western blot和实时荧光定量PCR研究结果表明G2/M期细胞环境有利于病毒DNA复制和蛋白的表达。
再次,本研究探讨了HHV-6诱导细胞G2阻滞的机制。体外激酶活性分析表明HHV-6感染抑制了cyclinB1-Cdk1(cdc2)激酶活性;Western blot结果显示磷酸化cdc2(Tyr15)表达量增加,总cdc2蛋白的表达也稍有增加;进一步研究表明HHV-6感染上调Weel蛋白表达,提高了Myt1激酶活性,并降低磷酸酶Cdc25C的活性。病毒感染使p53、p21、p27的蛋白和mRNA表达水平显著增加,并呈时间依赖性,并且磷酸化p53(Ser15)的表达也急剧增加;此外,HHV-6感染使磷酸化Chk2 (Thr68)和其下游分子磷酸化Cdc25C(Ser216)表达增加。以上结果提示,HHV-6感染可通过多途径,多分子协同诱导细胞G2阻滞。
另外,在本研究中发现HHV-6感染导致G2期阻滞之后诱导了细胞凋亡,因此我们对其诱导凋亡的途径进行了探讨。流式细胞术PI单标法和Annexin V-FITC/PI双标检测均表明HHV-6在感染晚期可诱导凋亡。电子显微镜观察显示感染细胞染色质高度凝集、边缘化,细胞核碎裂呈碎片状,线粒体形态也发生了变化,表现为体积增大,肿胀,嵴紊乱、断裂; HHV-6感染后caspase-3,caspase-9活性显著升高,而caspase-8活性无变化;线粒体膜电位下降,Bcl-2蛋白表达水平下降,Bax表达水平增加。以上表明线粒体凋亡途径参与HHV-6诱导的细胞凋亡。
综上所述,本研究结果提示HHV-6是通过多条途径、多种因子协同促进细胞的G2期阻滞,通过caspase依赖的线粒体途径诱导细胞凋亡。本研究为HHV-6感染相关疾病寻找新的治疗靶点提供了理论依据和新思路。
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.
引文
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