细胞周期蛋白激酶9(CDK9)对卡波氏肉瘤相关疱疹病毒(KSHV)裂解复制周期的影响

摘要

目的 探讨细胞周期蛋白激酶9(cyclin-dependent kinase 9,CDK9)在卡波氏肉瘤相关疱疹病毒(Kaposi′s sarcoma-associated herpesvirus,KSHV)裂解复制周期中的作用.方法 分别应用CDK9特异性抑制剂FIT-039处理或CDK9小干扰RNA(siRNA)转染iSLK.219及TREx-K-Rta BCBL-1细胞,四环素(Dox)或佛波酯(TPA)和丁酸钠刺激48 h后,分别采用荧光显微镜和实时定量PCR(qPCR)方法观察红色荧光蛋白(RFP,病毒裂解复制)阳性细胞数目及KSHV相关基因的mRNA转录水平.染色质免疫沉淀(ChIP)试验检测KSHV PAN启动子上RNA PolⅡ及其磷酸化CTD-S2的富集含量.结果 FIT-039处理和CDK9-siRNA转染后,iSLK.219细胞RFP阳性率显著降低、TREx-K-Rta BCBL-1细胞中KSHV裂解复制周期相关基因ORF50、PAN及K2的mRNA转录水平明显下降.且FIT-039处理后,TREx-K-Rta BCBL-1细胞中KSHV PAN启动子上结合的RNA PolⅡ及其磷酸化CTD-S2含量显著下降.结论 CDK9可能通过磷酸化RNA PolⅡ丝氨酸调控KSHV病毒裂解复制.%Objective To investigate the role of cyclin-dependent kinase 9 ( CDK9) in regulating lytic replication of Kaposi′s sarcoma-associated herpesvirus ( KSHV) .Methods Percentages of red fluores-cent protein (RFP) positive cells were counted after treating and stimulating iSLK .219 cells with FIT-039, a CDK9 specific inhibitor , and doxycycline ( Dox ) , respectively , or transfecting and stimulating them with CDK9 siRNA (si-CDK9) and Dox, respectively.Quantitative real-time PCR was used to detect the expres-sion of KSHV genes at mRNA level in TREx-K-Rta BCBL-1 cells treated with FIT-039 or transfected with si-CKD9 in the presence of Dox.Chromatin immunoprecipitation (ChIP) assay was used to examine the enrich-ment of RNA polymerase Ⅱ( RNA PolⅡ) and phosphorylated CTD-S2 of RNA PolⅡon the PAN promot-er in TREx-K-Rta BCBL-1 cells treated with FIT-039.Results Both FIT-039 intervention and CDK9 knockdown dramatically deceased the percentage of RFP-positive iSLK.219 cells and suppressed the expres-sion of ORF50, PAN and K2 in TREx-K-Rta BCBL-1 cells at mRNA level.Furthermore, FIT-039 signifi-cantly inhibited the enrichment of RNA Pol Ⅱand phosphorylated CTD-S2 of RNA Pol Ⅱon the PAN pro-moter in TREx-K-Rta BCBL-1 cells.Conclusion CDK9 is involved in regulating the lytic replication of KSHV through promoting the phosphorylation of CTD-S2 of RNA Pol Ⅱ.