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  

Effects of cyclin-dependent kinase 9 （CDK9） on lytic replication of Kaposi＇s sarcoma-associated herpesvirus （KSHV）

作　　者：张庆[1] 史震 李小翠 王晓天 薛敏 周峰 

机构地区：[1]徐州市肿瘤医院检验科,221002 [2]徐州医科大学江苏省免疫与代谢重点实验室,221004 [3]徐州医科大学生理学教研室,221004

出　　处：《中华微生物学和免疫学杂志》2017年第11期810-815,共6页Chinese Journal of Microbiology and Immunology

基　　金：江苏省自然科学基金面上项目（BK20141136）;国家自然科学基金青年基金项目（81500914）

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 expression of KSHV genes at mRNA level in TREx-K-Rta BCBL-1 ceils 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 Pol Ⅱ） and phosphorylated CTD-S2 of RNA Pol Ⅱ on the PAN promoter in TREx-K-Rta BCBL-I 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 expression of ORFS50, PAN and K2 in TREx-K-Rta BCBL-1 cells at mRNA level. Furthermore, FIT-039 signifi- cantly inhibited the enrichment of RNA Pol Ⅱ and phosphorylated CTD-S2 of RNA Pol Ⅱ on 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 ohosphorylation of CTD-S2 of RNA Pol Ⅱ.

关 键 词：细胞周期蛋白激酶9 卡波济肉瘤相关疱疹病毒 病毒裂解复制周期 基因转录延伸 

分 类 号：R373[医药卫生—病原生物学]