【作者】 贾雪梅；

【导师】 卢春；

【作者基本信息】 南京医科大学 ， 免疫学， 2010， 博士

【摘要】 目的:研究人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)病毒蛋白R(viral protein R,Vpr)对卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)裂解性周期复制的影响,并对此过程所涉及到的信号通路进行鉴定。方法:1、包装和鉴定含Vpr基因的重组慢病毒,并进行滴度测定。以重组慢病毒Vpr感染原发性渗出性淋巴瘤细胞系【primary effusion lymphoma,PEL,又称体腔淋巴瘤(body cavity-based lymphoma,BCBL)】来源的BCBL-1细胞,反转录RCR(reverse transcription PCR,RT-PCR)和免疫印迹(Western blot)分别检测Vpr基因的mRNA转录和蛋白表达。2、用病毒感染方法将Vpr基因导入PEL细胞系,采用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,real-time PCR)、RT-PCR和Western blot检测KSHV裂解期基因的mRNA转录和蛋白表达;进一步检测感染后细胞上清中KSHV病毒含量,并以RT-PCR和Western blot检测经上清感染的Vero细胞中KSHV裂解期和潜伏期基因mRNA转录和蛋白表达;将重组慢病毒Vpr感染PEL细胞系,然后转染含KSHV ORF50/ORF73基因启动子的虫荧光素酶(Luciferase)报告质粒,检测Luciferase活性。3、以重组慢病毒Vpr感染BCBL-1细胞,提取细胞总RNA进行人信号通路发现者PCR芯片操作,采用Western blot对基因芯片结果进行验证,加入信号通路上关键蛋白激酶抑制剂、过表达突变质粒后再检测KSHV裂解期蛋白的表达。结果:1、携带Vpr基因的慢病毒获得成功包装,病毒滴度为4×107Tu/ml。重组慢病毒Vpr感染BCBL-1细胞后可检测到Vpr基因的转录和表达。2、Vpr蛋白不但可以显著抑制BCBL-1细胞中KSHV裂解期基因ORF50(编码KSHV“分子开关”蛋白)、ORF26(编码KSHV次要衣壳蛋白)mRNA的转录,而且还能够显著降低KSHV裂解期蛋白病毒白细胞介素6(viral Interleukin-6,vIL-6)和KSHV复制与转录激活子(replication and transcription activator,Rta,即“分子开关”蛋白)的表达。BCBL-1细胞中过表达Vpr蛋白一方面能够显著下调KSHV的病毒拷贝数,同时明显地降低了Vero细胞中KSHV ORF73/LANA【潜伏相关核抗原,(latency-associated nuclear antigen)】、ORF26 mRNA的转录及vIL-6蛋白的表达。Vpr蛋白能够直接抑制ORF50和ORF73基因启动子的活化。3、Vpr蛋白降低了GSK-3β【糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK-3β)】信号通路中GSK-3β的磷酸化水平,应用GSK-3β抑制剂LiCl及过表达其突变质粒GSK-3β-S9A后,Vpr蛋白对KSHV vIL-6的抑制作用分别被进一步增强和部分阻断。Vpr蛋白下调了PTEN(phosphatase and tensin homolog deleted on chromosome ten)信号通路中PTEN的磷酸化水平,应用其过表达质粒pTEN后,Vpr蛋白对KSHV vIL-6的抑制作用被进一步增强。Vpr蛋白还降低了Ras/c-Raf/MEK/ERK信号通路中c-Raf、MEK1/2及ERK1/2的磷酸化水平。结论:1、含Vpr基因重组慢病毒可以有效地感染BCBL-1细胞,并在其中大量表达目的蛋白。2、Vpr蛋白能够显著抑制KSHV裂解性周期复制及子代病毒颗粒的释放,作用机制可能是通过抑制ORF50和ORF73基因启动子的活化。3、Vpr蛋白通过抑制GSK-3β及PTEN信号通路的活化下调KSHV复制,Ras/c-Raf/MEK/ERK信号通路可能参与调控Vpr蛋白抑制KSHV复制过程。推测在AIDS相关的KS(AIDS-KS)患者体内,Vpr蛋白有可能通过抑制KSHV裂解性周期复制来避免KSHV感染细胞后过早地被免疫清除,从而有利于潜伏感染的形成和肿瘤的发生。更多还原

【Abstract】 Objective: To evaluate the effect of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) on the lytic cycle replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) and identify the signaling pathway(s) involved in this process.Methods: 1. Recombinant lentivirus containing HIV-1 Vpr gene was packaged and identified, and simultaneously the viral titer was checked. The mRNA transcription and protein expression of Vpr gene in BCBL-1 (body cavity-based lymphoma, BCBL, or primary effusion lymphoma, PEL) cells infected with the recombinant lentivirus were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. 2. The mRNA transcription and protein expression of lytic phase genes of KSHV in PEL cells infected with the lentivirus were detected by real-time quantitative PCR (Real-time PCR) and Western blot, respectively. Then the supernatants of infected BCBL-1 were collected to measure KSHV viral particles as well as to infect vero cells. Subsequently the mRNA transcription and protein expression of lytic phase genes of KSHV in vero cells were also detected by RT-PCR and Western blot, respectively. Furthermore, the luciferase reporter assay was performed after PEL cells were infected with recombinant lentivirus carrying Vpr gene and further transfected with luciferase reporter plasmid carrying KSHV ORF50 or ORF73 gene promoter. 3. The human signal transduction pathwayfinder PCR array was performed using the RNA of BCBL-1 cells infected with recombinant lentivirus carrying Vpr gene and the results were verified by Western blot. Finally, the lytic protein expression of KSHV was further determined by Western blot after the addition of the specific inhibitor or overexpression of mutant plasmid.Results: 1. The recombinant lentivirus carrying Vpr gene was packaged successfully with the viral titer of 4×107 Tu/ml. And the mRNA transcription and protein expression of Vpr gene could be detected in BCBL-1 infected with the recombinant lentivirus. 2. The overexpression of Vpr protein remarkably decreased not only the lytic mRNA transcription of ORF50 (switch gene of KSHV cycle replication) and ORF26 (encoding KSHV minor capsid protein), but also the lytic proteins of viral interleukin-6 (vIL-6) and replication and transcription activator (Rta). Meanwhile, the overexpression of Vpr in BCBL-1 cells significantly downregulated the copies of KSHV viral particles as well as the mRNA transcription of ORF73/LANA (latent-associated nuclear antigen), ORF26 and the protein expression of vIL-6 in vero cells. In addition, the Vpr protein inhibited the activation of KSHV ORF50 and ORF73 promoters directly. 3. Overexpression of Vpr protein decreased the phosphorylation of glycogen synthase kinase 3β(GSK-3β) in BCBL-1 cells. Addition of LiCl, the specific inhibitor of GSK-3β, or transfection of GSK-3β-S9A construct, the overexpression plasmid of GSK-3β, reinforced or partially blocked the effect of Vpr-downregulated expression of KSHV vIL-6. Vpr protein also decreased the phosphorylation of PTEN and the transfection of overexpression plasmid of PTEN also reinforced the effect of Vpr-downregulated expression of KSHV vIL-6. Finally, Vpr protein also inhibited the phosphorylation of c-Raf、MEK1/2 and ERK1/2 of MAKP signaling pathway .Conclusions: 1. Recombinant lentivirus containing Vpr gene could efficiently infect BCBL-1 cells, and Vpr protein expressed in BCBL-1 cells. 2. The Vpr protein significantly downregulated the lytic cycle replication of KSHV by inhibiting the activation of ORF50 and ORF73 promoters. 3. Vpr repressed KSHV replication by inhibiting the activation of GSK-3βand PTEN signaling pathways. Ras/c-Raf/MEK/ERK MAPK pathway might also play a role in modulation of Vpr-inhibited KSHV replication. These data suggest that in AIDS-related KS (AIDS-KS) patients, Vpr protein may help KSHV to escape from the immune clearance by inhibiting KSHV lytic replication, which facilitate the establishment of latent infection and tumor formation.更多还原