开放读码框架50基因介导HIV-1感染相关的肝细胞生长因子诱导人类疱疹病毒8型复制

Reactivation of human herpesvirus 8 by hepatocyte growth factor/scatter factor similar to that produced by HIV-1-infected T cells via ORF50 promoter of HHV-8

目的 研究人类疱疹病毒 8型 (HHV 8)开放读码框架 (ORF) 5 0基因启动子在HIV 1感染相关细胞因子肝细胞生长因子 /驱散因子 (HGF/SF)诱导原发性渗出性淋巴瘤 (PEL)BC 3中潜伏感染的HHV 8复制过程中的作用。方法 将HGF/SF连续加入到体外培养的BC 3中 ,分别于培养第 3天和第 7天收集刺激细胞 ,电子显微镜观察成熟HHV 8病毒的形成 ;提取细胞总RNA ,North ernblot和定量聚合酶链反应 (PCR)法检查HHV 8次要衣壳蛋白ORF2 6mRNA转录。同时 ,将已构建的HHV 8ORF5 0启动子 +虫荧光素酶报告基因重组质粒和含HIV 1Tat基因重组表达质粒分别共转染BC 3和人脐静脉血管内皮细胞 (HUVECs) ,转染后 2 4h加入HGF/SF和 /或重组HIV 1gp12 0刺激 ,刺激后 2 4h收集细胞 ,进行虫荧光素酶活性检测。试验同时以佛波酯 (TPA)刺激为阳性对照。结果 HGF/SF可以上调HHV 8ORF2 6mRNA表达 ,且于刺激的第 7天 ,ORF2 6mRNA表达上升了 4 .1倍 ,BC 3细胞中同时可观察到成熟的HHV 8病毒粒子 ;HGF/SF能够诱导ORF5 0启动子活性 ,但HIV 1Tat和 gp12 0蛋白与HGF/SF协同上调ORF5 0启动子活性的能力较弱。 结论 HIV 1感染相关细胞因子HGF/SF诱导HHV 8复制的过程至少有部分由ORF5 0启动子介导。

Objective To investigate a potential role for ORF50 promoter of human herpesvirus 8(HHV 8)in reactivation of HHV 8 in primary effusion lymphoma(PEL)BC 3 cells by HIV 1 related inflammatory cytokine,hepatocyte growth factor/scatter factor(HGF/SF). Methods The persistent stimulation of BC 3 was performed by recombinant HGF/SF and treated BC 3 cells were collected at the 3 rd and 7 th day after persistent stimulation respectively. Northern blot, quantitative PCR(real time PCR), and electron microscopy(EM)were carried out to detect the expression of mRNA of minor capsid protein ORF26 and the presence of viral particles of HHV 8 from treated BC 3 cells. Co transfection of BC 3 and human umbilical veil endothelial cells (HUVECs) with HHV 8 ORF50 promoter driven luciferase construct built previously by us and recombinant expression plasmid named pEV containing HIV 1 Tat coding gene was performed and stimulation of co transfected cells with recombinant HGF/SF and/or HIV 1 recombinant gp120 was conducted at 24 hours after transfection to induce luciferase gene expression. Then relative luciferase activity unit(RLU) was calculated at 24 hours after stimulation. In the meantime, stimulation of co transfected cells with 12 O tetradecanoylphorbol 13 acetate(TPA) was performed to be used as positive control. Results It was found that HGF/SF induced an 4.1 folds increase in mRNA expression of ORF26 when it was added individually to BC 3 cells at the 7 th day after persistent stimulation. Viral particles of HHV 8 were readily identified in BC 3 cells stimulated with HGF/SF at the 7 th day with EM analysis. Recombinant HGF/SF could upregulate promoter activity of HHV 8 ORF50 promoter in BC 3 and HUVECs cells and however, HIV 1 Tat and gp120 failed to act synergistically with HGF/SF to enhance ORF50 promoter activity in HUVECs. Conclusions The results show that HIV 1 related inflammatory cytokine HGF/SF is responsible for the induction of HHV 8 lytic cycle replication and the induction may be, at least partly mediated by ORF50 promoter of HHV 8.