GENETIC INTERACTION OF HUMAN HERPESVIRUS 6 WITH HIV 1

人疱疹病毒6型(HHV-6)与人相互作用的研究 携带人类免疫缺陷病毒1型的巨细胞病毒 令人感兴趣的是HHV-6或 巨细胞病毒感染与艾滋病的发病机制。HHV-6和HIV-1可以 高效感染CD4T细胞和混合感染加速细胞病变 效果。研究是为了了解这些相互作用是如何 发生在分子水平上。我们测试了HHV-6基因，可能会影响 HIV-1复制。DNA导入人T细胞和猴体内的研究 肾细胞，我们已经证明了HHV-6转化基因片段， 此前在本实验室发现的ZVH14可以反式激活 HIV-1 LTR.这种反式激活是通过Sp1结合位点介导的 在HIV-1长末端重复。一个115个氨基酸的开放阅读框架， ZVH14DNA中的B115基因显示出类似于 ZVH14.体外表达研究发现，B115基因产物 预测的MR=18 kDa。该蛋白的突变体是由 定点突变以确认其在反式激活中的作用。一个 5‘端缺失5个核苷酸的特定突变体 ORF显示HIV-1 LTR的反式激活显著减少。 这一观察结果支持我们的发现，即B115编码反式激活子 蛋白。B115的体外表达蛋白或提取物 转染的Cos-7(SV40转化的猴肾细胞)细胞没有 凝胶移位分析显示与HIV-1 LTR有结合。在后续行动中 研究表明，B115还可以即时反式激活CMV 人成纤维细胞和猴肾细胞的早期启动子。SP1结合 网站似乎调解了这种转录激活，正如我们在 HIV-1 LTR激活。这些数据提示HHV-6转化基因 ZVH14片段编码一种反式激活蛋白，可以激活 异源启动子和Sp1结合位点的转录调控 对于这种激活是必不可少的。这项研究对 了解HHV-6在艾滋病和巨细胞病毒发病机制中的作用 视网膜炎。该项目还将为以下方面提供知识和工具 新的抗病毒药物和安全的病毒和质粒的发展 艾滋病基因治疗的载体。

Studies on interactions of human herpesvirus-6 (HHV-6) or human cytomegalovirus (HCMV) with human immunodeficiency virus type 1 (HIV-1) are of interest in view of the suggested relationship between HHV-6 or CMV infections and the pathogenesis of AIDS. HHV-6 and HIV-1 can productively infect CD4+T cells and coinfection accelerates cytopathic effects. Studies were conducted to understand how these interactions occur at the molecular level. We tested HHV-6 genes that may affect HIV-1 replication. By DNA transfection into human T-cells and monkey kidney cells, we have shown that an HHV-6 transforming gene segment, ZVH14, previously identified in this laboratory, can transactivate the HIV-1 LTR. This transactivation is mediated through Sp1 binding sites in the HIV-1 long terminal repeat. A 115-amino acid open reading frame, B115, in the ZVH14 DNA showed a similar transactivation capacity as ZVH14. In vitro expression studies found the B115 gene product of the predicted Mr = 18 kDa. Mutants of this protein were generated by site-specific mutagenesis to confirm its role in transactivation. A specific mutant having a deletion of 5 nucleotides from the 5' - end of the ORF showed significant reduction in transactivation of the HIV-1 LTR. This observation supports our finding that B115 encodes a transactivator protein. In vitro expressed protein or the extracts from B115 transfected Cos-7 (SV40 transformed monkey kidney cells) cells did not show any binding to the HIV-1 LTR in gel shift assays. In a follow-up study, we have shown that B115 can also transactivate the CMV immediate early promoter in human fibroblasts and monkey kidney cells. Sp1 binding sites seem to mediate this transactivation as we have reported with the HIV-1 LTR activation. These data suggest that HHV-6 transforming gene segment, ZVH14, encodes a transactivator protein which can activate transacription from heterologous promoters and Sp1 binding sites are essential for this transactivation. This study is important for understanding the role of HHV-6 in the pathogenesis of AIDS and CMV retinitis. The project will also provide knowledge and tools for development of novel antiviral therapies and safe viral and plasmid vectors for gene therapy of AIDS.