TRANSFORMING DOMAIN OF HHV 6 THAT TRANSACTIVATES HIV 1

越来越多的数据表明，HHV-6在HHV-6的进展中发挥了辅助因素的作用 在艾滋病和艾滋病相关的恶性肿瘤中。有意义的是，HHV-6和HIV-1 可以共同感染相同的CD4+靶T细胞，导致刺激 HIV-1复制。此外，HHV-6感染可诱导CD4的表达。 CD8+T淋巴细胞中，从而使这些细胞对HIV-1具有容许性 感染。我们的研究已经确定了3.9kbp的Sa/i-L DNA片段 HHV-6株U1102株来自反式激活的直接重复区 人类免疫缺陷病毒1型长末端重复序列(HIV-1 LTR)。 SA/I-L还能转化NIH3T3细胞并使原代仓鼠永生 胚胎成纤维细胞。反式激活和转化活动有 共定位于1.9kbp的Sa/i-L-Hindll亚片段(命名为 SA/I-L-SH)包含357个氨基酸(AA)开放阅读框， (指定为ORF-1)。ORF-1可以重新激活TAT缺陷的HIV-1前病毒 从潜伏感染的细胞中分离出ORF-1，表明ORF-1是HHV-6基因 可能对艾滋病进展过程中HIV-1的重新激活负责 活着。纯化的ORF-1蛋白足以反式激活HIV-1LTR。 为了扩大我们的观察范围，这项提议旨在剖析 Sa/i-L-SH内转型所需的基本区域和 激活，并确定它们在 在感染期间感染整个病毒。我们的具体目标是：1)塑造 ORF-1反式激活HIV-1的机制以及最低限度 HIV-1启动子内的HHV-6反式激活反应元件(S) 突变分析。将特别强调开放源码框架-1的作用 在整个病毒的背景下反式激活潜伏的艾滋病毒-1。2) Sa/i-L-SH片段内最小转换区的特征 无论是作为一个独立的实体，还是在整个病毒的背景下 体外细胞转化和致瘤性分析，以及3)确定 艾滋病相关淋巴和非艾滋病淋巴组织中ORF-1的存在和表达 Southern印迹、聚合酶链式反应和原位杂交检测恶性肿瘤 杂交分析。这些研究将提供重要的新的 中国人群中HHV-6特异性基因(S)的生物学相关性信息 通过测定艾滋病和艾滋病相关恶性肿瘤的进展 ORF-1转换和反式激活结构域在肺癌中的意义 病毒基因组的背景。

Accumulating data suggest a co-factor role for HHV-6 in the progression of AIDS and in AIDS-related malignancies. Of significance, HHV-6 and HIV-1 can co-infect the same CD4+ target T-cells resulting in the stimulation of HIV-1 replication. Moreover, HHV-6 infection can induce expression of CD4 in CD8+ T-lymphocytes, thus rendering these cells permissive for HIV-1 infection. Our studies have identified a 3.9 kbp Sa/I-L DNA fragment of HHV-6 strain U1102 from the direct repeat region which transactivates human immunodeficiency virus type 1 -long terminal repeat (HIV-1 LTR). Sa/I-L also transforms NIH 3T3 cells and immortalizes primary hamster embryo fibroblasts. The transactivating and transforming activities have been co-localized to a 1.9 kbp Sa/I-L-Hindlll subfragment, (designated Sa/I-L-SH) containing a 357 amino acid (aa) open reading frame, (designated ORF-1). ORF-1 can reactivate tat-defective HIV-1 provirus from latently infected cells, suggesting that ORF-1 is the HHV-6 gene putatively responsible for HIV-1 reactivation during AIDS progression in vivo. Purified ORF-1 protein is sufficient to transactivate the HIV-1 LTR. To extend our observations, this proposal is designed to dissect the essential regions within Sa/I-L-SH required for transformation and transactivation and to determine their relevance within the context of the whole virus during infection. Our specific aims are to: 1) Characterize the mechanism by which ORF-1 transactivates HIV-1 as well as the minimal HHV-6 transactivator responsive element(s) within the HIV-1 promoter by mutational analysis. Specific emphasis will be placed on the role of ORF-1 within the context of the whole virus to transactivate latent HIV-1. 2) Characterize the minimal transforming region within the Sa/I-L-SH fragment both as an independent entity and in the context of the whole virus by in vitro cell transformation and tumorigenicity assays, and 3) Determine the presence and expression of ORF-1 with AIDS-related and non-AIDS lymphoid malignancies by Southern blot, polymerase chain reaction, and in situ hybridization analyses. These studies will provide significant new information about the biologic relevance of specific HHV-6 gene(s) in the progression of AIDS and AIDS-related malignancies by determining the significance of the ORF-1 transforming and transactivating domains in the context of the viral genome.