RNA PROCESSING IN HUMAN RETROVIRUSES

人T细胞白血病病毒I型(HTLV-I)和II型(HTLV-II)与人类 免疫缺陷病毒I型(HIV-1)和II型(HIV-2)是不同的，但 与人类疾病有关的相关人类逆转录病毒。 这些逆转录病毒进化出了类似的反式作用调节蛋白， HTLV-I和-II Rex以及HIV-1和-2 Rev，是产生病毒所必需的 感染。REX和REV调节病毒RNA的加工和从 病毒对病毒结构基因表达的调节。此外，这些 病毒已经进化出顺式作用的RNA反应元件，从而实现 Rex和Rev.在介导基因调控中的类似作用 先前识别的顺式作用的REX反应元件(RxRE)和 相邻的抑制序列，或顺式作用抑制序列(CRS)， 在HTLV-II的5‘长末端重复序列(LTR)RNA中 Rex蛋白与RxRE RNA结合，并与Rex相关 功能。我们希望进一步定义HTLV-II的作用机制 Rex，通过关注顺式作用的病毒RNA反应元件的作用 以及雷克斯调控中的细胞因素。此外，我们希望 确定这些细胞因素是否也对REV有影响 对HIV-1的监管。 具体目标是：1.标识中的所有CRS和RxRE元素 通过检测HTLV-II RNA在瞬时基因表达实验中， 异源载体和含有缺失的前病毒构建体 或HTLV-II转录序列中的核苷酸替换。2.)至 确定CRS和RxRE元件影响病毒基因的机制 通过分析对病毒RNA加工的影响来表达，无论是否有 反式中的Rex，在几个阶段：RNA稳定性，RNA剪接，RNA 从细胞核到细胞质的运输，以及RNA多聚体的联合。3.) 鉴定和克隆细胞蛋白，这些蛋白参与 Rex对病毒基因表达的调控及其作用机制 使用体外结合分析与这些细胞蛋白相互作用 Lambdagt11表达文库的筛选或生物素化RNA-亲和素 纯化程序。4.)描述跨界监管机构的特征 影响HTLV-II的细胞蛋白对HIV-1基因表达的影响 REX对HTLV-II基因表达的调控 该提案涉及REX和REV中细胞因子的作用 病毒基因表达的调控。这些因素的特征 可能有助于更好地理解细胞RNA的调节。 加工，并可能揭示抗逆转录病毒治疗的新靶点。

Human T-cell leukemia viruses types I (HTLV-I) and II (HTLV-II) and human immunodeficiency viruses types I (HIV-1) and II (HIV-2), are distinct but related human retroviruses that have been associated with human disease. These retroviruses have evolved similar trans-acting regulatory proteins, HTLV-I and -II Rex and HIV-1 and -2 Rev, required for productive viral infection. Rex and Rev regulate viral RNA processing and the switch from viral regulatory to viral structural gene expression. In addition, these viruses have evolved cis-acting RNA response elements which fulfill analogous roles in mediating gene regulation by Rex and Rev. We have previously identified a cis-acting Rex responsive element (RxRE) and adjacent inhibitory sequences, or cis-acting repressive sequences (CRS), in HTLV-II 5' long terminal repeat (LTR) RNA and have demonstrated both that Rex protein binds to RxRE RNA and that binding correlates with Rex function. We wish to define further the mechanisms of action of HTLV-II Rex, by focusing on the role of cis-acting viral RNA response elements and of cellular factors in Rex regulation. In addition, we wish to determine whether these cellular factors also have effects on Rev regulation in HIV-1. The Specific aims are: 1.) To identify all CRS and RxRE elements within HTLV-II RNA by testing in a transient transfection gene expression assay, both heterologous vectors and proviral constructs containing deletions or nucleotide substitutions in HTLV-II transcribed sequences. 2.) To determine the mechanisms by which CRS and RxRE elements affect viral gene expression by analyzing effects on viral RNA processing, with or without Rex in trans, at several stages: RNA stability, RNA splicing, RNA transport from nucleus to cytoplasm, and RNA polysome association. 3.) To identify and clone cellular proteins that are involved in the regulation of viral gene expression by Rex and to determine how Rex interacts with those cellular proteins using in vitro binding assays and lambdagt11 expression library screening or a biotinylated RNA-avidin purification procedure. 4.) To characterize the crossover regulatory effects on HIV-1 gene expression of cellular proteins that affect HTLV-II Rex regulation of HTLV-II gene expression. This proposal addresses the role of cellular factors in Rex and Rev regulation of viral gene expression. Characterization of these factors may lead to a better understanding of the regulation of cellular RNA processing, and may reveal novel targets for antiretroviral therapy.