RNA PROCESSING IN HUMAN RETROVIRUSES

人T细胞白血病病毒I型（HTLV-I）和II型（HTLV-II）以及人 免疫缺陷病毒I型（HIV-1）和II型（HIV-2）是不同的，但 与人类疾病相关的人类逆转录病毒。 这些逆转录病毒已经进化出类似的反式作用调节蛋白， HTLV-I和-II雷克斯和HIV-1和-2 Rev，生产病毒所需 感染 雷克斯和Rev调节病毒RNA加工和从 病毒调节病毒结构基因表达。 另外这些 病毒已经进化出顺式作用RNA应答元件， 类似的作用，介导基因调控的雷克斯和Rev。 先前鉴定了顺式作用雷克斯应答元件（RxRE）， 相邻的抑制序列，或顺式作用抑制序列（CRS）， 在HTLV-II 5 ′长末端重复序列（LTR）RNA中， 该雷克斯蛋白与RxRE RNA结合并且该结合与雷克斯相关 功能 我们希望进一步确定HTLV-II的作用机制 雷克斯，通过关注顺式作用病毒RNA反应元件的作用， 和细胞因子在雷克斯调节中的作用。 此外，我们希望 确定这些细胞因子是否也对Rev 在HIV-1中的调节。 具体目标是：（1）。为了识别其中的所有CRS和RxRE元素， HTLV-II RNA通过在瞬时转染基因表达测定中测试， 含有缺失的异源载体和前病毒构建体 或HTLV-II转录序列中的核苷酸取代。 2.）的情况。到 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，有或没有 反式雷克斯，在几个阶段：RNA稳定性，RNA剪接，RNA 从细胞核到细胞质的转运，以及RNA多核糖体的结合。 3.）第三章 为了鉴定和克隆参与细胞凋亡的细胞蛋白质， 调节病毒基因表达的雷克斯，并确定如何雷克斯 使用体外结合测定与那些细胞蛋白相互作用， 生物素化的RNA-亲和素的表达文库筛选 纯化程序。 4.）为了表征交叉监管 影响HTLV-II的细胞蛋白对HIV-1基因表达的影响 雷克斯对HTLV-II基因表达的调控。 该建议涉及细胞因子在雷克斯和Rev 病毒基因表达的调控。 这些因素的特征 可能会导致更好地了解细胞RNA的调节 处理，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.