RNA PROCESSING IN HUMAN RETROVIRUSES

人类逆转录病毒中的 RNA 加工

• 批准号: 3460867
• 负责人: ALEXANDER C BLACK
• 金额: $ 10.24万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3460867
• 关键词: RNA binding protein; gene mutation; genetic library; genetic regulatory element; host organism interaction; human T cell lymphotropic virus type 2; human immunodeficiency virus 1; molecular cloning; platinum; posttranscriptional RNA processing; provirus; tissue /cell culture; transfection; ultraviolet radiation

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.

人类T细胞白血病病毒类型I（HTLV-I）和II（HTLV-II）和人类 I类型（HIV-1）和II（HIV-2）类型的免疫缺陷病毒是不同的，但 与人类疾病相关的相关人逆转录病毒。 这些逆转录病毒已经演变出类似的跨作用调节蛋白， HTLV -I和-II REX和HIV -1和-2 REV，生产性病毒所必需的 感染。 Rex和Rev调节病毒RNA处理，并从 病毒结构基因表达的病毒调节。 另外，这些 病毒已经发展出顺式作用的RNA响应元素 Rex和Rev的介导基因调节中的类似作用。我们有 先前确定的顺式REX响应元件（RXRE）和 相邻抑制序列或顺式作用抑制序列（CRS）， 在HTLV-II 5'长末端重复（LTR）RNA中，两者都证明了这两个 Rex蛋白与RXRE RNA结合，并且结合与REX相关 功能。 我们希望进一步定义HTLV-II的作用机制 雷克斯（Rex），专注于顺式作用病毒RNA响应元件的作用 和雷克斯调节中的细胞因子。 此外，我们希望 确定这些细胞因素是否也对REV有影响 HIV-1中的调节。 具体目的是：1。）确定其中的所有CRS和RXRE元素 通过在瞬时转染基因表达测定中测试HTLV-II RNA， 异源矢量和含有缺失的病毒构建体 HTLV-II转录序列中的核苷酸取代。 2.）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，有或没有 在反式的REX，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多质体关联。 3.） 识别和克隆的细胞蛋白 通过REX调节病毒基因表达并确定REX如何 使用体外结合测定法与这些细胞蛋白相互作用，并 lambdagt11表达式库筛选或生物素化的RNA-Avidin 纯化程序。 4.）表征交叉调节 对影响HTLV-II的细胞蛋白的HIV-1基因表达的影响 REX调节HTLV-II基因表达。 该提案解决了细胞因素在REX和REV中的作用 调节病毒基因表达。 这些因素的表征 可能会更好地理解细胞RNA的调节 加工，并可能揭示了抗逆转录病毒疗法的新靶标。