REGULATION OF HTLV II GENE EXPRESSION BY REX

REX 对 HTLV II 基因表达的调控

• 批准号: 3085905
• 负责人: ALEXANDER C BLACK
• 金额: $ 8.53万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3085905
• 关键词: Baculoviridae; RNase protection assay; antiviral antibody; binding proteins; chloramphenicol acetyltransferase; complementary DNA; crosslink; cytoplasm; gel filtration chromatography; gel mobility shift assay; gene expression; genetic mapping; genetic regulatory element; genetic terminator element; genome; human T cell lymphotropic virus type 2; messenger RNA; molecular cloning; mutant; nucleic acid repetitive sequence; nucleic acid sequence; nucleoproteins; polymerase chain reaction; precipitation; protein biosynthesis; protein purification; protein sequence; radioimmunoassay; site directed mutagenesis; transcription factor; transfection; viral leukemia; viral leukemogenesis; virus RNA; virus genetics; virus protein

Human T-cell leukemia virus type II (HTLV-II) has been associated with several cases of rare chronic T-cell leukemia, and has recently been found in a significant proportion of American intravenous drug abusers. HTLV-II encodes two trans-acting proteins, Tax and Rex, that regulate the viral life-cycle, and that may contribute to the malignant transformation of infected host cells. Recent studies in our laboratory have shown that Rex increases cytoplasmic levels of gag/pol mRNA, and that this effect requires cis-acting sequences in the 5' HTLV-II long terminal repeat (LTR), a Rex-responsive element (RxRE). If this RxRE is deleted, only negative regulation of LTR-linked expression by Rex occurs, which corresponds to a decrease in total LTR-linked mRNA levels. The overall objective of this proposal is to define the precise molecular and biochemical mechanisms of these competing Rex regulatory functions. The effects of Rex on the levels and subcellular distribution of gag/pol, env and tax/rex mRNAs will be measured in transient and stable transfections into lymphoid cells by S(1) nuclease protection or quantitative polymerase chain reaction (PCR) of RNA. Rex effects on HTLV-II mRNAs will also be compared to effects on expression of viral proteins, as determined by radioimmunoprecipitation assay (RIPA). Stable transfections will allow study of the steady-state of Rex regulation, and may provide an in vitro model of latent infection. The mechanisms underlying Rex regulation will be explored by mapping all cis-acting RNA RxREs within the HTLV-II genome, and by defining Rex protein functional domains through mutagenesis. All rex mutants and RxREs will be tested for effects on LTR-linked expression in co-transfections using either the wild-type LTR or LTR mutants containing one possible RxRE linked to the chloramphenicol acetyltransferase (CAT) indicator gene. Potential Rex protein-RxRE RNA interactions will be identified by RNA gel retardation assays using nucleoprotein extracts from HTLV-II-infected and -uninfected lymphoid cells and purified Rex protein from a baculovirus vector. If specific "retarded" bands are found, site-directed RxRE mutants will be created that disrupt the predicted RxRE RNA secondary structure. Changes in LTR-linked gene expression with RxRE mutants will be compared to changes in RNA gel shift patterns to correlate Rex-RxRE binding with Rex function. Understanding the mechanisms of Rex regulation in HTLV-II will help elucidate not only the controlling steps in the viral life-cycle, but also the processing of RNA in eukaryotic cells.

人类T细胞白血病II型（HTLV-II）与 几例罕见的慢性T细胞白血病，最近 在很大一部分美国静脉滥用药物中发现。 HTLV-II编码两个调节的反式蛋白质，即税收和REX 病毒生命周期，这可能导致恶性转化 感染的宿主细胞。 我们实验室的最新研究表明 雷克斯增加了cag/pol mRNA的细胞质水平，并且这种作用 需要在5'HTLV-II长时间重复中进行顺式作用序列 （LTR），REX响应元素（RXRE）。 如果此rxre被删除，仅 REX的LTR连接表达的负调节发生，这是 对应于总LTR连接的mRNA水平的降低。 总体 该建议的目的是定义精确的分子和 这些竞争性REX调节功能的生化机制。 雷克斯对水平和亚细胞分布的影响 插科打/pol，env和税收/雷克斯mRNA将以瞬态和稳定为准 S（1）核酸酶保护或 RNA的定量聚合酶链反应（PCR）。 雷克斯的影响 HTLV-II mRNA也将与对病毒表达的影响进行比较 蛋白质，由放射免疫沉淀测定法（RIPA）确定。 稳定的 转染将允许研究REX调节的稳态，并 可以提供潜在感染的体外模型。 机制 通过映射所有顺式作用RNA，将探索基础REX调节 HTLV-II基因组中的RXRE，并通过定义REX蛋白功能 通过诱变的结构域。 所有REX突变体和RXRE都将进行测试 对于使用任何一种 野生型LTR或LTR突变体，该突变体包含一个可能的RXRE 氯霉素乙酰转移酶（CAT）指示基因。 潜在的雷克斯 RNA凝胶延迟将鉴定蛋白质-RXRE RNA相互作用 使用HTLV-II感染的核蛋白提取物进行的测定 来自杆状病毒载体的淋巴样细胞和纯化的REX蛋白。 如果 发现特定的“延迟”频带，位于定位的RXRE突变体将是 创建破坏了预测的RXRE RNA二级结构。 更改 在LTR连接的基因表达中，将与RXRE突变体进行比较 RNA凝胶移位模式的变化将REX-RXRE结合与REX相关联 功能。 了解HTLV-II中REX调节的机制将 不仅阐明病毒生命周期的控制步骤， 还有真核细胞中RNA的加工。