REGULATION OF HTLV II GENE EXPRESSION BY REX

人类T细胞白血病病毒II型(HTLV-II)与 几例罕见的慢性T细胞白血病，最近被 在相当大比例的美国静脉注射吸毒者中发现了这种药物。 HTLV-II编码两种反式作用蛋白Tax和Rex，它们调节 病毒的生命周期，这可能有助于恶性转化 被感染的宿主细胞。我们实验室最近的研究表明 Rex增加了细胞质中Gag/polmRNA的水平，这一效应 需要5‘HTLV-II长末端重复序列中的顺式作用序列 (Ltr)，一个REX反应元件(RxRE)。如果删除此RxRE，则仅 Rex对LTR连接的表达进行负调控，这 对应于LTR相关的总mRNA水平的下降。整体而言 这项提议的目标是定义精确的分子和 这些相互竞争的霸王龙调控功能的生化机制。 REX对大鼠脑内血管紧张素转换酶水平及亚细胞分布的影响 GAG/POL、ENV和TAX/REX mRNAs将在瞬变和稳定状态下进行测量 S导入淋巴样细胞的研究(1)核酸酶保护或 RNA的定量聚合酶链式反应(PCR)。雷克斯效应对 HTLV-II的mRNAs也将与对病毒表达的影响进行比较 用放射免疫沉淀法(RIPA)测定蛋白质。稳定 转基因将允许研究Rex调控的稳定状态，以及 可能提供一种潜伏感染的体外模型。其作用机制 潜在的REX调控将通过绘制所有顺式作用的RNA来探索 HTLV-II基因组中的RxREs，并通过定义Rex蛋白的功能 通过诱变获得结构域。所有的REX突变体和RxREs都将被测试 对于使用以下两种方法之一的共转染中对LTR链表达的影响 含有一个可能与RxRE连锁的野生型Ltr或Ltr突变体 氯霉素乙酰转移酶(CAT)指示基因。潜在的霸王龙 蛋白质-RxRE RNA相互作用将通过RNA凝胶延迟来确定 HTLV-II感染和未感染核蛋白提取液的检测 淋巴样细胞和从杆状病毒载体中纯化的Rex蛋白。如果 特定的“迟缓”条带被发现，RxRE定点突变体将 破坏了预测的RxRE RNA二级结构。变化 在与RxRE突变体的LTR连锁的基因表达中将与 与Rex-RxRE结合相关的RNA凝胶移动模式的变化 功能。了解HTLV-II中Rex的调控机制将 不仅有助于阐明病毒生命周期中的控制步骤， 还包括真核细胞中RNA的加工。

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.