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 在美国静脉注射毒品滥用者中占很大比例。 HTLV-II编码两种反式作用蛋白Tax和雷克斯，它们调节HTLV-II的表达。 病毒的生命周期，这可能有助于恶性转化 感染的宿主细胞。 我们实验室最近的研究表明， 雷克斯增加gag/pol mRNA的细胞质水平， 需要5 ′ HTLV-II长末端重复序列中的顺式作用序列 (LTR)Rex响应元件（RxRE）。 如果删除此RxRE，则仅 雷克斯对LTR连锁表达进行负调控， 对应于总LTR连接的mRNA水平的降低。 整体 该提案的目的是定义精确的分子和 这些竞争性雷克斯调节功能的生化机制。 研究了雷克斯对大鼠海马神经元中 gag/pol、env和tax/雷克斯mRNA将在瞬时和稳定的细胞中测量。 通过S（1）核酸酶保护转染到淋巴样细胞中，或 RNA的定量聚合酶链反应（PCR）。 雷克斯对 还将比较HTLV-II mRNA对病毒表达的影响。 蛋白质，如通过放射免疫沉淀测定（RIPA）测定的。 稳定 转染将允许研究雷克斯调节的稳态， 可以提供潜伏感染的体外模型。 的机制 潜在的雷克斯调控将通过绘制所有顺式作用RNA HTLV-II基因组内的RxRE，并通过定义雷克斯蛋白功能 结构域通过诱变。 将检测所有雷克斯突变体和RxRE 对于在共转染中对LTR连接的表达的影响， 野生型LTR或含有一个可能的RxRE的LTR突变体，所述RxRE连接至 氯霉素乙酰转移酶（CAT）指示基因。 潜在的雷克斯 将通过RNA凝胶阻滞鉴定蛋白质-RxRE RNA相互作用 使用来自HTLV-II感染和未感染的核蛋白提取物的测定 淋巴样细胞和来自杆状病毒载体的纯化的雷克斯蛋白。 如果 如果发现特异性的"延迟"条带，则将发现定点RxRE突变体。 创建了破坏预测的RxRE RNA二级结构的分子。 变化 在LTR-连锁基因表达与RxRE突变体将进行比较， RNA凝胶位移模式的变化，以将Rex-RxRE结合与雷克斯相关联 功能 了解HTLV-II中雷克斯的调节机制将有助于 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.