CELLULAR STRESS RESPONSE AND HTLV-I REPLICATION

人类嗜T淋巴细胞病毒I型(HTLV-I)是引起 成人T细胞白血病/淋巴瘤(ATLL)和慢性脊髓病和 在全球范围内，感染被视为一个重要的公共卫生问题 美国。HTLV-I具有临床周期长的特点 特定主机控制的延迟和有限信息是已知的 调节HTLV-I表达的机制。HTLV-I病毒编码税务 蛋白质是一种强有力的反式激活剂，人们认为它扮演着重要的角色 在HTLV-I白血病发生中。总体目标是调查 HTLV-I复制过程中的细胞应激反应 必要的细胞反应可能最终影响病毒介导的 淋巴细胞转化。建议的研究是基于 以下两个假设：1)细胞应激反应的诱导 持续感染的HTLV-I淋巴细胞增强前病毒 转录；2)原核表达HSP73和诱导型HSP73 72与病毒蛋白复合体稳定和靶向征税 移位到细胞核。解决以下问题的具体目标 这些假设是：1)确定定性和定量模式 HTLV-I蛋白和RNA在细胞应激过程中的表达 响应，2)评估蛋白质相互作用和细胞运输 HSP 72、HSP 73和HTLV-I Tax蛋白在细胞应激中的表达 响应，以及3)评估HTLV-I Tax在 在细胞应激反应中HTLV-I LTR的激活。这个 实验将使用成功使用的技术来归纳和 监测慢性感染HTLV-I的细胞应激反应 转化的淋巴细胞。HTLV-I核糖核酸和蛋白质生产的评价 将使用各种互补的技术，包括间接 HTLV-I病毒蛋白的免疫荧光分析、Northern印迹和 缝隙印迹分析、核接合分析和合胞体形成 化验。特定目标2将利用本地免疫沉淀来 选择性沉淀HSP 73、HSP 72和HTLV-I蛋白，在 特殊的税收，这在压力反应中变得复杂。这些研究 将与单一和双重间接免疫荧光相关 通过锚定细胞分析和分类细胞仪进行分析。目标#3 将重点放在税收职能的转变上来激活税收 HTLV-I LTR.HeLa细胞或Hut 78细胞将与 报告质粒和HTLV-I LtR-Tax质粒。牢房将会是 进行细胞应激和CAT活性测定。离体 转录将在他在场和不在场的情况下进行 应激后的纳税蛋白质。这将是对转基因研究的补充 并增加转录速率变化的特异性 税金。应激反应是一种重要的生理反应，是由 通过多个生物相关的事件。可能诱发或抑制的刺激 HTLV-I Tax的增强表达在这一过程中发挥了重要作用 细胞转化的过程。这项提案将调查这些机制 细胞应激反应调控HTLV-I的表达 蛋白质和核糖核酸。

Human T-lymphotropic virus type I (HTLV-I) is the etiologic agent for both, adult T-cell leukemia/lymphoma (ATLL) and a chronic myelopathy and the infection recognized as an important public health problem in the United States. HTLV-I is characterized with long periods of clinical latency and limited information is known of specific host control mechanisms that regulate HTLV-I expression. The HTLV-I viral encoded Tax protein is a potent trans-activator and is felt to play an important role in HTLV-I leukemogenesis. The overall goal is to investigate the role of the cellular stress response in HTLV-I replication to clarify how this essential cellular response may ultimately influence viral mediated lymphocyte transformation. The proposed research is based on the following two HYPOTHESES: 1) Induction of the cellular stress response in persistently infected HTLV-I lymphocytes enhances proviral transcription and 2) The constitutive expressed hsp 73 and inducible hsp 72 complex with the viral protein Tax to stabilize and target Tax translocation to the nucleus. Specific objectives which will address these hypotheses are; 1) determine qualitative and quantitative patterns of expression of HTLV-I proteins and RNA during the cellular stress response, 2) evaluate protein interactions and the cellular trafficking of hsp 72, hsp 73, and the HTLV-I Tax protein during the cellular stress response, and 3) assess the functional activity of HTLV-I Tax on activation of the HTLV-I LTR during the cellular stress response. The experiments will use successfully employed techniques to induce and monitor the cellular stress response in chronically infected HTLV-I transformed lymphocytes. Evaluation of HTLV-I RNA and protein production will use a variety of complimentary techniques which include indirect immunofluorescence assay for HTLV-I viral proteins, northern blot and slot blot analysis, nuclear run-on analysis, and syncytia formation assays. Specific aim #2 will utilize native immunoprecipitation to selectively precipitate hsp 73, hsp 72, and HTLV-I proteins, in particular Tax, which complex during the stress response. The studies will be correlated with single and dual indirect immunofluorescence that are analyzed by a anchored cell analysis and sorting cytometer. Aim #3 will focus on the functional changes of Tax on the activation of the HTLV-I LTR. Hela cells or Hut 78 cells will be co-transfected with a reporter plasmid and a HTLV-I LTR-Tax plasmid. The cells will be subjected to cellular stress and CAT activity assayed. In vitro transcription will be performed in he presence and absence of a purified Tax protein after stress. This will complement the transfection studies and increase the specificity of the changes in transcriptional rates to Tax. The stress response is a vital physiologic response and is induced by multiple biologically relevant events. The stimuli that may induce or augment expression of HTLV-I Tax play an important role in the process of cellular transformation. This proposal will investigate the mechanisms by which the cellular stress response modulates the expression of HTLV-I proteins and RNA.