CELLULAR STRESS RESPONSE AND HTLV-I REPLICATION

细胞应激反应和 HTLV-I 复制

• 批准号: 2057312
• 负责人: JANICE M ANDREWS
• 金额: $ 8万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2057312
• 关键词: HeLa cells; cell sorting; gene expression; human T cell lymphotropic virus type 1; immunofluorescence technique; immunoprecipitation; intermolecular interaction; northern blottings; nuclear runoff assay; provirus; stress proteins; virus protein; virus replication

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.

人类T淋巴病毒I型（HTLV-I）是病因 两者都是成人T细胞白血病/淋巴瘤（ATLL）和慢性骨髓病和 感染被认为是重要的公共卫生问题 美国。 htlv-i的特征是长时间的临床 延迟和有限的信息已知特定的宿主控制 调节HTLV-I表达的机制。 HTLV-I病毒编码税 蛋白质是一种有效的反式激活剂，被认为起着重要作用 在HTLV-I白血病中。总体目标是调查 HTLV-I复制中的细胞应力反应，以阐明如何 必需的细胞反应最终可能影响病毒介导 淋巴细胞转化。拟议的研究基于 以下两个假设：1）诱导细胞应力反应 在持续感染的HTLV-I淋巴细胞中增强病毒 转录和2）本构表示HSP 73和诱导HSP 72与病毒蛋白税的复合物稳定和目标税 转移到核。要解决的特定目标 这些假设是； 1）确定定性和定量模式 在细胞应激期间HTLV-I蛋白和RNA的表达 反应，2）评估蛋白质相互作用和细胞运输 在细胞应激期间HSP 72，HSP 73和HTLV-I税蛋白 响应和3）评估HTLV-1税的功能活动 在细胞应力反应过程中HTLV-I LTR的激活。这 实验将使用成功采用的技术来诱导和 监测长期感染的HTLV-I的细胞应激反应 转化的淋巴细胞。 HTLV-I RNA和蛋白质产生的评估 将使用各种免费技术，包括间接 HTLV-I病毒蛋白，北印迹和 插槽印迹分析，核跑步分析和合成形成 测定。具体目标＃2将利用本机免疫沉淀 选择性沉淀HSP 73，HSP 72和HTLV-I蛋白 特定的税收，在压力反应中复杂。研究 将与单一和双重间接免疫荧光相关 通过锚定的细胞分析和分类细胞图仪进行分析。目标＃3 将重点介绍税收的功能变化对激活的激活 htlv-i ltr。 HeLa细胞或小屋78个细胞将与A共转染 报告基因质粒和HTLV-I LTR-TAX质粒。细胞将是 经过测定的细胞应激和CAT活性。体外 转录将在存在和不存在纯化的情况下进行 压力后的税收蛋白。这将补充转染研究 并将转录率变化的特异性提高到 税。压力反应是重要的生理反应，并被诱导 通过多个生物学相关事件。可能诱导或 增强HTLV-I税收的表达在此过程中起重要作用 细胞转化。该建议将调查机制 细胞应力反应调节HTLV-I的表达 蛋白质和RNA。