CELLULAR STRESS RESPONSE AND HTLV-I REPLICATION

细胞应激反应和 HTLV-I 复制

基本信息

批准号：
2671343
负责人：
JANICE M ANDREWS
金额：
$ 8.86万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-09-01 至 2000-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2671343
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

项目摘要

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 淋巴细胞病毒 I 型（HTLV-I）是下列疾病的病原体：成人 T 细胞白血病/淋巴瘤 (ATLL) 和慢性脊髓病该感染被认为是一个重要的公共卫生问题美国。 HTLV-I的特点是临床周期长特定主机控制的延迟和有限信息已知调节 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，被认为发挥着重要作用 HTLV-I 白血病发生过程中。总体目标是研究 HTLV-I 复制中的细胞应激反应，以阐明这是如何重要的细胞反应可能最终影响病毒介导的淋巴细胞转化。拟议的研究基于以下两个假设：1）诱导细胞应激反应在持续感染的 HTLV-I 淋巴细胞中增强前病毒转录和2)组成型表达的hsp 73和诱导型hsp 72 与病毒蛋白 Tax 形成复合物，以稳定和靶向 Tax 易位至细胞核。将解决的具体目标这些假设是； 1) 确定定性和定量模式细胞应激期间 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细胞应激期间 hsp 72、hsp 73 和 HTLV-I Tax 蛋白的变化响应，以及 3) 评估 HTLV-I Tax 的功能活动 HTLV-I LTR 在细胞应激反应过程中的激活。这实验将使用成功采用的技术来诱导和监测慢性感染 HTLV-I 的细胞应激反应转化的淋巴细胞。 HTLV-I RNA 和蛋白质生产的评估将使用各种补充技术，其中包括间接 HTLV-I 病毒蛋白的免疫荧光测定、Northern 印迹和槽印迹分析、核连续分析和合胞体形成化验。具体目标#2 将利用天然免疫沉淀选择性沉淀 hsp 73、hsp 72 和 HTLV-I 蛋白，特别是税收，在压力反应过程中会变得复杂。研究将与单间接免疫荧光和双间接免疫荧光相关通过锚定细胞分析和分选细胞仪进行分析。目标#3 将重点关注税务激活后的功能变化 HTLV-I LTR。 Hela 细胞或 Hut 78 细胞将与报告质粒和 HTLV-I LTR-Tax 质粒。细胞将是经受细胞应激并测定 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补充蛋白质。这将补充转染研究并增加转录率变化的特异性税。应激反应是一种重要的生理反应，是由应激反应引起的由多个生物学相关事件引起。可能引起或 HTLV-I Tax 的增强表达在此过程中发挥重要作用的细胞转化。该提案将研究机制细胞应激反应通过其调节 HTLV-I 的表达蛋白质和RNA。