CELLULAR STRESS RESPONSE AND HTLV-I REPLICATION

人类嗜T淋巴细胞病毒I型（HTLV-I）是 成人T细胞白血病/淋巴瘤（ATLL）和慢性脊髓病， 这种感染被认为是一个重要的公共卫生问题， 美国的HTLV-I的特点是具有长时间的临床症状， 特定主机控制器延迟和有限信息是已知的 调节HTLV-I表达的机制。HTLV-I病毒编码的Tax 蛋白质是一种有效的反式激活剂， 在HTLV-I白血病发生中的作用总的目标是研究 HTLV-I复制中的细胞应激反应，以阐明这是如何 必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是基于 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， 2）组成型表达的hsp 73和诱导型hsp 72与病毒蛋白Tax复合以稳定和靶向Tax 转移到细胞核。具体目标将涉及 这些假设是：1）确定定性和定量模式 细胞应激过程中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 热休克蛋白72，热休克蛋白73，和HTLV-Ⅰ Tax蛋白在细胞应激过程中的作用 反应，和3）评估HTLV-I税的功能活性， 在细胞应激反应期间HTLV-I LTR的活化。的 实验将使用成功采用的技术， 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和蛋白质生产的评价 将使用各种各样的补充技术，其中包括间接 HTLV-1病毒蛋白免疫荧光测定、北方印迹和 狭缝印迹分析、核连续分析和合胞体形成 测定。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-1蛋白， 特别是税，其中复杂的压力反应。研究 将与单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 将重点放在税收功能的变化上， HTLV-I LTR. Hela细胞或Hut 78细胞将与一种或多种细胞共转染。 报告质粒和HTLV-I LTR-Tax质粒。将细胞 进行细胞应激并测定CAT活性。体外 转录将在存在和不存在纯化的 压力后的蛋白质税。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， 多个生物学相关事件的影响可能诱发或 HTLV-I Tax的增强表达在这一过程中发挥了重要作用 细胞的转化。本提案将研究 细胞应激反应通过其调节HTLV-I的表达 蛋白质和RNA。

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.