CELLULAR STRESS RESPONSE AND HTLV-I REPLICATION

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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.