HTLV-1 & Cellular Factors in Neuroinflammatory Disease

描述(申请人提供)：人类T细胞白血病病毒1型(HTLV-1)是成人T细胞白血病(ATL)和神经疾病HTLV-1相关性脊髓病/热带痉挛麻痹(HAM/TSP)的病原体。该病毒主要针对CD4T细胞，但也感染其他次级细胞类型，如CD8T细胞、骨髓前体细胞、单核-巨噬细胞系细胞和常驻CNS细胞群。我们和其他人之前的研究表明，病毒诱导的这些细胞室的变化在进行性神经疾病HAM/TSP的发生中起着重要作用。然而，关于HTLV-1启动子(长末端重复序列或LTR)在这些细胞中调控的分子机制还知之甚少。此外，几乎所有强调细胞转录因子在HTLV-1Tax介导的LTR激活中的重要性以及Tax蛋白与这些因子独立相互作用的能力的研究都是使用瞬时转染的病毒报告质粒或在不代表HTLV-1体内主要靶点的细胞系中进行的。用HIV-1进行的研究表明，整合的前病毒在物理上和对某些细胞因子的需求上都不同于转染的病毒质粒，特别是那些属于染色质重塑组蛋白乙酰转移酶(HAT)家族的因子。因此，为了更好地了解HTLV-1基因的调控以及与整合前病毒的复杂相互作用，我们先前鉴定了一些稳定的CD4T细胞克隆(Jurkat)、单核细胞(U-937)和祖细胞(TF-1)细胞系携带HTLV-1 LTR的整合拷贝来驱动荧光素酶基因的表达。使用这些克隆进行的初步研究突出了Tax蛋白和参与调控染色质重塑的宿主microRNAs之间的新的相互作用，从而导致了拟议的研究。我们的结果与一个新的假设是一致的，即Tax可以以细胞类型特异性的方式调节细胞miRNA机制，涉及染色质重塑影响HTLV-1 LTR控制的病毒基因表达。验证这一概念和检验我们的假设的具体目的是：(1)研究病毒病程中初级和次级靶细胞染色质背景下Tax介导的DNA-蛋白质相互作用的机制；(2)确定HTLV-1Tax调控的宿主miRNA表达与HTLV-1靶向初级和次级细胞群染色质重塑有关；以及(3)在患者队列中验证Tax-miRNA-染色质相互作用在HTLV-1疾病中的作用(S)。所提出的研究可能回答与HTLV-1致病相关的基本关键问题，并将为许多携带HTLV-1前病毒序列的感染者的CD4T细胞不表达病毒蛋白提供新的见解。此外，这些研究还将对T细胞和骨髓前体细胞以及单核巨噬细胞系细胞中与HTLV-1基因调控相关的分子机制(S)进行比较，后者是逆转录病毒神经发病机制中的关键细胞成分。

DESCRIPTION (provided by applicant): Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL) and the neurological disorder, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus preferentially targets CD4+ T cels, but also infects other secondary cell types such as CD8+ T cells, bone marrow progenitor cells, cells of the monocyte-macrophage lineage, and resident CNS cell populations. Previous studies from us and others have suggested that virus-induced alterations in these cell compartments play important roles in the genesis of the progressive neurological disorder HAM/TSP. However, little information exists concerning the molecular mechanisms of HTLV-1 promoter (long terminal repeat or LTR) regulation in these cells. Moreover, nearly all studies highlighting the importance of the cellular transcription factors in HTLV-1 Tax-mediated LTR activation and the ability of Tax protein to interact with these factors independently have been performed using transiently transfected viral reporter plasmids or in cell lines that do not represent the primary target for HTLV-1 in vivo. Studies performed with HIV-1 have indicated that the integrated provirus differs from a transfected viral plasmid both physically and in the requirement for certain cellular factors, especially those belonging to the chromatin remodeling histone acetyltransferase (HAT) family. Hence, to better understand HTLV-1 gene regulation and the complex interplay with the integrated provirus, we previously characterized a number of stable clones of CD4+ T-cell (Jurkat), monocyte (U-937), and progenitor (TF-1) cell lines carrying integrated copies of the HTLV-1 LTR driving luciferase gene expression. Preliminary investigations using these clones have highlighted novel interactions between the Tax protein and host microRNAs involved in regulating chromatin remodeling leading to the proposed studies. Our results are consistent with a novel hypothesis that Tax can modulate the cellular miRNA machinery in a cell-type specific manner involving chromatin remodeling effecting viral gene expression controled by the HTLV-1 LTR. The Specific Aims to validate this concept and to test our hypothesis are to (1) Investigate the mechanism of Tax- mediated DNA-protein interactions in the context of chromatin in primary and secondary target cell populations during the course of viral disease, (2) Define HTLV-1 Tax-modulated host miRNA expression linked to chromatin remodeling in primary and secondary cell populations targeted by HTLV-1, and (3) Validate role of Tax-miRNA-chromatin interactions in HTLV-1 disease in patient cohort(s). The proposed studies ofer the potential to answer basic key questions related to HTLV-1 pathogenesis and will provide novel insight into why many CD4+ T cells in infected individuals containing HTLV-1 proviral sequences do not express viral proteins. In addition, these studies will provide a comparative account of molecular mechanism(s) related to HTLV-1 gene regulation in T cells versus bone marrow progenitor cells and cells of the monocyte-macrophage lineage, which are critical cellular components involved in retroviral neuropathogenesis.