Regulation of HTLV-I replication by the viral p30

描述(申请人提供)：人类T细胞嗜淋巴病毒1型(HTLV-I)在流行病学上与一种侵袭性和致命性T细胞型白血病/淋巴瘤有关，命名为成人T细胞白血病/淋巴瘤(ATLL)。HTLV-I感染也与一种进行性脊髓病有关，称为热带痉挛截瘫/HTLV-I相关性脊髓病(TSP/HAM)。据估计，全球有2000万至3000万人感染了HTLV-I。HTLV-I具有不寻常的特征，不能预测其在具有免疫能力的宿主中的存活率：该病毒传染性较差，但会引起强烈的体液和细胞宿主免疫反应。此外，HTLV-I主要通过感染细胞的分裂在体内复制，因此呈现出非常低的抗原性变异性。然而，尽管有这些明显的缺点，该病毒在人类体内已经持续了10万多年，这表明HTLV-I已经有效地适应了它的宿主。我们最近发现HTLV-I已经进化出一种蛋白质P30，它与编码病毒表达正调控因子的Tax/Rex病毒RNA特异性地相互作用。由于P30不能穿梭出细胞核，Tax/Rex RNA被困在细胞核中，这些蛋白的表达受到抑制。 本研究的目的是揭示P30与病毒RNA相互作用的分子机制，以及P30对转录的影响，从而抑制病毒复制和潜伏时间。提出了四个具体目标。目的1)研究P30-RNA相互作用的生化特征。定义HTLV-I前病毒内最小的P30反应RNA序列，并确定参与这些相互作用的P30蛋白的结构域(S)。目的2)分析P30在体内外转录和转录后对HTLV-I复制的影响。将产生不能与转录调控因子CBP/p300或病毒RNA相互作用的特定突变体，以区分P30的每一种活性。目的3)研究P30核仁滞留的分子决定因素以及P30对Rex-RNA结合活性或Rex功能的影响，反之亦然。目的4)利用突变为P30的分子克隆，研究P30和TAX/REX RNA的体外表达与病毒表达的关系。将在体内研究从感染松鼠猴模型纵向收集的不同淋巴组织中病毒RNA表达的动力学，以及无症状或HTLV-I感染的神经系统或血液学疾病患者。

DESCRIPTION (provided by applicant): The human T-cell lymphotropic virus type 1 (HTLV-I) is epidemiologically associated with an aggressive and fatal T-cell type leukemia/ lymphoma designated Adult T-cell Leukemia/ Lymphoma (ATLL). HTLV-I infection is also associated with a progressive myelopathy designated Tropical Spastic Paraparesis/ HTLV-I Associated Myelopathy (TSP/HAM). It is estimated that 20 to 30 million people worldwide are infected with HTLV-I. HTLV-I possesses unusual features that would not predict its survival in an immune-competent host: The virus is poorly infectious but elicits a vigorous humoral and cellular host immune response. In addition, HTLV-I is mainly replicated in vivo through division of infected cells and therefore presents a very low antigenic variability. However, in spite of these apparent disadvantages the virus has persisted in humans for more than 100,000 years, indicating that HTLV-I has efficiently adapted to its host. We have recently found that HTLV-I has evolved a protein, p30, that interacts specifically with the tax/rex viral RNA encoding positive regulators of virus expression. Because p30 is unable to shuttle out of the nucleus, tax/rex RNA is trapped in the nucleus and expression of these proteins is inhibited. The goals of this study are to uncover the molecular mechanisms involved in p30-viral RNA interactions and p30 effects on transcription leading to inhibition of virus replication and latency. Four specific aims are proposed. Aim 1) Investigate biochemical characteristics of p30-RNA interactions. Define the minimal p30-response RNA sequence within HTLV-I provirus and identify the domain(s) of the p30 protein involved in these interactions. Aim 2) Analyze transcriptional and post transcriptional effects of p30 on HTLV-I replication in vivo and in vitro. Specific mutants unable to interact with transcriptional regulator CBP/p300 or with viral RNA will be generated in order to discriminate each activity of p30. Aim 3) Investigate molecular determinants for the nuclear/nucleolar retention of p30 and the effects of p30 on Rex-RNA binding activity or Rex functions and vice versa. Aim 4) Study the kinetics of p30 and tax/rex RNA expression in relation to virus expression in vitro using molecular clones mutated for p30. Kinetics of viral RNA expression will be studied in vivo in different lymphoid tissues collected longitudinally from an infected squirrel monkey model and in asymptomatic or HTLV-I-infected patients with neurological or hematological disorders.