LOCAL CHANGES IN GENE EXPRESSION DURING SIV ENCEPHALITIS

SIV 脑炎期间基因表达的局部变化

• 批准号: 6539063
• 负责人: TODD A REINHART
• 金额: $ 30.31万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6539063
• 关键词: AIDS dementia complex; Macaca mulatta; astrocytes; biological models; biomarker; complementary DNA; disease /disorder model; disease /disorder onset; gene expression; genetic library; immunocytochemistry; in situ hybridization; messenger RNA; neuropathology; northern blottings; protein protein interaction; protein structure function; psychomotor function; regulatory gene; simian immunodeficiency virus; virus genetics; virus infection mechanism; virus load; virus replication; yeast two hybrid system

In addition to the severe immunodeficiency resulting from infection with the human immunodeficiency virus type-1 (HIV-1), a significant proportion of individuals develop neurologic dysfunctions referred to collectively as the HIV-1-associated motor/cognitive disorder (HMCD). Simian immunodeficiency virus (SIV) infection of rhesus macaques serves as an excellent model system for HIV-1 entry into and replication within the central nervous system (CNS). How infection of the CNS by HIV-1 or SIV leads to neurologic impairment is not fully understood. The overall objective of this proposal is to identify virus/host interactions within the CNS of SIV infected rhesus macaques that contribute to motor/cognitive deficits. Our hypothesis is that there are brain-specific cellular genes that are differentially expressed during SIV encephalitis (SIVE) and there are brain- specific cellular proteins whose activities are modulated by interaction with the SIV Nef protein. Furthermore, we propose that these changes in gene expression and protein function contribute to the neurologic impairments associated with infection by SIV. In order to address these issues, gene expression profiles of brain tissues from macaques without SIVE or in the early stages of SIVE, will be generated using the recently developed serial analysis of gene expression (SAGE) technology. This approach will determine the relative levels of expression of individual mRNAs present in macaque brains and will identify genes that are differentially expressed in brain cells during early SIVE relative to non-encephalitic brain tissues. The numbers, locations, and types of brain cells actively transcribing differentially expressed mRNAs will be determined by in situ hybridization/immunohistochemistry, and their relationship to SIV productively infected cells with be examined. In parallel, macaque brain proteins that interact with SIV Nef will be identified by screening a brain cDNA expression library for Nef binding partners using established yeast 2-hybrid methods. Modulation of intracellular locale or enzymatic activity of interacting proteins by Nef will be comprehensively characterized. These data will provide mechanistic insight into how SIV alters the genetic and biochemical environment within the brain, thereby identifying genes and gene products that might be altered by HIV-1 replication in humans and which might serve as eventual targets for therapeutic interventions.

除了由人类免疫缺陷病毒1型（HIV-1）感染引起的严重免疫缺陷外，相当大比例的个体发展神经功能障碍，统称为HIV-1相关运动/认知障碍（HMCD）。 猴免疫缺陷病毒（SIV）感染恒河猴是HIV-1进入中枢神经系统（CNS）并在其中复制的一个极好的模型系统。 HIV-1或SIV感染CNS如何导致神经功能损害尚不完全清楚。 本提案的总体目标是确定SIV感染恒河猴CNS内导致运动/认知缺陷的病毒/宿主相互作用。 我们的假设是存在脑特异性细胞基因，其在SIV脑炎（SIVE）期间差异表达，并且存在脑特异性细胞蛋白，其活性通过与SIV Nef蛋白的相互作用来调节。 此外，我们认为这些基因表达和蛋白质功能的变化有助于与SIV感染相关的神经功能障碍。 为了解决这些问题，将使用最近开发的基因表达系列分析（SAGE）技术，从猕猴脑组织中产生的基因表达谱没有SIVE或在SIVE的早期阶段。 该方法将确定猕猴脑中存在的单个mRNA的相对表达水平，并将鉴定相对于非脑炎脑组织在早期SIVE期间在脑细胞中差异表达的基因。将通过原位杂交/免疫组织化学确定活跃转录差异表达mRNA的脑细胞的数量、位置和类型，并检查它们与SIV有效感染细胞的关系。与此同时，猕猴脑蛋白与SIV Nef相互作用将通过使用已建立的酵母双杂交方法筛选脑cDNA表达文库的Nef结合伴侣来鉴定。 Nef对相互作用蛋白的细胞内区域或酶活性的调节将被全面表征。 这些数据将为SIV如何改变大脑内的遗传和生物化学环境提供机制性见解，从而确定可能被人类HIV-1复制改变的基因和基因产物，并可能作为治疗干预的最终目标。