Sam68 and HIV-1 replication control in astrocytes

描述（由申请人提供）：HIV-1复制是病毒生命周期多个步骤（包括进入、基因表达、组装和出芽）中发生的宿主-病毒相互作用的综合结果。我们的长期目标是了解宿主因素对HIV-1基因表达和复制的调控，从而制定治疗艾滋病患者并最终预防和消除HIV-1感染的策略。与原代CD 4 + T淋巴细胞和巨噬细胞/单核细胞不同，CD 4-星形胶质细胞对HIV-1感染的敏感性较低，并且不支持进入后的生产性HIV-1复制。因此，星形胶质细胞被认为是一个理想的天然平台，以确定宿主因子是必不可少的HIV-1的进入，基因表达和复制。使用基于逆转录病毒的表达cDNA文库，我们揭示了HIV-1以相对低效的CD 4非依赖性和内吞依赖性方式通过人甘露糖受体进入星形胶质细胞。此外，使用消减克隆策略，我们已经鉴定出Sam 68至少部分地负责星形胶质细胞中的进入后非生产性HIV-1复制。该提案的总体目标是表征Sam 68在星形胶质细胞以及其他HIV-1天然靶细胞中HIV-1复制中功能的分子机制。该建议基于我们的三个主要发现：（1）Sam 68是HIV-1 Rev核输出的重要细胞辅因子;（2）缺乏核定位信号的Sam 68突变体对HIV-1复制的抑制与组成型Sam 68的细胞质定位相关;（3）Sam 68调节HIV-1 mRNA在细胞质中的翻译。该提议的基本假设是，Sam 68在细胞核和细胞质中调节HIV-1病毒生命周期的多个步骤的复制。换句话说，Sam 68是HIV-1复制和发病机制的重要细胞辅因子。为了验证这一假设，我们提出了三个相互关联的具体目标：（1）确定Sam 68表达和HIV-1复制之间的关系;（2）表征Sam 68在HIV-1 Rev核输出中的功能;（3）确定Sam 68在HIV-1复制中的胞质功能。我们将在整个研究中使用各种最先进的生化，细胞和分子方法。寻求的答案对于理解HIV-1复制中这种关键和普遍的蛋白质Sam 68具有根本意义。它们还应有助于发展抗艾滋病毒治疗战略。公共卫生相关性： HIV-1感染通常会导致一些脑部疾病，并影响人们照顾自己的能力，从而影响他们的日常生活质量。其社会和经济影响怎么强调都不过分。目前的研究旨在更好地了解HIV-1传播所需的宿主因素，然后利用所获得的知识为HIV/AIDS患者开发新的治疗方案。

DESCRIPTION (provided by applicant): HIV-1 replication is a combined result of host-virus interactions occurring at multiple steps of the virus life cycle including entry, gene expression, assembly, and budding. It is our long-term goal to understand regulation of HIV-1 gene expression and replication by host factors, thereby develop strategies for treating AIDS patients and ultimately preventing and eliminating HIV-1 infection. Unlike primary CD4+ T lymphocytes and macrophages/momocytes, CD4- astrocytes are less susceptible to HIV-1 infection and do not support post- entry productive HIV-1 replication. Therefore, astrocytes have been regarded as an ideal natural platform to define the host factors that are essential for HIV-1 entry, gene expression and replication. Using a retrovirus- based expression cDNA library, we have revealed that HIV-1 gains access to astrocytes through human mannose receptor in a relatively inefficient CD4-independent and endocytosis-dependent manner. In addition, using the subtractive cloning strategy, we have identified Sam68 to be, at least in part, responsible for post- entry non-productive HIV-1 replication in astrocytes. The overall goal of this proposal is to characterize the molecular mechanisms of Sam68 function in HIV-1 replication in astrocytes as well as other HIV-1 natural target cells. This proposal is based on our three major findings (1) Sam68 is an essential cellular co-factor for HIV-1 Rev nuclear export; (2) Inhibition of HIV-1 replication by Sam68 mutants lacking a nuclear localization signal correlates with the cytoplasmic localization of constitutive Sam68; (3) Sam68 regulates translation of HIV-1 mRNAs in cytoplasm. The underlying hypothesis for this proposal is that Sam68 regulates HIV-1 replication at multiple steps of viral life cycle in both the nucleus and the cytoplasm. In other words, Sam68 is an important cellular co-factor for HIV-1 replication and pathogenesis. To test this hypothesis, we propose three interrelated specific aims: (1) To determine the relationship between Sam68 expression and HIV-1 replication; (2) To characterize Sam68 function in HIV-1 Rev nuclear export; and (3) To define the cytoplasmic function of Sam68 in HIV-1 replication. We will use a variety of state-of-art biochemical, cellular, and molecular approaches throughout the studies. The answers sought have fundamental significance for understanding of this critical and pervasive protein Sam68 in HIV-1 replication. They should also aid in the development of anti- HIV therapeutic strategies. PUBLIC HEALTH RELEVANCE: HIV-1 infection often causes a number of brain diseases and affects the ability of people to care for themselves and thus the quality of their daily life. The social and economic impact can not be overemphasized. The current study seeks to have a better understanding of the host factors that are required for HIV-1 propagation and then use the knowledge acquired to develop new treatment options for HIV/AIDS patients.