Sam68 and HIV-1 replication control in astrocytes

描述(申请人提供)：HIV-1复制是宿主-病毒相互作用的综合结果，发生在病毒生命周期的多个步骤，包括进入、基因表达、组装和萌芽。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感染的策略是我们的长期目标。与原始的CD4+T淋巴细胞和巨噬细胞/母细胞不同，CD4-星形胶质细胞对HIV-1感染的敏感性较低，并且不支持进入后生产性的HIV-1复制。因此，星形胶质细胞被认为是确定HIV-1进入、基因表达和复制所必需的宿主因子的理想天然平台。利用基于逆转录病毒的表达cDNA文库，我们揭示了HIV-1通过人类甘露糖受体以相对低效的CD4非依赖性和内吞依赖性的方式获得与星形胶质细胞的结合。此外，使用消减克隆策略，我们已经确定Sam68至少部分地对进入星形胶质细胞后的非生产性HIV-1复制负责。这项建议的总体目标是表征Sam68在星形胶质细胞和其他HIV-1天然靶细胞中复制HIV-1的分子机制。这一建议基于我们的三个主要发现：(1)Sam68是HIV-1 Rev核输出所必需的细胞辅助因子；(2)缺乏核定位信号的Sam68突变体对HIV-1复制的抑制与组成部分Sam68的细胞质定位有关；(3)Sam68调节HIV-1mRNAs在细胞质中的翻译。这一提议的基本假设是，Sam68在病毒生命周期的多个阶段调节细胞核和细胞质中的HIV-1复制。换句话说，Sam68是HIV-1复制和致病的重要细胞辅助因子。为了验证这一假设，我们提出了三个相互关联的特定目标：(1)确定Sam68表达与HIV-1复制之间的关系；(2)确定Sam68在HIV-1 Rev核输出中的功能；(3)确定Sam68在HIV-1复制中的细胞质功能。在整个研究过程中，我们将使用各种最先进的生化、细胞和分子方法。所寻求的答案对于理解这种在HIV-1复制中关键且普遍存在的蛋白Sam68具有基本意义。它们还应协助制定抗艾滋病毒治疗战略。公共卫生相关性： 艾滋病毒-1感染常常导致一些脑部疾病，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为过。目前的研究试图更好地了解艾滋病毒-1传播所需的宿主因素，然后利用所获得的知识为艾滋病毒/艾滋病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.