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。

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.