Sam68 and HIV-1 replication control in astrocytes

描述（由申请人提供）：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、组装和出芽。我们的长期目标是了解宿主因子对HIV-1基因表达和复制的调控，从而制定治疗艾滋病患者的策略，最终预防和消除HIV-1感染。与原发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感染的易感性较低，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定义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，我们发现HIV-1通过人甘露糖受体以相对低效的cd4独立和内吞依赖的方式进入星形胶质细胞。此外，使用减法克隆策略，我们已经确定Sam68至少在一定程度上负责星形胶质细胞进入后非生产性HIV-1复制。本提案的总体目标是表征Sam68在星形胶质细胞以及其他HIV-1自然靶细胞中HIV-1复制中的分子机制。这一建议是基于我们的三个主要发现：(1)Sam68是HIV-1 Rev核输出的重要细胞辅助因子；(2)缺乏核定位信号的Sam68突变体对HIV-1复制的抑制与构成Sam68的细胞质定位有关；(3) Sam68调控细胞质中HIV-1 mrna的翻译。这一提议的基本假设是Sam68在病毒生命周期的多个步骤中在细胞核和细胞质中调节HIV-1的复制。换句话说，Sam68是HIV-1复制和发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)研究Sam68在HIV-1 Rev核输出中的功能；(3)明确Sam68在HIV-1复制中的细胞质功能。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。所寻求的答案对于理解这种在HIV-1复制中至关重要且普遍存在的蛋白质Sam68具有根本意义。它们也应该有助于抗HIV治疗策略的发展。公共卫生相关性：

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.