Cellular Proteins Involved in Trafficking of HIV-1

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)是国家和国际大流行的病原体。目前还没有疫苗可用，对当前以药物为基础的抗病毒疗法产生抗药性的病毒变种正在全球范围内出现。这些事实使得确定抗病毒药物开发的新目标和新战略势在必行。HIV和其他逆转录病毒的Gag基因编码病毒结构前体多蛋白Gag，足以组装和释放病毒样颗粒。许多细胞蛋白参与了这一过程，并代表了潜在的新靶点。我们的初步研究表明，肌醇(1，4，5)-三磷酸受体(IP3R)参与了HIV-1 Gag和相关的禽劳斯肉瘤病毒(RSV)Gag的转运到质膜上的释放部位。IP3R蛋白以前没有与逆转录病毒贩运有关。IP3R形成一个配体门控的钙通道，调节细胞内钙库的释放。钙释放调节许多细胞过程，包括细胞内转运和胞吐。IP3R的抑制剂干扰了GAG的贩运。对IP3R抑制剂的敏感性需要HIV和RSV Gag蛋白中的晚期结构域，这两个区域分别与Tsg101和Nedd4结合。Tsg101和Nedd4是细胞蛋白，分别促进HIV和RSV Gag的运输到质膜上的位置，在那里Gag组装成病毒样颗粒(VLP)和芽进入细胞外环境。这些研究的目标是(目的1)：确定IP3R在HIV Gag释放中的作用；(AIM 2)：确定IP3R在RSV Gag释放中的作用；(AIM 3)：测试IP3R与Gag在细胞中的相互作用，并确定与已知参与细胞内转运的蛋白质的关系。将使用遗传、生化和细胞生物学方法，包括使用显微注射和短杂交干扰RNA的靶向蛋白耗竭；靶向耗竭与突变的IP3R蛋白的替代；IP3R拮抗剂和激动剂的使用；以及基于荧光的方法。这项研究可能会将逆转录病毒Gag蛋白的胞内运输与钙信号联系起来，并为开发干扰病毒组装和传播的新型抗病毒策略提供新的机会。

DESCRIPTION (provided by applicant): The human immunodeficiency virus type 1 (HIV-1) is the causative agent of a national and international pandemic. No vaccine is currently available and viral variants resistant to current drug-based antiviral therapies are emerging world-wide. These facts make it imperative to identify new targets and strategies for development of anti-viral drugs. The gag gene of HIV and other retroviruses encode the viral structural precursor polyprotein, Gag, and is sufficient for assembly and release of virus-like particles. Many cellular proteins contribute to this process and represent potential novel targets. Our preliminary studies suggest that the inositol (1,4,5)-triphosphate receptor (IP3R) is involved in trafficking of HIV-1 Gag and the related avian Rous Sarcoma Virus (RSV) Gag to release sites on the plasma membrane. The IP3R protein has not been previously linked to retroviral trafficking. IP3R forms a ligand-gated calcium channel that regulates the release of intracellular calcium stores. Calcium release regulates many cellular processes, including intracellular trafficking and exocytosis. Inhibitors of IP3R interfere with Gag trafficking. Susceptibility to IP3R inhibitors requires the Late domains in the HIV and RSV Gag proteins, which are regions that bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 are cellular proteins that facilitate trafficking of HIV and RSV Gag, respectively, to sites on the plasma membrane where Gag assembles into virus-like particles (VLPs) and buds into the extracellular environment. The goals of the proposed studies are (AIM 1): To determine the role of IP3R in HIV Gag release; (AIM 2): To determine the role of IP3R in RSV Gag release; (AIM 3): To test for IP3R-Gag interaction in cells and define the relationship to proteins known to be involved in endocytic trafficking. Genetic, biochemical, and cell biological approaches will be used that will include targeted protein depletion using microinjection and short hybrid interfering RNAs; targeted depletion coupled to replacement with mutated IP3R proteins; use of IP3R antagonists and agonists; and fluorescence-based approaches. This investigation will potentially link endocytic trafficking of retroviral Gag proteins to calcium signaling and provide new opportunities for development of novel antiviral strategies that interfere with virus assembly and transmission.