HIV ENV TRANSMEMBRANE DOMAIN AND FUSION

这项建议的长期目标是确定 HIV包膜糖蛋白(Env)催化 生物膜的融合。在这项研究中，我们将重点关注角色 包膜的gp41亚单位的跨膜(TM)结构域 蛋白质在融合过程中发挥作用。具体目标是：(1) 确定的膜跨结构域的N-末端边界 HIV包膜蛋白。(2)确定建筑物的结构特征 膜所需的HIV包膜蛋白的跨膜区 融合活性，以及(3)确定影响细胞的突变 融合活性对病毒的渗透性和传染性也有影响。 鉴于在病毒复制中的关键作用，HLV包膜糖蛋白 是抗病毒药物开发和治疗的合适靶点；因此， 特定干扰病毒诱导的化合物或策略 膜融合技术提供了治疗的潜力。然而，在这方面的进展 这一领域的抗病毒研究一直很缓慢，主要是因为 病毒融合蛋白发挥作用的确切机制尚不清楚 下定决心。HIV env蛋白具有不常见的TM结构域，但几乎没有 已知它们的结构和功能意义。因为 TM领域的边界尚未被充分绘制，第一 将进行一系列实验来确定N端边界 直接用蛋白酶消化胞外结构域，然后再用氨基酸 膜环境保护的残基的测序。 分子模拟显示HIV-1TM结构域具有潜在的 形成一个两亲性的α-螺旋。我们将检验这一假设 环状病毒的融合功能需要两亲性的TM结构域 定点突变。最后，每个突变的包膜蛋白 已经在初步研究中产生了，而那些 将分析其支持病毒的能力 使用复制缺陷的艾滋病毒互补试验感染，以及 复制能力强的艾滋病毒系统。

The long term objectives of this proposal are to determine of the mechanism by which the HIV envelope glycoprotein (Env) catalyzes the fusion of biological membranes. In this study we will focus on the role that the membrane-spanning (TM) domain of the gp41 subunit of the envelope protein plays in the fusion process. The specific aims are: (1) To determine the N-terminal boundary of the membrane-spanning domain of the HIV envelope protein. (2) to determine the structural features in the membrane-spanning domain of the HIV Env protein required for membrane fusion activity, and (3) To determine whether mutations that affect cell fusion activity also have an effect on virus penetration and infectivity. Given the critical roles in virus replication, the HlV Env glycoproteins are suitable targets for antiviral drug development and therapy; thus, compounds or strategies that specifically interfere with the virus-induced membrane fusion process offer therapeutic potential. However, progress in this area of antiviral research has been slow, primarily because the precise mechanism by which viral fusion proteins function has not been determined. The HIV Env proteins have uncommon TM domains, yet little is known about their structural and functional significance. Because the boundaries of the TM domain have not yet been adequately mapped, the first series of experiments will be to determine the N-terminal boundary directly by protease digestion of the ectodomain followed by amino acid sequencing of the residues protected by the membrane environment. Molecular modeling reveals that the HIV-1 TM domain has the potential to form an amphipathic alpha-helix. We will test the hypothesis that an amphipathic TM domain is required for the fusion function of Env using site-directed mutagenesis. Finally, each of the mutant envelope proteins that have already been generated in the preliminary studies, and those that are proposed, will be analyzed for their ability to support virus infection using a replication-defective HIV complementation assay, and a replication competent HIV system.