Cellular Protein Involved in Trafficking of HIV-1

项目摘要(申请人提供)：Tsg101，ESCRT-I的一个组成部分(内涵体 运输所需的分选复合体-I)，是传染性艾滋病毒和几个 其他人类病原体。病毒编码的结构前体中的富含脯氨酸的序列PTAP 多蛋白，GAG，通过以下方式将细胞因子招募到质膜上的病毒组装位置 与Tsg101 N端UEV[泛素(Ub)E2变异体]中的PTAP结合口袋相互作用 域。UEV结构域也结合了Ub，然而，Ub在萌芽和关系中的作用 UEV Ub结合到PTAP介导的ESCRT机制的招募尚不清楚。 最近，利用高通量鉴定的小分子筛选了一个已知的文库 能够结合UEV的药物，我们发现了通过干扰Ub-1来抑制HIV-1萌芽的药物。 在UEV中的已知口袋结合而不干扰PTAP结合口袋。我们的核磁共振 研究发现Tsg101 UEV结构域中还有一个Ub结合位点，并证明 该结构域能够结合先前显示的参与两个内吞的二-Ub部分 贩卖和萌芽。本申请建议使用诱变剂、诱变剂和结构剂 分析以确定原始和新发现的UEV Ub结合位点的机制 通过实现3个目标来促进萌芽过程。目标1的目标是结构- UEV-Ub复合体的定点突变和小分子功能分析 分子探测器。我们和其他人发现，在必要时，PT/SAP调解的招聘 仅有Tsg101对于萌芽是不够的，AIM 2的目标是测试以下假设 Tsg101 Ub绑定功能需要与PTAP绑定功能结合使用，以实现高效 蛋白质在质膜上的募集。AIM 3的目标是确定 Tsg101的Ub结合功能对于Tsg101不直接存在的运输途径很重要 由病毒结构蛋白参与，即由ESCRT适配器Alix和Alix促进的通路 Nedd4.众所周知，当Gag相互作用时，HIV利用替代的萌发途径 Tsg101被破坏：因此，重要的是了解所需的Tsg101-Ub绑定 功能是多余的。总的来说，拟议的研究将提供机械性的理解 Tsg101如何促进HIV-1病毒的贩运和病毒萌芽，并可能揭示新的 抗病毒药物开发的目标。

PROJECT ABSTRACT (provided by applicant): Tsg101, a component of ESCRT-I (endosomal sorting complex required for transport-I), is required for budding of infectious HIV and several other human pathogens. A proline-rich sequence, PTAP, in the viral-encoded structural precursor polyprotein, Gag, recruits the cellular factor to virus assembly sites on the plasma membrane by interacting with a PTAP-binding pocket in the Tsg101 N-terminal UEV [ubiquitin (Ub) E2 variant] domain. The UEV domain also binds Ub, however, the role of Ub in budding and the relationship of UEV Ub-binding to the PTAP-mediated recruitment of the ESCRT machinery is not clear. Recently, using small molecules identified by high-throughput screening of a library for known drugs capable of binding the UEV, we found agents that inhibit HIV-1 budding by disrupting Ub- binding at the known pocket in the UEV without disturbing the PTAP-binding pocket. Our NMR studies identified an additional Ub-binding site in the Tsg101 UEV domain and demonstrate that the domain is capable of binding di-Ub moieties previously shown to participate in both endocytic trafficking and budding. This application proposes to use the agents, mutagenesis and structural analysis to define the mechanism by which the original and newly identified UEV Ub-binding sites facilitate the budding process through achievement of 3 goals. The goal of AIM 1 is structure- function analysis of the UEV-Ub complex employing site-directed mutagenesis and small molecule probes. As we and others find that, while necessary, PT/SAP-mediated recruitment of Tsg101 alone is not sufficient for budding, the goal of AIM 2 is to test the hypothesis that the Tsg101 Ub- binding function is required in conjunction with the PTAP-binding function for efficient recruitment of the protein to the plasma membrane. The goal of AIM 3 is to determine whether the Tsg101 Ub-binding function is important for trafficking pathways where Tsg101 is not directly engaged by a viral structural protein, i.e., pathways facilitated by the ESCRT adaptors Alix and Nedd4. It is well-established that HIV utilizes alternative budding pathways when Gag interaction with Tsg101 is disrupted: It is therefore important to understand if the required Tsg101-Ub binding function is redundant. Collectively, the proposed studies will provide mechanistic understanding of how Tsg101 facilitates HIV-1 Gag trafficking and virus budding and possibly also reveal new targets for anti-viral drug development.