Cellular Protein Involved in Trafficking of HIV-1

项目摘要（申请人提供）：Tsg 101，ESCRT-1（内体）的组分 运输所需的分选复合物-I），是传染性HIV萌芽所需的， 其他人类病原体病毒编码的结构前体中富含脯氨酸的序列PTAP 多蛋白Gag通过以下方式将细胞因子募集到质膜上的病毒组装位点： 与Tsg 101 N-末端UEV中的PTAP结合口袋相互作用[泛素（Ub）E2变体] 域UEV结构域也与Ub结合，然而，Ub在出芽中的作用以及UEV结构域与Ub的关系是未知的。 UEV Ub与PTAP介导的ESCRT募集机制的结合尚不清楚。 最近，使用通过高通量筛选文库鉴定的小分子， 在能够结合UEV的药物中，我们发现了通过破坏Ub-1来抑制HIV-1出芽的药物。 在UEV中的已知口袋处结合，而不干扰PTAP结合口袋。我们的NMR 研究在Tsg 101 UEV结构域中鉴定了另外的Ub结合位点，并证明， 所述结构域能够结合先前显示参与内吞和内分泌的di-Ub部分， 贩运和萌芽。本申请提出使用所述试剂、诱变剂和结构修饰剂， 分析以确定原始和新鉴定的UEV Ub结合位点的机制 通过实现三个目标促进萌芽进程。AIM 1的目标是结构- 采用定点突变和小分子标记对UEV-Ub复合物进行功能分析 分子探针正如我们和其他人发现的那样，虽然必要，PT/SAP介导的招募， 单独的Tsg 101是不足以出芽的，AIM 2的目标是测试假设， 需要Tsg 101 Ub结合功能与PTAP结合功能结合，以实现有效的免疫调节。 将蛋白质募集到质膜。AIM 3的目标是确定 Tsg 101 Ub结合功能对于Tsg 101不直接 由病毒结构蛋白接合，即，ESCRT衔接子阿利克斯和 Nedd4.众所周知，当Gag相互作用时，HIV利用替代出芽途径 与Tsg 101的结合被破坏：因此，了解所需的Tsg 101-Ub结合是否 功能是多余的。总的来说，拟议的研究将提供机械的理解， Tsg 101如何促进HIV-1 Gag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.