Biochemistry of HIV-1 Membrane Recognition and Budding

HIV-1 膜识别和出芽的生物化学

• 批准号: 6450627
• 负责人: WESLEY I. SUNDQUIST
• 金额: $ 37.5万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6450627
• 关键词: binding sites; biochemistry; cell biology; cell membrane; conformation; electron microscopy; fluorescence microscopy; gag protein; genetic regulation; high throughput technology; human genetic material tag; human immunodeficiency virus 1; molecular biology; myristates; nuclear magnetic resonance spectroscopy; protein protein interaction; protein purification; protein structure; protein structure function; structural biology; virus infection mechanism; virus protein; virus replication

DESCRIPTION (provided by applicant): HIV-1 Gag is translated in the cytoplasm and trafficked to the plasma membrane, where it assembles into spherical particles that bud from the cell. Proposed studies will address the structural biology, biochemistry,and molecular virology of viral membrane targeting and budding. The first aim is to determine the solution structure, dynamics, and membrane biochemistry of the myristoylated Gag MA domain. These studies, together with our previous structure of myristoylated MA, should reveal the molecular mechanism of the "myristoyl switch" that governs Gag targeting to the plasma membrane. The second aim is to analyze the biochemistry of virus budding. We have identified human Tsg101 as an attractive candidate for the cellular factor that binds the Gag p6 "Late" domain and facilitates virus release. Tsg101 appears to coordinate the cellular pathways of endocytosis, exocytosis, and vacuolar protein sorting (Vps), suggesting that machinery from these pathways may be recruited to assist in the viral release. We will now fully characterize the network of cellular proteins that define the different Tsg101 pathways. These studies will employ high throughput approaches for quantitating putative protein-protein interactions initially identified in automated two-hybrid screens. The third aim is to determine the solution structure of the N-terminal, p6 binding domain of Tsg101, both free and in complex with its p6 binding site. Our preliminary NMR and biochemical analyses indicate that this domain is structurally similar to E2 enzymes that function in the transfer of ubiquitin (denoted Tsg101 E2*) and that peptides spanning the "PTAP" sequence motif of ID:V-1 p6 bind specifically to a site surrounding Tsg101 E2* beta-strand 4. The structure of Tsg101 in complex with its p6 binding site should reveal how the virus can recruit Tsg101 to assist in virus budding and may serve as the basis for structure-based design of inhibitors of viral egress. The final aim is to investigate the molecular virology of HIV-1 budding. We have demonstrated that dominant negative constructs of Vps4, which mislocalize Tsg101 and inhibit vacuolar protein sorting, also block HIV-1 release. We will now characterize the functional role of Tsg101 and the Vps pathway in the viral budding process.

描述（由申请人提供）：HIV-1 Gag 在细胞质中翻译 并被运输到质膜，在那里组装成球形 从细胞中萌芽的颗粒。拟议的研究将解决结构性问题 病毒膜靶向的生物学、生物化学和分子病毒学 正在萌芽。第一个目标是确定解决方案的结构、动态和 肉豆蔻酰化 Gag MA 结构域的膜生物化学。这些研究， 与我们之前的肉豆蔻酰化 MA 结构一起，应该揭示 控制 Gag 靶向的“肉豆蔻酰开关”的分子机制 质膜。第二个目的是分析病毒的生化 正在萌芽。我们已经确定人类 Tsg101 是一个有吸引力的候选者 结合 Gag p6“Late”结构域并促进病毒传播的细胞因子 发布。 Tsg101 似乎协调细胞内吞作用的途径， 胞吐作用和液泡蛋白分选（Vps），表明机械 这些途径可以被招募来协助病毒释放。我们现在将 充分表征细胞蛋白质网络，这些蛋白质定义了不同的 Tsg101 途径。这些研究将采用高通量方法 定量最初确定的假定蛋白质-蛋白质相互作用 自动化双混合筛选。第三个目标是确定解决方案 Tsg101 的 N 端 p6 结合结构域的结构，包括游离的和在 与其 p6 结合位点形成复合物。我们的初步核磁共振和生化分析 表明该结构域在结构上与 E2 酶相似，其功能 泛素（表示为 Tsg101 E2*）的转移以及跨肽 ID:V-1 p6 的“PTAP”序列基序特异性结合到周围的位点 Tsg101 E2* beta 链 4. Tsg101 与其 p6 结合复合物的结构 网站应揭示病毒如何招募 Tsg101 来协助病毒萌芽 并可作为基于结构的病毒抑制剂设计的基础 出口。最终目的是研究 HIV-1 的分子病毒学 正在萌芽。我们已经证明了 Vps4 的显性负结构， 错误定位 Tsg101 并抑制液泡蛋白分选，还可阻断 HIV-1 发布。我们现在将描述 Tsg101 和 Vps 的功能角色 病毒出芽过程中的途径。