Biochemistry of HIV-1 Membrane Recognition and Budding

HIV-1 膜识别和出芽的生物化学

• 批准号: 7121006
• 负责人: WESLEY I. SUNDQUIST
• 金额: $ 2.06万
• 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/7121006
• 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(申请人提供)：HIV-1 Gag在细胞质中翻译 并运输到质膜，在那里它组装成球形 从细胞中发芽的颗粒。拟议的研究将涉及结构上的 病毒膜靶向和分子病毒学的生物学、生化和分子病毒学 萌芽中。第一个目标是确定解决方案的结构、动力学和 肉豆蔻酰化Gag MA结构域的膜生物化学。这些研究， 结合我们以前的肉豆蔻酰化MA的结构，应该可以揭示 调控GAG靶向的“肉豆蔻基开关”的分子机制 质膜。第二个目标是分析病毒的生物化学。 萌芽中。我们已经确定人类Tsg101是一个有吸引力的候选者 结合Gag p6“晚期”结构域并促进病毒传播的细胞因子 放手。Tsg101似乎协调了细胞内吞作用的途径， 胞吐作用和空泡蛋白分选(VPS)，表明机械从 这些途径可能被招募来帮助病毒的释放。我们现在就会 充分描述定义不同的细胞蛋白质网络 Tsg101通路。这些研究将采用高吞吐量方法来 对最初确定的假定的蛋白质-蛋白质相互作用进行定量 自动双混合屏幕。第三个目标是确定解决方案 Tsg101的N-末端p6结合结构域的结构 具有p6结合位点的复合体。我们的初步核磁共振和生化分析 表明该结构域在结构上类似于起作用的E2酶 在泛素(表示为Tsg101 E2*)的转移中以及跨越 ID：v-1 p6“PTAP”序列基序与周围的位点特异结合 Tsg101 E2*β链4.Tsg101的结构及其与p6的结合 网站应该揭示病毒如何招募Tsg101来帮助病毒萌芽 可作为基于结构的病毒抑制剂设计的基础 出口。最终目标是研究HIV-1的分子病毒学。 萌芽中。我们已经证明了Vps4的显性否定结构，它 错配Tsg101并抑制空泡蛋白分离，也阻断HIV-1 放手。我们现在将描述Tsg101和VP的功能角色 病毒萌发过程中的一条途径。