NMR Studies of HIV-1 Proteins

拟议的研究旨在了解HIV-1 Gag多蛋白及其组分 基质（MA）和衣壳（CA）结构域在病毒复制期间起作用。本供资期间 我们（i）确定了HIV-1 Gag的N-末端半部分和中心部分的结构，（ii）鉴定了一个 结构开关CA似乎触发衣壳组装，（iii）确定的结构， 肉豆蔻酰化的HIV-1 MA蛋白，（iv）确定了一种新的熵肉豆蔻基转换机制， 膜结合，（v）确定来自相关人T细胞的完整衣壳蛋白的结构 白血病（HTLV-I）和劳斯肉瘤（RSV）病毒，提供了完整逆转录病毒的第一个3D结构 衣壳蛋白，（vi）发现了第一个HIV-1衣壳组装的抗病毒抑制剂。 我们现在准备确定Gag、MA和CA如何相互作用，以及如何与病毒和 细胞成分，并提高抑制衣壳组装的抗病毒剂的功效。一是 了解调节特定膜结合和细胞内运输，相互作用的机制 MA与肌醇磷酸（IP）和AP-3运输复合物的研究。初步研究 表明IP结合MA并触发肉豆蔻酸酯暴露，这可以解释Gag如何靶向IP丰富 膜组装。影响核定位的MA突变和磷酸化事件也将 被研究。接下来，为了深入了解促进病毒组装的Gag-Gag相互作用，我们将确定 含有三聚体促进GCN 4衍生肽的嵌合Gag构建体的结构被取代， N-末端肉豆蔻基。（肉豆蔻酰化Gag三聚体的研究被高阶聚合物排除在外。 在NMR浓度下的聚集）。初步研究表明，GCN 4-Gag构建体形成良好- 表现出具有以前未表征的三聚体CA界面的三聚体。三是着力 提高衣壳组装抑制剂的功效。我们现在已经鉴定出了500倍于 改善的结合亲和力（Kd ~ 1 μ M）和>100倍改善的EC 50值（~20 μ M）， 初步线索CA：抑制剂复合物获得的热力学和结构信息将用于 结合体内研究，以鉴定具有潜在临床效用的更有效的抗病毒剂。 多组分体系的研究在技术上比我们以前对孤立体系的研究更具挑战性。 逆转录病毒蛋白和结构域，但潜在的回报显着更高，并可能导致 开发用于治疗新出现的艾滋病毒多药耐药株的新药。

The proposed studies are aimed at understanding how the HIV-1 Gag polyprotein and its constituent matrix (MA) and capsid (CA) domains function during viral replication. During the current funding period we (i) determined the structures of the N-terminal half and central portions of HIV-1 Gag, (ii) identified a structural switch in CA that appears to trigger capsid assembly, (iii) determined the structure of the myristoylated HIV-1 MA protein, (iv) identified a novel entropic myristyl switch mechanism that regulates membrane binding, (v) determined the structures of the intact capsid proteins from the related human T-cell leukemia (HTLV-I) and Rous Sarcoma (RSV) viruses, providing the first 3D structures of intact retroviral capsid proteins, and (vi) discovered the first antiviral inhibitors of HIV-1 capsid assembly. We are now poised to determine how Gag, MA and CA interact with each other and with viral and cellular constituents, and to improve the efficacy of antiviral agents that inhibit capsid assembly. First, to understand the mechanisms that regulate specific membrane binding and intracellular trafficking, interactions of MA with inositol phosphates (IPs) and the AP-3 trafficking complex will be studied. Preliminary studies indicate that IPs bind MA and trigger myristate exposure, which may explain how Gag is targeted to IP-rich membranes for assembly. MA mutations and phosphorylation events that affect nuclear localization will also be studied. Next, to gain insights into Gag-Gag interactions that promote virus assembly, we will determine the structures of chimeric Gag constructs containing trimer-promoting GCN4-derived peptides substituted for the N-terminal myristyl group. (Studies of myristoylated Gag trimers are precluded by higher-order aggregation at NMR concentrations). Preliminary studies indicate that GCN4-Gag constructs form well- behaved trimers with previously uncharacterized trimeric CA interfaces. Third, efforts will be made to improve the efficacy of capsid assembly inhibitors. We have now identified compounds with 500-fold improved binding affinities (Kd ~ 1 ^M) and >100-fold improved EC50 values (~20 (j,M) compared to our initial leads. Thermodynamic and structural information obtained for CA:inhibitor complexes will be used in conjunction with in vivo studies to identify more potent antiviral agents with potential clinical utility. Studies of multi-component systems are technically more challenging than our previous studies of isolated retroviral proteins and domains, but the potential rewards are significantly higher, and could lead to the development of new drugs for the treatment of emerging multi-drug resistant strains of HIV.