Retrovirus Structure and Assembly with Inositol Phosphates

逆转录病毒结构和用磷酸肌醇组装

• 批准号: 10451534
• 负责人: Robert A Dick
• 金额: $ 26.86万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-05 至 2023-05-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451534
• 关键词: AIDS/HIV problem; Affect; Amino Acids; Anti-Retroviral Agents; Antiviral Agents; Binding; Binding Sites; Biochemical; Biological Assay; CRISPR/Cas technology; Cells; Cryo-electron tomography; Cryoelectron Microscopy; Data; Development; Drug Screening; Drug Targeting; Enzymes; Equine Infectious Anemia Virus; Excision; Foundations; GAG Gene; Genus Alpharetrovirus; Goals; HIV; HIV-1; Health; Human; Human T-Cell Leukemia Viruses; In Vitro; Inositol; Inositol Phosphates; Investigation; Lentivirus; Life Cycle Stages; Methods; Micro Electron Diffraction; Modeling; Molecular; Peptide Hydrolases; Pharmaceutical Preparations; Phytic Acid; Process; Production; Proteins; Research Personnel; Resistance; Resolution; Retroviridae; Reverse Transcription; Role; Rous sarcoma virus; Sequence Analysis; Series; Site; Structure; Testing; Time; Viral; Virion; Virus; Virus Assembly; Virus-like particle; Work; cofactor; design; experimental study; in vivo; inhibitor; mutant; new therapeutic target; screening; small molecule

Project Summary Assembly of Gag into an immature virus particle is a critical step in the viral life cycle. After assembly the immature virus particle goes through a process called maturation which is required to produce infectious virus particles. We recently showed that the small cellular molecule, inositol hexakisphosphate (IP6) is required for both assembly and maturation, and that decreasing IP6 levels in cells severely reduces the formation of infectious virus. While we know have a “big picture” view of how IP6 affects HIV, there is still a great deal we don’t understand. This proposal seeks to characterize the mechanism of IP6 induced immature assembly, how IP6 promotes mature assembly, and if these assembly steps can be targeted by antiretrovirals. Furthermore, preliminary work suggests that IP6 is a cofactor for other retroviruses, and so this proposal will test the hypothesis that IP6 as a retroviral cofactor is evolutionarily conserved. Specific Aim 1 is to further characterize how IP6 binds and promotes HIV-1 assembly and maturation. High resolution cryo-EM analysis will be performed on IP6 assembled mature virus-like particles with and without cellular proteins that are known to bind to the viral core in cells. The effect of maturation inhibitors Bevirimat and PF-46396, which bind to the same region as IP6, on IP6 binding and enhanced assembly will be determined using a series of biochemical assays. In addition, this aim will test the hypothesis that IP6 is utilized by other lentiviruses. For example, preliminary data suggest that the Equine Infectious Anemia Virus also employs IP6 for the production of infectious virus. Specific Aim 2 is to determine if and how IP6 is utilized by retroviruses outside of the lentivirus genus. Preliminary data shows that the alpha retrovirus Rous sarcoma virus (RSV), and the delta retrovirus Human T-Cell Leukemia Virus (HTLV) both utilize IP6. We will identify the site of IP6 action on both viruses using biochemical and structural approaches. We will also screen viruses from the other retroviral genera to determine if they also utilize IP6. Specific Aim 3 is to determine if and how IP6 related compounds affect HIV-1 immature assembly, maturation, and mature assembly. Working with a collaborator, we will synthesize and screen IP6-like compounds in assembly and maturation assays. This aim will help to determine if compounds that target the IP6 binding site are a feasible antiretroviral strategy.

项目概要 将 Gag 组装成未成熟的病毒颗粒是病毒生命周期中的关键步骤。组装后 未成熟的病毒颗粒要经历一个称为成熟的过程，这是产生传染性病毒所必需的 颗粒。我们最近表明，细胞小分子六磷酸肌醇 (IP6) 是 组装和成熟过程中，细胞中 IP6 水平的降低会严重减少 传染性病毒。虽然我们知道 IP6 如何影响 HIV 有一个“大局”观点，但我们仍然有很多问题需要了解。 不明白。该提案旨在描述 IP6 诱导不成熟组装的机制，如何 IP6 促进成熟组装，如果这些组装步骤可以成为抗逆转录病毒药物的目标。此外， 初步工作表明IP6是其他逆转录病毒的辅助因子，因此该提案将测试 假设 IP6 作为逆转录病毒辅助因子在进化上是保守的。具体目标 1 是进一步 描述 IP6 如何结合并促进 HIV-1 组装和成熟。高分辨率冷冻电镜分析 将在具有或不具有已知细胞蛋白的 IP6 组装的成熟病毒样颗粒上进行 与细胞中的病毒核心结合。成熟抑制剂 Bevirimat 和 PF-46396 的作用，它们与 与IP6相同的区域，将使用一系列生化测定对IP6的结合和增强的组装 化验。此外，该目标将检验 IP6 被其他慢病毒利用的假设。例如， 初步数据表明，马传染性贫血病毒也采用 IP6 来生产 传染性病毒。具体目标 2 是确定 IP6 是否以及如何被外部逆转录病毒利用。 慢病毒属。初步数据显示，α逆转录病毒劳斯肉瘤病毒（RSV）和δ 逆转录病毒人类 T 细胞白血病病毒 (HTLV) 均利用 IP6。我们将在两者上确定 IP6 作用的位置 使用生化和结构方法的病毒。我们还将从其他逆转录病毒中筛选病毒 来确定它们是否也使用 IP6。具体目标 3 是确定 IP6 是否以及如何与化合物相关 影响 HIV-1 未成熟组装、成熟和成熟组装。与合作者合作，我们将 在组装和成熟测定中合成和筛选类似 IP6 的化合物。这一目标将有助于确定 靶向 IP6 结合位点的化合物是否是可行的抗逆转录病毒策略。