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: 10214490
• 负责人: Robert A Dick
• 金额: $ 39.25万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214490
• 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/antagonist; 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.

项目摘要 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 未成熟的病毒粒子经历了一个称为成熟的过程，该过程是产生传染病所必需的 颗粒。我们最近表明，需要小的细胞分子，肌醇六磷酸（IP6） 组装和成熟都降低了细胞中的IP6水平，严重降低了形成 传染病。虽然我们知道对IP6如何影响艾滋病毒有一个“大图”观点，但我们仍然有很多事情 不明白。该建议旨在表征IP6诱发的未成熟组装机制，如何 IP6促进成熟的组件，如果这些组装步骤可以由抗逆转录病毒靶向。此外， 初步工作表明IP6是其他逆转录病毒的辅助因子，因此该建议将测试 假设IP6作为逆转录病毒辅助因子是进化配置的。具体目标1是进一步 表征IP6结合和促进HIV-1组装和成熟的方式。高分辨率冷冻EM分析 将在IP6组装的成熟病毒样颗粒上进行，有或没有细胞蛋白已知 与细胞中的病毒核结合。成熟抑制剂bevirimat和pf-46396的影响，与 与IP6相同的区域，在IP6结合和增强组装上，将使用一系列生化确定 测定。此外，此目的将检验以下假设：其他慢病毒利用IP6。例如， 初步数据表明，马传染性贫血病毒还采用IP6来生产 传染病。具体目的2是确定逆转录病毒是否以及如何利用IP6。 慢病毒属。初步数据表明，α逆转录病毒ROUS肉瘤病毒（RSV）和三角洲 逆转录病毒人类T细胞白血病病毒（HTLV）都利用IP6。我们将在这两个方面确定IP6动作的网站 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 属确定它们是否也使用IP6。特定目标3是确定IP6是否以及如何相关化合物 影响HIV-1不成熟的组装，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屏幕类似IP6的化合物。这个目标将有助于确定 如果针对IP6结合位点的化合物是可行的抗逆转录病毒策略。