Application of New Tools for Probing the Roles of Sphingolipids and Cholesterol in Influenza Virus Infection

应用新工具探索鞘脂和胆固醇在流感病毒感染中的作用

• 批准号: 10678459
• 负责人: Melanie Brunet Torres
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678459
• 关键词: Affect; Antiviral Agents; Binding; Cell Line; Cell membrane; Cell physiology; Cells; Cellular Structures; Cessation of life; Cholesterol; Data; Development; Drug resistance; Endosomes; Fibroblasts; Goals; Health; Hemagglutinin; Herd Immunity; Human; Image; Imaging Techniques; Individual; Infection; Influenza A virus; Influenza Hemagglutinin; Label; Life Cycle Stages; Lipid Bilayers; Lipids; MDCK cell; Membrane; Membrane Lipids; Metabolic; Modeling; Morbidity - disease rate; Mutate; Normal Cell; Organelles; Peptides; Persons; Pharmaceutical Preparations; Plasma Cells; Play; Prevention; Production; Public Health; Publishing; Reporting; Research; Resolution; Role; Site; Spectrometry, Mass, Secondary Ion; Sphingolipids; Sphingomyelins; System; Techniques; Testing; Three-Dimensional Image; Transfection; Vaccination; Vaccines; Variant; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Visualization; combat; endosome membrane; env Gene Products; image reconstruction; influenza infection; influenzavirus; innovation; insight; instrument; isotope incorporation; mass spectrometric imaging; membrane assembly; mimetics; models and simulation; molecular dynamics; mortality; novel; pandemic disease; stable isotope; tool; trafficking; virus envelope

PROJECT SUMMARY Influenza A virus (IAV) is a major public health threat that causes 290,000 to 650,000 deaths per year worldwide, while pandemic strains have caused millions of deaths. Despite the existence of antiviral drugs and vaccines, IAV still causes high mortality and morbidity due to its ability to rapidly mutate and escape herd immunity. Identification of the host cell components that promote infection when present at high levels but sustain normal cell function at lower levels could lead to the development of new types of antiviral therapeutics with universal strain potency and decreased drug resistance. Sphingolipids and cholesterol are potential targets because IAV replication and infectivity is correlated with their abundances in the host cell. Why high levels of sphingolipids and cholesterol seem to promote IAV infection remains a mystery. Likewise, which steps in the virus lifecycle require these membrane components have not been identified. The research proposed herein will use new modeling and experimental strategies to investigate the roles of sphingolipids and cholesterol in the IAV lifecycle. These cutting-edge strategies include molecular dynamic (MD) simulations for modeling IAV fusion peptide (HAfp) insertion into membranes, and a high-resolution secondary ion mass spectrometry (SIMS) technique for imaging cholesterol and sphingolipids on and within cells. Three aims that use these techniques are proposed. In Aim 1 the molecular interactions between different lipid species, cholesterol, and the HAfp will be characterized by using MD simulations to determine how these cholesterol and sphingolipids affect HAfp insertion into the endosomal membrane. Completion of this aim will provide understanding of how the HAfp interacts with distinct membrane lipids, which is a necessary perquisite for elucidating the roles of membrane composition in IAV fusion during entry. In Aim 2, the sphingolipid and cholesterol abundances at the sites where the IAV assembles and buds from the host cell plasma membrane will be identified by using a combination of metabolic rare stable isotope incorporation, immunolabeling, and high-resolution SIMS imaging. This will be a decisive test of the hypothesis that the influenza A virus assembles and buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. In Aim 3, high-resolution SIMS depth profiling, an innovative 3D image reconstruction tool developed by sponsor’s lab, and statistical hypothesis tests will be used to visualize and compare the relative abundances of cholesterol in compartments that contain the influenza virus envelope protein, hemagglutinin (HA), within IAV-infected and uninfected cells. These studies will test the hypothesis that cholesterol levels are elevated in the compartments involved in trafficking newly synthesized IAV proteins to the plasma membrane for assembly into progeny virus particles. Successful completion of these aims may identify key host cell factors that might be exploited to combat IAV infection.

项目概要 甲型流感病毒 (IAV) 是一种主要的公共卫生威胁，每年在全球造成 290,000 至 650,000 人死亡， 尽管存在抗病毒药物和疫苗，但大流行毒株已导致数百万人死亡。 由于 IAV 具有快速变异和逃避群体免疫的能力，它仍然导致很高的死亡率和发病率。 鉴定在高水平存在但维持正常时促进感染的宿主细胞成分 较低水平的细胞功能可能会导致新型通用抗病毒疗法的开发 菌株效力和耐药性降低是 IAV 的潜在目标。 复制和感染性与其在宿主细胞中的丰度相关。 胆固醇似乎会促进 IAV 感染，同样，病毒生命周期中的哪个步骤仍然是个谜。 需要这些膜组件尚未被识别，本文提出的研究将使用新的。 研究鞘脂和胆固醇在 IAV 生命周期中的作用的建模和实验策略。 这些尖端策略包括用于 IAV 融合肽建模的分子动力学 (MD) 模拟 （HAfp）插入膜中，以及高分辨率二次离子质谱（SIMS）技术 提出了使用这些技术的三个目标。 在目标 1 中，不同脂质种类、胆固醇和 HAfp 之间的分子相互作用将是 其特点是使用 MD 模拟来确定这些胆固醇和鞘脂如何影响 HAfp 完成这一目标将有助于了解 HAfp 是如何插入内体膜的。 与不同的膜脂相互作用，这是阐明膜的作用的必要条件 在目标 2 中，IAV 融合的组成位点的鞘脂和胆固醇丰度。 来自宿主细胞质膜的 IAV 组装和芽将通过使用以下组合来识别 代谢稀有稳定同位素掺入、免疫标记和高分辨率 SIMS 成像。 确定了甲型流感病毒从质膜结构域组装和出芽的假设的检验 富含胆固醇和鞘脂 在 Aim 3 中，高分辨率 SIMS 深度分析是一项创新。 由赞助商实验室开发的3D图像重建工具，将使用统计假设检验来可视化 并比较含有流感病毒的隔室中胆固醇的相对丰度 这些研究将检验 IAV 感染和未感染细胞内的蛋白质、血凝素 (HA)。 在参与将新合成的 IAV 蛋白运输到体内的区室中，胆固醇水平升高。 用于组装成子代病毒颗粒的质膜可以成功完成这些目标。 可用于对抗 IAV 感染的关键宿主细胞因子。