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

项目摘要 甲型流感病毒（IAV）是一种主要的公共卫生威胁，每年在全世界造成290，000至650，000人死亡， 而大流行病毒株已导致数百万人死亡。尽管存在抗病毒药物和疫苗， 由于IAV能够迅速变异并逃避群体免疫，因此它仍然导致高死亡率和发病率。 鉴定当以高水平存在时促进感染但维持正常水平的宿主细胞组分 细胞功能处于较低水平可能导致开发新型抗病毒疗法， 菌株效力和降低的耐药性。鞘脂和胆固醇是潜在的目标，因为IAV 复制和感染性与它们在宿主细胞中的丰度相关。为什么高水平的鞘脂 和胆固醇似乎促进IAV感染仍然是一个谜。同样，病毒生命周期中的哪些步骤 要求这些膜组件尚未确定。本文提出的研究将使用新的 建模和实验策略，以研究鞘脂和胆固醇在IAV生命周期中的作用。 这些尖端策略包括分子动力学（MD）模拟IAV融合肽 （HAfp）插入到膜中，以及高分辨率二次离子质谱（西姆斯）技术， 对细胞上和细胞内的胆固醇和鞘脂进行成像。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被描述。 其特征在于使用MD模拟来确定这些胆固醇和鞘脂如何影响HAfp 插入内体膜。完成这一目标将提供理解如何HAFP 与不同的膜脂相互作用，这是阐明膜作用的必要前提 在进入IAV融合过程中的成分。在目标2中，鞘脂和胆固醇在以下部位的丰度： IAV从宿主细胞质膜上组装和出芽将通过使用以下组合来鉴定： 代谢稀有稳定同位素掺入、免疫标记和高分辨率西姆斯成像。这将是一 对甲型流感病毒从质膜结构域组装和出芽的假设的决定性检验 富含胆固醇和鞘脂。在目标3中，高分辨率西姆斯深度剖面， 申办者实验室开发的3D图像重建工具和统计假设检验将用于可视化 并比较含有流感病毒包膜的隔室中胆固醇的相对丰度 蛋白质，血凝素（HA），在IAV感染和未感染的细胞。这些研究将检验以下假设： 胆固醇水平在参与运输新合成的IAV蛋白质的隔室中升高， 用于组装成子代病毒颗粒的质膜。成功完成这些目标可以确定 关键的宿主细胞因子，可能被利用来对抗IAV感染。

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.