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

项目总结 甲型流感病毒(IAV)是一种主要的公共卫生威胁，每年导致全球29万至65万人死亡， 而大流行的毒株已经导致数百万人死亡。尽管存在抗病毒药物和疫苗， 由于IAV具有快速变异和逃避群体免疫的能力，因此仍会导致较高的死亡率和发病率。 在高水平存在但维持正常时促进感染的宿主细胞成分的鉴定 较低水平的细胞功能可能导致具有普遍意义的新型抗病毒疗法的开发 菌株效力和抗药性降低。鞘脂和胆固醇是潜在的靶标，因为IAV 复制和感染性与它们在宿主细胞中的丰度有关。为什么会有高水平的鞘脂 而胆固醇似乎促进了IAV感染，这仍然是一个谜。同样，病毒生命周期中的哪些步骤 要求这些膜组分还没有被鉴定。这里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。 这些前沿策略包括用于模拟IAV融合肽的分子动力学(MD)模拟 (HAfp)插入膜，以及高分辨率二次离子质谱仪(SIMS)技术 对细胞内外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，不同脂质物种、胆固醇和HAfp之间的分子相互作用将是 其特征是使用MD模拟来确定这些胆固醇和鞘脂如何影响HAfp 插入到内体膜中。完成这一目标将有助于理解HAfp如何 与不同的膜脂相互作用，这是阐明膜的作用的必要前提。 进入时IAV融合的成分。在目标2中，鞘脂和胆固醇在以下位置丰富 来自宿主细胞质膜的IAV组装和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SIMS成像。这将是一个 甲型流感病毒从质膜区组装和发芽假说的决定性检验 富含胆固醇和神经鞘脂脂。在Aim 3中，高分辨率SIMS深度剖析，一个创新的 由赞助商实验室开发的3D图像重建工具和统计假设检验将用于可视化 并比较包含流感病毒包膜的隔间中胆固醇的相对丰度 血凝素(HA)，在IAV感染和未感染的细胞中。这些研究将检验这一假设 胆固醇水平在运输新合成的IAV蛋白到 用于组装成子代病毒颗粒的质膜。成功完成这些目标可能会确定 可能被利用来对抗IAV感染的关键宿主细胞因子。

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.