Super-resolution studies of the entry mechanisms of influenza viruses

流感病毒侵入机制的超分辨率研究

• 批准号: 8392262
• 负责人: XIAOWEI ZHUANG
• 金额: $ 32.56万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-22 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8392262
• 关键词: Actins; Adaptor Signaling Protein; Area; Behavior; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Biomedical Research; Cell Communication; Cell physiology; Cell surface; Cells; Cellular Structures; Cellular biology; Clathrin; Clathrin-Coated Vesicles; Complement; Drug Targeting; Electron Microscopy; Endocytosis; Endosomes; Event; Glycolipids; Glycoproteins; Goals; HIV; Health; Human; Human Influenza A Virus; Human Virus; Image; Imaging Techniques; Imaging technology; Individual; Infection; Influenza; Influenza A Virus, H5N1 Subtype; Influenza A virus; Integration Host Factors; Investigation; Laboratories; Lead; Length; Life; Lipids; Mediating; Microscopy; Molecular; Optics; Parasites; Pathway interactions; Patients; Population; Proteins; RNA Interference; Research; Resolution; Shapes; Sialic Acids; Specificity; Structure; TFAP2A gene; Techniques; Testing; Time; Vesicle; Vesicular stomatitis Indiana virus; Viral; Viral Envelope Proteins; Viral Genome; Virion; Virus; Virus Diseases; Virus Replication; anti-influenza; base; coated pit; epsin 1; flu; fluorescence imaging; genome-wide; imaging modality; improved; influenza epidemic; influenzavirus; insight; meetings; molecular scale; nanoscale; novel; pandemic disease; pandemic influenza; pathogen; polymerization; preference; protein complex; public health relevance; receptor; reconstruction; research study; trafficking; ultra high resolution; virology

DESCRIPTION (provided by applicant): Viral infections pose major threats to human health. As parasites that hijack endogenous cellular functions for replication, viruses are also excellent probes for studying cellular processes. Therefore investigations of viral infection mechanisms may not only lead to new therapies against viral diseases but also to a better understanding of fundamental cell biology and host cell-pathogen interactions. This application focuses on the influenza A virus. Our long-term goal is to dissect the influenza infection pathway and to understand the molecular mechanisms underlying individual steps along the infection pathway. In this project, we will focus on the viral entry step, which is a promising target for drug inhibition and for anti-flu therapies. Given the small sizes of the cellular endocytic structures and the virus, imaging with both ultra-high spatial resolution and molecular specificity is required to visualize the molecular details of viral entry. This requirement is difficult to meet with conventional imaging methods especially for living cells. We have recently developed a super-resolution fluorescence imaging technology, stochastic optical reconstruction microscopy (STORM), which provides near-molecular-scale resolution with high molecular specificity for imaging cellular structures. In this project, we will further improve the resolution of STORM and apply this novel technique to investigate the internalization mechanisms of influenza viruses for the first time. These high-resolution fluorescence imaging studies will be complemented by electron microscopy (EM), biochemical, cell biology, and virology assays. We will tackle the following four specific aims. In Aim 1, we will determine the spatio-temporal organization of the endocytic structures internalizing influenza virus, and compare the results with those obtained for other endocytic cargos, to elucidate the molecular mechanisms underlying virus internalization specifically and clathrin-mediated endocytosis in general. While influenza virus binds to cells through cell-surface sialic acids, various experiments suggest that influenza virus entry may require specific protein receptors in addition to sialic acids. In Aim 2, we will explore the molecular identity of the cellular receptor(s) required for influenza infection and investigate how such receptor(s) may guide viral entry. In Aims 3 and 4, we will determine the internalization mechanisms of two pathologically important influenza virus types. In Aim 3, we will investigate the internalization mechanism of the avian H5N1 influenza virus, which poses a pandemic threat, and compare the results to those on the human virus strains. In Aim 4, we will investigate the entry mechanism of filamentous influenza virus, which represents a prominent form of virus isolated from flu patients. We anticipate that results obtained from these studies will not only elucidate the entry mechanisms of influenza virus, but will also provide new insights into fundamental cellular processes, such as endocytosis, as well as host cell-pathogen interactions.

描述（由申请人提供）：病毒感染对人类健康构成了重大威胁。作为劫持内源性细胞功能复制的寄生虫，病毒也是研究细胞过程的极好探针。因此，对病毒感染机制的研究不仅可能导致针对病毒疾病的新疗法，而且还可以更好地了解基本细胞生物学和宿主细胞病原体相互作用。该应用集中在流感病毒上。我们的长期目标是剖析流感感染途径，并了解沿着感染途径的单个步骤的分子机制。在这个项目中，我们将重点关注病毒进入步骤，这是药物抑制和抗FLU疗法的有希望的靶标。鉴于细胞内吞结构和病毒的尺寸很小，需要具有超高空间分辨率和分子特异性的成像来可视化病毒进入的分子细节。这种要求很难满足常规成像方法，尤其是对于活细胞而言。我们最近开发了一种超分辨率荧光成像技术，随机光学重建显微镜（Storm），该技术提供了近分子尺度分辨率，具有高分子特异性，用于成像细胞结构。在这个项目中，我们将进一步改善风暴的分辨率，并将这种新颖的技术首次研究流感病毒的内在化机制。这些高分辨率的荧光成像研究将由电子显微镜（EM），生化，细胞生物学和病毒学测定法补充。我们将解决以下四个特定目标。在AIM 1中，我们将确定内吞结构内部化流感病毒的时空组织，并将结果与​​其他内吞cargos获得的结果进行比较，以阐明一般来说，病毒内在化的分子机制，总体而言。虽然流感病毒通过细胞表面唾液酸与细胞结合，但各种实验表明，除唾液酸外，流感病毒进入还需要特定的蛋白质受体。在AIM 2中，我们将探索流感感染所需的细胞受体的分子认同，并研究该受体如何指导病毒进入。在目标3和4中，我们将确定两种具有病理上重要的流感病毒类型的内在化机制。在AIM 3中，我们将研究禽类H5N1流感病毒的内在化机制，该病毒构成了大流行威胁，并将结果与​​人类病毒菌株的结果进行比较。在AIM 4中，我们将研究丝状流感病毒的进入机制，该病毒是从流感患者中分离出的一种突出形式的病毒。我们预计从这些研究获得的结果不仅会阐明流感病毒的进入机制，而且还将为基本细胞过程（例如内吞作用以及宿主细胞病原体的相互作用）提供新的见解。