Super-resolution studies of the entry mechanisms of influenza viruses

描述(申请人提供)：病毒感染对人类健康构成重大威胁。作为劫持内源性细胞功能进行复制的寄生虫，病毒也是研究细胞过程的极佳探针。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的治疗方法，而且还可以更好地了解基础细胞生物学和宿主细胞与病原体的相互作用。这个应用的重点是甲型流感病毒。我们的长期目标是剖析流感感染途径，并了解感染途径上各个步骤背后的分子机制。在这个项目中，我们将重点放在病毒进入步骤上，这是药物抑制和抗流感治疗的一个有希望的目标。鉴于细胞内吞结构和病毒的小尺寸，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。这一要求很难用传统的成像方法来满足，特别是对于活细胞。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)，它为成像细胞结构提供了近分子尺度的分辨率和高分子特异性。在本项目中，我们将进一步提高STORM的分辨率，并首次应用这一新技术来研究流感病毒的内化机制。这些高分辨率荧光成像研究将得到电子显微镜(EM)、生化、细胞生物学和病毒学分析的补充。我们将努力实现以下四个具体目标。在目标1中，我们将确定内化流感病毒的内吞结构的时空组织，并将所获得的结果与其他内吞货物的结果进行比较，以阐明病毒特异性内化和笼蛋白介导的内吞作用的分子机制。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，流感病毒进入细胞可能除了唾液酸外还需要特定的蛋白质受体。在目标2中，我们将探索流感感染所需的细胞受体(S)的分子特性，并研究这种受体(S)如何引导病毒进入。在目标3和4中，我们将确定两种病理上重要的流感病毒类型的内化机制。在目标3中，我们将研究构成大流行威胁的H5N1禽流感病毒的内化机制，并将结果与人类病毒株的结果进行比较。在目标4中，我们将调查丝状流感病毒的进入机制，丝状流感病毒是从流感患者中分离出来的一种主要病毒形式。我们预计，这些研究的结果不仅将阐明流感病毒的进入机制，还将为基本的细胞过程，如内吞作用，以及宿主细胞与病原体的相互作用提供新的见解。

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.