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.