Understanding the pathway(s) that a virus follows during infectious penetration will greatly facilitate the development of useful entry inhibitors. We outline plans to use contemporary tools of live-cell and single- molecule imaging to provide quantitative descriptions of the invasion of mammalian cells by viral pathogens, including analysis of molecular mechanisms of membrane fusion. We will use an advanced live-cell, spinning-disk confocal fluorescence imaging configuration, with very sensitive optics and optimized illumination, for long time-course studies of viral entry and penetration, and we will develop software for 3D multi-tracking to enable analysis of all particles within the field of view in a given experiment. We will apply these technologies in close collaboration with Projects 1, 2 and 3 to analyse the pathways of entry of pseudotyped VSV particles bearing the glycoproteins from representatives of a broad range of enveloped RNA viruses and to determine how candidate entry inhibitors block progression along the normal entry route. We will use genome-edited cells expressing at endogenous markers for various compartments of endocytic pathways (clathrin, Rab proteins, etc.) to avoid the perturbations often associated with ectopic overexpression, and we will generate such cell lines for a number of additional proteins identified as potential targets in Project 3. Our long-term goals are to contribute a 3D time course in vivo to the characterization of candidate inhibitors and in so doing to achieve a substantially more detailed molecular description of endosomal pathways and of the proteins along those pathways that regulate or subvert viral entry.