Application of fluorescence microscopy and live-cell imaging to elucidate the dynamics of viral assembly and budding

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Advances in the development of fluorescent markers, fluorescence methods, and sensitive optical detection have contributed to making fluorescence live-cell imaging a powerful method for investigating cellular processes. In this proposal, we will apply our experience with ultra-sensitive fluorescence techniques and single-molecule studies to initially investigate the budding process of HIV-1 virions and later on, extend these techniques to other viruses of the SPP 1175. Our specific aims are: 1) to characterize the oligomerization of Gag proteins within the cytosol as a starting point, 2) to investigate the assembly of the Gag proteins at the plasma membrane by studying the interaction of Gag with Gag, lipids, envelope proteins, and with ESCRT complexes and 3) to observe and measure the kinetics of the budding process and the release of virus particles. To perform these investigations with high temporal and spatial resolution, we will utilize four different fluorescence techniques: wide-field imaging and two-dimensional tracking, optical sectioning, total internal reflection fluorescence microscopy and fluorescence correlation and cross correlation spectroscopy. These investigations promise to increase our understanding of the envelopment and budding of viruses as well as the cellular machinery that is needed for these processes.