Dynamics of early ESCRT recruitment during HIV budding

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DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) hijacks endosomal sorting complexes required for transport (ESCRT) to facilitate release from the host plasma membrane. ESCRT assembly is initiated by recruitment of ALIX and TSG101, which bind directly to the viral Gag protein. The temporal and spatial details of ALIX and TSG101 recruitment are not yet well understood. We have recently observed that ALIX is recruited transiently at the end of Gag assembly during virion formation. We have also developed super resolution imaging approaches through which we have localized the residual ALIX within released virus like particles. Our super resolution images show ALIX asymmetrically located within released VLPs. Here we will establish the sequence of events that underlie ALIX recruitment to HIV virus like particles (VLP) and create tools and methodologies that can be used to define how other ESCRT components are recruited during HIV budding. This information is critical for understanding the mechanism of HIV budding and will also inform our understanding of membrane protein trafficking and cell division because ESCRTs facilitate MVB vesicle formation and the abscission stage of cytokinesis. The single virion and single cell experiments proposed here have been enabled through recent technological advances in single molecule imaging and optical high-resolution techniques. These experiments provide an important set of information complementary to what is readily available through population-based assays. Together, the information gathered from traditional population methods and from single virion and single cell methodologies have the potential to revolutionize our understanding of viral machines.