Tracking Retrovirus Particles at Different Stages of the Viral Life Cycle using 3-Dimensional Superresolution Microscopy

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In the lifecycle of disease-causing retroviruses such as human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1), the viral structural protein Gag is recruited to assembly sites at the inner leaflet of the plasma membrane. Upon the completion of virus particle assembly, infectious particles bud from the surface of the producer cell, and migrate to a new target cell, where they attach to surface receptors at the plasma membrane. However, biophysical aspects of these processes remain poorly characterized. Here, we employ a double helix point spread function (DHPSF) to achieve 3-dimensional sub-diffraction localization of viral particles with increased axial range. We began by characterizing and optimizing our optical system in preparation for measurements on virus particles in live cells. To this end, we extended the axial range of the DHPSF to cover the complete depth of cells. We next characterized and optimized the temporal resolution of our optical system using 3-dimensional superresolved particle tracking experiments on fluorescent spheres and purified virus particles in aqueous solution. Upon analysis of the data, we identified biases in the axial density profile of recovered localizations that are related to standard calibration procedures and required correction. Finally, we applied 3-dimensional superresolved particle tracking to study viral mobility in cells. Across the virus lifecycle, particle mobility spans a large dynamic range from the slow-moving membrane-associated assembly sites to the freely diffusing particles immediately post-budding. We present preliminary results on viral particle mobility during the events surrounding entry and particle budding, thereby providing insights into the dynamic aspects of the viral lifecycle. This work is supported by grants from the National Institutes of Health (RO1 GM64589, RO1 GM098550, RO1 GM124279).