Steps within the Assembly of HIV Gag Virus Like Particles

Abstract

HIV gag assembles at the plasma membrane and results of budding of HIV particles. We have monitored assembly of fluorescently labeled Gag molecules at the bottom surface of cells using TIRF microscopy with variable penetration depths. In this work we present quantitative analysis of intensity of vesicle like particles (VLPs) which form with fluorescently-tagged Gag on the cell membrane. To verify that these vesicles actually represent fully coated VLPs that separate from the host cell we have monitored the recruitment of VPS4-mCherry which is a critical member of the Endosomal Sorting Complexes Required for Transport (ESCRT) and its recruitment indicates fission from the host cell. Since the polymerization of Gag is a constant process, we expect the fluorescence intensities to increase continuously over time until it reaches a maximum, which signifies that the VLP is complete. However, we have found that the intensities of these VLPs increase in discrete steps. In order to further investigate the frequency and distribution of these pauses we have developed a mathematical model of Gag polymerization during virus assembly. This model is based on the spherical geometry of a VLP and how the total number of Gag proteins changes as the sphere forms. We have used least squares regression to determine the best fit of the model to the data, which we have then used to consider how these pauses have affected the total assembly time of the VLPs.