Developing a fluorescent derivative of GagPol as a tool for live-cell imaging of HIV-1 assembly

The HIV-1 assembly process is driven by the structural protein Gag, which forms small cytosolic oligomers that are later trafficked to the cell membrane. While Gag alone is sufficient to drive particle formation, the incorporation of GagPol, a polyprotein comprising Gag and viral enzymes, is essential for productive infection. Maintenance of the proper Gag:GagPol ratio is essential for infectivity. Yet, the mechanisms regulating their cytosolic interactions remain incompletely understood, in part because most studies rely on ensemble biochemical assays that hide cell-to-cell heterogeneity and lack spatial and temporal resolution. To overcome these limitations and investigate the dynamics of GagPol at the molecule level, we systematically tested different approaches to fluorescently label it. We successfully produced two functional fluorescent GagPol variants: one single-labeled GagPol, and a second double-labeled variant with a fluorescent protein added within Gag, that allows concurrent visualization of Gag and GagPol. These labeled versions, in combination with the use of raster image (cross-) correlation spectroscopy, enabled the quantification of Gag and GagPol relative concentrations and intermolecular interactions at the single-cell level. Overall, these variants set the stage for in-depth investigations of GagPol during HIV assembly providing insights into cytoplasmic trafficking, particle assembly, and the kinetics of these processes.