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 polyprotein Gag, which forms small cytosolic oligomers that traffic to the plasma 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 crucial for virion 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. ImportanceThe participation of Gag and GagPol in HIV-1 assembly is central to the generation of infectious virions. While the structural role of Gag and the enzymatic functions of GagPol are well described, little is known regarding the mechanisms underlying their cytosolic interactions, spatial organization, and relative incorporation into assembling particles. Here, we successfully developed two stable fluorescently labeled variants of GagPol; a single labeled variant with a label in the Pol domain and double-labeled construct in which the Gag domain is also tagged. Using these constructs, we can quantitatively investigate Gag and GagPol interactions on the single-cell level. Hence, they provide a framework for a comprehensive study of the oligomerization properties and trafficking of these polyproteins at different stages of HIV-1 assembly, including membrane recruitment and incorporation into nascent virions.