Identification of an Intracellular Trafficking and Assembly Pathway for HIV‐1 Gag

Retroviral Gag proteins are membrane‐bound polyproteins that are necessary and sufficient for virus‐like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV‐1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)‐like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amounts of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV‐1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag association with MVB‐like compartments regulates the site of HIV‐1 budding and particle formation.