title: Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane.
abstract: Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-beta-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.
authors.affiliation: Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal (IRCM), Univerisité de Montréal, Montreal, Quebec, Canada.
authors.forename: Andrés
authors.initials: A
authors.lastname: Finzi
authors.forename: Alexandre
authors.initials: A
authors.lastname: Orthwein
authors.forename: Johanne
authors.initials: J
authors.lastname: Mercier
authors.forename: Eric A
authors.initials: EA
authors.lastname: Cohen
mesh_terms: D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000821:virology*; D004705:Endocytosis; D015683:Gene Products, gag / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D019065:Virus Assembly
chemical_list: D015683:Gene Products, gag