Open AccessProductive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane
Author(s) -
Andrés Finzi,
Alexandre Orthwein,
Johanne Mercier,
Éric A. Cohen
Publication year - 2007
Publication title -
journal of virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.617
H-Index - 292
eISSN - 1070-6321
pISSN - 0022-538X
DOI - 10.1128/jvi.00308-07
Subject(s) - biology , endosome , budding , microbiology and biotechnology , hek 293 cells , cell fractionation , intracellular , cell membrane , cell , membrane , cell culture , biochemistry , genetics
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-β-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.