Recent results by us and others have shown that the accessory protein Vpu determines plasma membrane versus endosomal accumulation of the HIV-1 core protein Gag and progeny virions in the HeLa model of HIV-1 infection, since Vpu suppresses endocytosis of cell surface-associated Gag. In this report, we used pulse-chase studies and subcellular fractionations to investigate endocytosis of newly synthesized Gag in HeLa HI cells. The uptake of Gag in Delta Vpu-virus background was not blocked by inhibitors of clathrin-mediated endocytosis and macropinocytosis. The cholesterol-sequestering drug filipin inhibited the uptake, but only if the drug was applied before extensive multimerization of Gag had taken place. Thus, the uptake mechanism most likely is only indirectly dependent on cholesterol. Our results also indicated that targeting phenotype of Gag was different in confluent versus subconfluent cell cultures, which could perhaps explain some of the controversies in intracellular targeting of Gag. (c) 2007 Elsevier Inc. All rights reserved.