Vpu and Tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor Pr55gag

Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55(gag). Depending on the cell type, Pr55(gag) accumulates either at the plasma membrane or on late endosomes/ multivesicular bodies. Intracellular localization of Pr55(gag) determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55(gag) are poorly characterized. We have analyzed targeting of newly synthesized Pr55(gag) in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55(gag) was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55(gag) remained at the plasma membrane and virions assembled at this site. In contrast, Pr55(gag) expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55(gag) required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55(gag) and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55(gag).