Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55^gag associates with membrane domains that are largely resistant to Brij98 but sensitive to triton X-100

The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55^gag. We have analyzed whether Pr55^gag has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55^gag has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55^gag particles (VLPs) yield buoyant Pr55^gag complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55^gag complexes cannot be taken as a proof for raft association of Pr55^gag, since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55^gag. However, Pr55^gag might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55^gag. Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55^gag complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55^gag localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55^gag VLPs.