segregation of cd4 and cxcr4 into distinct lipid microdomains in t lymphocytes suggests a mechanism for membrane destabilization by human immunodeficiency virus recent evidence has suggested that plasma membrane sphingolipids and cholesterol spontaneously coalesce into raftlike microdomains and that specific proteins including cd4 and some other tcell signaling molecules sequester into these rafts in agreement with these results we found that cd4 and the associated lck tyrosine kinase of peripheral blood mononuclear cells and h9 leukemic t cells were selectively and highly enriched in a lowdensity lipid fraction that was resistant at 0 degrees c to the neutral detergent triton x100 but was disrupted by extraction of cholesterol with filipin or methylbetacyclodextrin in contrast the cxcr4 chemokine receptor a coreceptor for x4 strains of human immunodeficiency virus type 1 hiv1 was almost completely excluded from the detergentresistant raft fraction accordingly as determined by immunofluorescence with confocal microscopy cd4 and cxcr4 did not coaggregate into antibodyinduced cell surface patches or into patches of cxcr4 that formed naturally at the ruffled edges of adherent cells the cxcr4 fluorescent patches were extracted with cold 1 triton x100 whereas the cd4 patches were resistant in stringent support of these data cd4 colocalized with patches of cholera toxin bound to the raftassociated sphingoglycolipid gm1 whereas cxcr4 did not addition of the cxcr4activating chemokine sdf1 alpha did not induce cxcr4 movement into rafts moreover binding of purified monomeric gp120 envelope glycoproteins from strains of hiv1 that use this coreceptor did not stimulate detectable redistributions of cd4 or cxcr4 between their separate membrane domains however adsorption of multivalent gp120containing hiv1 virion particles appeared to destabilize the local cd4containing rafts indeed adsorbed hiv1 virions were detected by immunofluorescence microscopy and were almost all situated in nonraft regions of the cell surface we conclude that hiv1 initially binds to cd4 in a raft domain and that its secondary associations with cxcr4 require shifts of proteins and associated lipids away from their preferred lipid microenvironments our evidence suggests that these changes in proteinlipid interactions destabilize the plasma membrane microenvironment underlying the virus by at least several kilocalories per mole and we propose that this makes an important contribution to fusion of the viral and cellular membranes during infection thus binding of hiv1 may be favored by the presence of cd4 in rafts but the rafts may then disperse prior to the membrane fusion reaction