Cooperation of multiple CCR5 coreceptors is required for infections by human immunodeficiency virus type 1

In addition to the primary cell surface receptor CD4, CCR5 or another corec eptor is necessary for infections by human immunodeficiency virus type 1 (H IV-1), yet the mechanisms of coreceptor function and their stoichiometries in the infection pathway remain substantially unknown. To address these iss ues, we studied the effects of CCR5 concentrations on HIV-1 infections usin g wild-type CCRS and two attenuated mutant CCR5s, one with the mutation Y14 N at a critical tyrosine sulfation site in the amino terminus and one with the mutation G163R in extracellular loop 2. The Y14N mutation converted a Y YT sequence at positions 14 to 16 to an NYT consensus site for N-linked gly cosylation, and the mutant protein was shown to be glycosylated at that pos ition. The relationships between HIV-1 infectivity values and CCRS concentr ations took the form of sigmoidal (S-shaped) curves, which were dramaticall y altered in different ways by these mutations. Both mutations shifted the curves by factors of approximately 30- to 150-fold along the CCRS concentra tion axis, consistent with evidence that they reduce affinities of virus Fo r the coreceptor. In addition, the Y14N mutation specifically reduced the m aximum efficiencies of infection that could be obtained at saturating CCR5 concentrations, The sigmoidal curves For all R5 HIV-1 isolates were quantit atively consistent with a simple mathematical model, implying that CCR5s re versibly associate with cell surface HIV-1 in a concentration-dependent man ner, that approximately four to six CCR5s assemble around the virus to form a complex needed for infection, and that both mutations inhibit assembly o f this complex but only the Y14N mutation also significantly reduces its ab ility to successfully mediate HIV-1 infections. Although several alternativ e models would be compatible with our data, a common feature of these alter natives is the cooperation of multiple CCR5s in the HIV-1 infection pathway . This cooperativity will need to be considered in future studies to addres s in detail the mechanism of CCR5-mediated HIV-1 membrane fusion.