CCR5 HIV-1 coreceptor activity. Role of cooperativity between residues in N-terminal extracellular and intracellular domains.

Human (H-) CCR5 is the primary coreceptor for ENV-mediated fusion by R5 strains of human immunodeficiency virus type 1, whereas mouse (M-) CCR5 lacks this function. An array of 23 H/M-CCR5 hybrids containing increasing amounts of H-CCR5 extending from the N terminus generated by random chimeragenesis had a biphasic pattern of coreceptor activity with JRFL and 89.6, revealing active regions in the N-terminal extracellular domain (N-ED) and at the junction of cytoplasmic loop 3. The M-CCR5 mutant in which divergent residues were replaced with the corresponding H-CCR5 N-ED sequence (NyYTsE) gained coreceptor function in fusion but not infection experiments. A M-CCR5 double mutant with substitution of human sequences for divergent residues from the N-ED and cytoplasmic loop 3 had augmented coreceptor activity in fusion assays and gain of function in infection experiments. The SIV-251 ENV utilized H- and M-CCR5 and variants. Flow cytometric analysis of M-CCR5 mutants and bifunctional receptors composed of CD4 domains fused to M-CCR5 mutants excluded the possibility that differences in coreceptor activity resulted from variations in cell surface expression. These results demonstrate that the coreceptor activity of the H-CCR5 N-ED is modulated by intracellular residues, illustrating the complexity of CCR5 requirements for interaction with ENV.