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CC chemokine receptor 5 (CCR5), the major HIV coreceptor, is a G protein-coupled receptor (GPCR) involved in cell activation and migration in response to chemokines. Blockade of CCR5 is an effective anti-HIV strategy, and potent anti-HIV chemokine analogs such as PSC-RANTES have been developed. These inhibitors act by interfering with receptor trafficking, thereby inducing prolonged intracellular sequestration of CCR5. Like many GPCRs, CCR5 is desensitized following agonist activation. The initial steps in this process are well understood, but later stages, including where CCR5 is sequestered during desensitization, and how anti-HIV chemokine analogs intervene to achieve prolonged sequestration, have yet to be elucidated in detail. In this study we demonstrate that CCR5 cycles to and from the cell surface via the endosome recycling compartment and the trans-Golgi network during desensitization, accumulating in the trans-Golgi network following internalization by both PSC-RANTES and CCL5, the native ligand from which it was derived. In addition, we show that unlike CCR5 sequestered by CCL5, CCR5 sequestered by PSC-RANTES cannot be induced to return to the cell surface by addition of the small molecule CCR5 inhibitor, TAK-779, and that association of PSC-RANTES with CCR5 is more durable than that of native CCL5 during desensitization. Our findings reconcile the previously conflicting descriptions of the location of sequestered CCR5 during desensitization, as well as providing more general insights into potential trafficking routes for endocytosed GPCRs and further elucidation of the unusual inhibitory mechanism of chemokine analogs with potent anti-HIV activity.

The HIV coreceptor CCR5 is a validated target for both the prevention and therapy of HIV infection. PSC-RANTES, an N-terminally modified analogue of one of the natural chemokine ligands of CCR5 (RANTES/CCL5), is a potent inhibitor of HIV entry into target cells. Here, we set out to engineer the anti-HIV activity of PSC-RANTES into another natural CCR5 ligand (MIP-1beta/CCL4), by grafting into it the key N-terminal pharmacophore region from PSC-RANTES. We were able to identify MIP-1beta/CCL4 analogues that retain the receptor binding profile of MIP-1beta/CCL4, but acquire the very high anti-HIV potency and characteristic inhibitory mechanism of PSC-RANTES. Unexpectedly, we discovered that in addition to N-terminal structures from PSC-RANTES, the side chain of Lys33 is also necessary for full anti-HIV potency.