Background:  CCR5 represents a major chemokine receptor for R5-HIV entry to target cells, serving as an attractive target for intervention of R5-HIV infection. However, it is concerned that long-term disruption of otherwise physiologic CCR5 interactions could compromise certain biologic defense systems. Here, we generated yellow fluorescent protein (YFP)-tagged CCR5-expressing U373-MAGI cells and examined whether CCR5 dynamics are altered by experimental CCR5 inhibitors, aplaviroc (AVC, anti-HIV IC₅₀ ~0.3 nM) and TAK-779 (anti-HIV IC₅₀ 2-5 nM).

Methods:  We generated a plasmid carrying CCR5 gene connected to YFP at its C-terminus. U373-MAGI cells were transfected with the plasmid, cloned using magnetic sorting and the limiting dilution technique. Real time CCR5 internalization was visualized with confocal laser microscopy by incubating the cells with chemokines for 1 h at 37^oC. We also examined the influence of CCR5 inhibitors on the mobility of CCR5 using fluorescence recovery after photobleaching (FRAP).

Results:  We obtained clones highly expressing membranous CCR5 (~93% fluorometry-positive). When we cultured such cells with 100 ng/mL RANTES, intracellular fluorescence intensity readily increased with membranous fluorescence vanished, signifying CCR5 internalization. Exposure of the cells to AVC or TAK-779 alone (up to 1 mM) did not elicit the internalization. When pretreated with 100 nM AVC for 1 hour, RANTES exposure fully elicited the internalization, whereas 100 nM TAK-779 blocked the internalization by ~40%. The result was that 1  mM AVC blocked the internalization by ~20%, while 1 mM TAK-779 completely did so. Both AVC and TAK-779 (at 100 nM) completely blocked MIP-1a-triggered CCR5 internalization. When the effects of AVC and TAK-779 on the mobility of membranous CCR5 were examined using the FRAP technique, no changes in CCR5 mobility were seen even at a high concentration of AVC (1 µM), suggesting that the binding of AVC or TAK-779 to CCR5 does not alter CCR5 mobility per se.

Conclusions:  The present data demonstrate that while the moderately potent TAK-779 blocks CCR5 internalization, the highly potent AVC only partially does so even at 1 µM, suggesting that development of highly HIV-specific CCR5 inhibitors, which do not significantly affect the chemokine-CCR5 interactions, is feasible. The data also show that the YFP-tagged CCR5-expressing U373-MAGI cells should be useful in examining the dynamics of CCR5 and its interactions with chemokines.