Mutation of Asp(171) and Asp(262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investi gated the interaction of AMD3100 with CXCR4 at the molecular level by mutat ional analysis. We established a set of stably transfected U87.CD4 cell lin es expressing different mutant forms of CXCR4 (i.e., CXCR4 [WT], CXCR4[D171 N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the act ivity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4-as assessed by the inhibi tory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5-was greatly redu ced by substitution of Asp(171) and/or Asp(262) by neutral asparagine resid ue(s). Both aspartates, but most particularly Asp(262), also proved essenti al for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His(281) by a neutral alanine potentia ted the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D2 62N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domai ns 4 and 6 of the 7-transmembrane receptor, respectively, may represent cru cial sites for electrostatic interaction of the positive charges of the bic yclams, as well as for the highly basic V3 loop of the gp120 envelope prote in of certain HIV-1 strains.