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. 2005 Oct;79(20):12667-73.
doi: 10.1128/JVI.79.20.12667-12673.2005.

Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains

Shaomin Tian  1 , Won-Tak Choi, Dongxiang Liu, James Pesavento, Youli Wang, Jing An, Joseph G Sodroski, Ziwei Huang
Affiliations

Distinct functional sites for human immunodeficiency virus type 1 and stromal cell-derived factor 1alpha on CXCR4 transmembrane helical domains

Shaomin Tian et al. J Virol. 2005 Oct.

Abstract

The entry of human immunodeficiency virus type 1 (HIV-1) into the cell is initiated by the interaction of the viral surface envelope protein with two cell surface components of the target cell, CD4 and a chemokine coreceptor, usually CXCR4 or CCR5. The natural ligand of CXCR4 is stromal cell-derived factor 1alpha (SDF-1alpha). Whereas the overlap between HIV-1 and SDF-1alpha functional sites on the extracellular domains of CXCR4 has been well documented, it has yet to be determined whether there are sites in the transmembrane (TM) helices of CXCR4 important for HIV-1 and/or SDF-1alpha functions, and if such sites do exist, whether they are overlapping or distinctive for the separate functions of CXCR4. For this study, by employing alanine-scanning mutagenesis, (125)I-SDF-1alpha competition binding, Ca(2+) mobilization, and cell-cell fusion assays, we found that the mutation of many CXCR4 TM residues, including Tyr(45), His(79), Asp(97), Pro(163), Trp(252), Tyr(255), Asp(262), Glu(288), His(294), and Asn(298), could selectively decrease HIV-1-mediated cell fusion but not the binding activity of SDF-1alpha. Phe(87) and Phe(292), which were involved in SDF-1alpha binding, did not play a significant role in the coreceptor activity of CXCR4, further demonstrating the disconnection between physiological and pathological activities of CXCR4 TM domains. Our data also show that four mutations of the second extracellular loop, D182A, D187A, F189A, and P191A, could reduce HIV-1 entry without impairing either ligand binding or signaling. Taken together, our first detailed characterization of the different functional roles of CXCR4 TM domains may suggest a mechanistic basis for the discovery of new selective anti-HIV agents.

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Figures

FIG. 1.
FIG. 1.
Mutation sites on CXCR4. The sequence and predicted topology of the extracellular, TM, and cytoplasmic regions are shown, with shaded residues indicating the sites of mutations.
FIG. 2.
FIG. 2.
SDF-1α ligand binding curve of a representative CXCR4 mutant, D97E. The 50% inhibitory concentration (IC50) was determined for each mutant by using radioisotope competition binding assays with a single concentration of 125I-SDF-1α (0.2 nM) in the presence of various concentrations of unlabeled SDF-1α. The data shown here are representative of at least three independent experiments.
FIG. 3.
FIG. 3.
SDF-1α signaling activities of CXCR4 mutants. The intracellular Ca2+ influx in 293 cells was measured in response to 50 nM SDF-1α. (A) Changes in [Ca2+]i for all mutants, expressed as percentages of wild-type CXCR4 value. (B) Ca2+ signals of representative mutants. The data shown are representative of at least three independent experiments.
FIG. 4.
FIG. 4.
HIV-1 coreceptor activities of CXCR4 mutants. (A) Coreceptor activities of TM mutants, as determined by cell-cell fusion assays with the 89.6 dual-tropic isolate. (B) Coreceptor activities of ECL2 mutants, as determined by cell-cell fusion assays with the IIIB T-tropic and 89.6 dual-tropic isolates. The results are presented as average percentages of the luciferase activity of wild-type CXCR4. All data shown are means ± standard deviations from at least three independent experiments.
FIG. 5.
FIG. 5.
Distinct functional sites for HIV-1 gp120 and SDF-1α on a hypothetical structural model of CXCR4. The TM residues Phe87 and Phe292, required for SDF-1α binding only, are highlighted in a lighter color and represented as ball-and-stick figures with asterisks. Asp171, the only residue found to be involved in both HIV-1 gp120 and SDF-1α interactions, is highlighted in a lighter color with an asterisk. The TM residues that are involved in HIV-1 coreceptor activity only are highlighted in the darker color. Only the TM domains of CXCR4, with side (A) and top (B) views, are shown for simplicity. (C) Schematic illustration of the locations of residues found to be important for HIV-1 or SDF-1α on CXCR4 TM and ECL2 domains. The residues required for SDF-1α binding are shown as white spots, while those that are involved in HIV-1 coreceptor activity are shown as black spots. Asp171 is highlighted as a white spot.
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