Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains

Abstract

The chemokine receptor CXCR4 functions as a fusion coreceptor for T cell tropic and dual-tropic HIV-1 strains. To identify regions of CXCR4 that are important for coreceptor function, CXCR4-CXCR2 receptor chimeras were tested for the ability to support HIV-1 envelope (env) protein-mediated membrane fusion. Receptor chimeras containing the first and second extracellular loops of CXCR4 supported fusion by T tropic and dual-tropic HIV-1 and HIV-2 strains and binding of a monoclonal antibody to CXCR4, 12G5, that blocks CXCR4-dependent infection by some virus strains. The second extracellular loop of CXCR4 was sufficient to confer coreceptor function to CXCR2 for most virus strains tested but did not support binding of 12G5. Truncation of the CXCR4 cytoplasmic tail or mutation of a conserved DRY motif in the second intracellular loop did not affect coreceptor function, indicating that phosphorylation of the cytoplasmic tail and the DRY motif are not required for coreceptor function. The results implicate the involvement of multiple CXCR4 domains in HIV-1 coreceptor function, especially the second extracellular loop, though the structural requirements for coreceptor function were somewhat variable for different env proteins. Finally, a hybrid receptor in which the amino terminus of CXCR4 was replaced by that of CCR5 was active as a coreceptor for M tropic, T tropic, and dual-tropic env proteins. We propose that dual tropism may evolve in CCR5-restricted HIV-1 strains through acquisition of the ability to utilize the first and second extracellular loops of CXCR4 while retaining the ability to interact with the CCR5 amino-terminal domain.

Figure 1

CXCR4–CXCR2 chimeras. Chimeric receptors based on CXCR4 and CXCR2 are depicted schematically. Amino-terminal domain exchanges were performed at the conserved cysteine residue at position 28 of CXCR4.

Figure 4

CXCR4–CCR5 chimeras. A receptor chimera containing the amino-terminal domain of CCR5 (up to the cysteine residue) in a CXCR4 background (5444) and its reciprocal (4555) were tested for coreceptor function in the cell–cell fusion assay described in Fig. 2. Results are expressed in relative light units.

Figure 2

Effects of single CXCR2 domains in CXCR4 on coreceptor function. Receptor chimeras composed of single CXCR2 domains in a CXCR4 background were tested for function. HeLa cells expressing the BH8, 89.6, BK132, RF, or HIV-2_SBL6669 env proteins in conjunction with T7 RNA polymerase as a consequence of infection with the appropriate recombinant vaccinia virus vectors were mixed with quail QT6 cells transfected with plasmids expressing CD4, the indicated chemokine receptor, and luciferase under control of the T7 promoter. After 8 h at 37°C, the cells were lysed, and the amount of luciferase activity was measured in relative light units and expressed relative to wild-type CXCR4. The signal/noise values for wild-type CXCR4 were typically greater than 50:1.

Figure 3

Effects of multiple domain substitutions on coreceptor function. The ability of receptor chimeras containing multiple domain substitutions between CXCR4 and CXCR2 to support env-mediated cell–cell fusion by the BH8, 89.6, BK132, RF, or HIV-2_SBL6669 env proteins was determined with the assay described in Fig. 2.