Mutagenesis of CXCR4 identifies important domains for human immunodeficiency virus type 1 X4 isolate envelope-mediated membrane fusion and virus entry and reveals cryptic coreceptor activity for R5 isolates

Abstract

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.

FIG. 1

Bubble diagram of CXCR4. Predicted extracellular, transmembrane, and cytoplasmic regions are indicated. Residues that have been altered are numbered. Acidic residues are lightly shaded; basic residues are darkly shaded. Extracellular cysteines are highlighted with bold circles. Dashed lines indicate positions where N-terminal deletions were made.

FIG. 2

Coreceptor function of mutant CXCR4s in cell-cell fusion assays with T-tropic (X4) and dual-tropic (R5X4) HIV-1 Envs. U373 target cells were transfected with a plasmid encoding the wild-type or a mutant CXCR4 gene linked to a vaccinia virus promoter and infected with vCB21R-LacZ and vCB-3 (CD4). HeLa Env-expressing effector cells were infected with a vaccinia virus encoding T7 polymerase (vTF1-1) and one encoding LAV (vCB-41) (A), IIIB/BH10 (vCB-40) (B), SF2 (vCB-34) (C), or 89.6 (vDC-1) (D). Duplicate cell mixtures were incubated at 37°C for 2.5 h. Fusion was assessed by measurement of β-Gal in detergent lysates of cells. The activities of the mutant CXCR4s are presented as percentages of wild-type CXCR4 activity after adjustment for the level of cell surface expression as detailed in Materials and Methods. Each mutant CXCR4 construct was tested in duplicate three times. The averages of these results are shown in the figure. The error bars in the figure represent the ranges of those three calculated percentages.

FIG. 3

Coreceptor function of mutant CXCR4 in virus infection assays with X4 HIV-1 Envs. U373-CD4⁺ cells were transfected with a plasmid (pCDNA3) encoding the indicated wild-type coreceptor (CXCR4 or CCR5) or mutant CXCR4. Wells of cells (in triplicate) were infected with the indicated HIV-1 Env luciferase reporter virus. Infection was assessed on day 4 by measuring the amounts of luciferase activity in cell lysates. The luciferase activities shown were obtained from separate samples in the same experiment. Error bars indicate the standard deviations of the mean values for triplicate wells. This experiment was repeated three times, and the data from a representative experiment are shown in the figure.

FIG. 4

Coreceptor function of mutant CXCR4s that support R5 Env fusion. BS-C-1 target cells were transfected with a plasmid encoding wild-type CXCR4 or the indicated mutant CXCR4 construct linked to a vaccinia virus promoter and infected with vCB21R-LacZ and vCB-3 (CD4). HeLa effector cells were infected with vTF-1 and either LAV (vCB-41), IIIB/BH10 (vCB-40), 89.6 (vDC-1), Ba-L (vCB-43), JR-FL (vCB-28), or SF162 (vCB-32). Duplicate cell mixtures were incubated at 37°C for 2.5 h. Fusion was assessed by measurement of β-Gal in detergent lysates of cells. The rates of β-Gal activity shown were obtained from separate samples in the same experiment. Error bars indicate the standard deviations of the mean values obtained for duplicate fusion assays. This experiment was performed three times, and the data from a representative experiment are shown in the figure. OD, optical density.

FIG. 5

Coreceptor function of mutant CXCR4s that support R5 Env fusion in a syncytium assay with the R5 isolate JR-FL Env. U373 target cells were transfected with a plasmid encoding wild-type CXCR4 or the indicated mutant CXCR4 construct linked to a vaccinia virus promoter and infected with vCB21R-LacZ and vCB-3 (CD4). (A) Wild-type CXCR4; (B) CXCR4 D187A; (C) CXCR4 N11A D187A; (D) wild-type CCR5. HeLa effector cells were infected with vaccinia virus encoding the JR-FL Env (vCB28). Duplicate cell mixtures were incubated at 37°C for 6 h. Fusion was assessed by light microscopy after staining cells with crystal violet. Magnification, ×200.

FIG. 6

Coreceptor function of mutant CXCR4s in virus infection assays with R5 HIV-1 Envs. U373-CD4⁺ cells were transfected with a plasmid (pCDNA3) encoding the indicated wild-type coreceptor (CXCR4 or CCR5) or mutant CXCR4. Wells of cells (in triplicate) were infected with the indicated HIV-1 Env luciferase reporter virus. Infection was assessed on day 4 by measuring the amounts of luciferase activity in cell lysates. The luciferase activities shown were obtained from separate samples in the same experiment. Error bars indicate the standard deviations of the mean values for triplicate wells. This experiment was performed three times, and the data from one experiment are shown in the figure.

FIG. 7

Coreceptor function of mutant CXCR4s in cell-cell fusion assays with low-level expression. U373-CD4⁺ cells were transfected with a plasmid (pCDNA3) encoding the indicated wild-type coreceptor (CXCR4 or CCR5) or mutant CXCR4, and after overnight expression the cells were infected with vCB21R-LacZ. HIV-1 effector cells were infected with vTF1.1 (T7 polymerase) and vaccinia virus encoding either the JR-FL Env (vCB28) or the IIIB/BH10 Env (vCB40). Fusion was assessed by measurement of β-Gal in detergent lysates of cells. Error bars represent the standard deviations for duplicate fusion assays. OD, optical density.

FIG. 8

Coreceptor function of cysteine mutant CXCR4s in cell-cell fusion assays with R5, X4, and R5X4 HIV-1 Envs. U373 target cells were transfected with a plasmid encoding the wild-type coreceptor (CXCR4 or CCR5) or mutant CXCR4 construct linked to a vaccinia virus promoter and infected with vCB21R-LacZ and vCB-3 (CD4). HeLa effector cells were infected with vTF1-1 (T7 polymerase) and either vCB-32 (SF162), vCB-43 (Ba-L), vCB-28 (JR-FL), vDC-1 (89.6), vCB-34 (SF2), vCB-41 (LAV), or vCB-40 (IIIB/BH10). Fusion was assessed by measurement of β-Gal in detergent lysates of cells. The rates of β-Gal activity shown were obtained from separate samples in the same experiment. Error bars indicate the standard deviations of the mean values obtained for duplicate fusion assays. This experiment was performed three times, and the data from a representative experiment are shown in the figure. OD, optical density.