Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses

Abstract

CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1NDK or by FIVPET (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-189.6 (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-189.6. In contrast, its inactivity toward HIV-1NDK seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIVPET. Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1NDK infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue.

FIG. 1

Infection of U373MG-CD4 cells expressing human or rat CXCR4 or chimeric receptors by the different HIV-1 strains. HIV-1_LAI and HIV-1_NDK are cell line-adapted X4 strains; HIV-1_OUA and HIV-1_ATE are primary X4 isolates; and HIV-1_89.6 is a molecularly cloned R5X4 strain. HHRH is human CXCR4 with the rat CXCR4 ECL2; RRHR is the reciprocal chimera. Bars represent infected cells expressed as a percentage of infection upon transfection with WT human CXCR4. The target cell line bears a Tat-inducible lacZ gene, allowing detection of HIV-infected cells by their high β-galactosidase activity (blue staining with X-Gal). Cells were infected in 12-well trays 24 h after transfection with CXCR4 expression vectors, and X-Gal staining was performed 48 h later. Approximately 1,000 infected cells per well were detected in the case of WT human CXCR4, except for infections with HIV-1_ATE (∼200 cells).

FIG. 2

Effect of ECL2 substitutions between human and rat CXCR4 on the efficiency of HIV-1 infection. (A) Alignment of the ECL2 domains. The numbering corresponds to the human CXCR4 sequence. (B) Schematic organization of the different chimeric constructs and their efficiency at mediating HIV-1_LAI, HIV-1_NDK, and HIV-1_89.6 infection relative to WT human CXCR4. Symbols ++++, 100% (∼10³ infected cells per well); +++, >50%; ++, 20 to 50%; +, 10 to 20%; and −, <5%. Infections were performed as described in the legend to Fig. 1. The results are the means of three independent experiments.

FIG. 3

Effect of reciprocal exchanges of ECL2 residues between human and rat CXCR4 on infection by HIV-1_LAI and HIV-1_NDK. Human CXCR4 mutants (A) or rat CXCR4 mutants (B) were expressed in U373MG-CD4 cells, and infections were performed as described in the legend to Fig. 1. Approximately 4,000 infected cells per well were detected for the WT human CXCR4 (100%). The values represent the means of three independent experiments. Mutants are designated by the position of the corresponding residue in the human CXCR4 sequence. For convenience, the same numbering scheme was used for the rat CXCR4 mutants. The WT human and rat CXCR4 and all derived mutants (excepted D182G) bear a MYC epitope tag at their amino terminus.

FIG. 4

Effect of Asp193 substitutions in human CXCR4 ECL2 on infection by different HIV-1 strains. The experiment was performed and results are represented as described in Fig. 1. The inocula yielded ∼1,000 infected cells per well (∼200 with HIV-1_ATE) for WT human CXCR4. The D193A and D193S mutants are MYC tagged.

FIG. 5

Effect of single amino acid substitutions in human CXCR4 ECL2 on infection by different HIV-1 strains. The experiment was performed and the results are represented as described in Fig. 1. The inocula yielded 600 to 1,000 infected cells per well for WT human CXCR4. All mutants (excepted D182G) and WT were MYC tagged.

FIG. 6

Formation of syncytia between FIV_PET-infected cells and cells expressing WT or mutant CXCR4. Feline CCC cells were transfected with WT or mutant CXCR4 expression vectors and cocultured (1:1 ratio) with FIV_PET-infected CrFKcells in 24-well trays. Cells were fixed after 24 h, and syncytia were scored in five independent fields. Results represent the mean number of syncytia and the standard error in three experiments. The rat CXCR4 mutants (a) and human CXCR4 mutants (b and c) correspond to reciprocal exchanges of ECL2 residues (a and b) or to substitutions of Ala for other human CXCR4 ECL2 residues (c). All mutants (excepted D182G) are MYC tagged.

FIG. 7

Cell surface expression of human and rat CXCR4 mutants. Flow cytometry analysis was performed on COS cells cotransfected with CXCR4 and GFP expression vectors (6:1) and stained with the 9E10 (anti-c-MYC) or the 12G5 (anti-CXCR4) MAb as indicated. The WT human (HU) and rat CXCR4 and the mutants tested for reactivity with 9E10 bear a c-MYC epitope tag at their amino terminus. The asterisk indicates that the 6H8 anti-CXCR4 MAb was used instead of 9E10. NT, not tested. After the staining with a secondary PE-conjugated antibody, cells were analyzed for green (GFP) and red (PE) fluorescence. The fractions of GFP⁺ cells, indicating efficient transfection, ranged from 20 to 40%. The bars represent the fractions of GFP⁺ cells that were stained by 9E10, 12G5, or 6H8 as indicated. The results are expressed relative to cells transfected with WT human CXCR4 (100%).