Patterns of CCR5, CXCR4, and CCR3 usage by envelope glycoproteins from human immunodeficiency virus type 1 primary isolates

Abstract

Coreceptor usage by Envs from diverse primary human immunodeficiency virus type 1 isolates was analyzed by a vaccinia virus-based expression and assay system. Usage of recombinant CCR5 and CXCR4 correlated closely with fusogenicity toward macrophages and T-cell lines expressing endogenous coreceptors. Surprisingly, recombinant CCR3 was utilized by most primary and T-cell-line-adapted Envs. Endogenous CXCR4 in macrophages was functional as a coreceptor.

FIG. 1

Surface expression of coreceptors. NIH 3T3 cells were transfected with pSC59-based plasmids containing a synthetic vaccinia virus promoter linked to the indicated coreceptor genes and coinfected with vCB-3 (CD4) and vCB21R-LacZ; control cells were transfected with the empty pSC59 plasmid and infected identically. Following overnight incubation to allow expression of vaccinia virus-encoded proteins, the cells were stained with the corresponding antibodies as follows: for CCR5, rabbit polyclonal antisera against a synthetic peptide representing the CCR5 extracellular N terminus (1:50 dilution) (4); for CXCR4, the 12G5 monoclonal antibody (23 μg/ml) (22), donated by J. Hoxie, University of Pennsylvania; and for CCR3, the 7B11 monoclonal antibody (20 μg/ml) (29), donated by C. Mackay, Leukosite. Detection was achieved with the following secondary antibodies (10 μg/ml; Boehringer Mannheim): for CCR5, fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G; and for CXCR4 and CCR3, fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G. The cells were washed, treated with 1 μg of ethidium bromide homodimer per ml, fixed with 0.1 ml of 4% paraformaldehyde, and analyzed with a FACSCAN flow cytometer (Becton Dickinson, Menlo Park, Calif.). Analyses of forward and side scatter as well as ethidium bromide homodimer fluorescence indicated nearly homogeneous populations of viable cells. Shaded profiles indicate coreceptor-expressing cells; unshaded profiles indicate control cells transfected with the empty pSC59 plasmid. The mean fluorescence intensities with coreceptor-expressing versus control cells were as follows: CCR5, 92 versus 21; CXCR4, 167 versus 4; and CCR3, 57 versus 9. In separate experiments, similar distinctions were seen when coreceptor-expressing cells were stained with either an anticoreceptor monoclonal antibody or an isotype-matched control monoclonal antibody (data not shown).

FIG. 2

Fusogenic activities of each Env with recombinant coreceptors. Target NIH 3T3 cells coexpressing coreceptors and CD4 (and containing the lacZ gene linked to the T7 promoter) as well as control cells lacking coreceptors were prepared as described in the legend for Fig. 1. Effector HeLa cells were transfected with plasmids encoding the indicated Envs and then infected with vP11T7gene1 (T7 polymerase). After overnight incubation to allow recombinant protein expression, cells were mixed and fusion was scored after 3 h. The low background values for each Env obtained with the control target cells expressing CD4 but no coreceptors were subtracted to give the data shown. Error bars indicate the sample standard deviations of the mean values obtained from duplicate samples. OD, optical density.

FIG. 3

(A) Correlation between coreceptor usage profiles and fusion specificities for natural target cells. For each Env, the ratio of the fusion activity with CCR5 versus CXCR4 was calculated from the data shown in Fig. 2 (open triangles); the ratio of the relative fusion activity obtained with macrophage versus Jurkat cell targets was calculated from the data in Table 1 (closed circles). All ratio values below 0.1 or above 10 were grouped together. (B) Functional CXCR4 coreceptor on macrophages. Macrophages coinfected with vTF7-3 (T7 RNA polymerase) and vCB-3 (CD4) were preincubated for 45 min at 37°C without antibody or with 1 mg of preimmune or immune immunoglobulin per ml purified from a rabbit immunized with a peptide representing the extracellular N terminus of CXCR4 (24). Effector HeLa cells were coinfected with vCB21RLacZ and either vCB-41 (LAV Env), vCB-43 (Ba-L Env), or vCB-16 (Unc Env). The Ba-L Env infection was performed with AraC to reduce the fusion activity so that it was comparable to that of the LAV Env; similar results were obtained when the Ba-L Env was expressed without AraC (i.e., no inhibition; data not shown). For the LAV and Ba-L Envs, the minimal values obtained with Unc Env were subtracted and the results are expressed as the percentage of activity obtained in the absence of antibody (set at 100%). Error bars indicate the sample standard deviations of the mean values obtained from duplicate samples.