G-protein signaling triggered by R5 human immunodeficiency virus type 1 increases virus replication efficiency in primary T lymphocytes

Abstract

The binding of R5 envelope to CCR5 during human immunodeficiency virus type 1 (HIV-1) entry provokes cell activation, which has so far been considered to have no effect on virus replication, since signaling-defective CCR5 molecules have been shown to function normally as HIV-1 coreceptors on transformed cells or mitogen-stimulated T lymphocytes. As the background state of activation of these cells might have biased the results, we performed experiments using the same approach but with nonactivated primary T lymphocytes. We now report that the single R126N mutation in the DRY motif, involved in G-protein coupling, results in a signaling-defective CCR5 coreceptor with a drastically impaired capacity to support HIV-1 infection.

FIG.1.

CCR5 expression on Δ32/Δ32 mononuclear cells transduced with LacZ (A), the wild-type CCR5 (B) or the R126N-CCR5 (C) gene. Cells were labeled indirectly with the anti-CCR5 monoclonal antibody 2D7 (-) or with a negative control antibody (^…) as described previously (14), and analyzed by flow cytometry. The percentages of transduced cells and the mean fluorescence intensities (MFI) are indicated.

FIG. 2.

Cell size and structure and the expression of the activation markers CD25 and CD69 10 days after the transduction of PBMC with LacZ, wild-type CCR5, or R126N-CCR5 as determined by flow cytometry. The percentages of labeled cells, measured by comparisons with the signal obtained with a negative control antibody, are indicated. WT, wild type.

FIG. 3.

(A, B, C) Infectibility of Δ32/Δ32 mononuclear cells transduced with the wild-type CCR5 or the R126N-CCR5 gene. Cells were exposed to 10 ng of the laboratory R5 strain Ada-M (A), to 10 ng of a primary R5 strain (B), or to 20 ng of the laboratory X4 strain NL4-3 (C) for 18 h, extensively washed, and cultured for 10 days. Virus production was evaluated by measuring p24 Gag antigen concentration in the culture supernatant (▪, wild-type CCR5-transduced cells; ▴, R126N-CCR5-transduced gene; •, LacZ-transduced cells). (D) Effect of pertussis toxin on the infectibility of Δ32/Δ32 mononuclear cells transduced with LacZ, the wild-type CCR5 or the R126N-CCR5 gene. Cells were exposed (open bars) or not exposed (closed bars) to 0.1 ng/ml of pertussis toxin for 18 h, infected with 10 ng of the laboratory R5 strain Ada-M as described above, and cultured in the presence or absence of toxin. Virus production was evaluated by measuring p24 Gag antigen concentration in the culture supernatant at day 10.