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. 2003 Feb;77(4):2550-8.
doi: 10.1128/jvi.77.4.2550-2558.2003.

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

Ali Amara  1 , Aurore Vidy, Genevieve Boulla, Karine Mollier, Javier Garcia-Perez, Jose Alcamí, Cedric Blanpain, Marc Parmentier, Jean-Louis Virelizier, Pierre Charneau, Fernando Arenzana-Seisdedos
Affiliations

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

Ali Amara et al. J Virol. 2003 Feb.

Abstract

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.

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Figures

FIG. 1.
FIG. 1.
Functional characterization of lentivirus-expressed wt CCR5 and CCR5-R126N in T-cell lines. (A) The transduction efficiency of the TRIP ΔU3 vector was evaluated in MT4 cells and PHA-activated PBMCs. Cells were exposed to VSV-G-pseudotyped TRIP ΔU3-CMV-EGFP vector particles for a 12-h period, washed, and cultured for 2 days to ensure optimal gene expression. GFP expression was analyzed by flow cytometry. (B) MT4 cells were incubated with wt TRIP ΔU3-CCR5 or TRIP ΔU3-CCR5-R126N vector particles for 12 h. CCR5 expression was evaluated 48 h later by flow cytometry with the anti-CCR5 2D7 MAb. (C) Calcium mobilization in response to MIP-1β. Parental MT4, MT4 wt CCR5, and MT4 CCR5-R126N cells were loaded with 1.5 μM fura-2-AM and stimulated with 50 nM MIP-1β at the time points indicated by the arrows. (D) Parental MT4, MT4 wt CCR5, and MT4 CCR5-R126N cells were infected with the replication-competent luciferase reporter HIV JR-CSF virions. Cell lysates were prepared at 72 h postinfection and analyzed for luciferase activity (in relative light units [RLU]). Results from one representative experiment out of two are shown. Error bars indicate standard deviations.
FIG. 2.
FIG. 2.
wt CCR5 and CCR5-R126N HIV coreceptor function in PHA-activated PBMCs. (A) PHA activated-PBMCs from a ccr5 Δ32/Δ32 individual (donor A) were inoculated with wt TRIP ΔU3-CCR5 or TRIP ΔU3-CCR5-R126N vector particles. Cells were maintained in medium containing 20 ng of IL-2 per ml for 5 days, and CCR5 transgene expression was monitored by flow cytometry analysis with the PE-conjugated 2D7 anti-CCR5 MAb. (B to D) At day 5 posttransduction, parental (/) or wt CCR5- or CCR5-R126N-transduced PBMCs were infected with R5 HIV-1 JR-CSF luc reporter virus (B), R5 HIV-1 strain Ba-L (C), or HIV-GFP reporter virions pseudotyped with the R5 HIV-1 Ba-L envelope glycoprotein (D). In panels B and C, HIV replication was evaluated by measuring luciferase activity in cell lysates and p24 production in the culture supernatants, respectively. In panel D, infected cells were simultaneously stained with anti-CD4-APC and anti-CD45RO-PE MAbs. HIV replication was assessed by detection of GFP by flow cytometry on gated CD4 T cells. Data are representative of those from two independent experiments. RLU, relative light units. Error bars indicate standard deviations.
FIG. 3.
FIG. 3.
PBMCs from a ccr5 Δ32/Δ32 individual (donor B) were depleted of monocytes and thereafter incubated with IL-15 and IL-2 for 5 days. Cytokine-treated cells were transduced with TRIP ΔU3 vector particles encoding EGFP, wt CCR5, or CCR5-R126N. (A) CCR5 and GFP expression in CD3 T cells from parental (/) and transduced cells was evaluated 4 days later by flow cytometry. (B) HIV infection was assessed as described for Fig. 2. +/+, PBMCs isolated from an individual carrying a wt CCR5 genotype, used as control. Data are representative of those from two independent experiments. RLU, relative light units. Error bars indicate standard deviations.
FIG. 4.
FIG. 4.
Macrophages from a ccr5 Δ32/Δ32 individual (donor C) were inoculated with wt TRIP ΔU3-CCR5 or TRIP ΔU3-CCR5-R126N vector. (A) CCR5 expression was evaluated 4 days later as described for Fig. 1. (B) Transduced macrophages were infected with R5 HIV-1 Ba-L, and cell culture supernatants were sampled for detection of p24 production. Data shown are representative of those from two independent experiments (donors B and C). +/+, macrophages isolated from an individual carrying a wt CCR5 genotype, used as control.
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