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Journal of Virology, June 2005, p. 7938-7941, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7938-7941.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

G-Protein Signaling Triggered by R5 Human Immunodeficiency Virus Type 1 Increases Virus Replication Efficiency in Primary T Lymphocytes

Yea-Lih Lin,1 Clément Mettling,1 Pierre Portalès,2 Brigitte Réant,1,2 Jacques Clot,2 and Pierre Corbeau1,2*

Institut de Génétique Humaine, CNRS UPR1142,1 Laboratoire d'Immunologie, Hôpital Saint Eloi, Montpellier, France2

Received 15 March 2004/ Accepted 18 February 2005


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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.


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TEXT
 
Most human immunodeficiency virus type 1 (HIV-1) strains use CCR5 as a coreceptor in addition to the CD4 molecule (8). The interaction between the gp120 surface envelope of these R5 isolates and the C-C chemokine receptor CCR5 results in numerous cell activation signals, which include phosphorylation of CCR5 itself and the focal adhesion kinase as well as their association (5), activation of ion channels (12), calcium mobilization (4), tyrosine phosphorylation of the protein tyrosine kinase Pyk2 (6), downregulation of intracellular cAMP (10), and induction of chemotaxis (16). We wondered whether these activation signals might facilitate the replication of the virus in peripheral blood CD4+ T cells. So far, various reports argue against this hypothesis. Actually, using signaling-defective CCR5 receptors, many groups have failed to demonstrate that cell activation consecutive to CCR5 engagement by R5 gp120 has any impact on HIV replication. Thus, the truncation of the CCR5 intracytoplasmic terminal carboxyl tail, which results in the abolition of CCL4-mediated calcium mobilization (1, 9), inositol phosphate release, and the inhibition of adenylyl cyclase (9), did not modify CCR5 function as an HIV-1 coreceptor (1, 9). Likewise, CCR5 receptors mutated in the second (7) or seventh (3) transmembrane domain or in the second intracellular loop (7, 9), which also failed to induce an intracellular increase in calcium concentration (7, 9) or to inhibit adenylyl cyclase (3) secondary to exposition to C-C chemokines, fully supported R5 infection (3, 7, 9). One reason for these conflicting results might have been that until recently, none of the works on signaling defective CCR5 receptors had been performed with primary cells, the basic state of activation of which differs widely from the one of the transformed cells used in these works. For this reason, Amara et al. used the same approach, but with primary T cells (2). They activated primary mononuclear cells from subjects who were homozygous for the 32-base deletion ({Delta}32) in the CCR5 gene and who express no CCR5 molecule at the surface of their cells, in order to efficiently transduce them with a wild-type CCR5 gene or a mutated CCR5 gene encoding a receptor unable to couple with G proteins. Infecting both cell preparations right after these transductions, they observed no difference in virus production and concluded that CCR5 signaling definitely played no role in the efficiency of R5 infection, even in primary cells. Nonetheless, it may be argued that in these experiments, the full cell activation induced prior to the transduction in order to optimize the gene transfer and the activating effect of the transduction per se might have concealed any enhancing effect of CCR5 signaling on the virus life cycle. Therefore, we repeated the same experiment with two modifications. First, we did not activate the cells before transducing them, and second, we left them at rest for 10 days after the gene transfer before testing their infectivity.

For this purpose, we created the same R126N mutation in the highly conserved DRY motif of the second intracellular loop of CCR5, which has been shown to be involved in the coupling of the C-C chemokine receptor with G proteins, and thereby in calcium mobilization (13). The mutation (AGG versus AAC) was introduced by a two-step PCR with flanking oligonucleotides carrying SalI and SpeI sites. After sequencing, wild-type and mutant CCR5 (from nucleotide 345 to 1436 in NCBI sequence NM_000579) were subcloned in pHR-BX lentiviral vector to produce virions as previously described (11). We confirmed by flow cytometry that R126N-CCR5 was unable to mobilize calcium in the presence of the CCR5 agonist CCL5 (data not shown). We introduced the wild-type and mutant CCR5 genes and the negative control gene LacZ into HIV vectors and transduced {Delta}32/{Delta}32 peripheral blood mononuclear cells (PBMCs), isolated by Ficoll-Hypaque gradient centrifugation and cultured for 72 h in RPMI 1640 supplemented with 10% fetal calf serum, with these vectors without any preactivation. Figure 1A shows that we obtained comparable CCR5 cell surface expression at the surface of resting mononuclear cells transduced with the wild-type or mutated CCR5 gene. Cells were cultured for 10 days in the same culture medium without any additive, in order to minimize their activation. After 10 days, over 50% of the cells were viable. Likewise, Stevenson et al. have previously reported that the viability of quiescent primary mononuclear cells infected with HIV-1 can be maintained at >75% for over 2 weeks in culture (15). Less than 15% of the cells expressed the activation markers CD25 or CD69 (Fig. 2). We then exposed the cells to the R5 laboratory strain Ada-M, and monitored virus production in the cell supernatant at different time points. In these conditions, the infectibility conferred by wild-type CCR5 is much more important than that conferred by the R126N-CCR5 receptor (Fig. 3A). A similar difference was observed with another donor (data not shown). Figure 3B shows that we obtained the same result with a primary R5 strain. By contrast, the X4 strain NL4-3 replicated with the same efficiency in the three cell subpopulations, as represented in Fig. 3C.



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  		FIG.1. CCR5 expression on {Delta}32/{Delta}32 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.



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  		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.



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  		FIG. 3. (A, B, C) Infectibility of {Delta}32/{Delta}32 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 ({blacksquare}, wild-type CCR5-transduced cells; {blacktriangleup}, R126N-CCR5-transduced gene; •, LacZ-transduced cells). (D) Effect of pertussis toxin on the infectibility of {Delta}32/{Delta}32 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.

The fact that a G protein signaling-defective CCR5 molecule is impaired as an R5 coreceptor suggests that this signaling facilitates the R5 replicative cycle. CCR5 is known to signal through G{alpha}i and G{alpha}q proteins. Guntermann et al. have previously reported that preincubation of PBMCs with the G{alpha}i inhibitor pertussis toxin inhibited HIV-1 replication (10). In order to definitively demonstrate the role of G protein activation in R5 infection, we tested the effect of this inhibitor in our system. We observed that the addition of pertussis toxin in the cell culture drastically reduced the difference in infectivity observed between wild-type- and R126N-CCR5-transduced PBMCs (Fig. 3D).

Our results show that G-protein signaling triggered during R5 infection drastically facilitates this infection in primary mononuclear cells. They shed a new light on the physiopathology of HIV-1 infection. The permissiveness of a cell might depend not only on the presence of the right receptors at its surface but also on the capacity of the virion to efficiently trigger the right activation pathways through these receptors. Finally, a consequence of our findings is that the G protein pathway represents a new therapeutic target for anti-HIV drugs.


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ACKNOWLEDGMENTS
 
This study was funded by the Agence Nationale de Recherche contre le SIDA (ANRS). Y.-L.L. was supported by a fellowship from SIDACTION.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire d'Immunologie, Hopital Saint Eloi, 80 avenue Augustin Fliche, 34295 Montpellier Cedex 5, France. Phone: 33-4-67-33-71-35. Fax: 33-4-67-33-71-29. E-mail: pierre.corbeau{at}igh.cnrs.fr. Back


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Journal of Virology, June 2005, p. 7938-7941, Vol. 79, No. 12
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.12.7938-7941.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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