CCR5-using (R5) HIV-1 strains are present during the whole course of the infection in all subjects, whereas CXCR4-using (X4) HIV-1 strains appear only in the late stages of the infection in some subjects. In this study, we tested the hypothesis that this phenomenon might be the result of a replicative advantage of R5 over X4 strains. We compared the infectivity of an R5 and an X4 strain that differ only in their env gene in peripheral blood mononuclear cells. CD4+ T cells in culture, where the CXCR4 ligand SDF-1 is absent, overexpress CXCR4 at their surface. Therefore, a cell line producing the chemokine SDF-1, that binds to and induces the internalization of CXCR4, was established by transfer of the SDF-1 gene. We cocultured peripheral blood mononuclear cells with this SDF-1-producing cell line to obtain SDF-1 concentrations that maintained the CD4+ T cell surface CXCR4 densities observed in vivo. Under these conditions, the R5 strain appeared to replicate more efficiently than the X4 strain. Thus, in vitro, when CD4+ T cells express physiological levels of CXCR4 coreceptors, R5 virions are more fit for replication than X4 virions and in vivo that limited surface expression of CXCR4 on cell targets could contribute to the preponderance of R5 viruses.
From the *Institut de Génétique Humaine, CNRS UPR1142 and †Laboratoire d'Immunologie, Universitary Hospital, Montpellier, France.
Received for publication September 16, 2008; accepted October 13, 2009.
Correspondence to: Anne-Laure Fiser, PhD, IGH, CNRS-UPR 1142 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France (e-mail: email@example.com).
HIV-1 strains use either the CC-chemokine receptor CCR5 (R5 strains) or the CXC-chemokine receptor CXCR4 (X4 strains), in addition to the CD4 receptor, to infect target cells. Yet, X4 strains emerge in only 30% to 50% of the infected subjects and at late stages of the disease, whereas R5 strains are permanently harbored by almost all infected subjects.1 The reason why an individual, even if he or she is contaminated with X4 strains, develops initially mostly R5 strains remains so far unclear. It is important to understand this phenomenon because R5 to X4 switch is linked to an increase in disease progression1 and because it would give a major insight into the physiopathology of the infection. Various reasons have been proposed to account for this predominance of R5 strains over X4 strains. Some authors have argued that R5 strains are favored at the beginning of the infection because they are fit for the compartment where most of the viral replication initially occurs, the lymphoid tissue associated with the gastrointestinal tract.2 Other authors think that it is the humoral3 or the cellular4 immune response that makes the difference, being more efficient against X4 strains than against R5 strains. Yet, this hypothesis does not explain why all infected subjects do not develop X4 strains at late stages. Finally, an alternative possibility is that the X4 strains are counterselected because they are less fit for infection than the R5 strains. However, the comparison between R5 and X4 infectabilities has led so far to inconsistent results. Initially, monocytotropic nonsyncitium-inducing strains, corresponding mostly to R5 strains, isolated from early-stage individuals, were presented as replicating less efficiently (“slow/low”) than lymphotropic syncitium-inducing strains, corresponding mostly to X4 strains isolated from late-stage individuals (“rapid/high”).5 Likewise, Kaneshima et al, comparing the capacity of nonsyncitium-inducing and syncitium-inducing isolated from the same patients to replicate in human thymus engrafted in SCID mice, observed the same hierarchy.6 Opposite results have been reported by various groups. Exposing activated CD4+ T cells or lymphoid tissues to isogenic strains as well as primary isolates, two groups have noted a better replication of R5 strains compared with X4 strains.7-9 Moreover, Pastore et al published that most X4 variants show reduced replication compared with their parental R5 isolate.10 A better replication of laboratory and primary R5 strains has also been reported in SCID mice reconstituted with human peripheral blood mononuclear cells (PBMCs)11,12 and in chimpanzees.13
The infectivity of CD4+ T cells is dependent on their surface coreceptor density. It has actually been shown that the level of CCR5 expression on lymphocytes cultured in vitro determines the level of their infectability by R5 strains.14 Likewise, CD4+ T lymphocyte surface CXCR4 density has been linked to susceptibility to X4 infection,15,16 and X4 strains are more often present in subjects presenting with high CD4+ T cell surface CXCR4 densities.17 Therefore, it is of major importance to monitor the level of expression of the coreceptors in vitro in the system used to test HIV-1 infectivity to ascertain that this level is physiological. For these reasons, we followed CCR5 and CXCR4 expression at the surface of cultured primary CD4+ T lymphocytes and established an in vitro system guaranteeing a physiological level of coreceptor expression. We used this system to test the hypothesis that the predominance of R5 over X4 strains in vivo might actually stem from a replicative advantage of these strains.
Cells, Medium, and Reagents
293T and HOS (AIDS Reagent Program, Rockville, MD) cells were grown in DMEM medium supplemented with 10% FCS, 10 mM of glutamax-1, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen, Cergy pontoise, France). PBMCs isolated by density-gradient centrifugation, Jurkat CD4+-CCR5+ (AIDS Reagent Program), 293T/SDF1, and 293T/EGFP cells were grown in RPMI-1640 medium supplemented with 10% FCS, 10 mM of glutamax-1, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Invitrogen). SDF-1 was purchased from R&D Systems (Abingdon, UK).
CXCR4 and CCR5 Phenotyping
CXCR4 and CCR5 densities at the surface of CD4+ T cells were measured by quantitative flow cytometry as previously described.18
HIV Vector Production
The pHR-EGFP plasmid was obtained by opening the pHR plasmid19 containing the cPPT sequence downstream of the internal CMV promoter and inserting there an IRES-EGFP cassette obtained from the pEGFP-N1 and pIRES1 neo sequence (Clontech, Heidelberg, Germany). The pHR-SDF-1 plasmid was obtained by inserting the full-length human CXCL12 cDNA (image ID 5729604; ImaGenes, Mountain View, CA) in pHR-EGFP between the internal CMV promoter and the IRES-EGFP cassette. 293T cells plated into 75-cm2 flasks were cotransfected by the calcium phosphate method with three plamsids: pMD.G, encoding the vesicular stomatitis virus envelope glycoprotein, the HIV packaging plasmid p8.2,19 and either the pHR-EGFP or the pHR-SDF1 transfer plasmids. Supernatants were collected at Day 3 and concentrated by ultracentrifugation at 50,000 g for 90 minutes.
Establishment of SDF-1-Producing Cells
293T cells (150 × 103) plated into 12-well plates were transduced with 300 ng of p24 equivalents of HIV vector in the presence of 8 μg/mL of polybrene. Twenty-four hours later, cells were washed and cultured in fresh medium.
HIV Infection Assays
293T cells were transfected with 6 μg of pNL4.3 or of pAd8. Cell supernatants were collected at Day 3 and frozen. NL4.3 was amplified in Jurkat CD4+-CCR5+ cell and Ad8 in HOS cells for 10 days. PBMCs were exposed for 24 hours to 100 ng of p24 equivalents of NL4.3 or of Ad8, washed extensively, and cultured at 2 × 106 cells/mL in 200 μL of culture medium.
Determinations of Chemokine Concentrations
Supernatants stored at -80°C were thawed and analyzed for RANTES and SDF1 concentrations using commercial immunoassays (R&D Systems).
Establishment of a Cell Culture System Preserving Peripheral Blood Mononuclear Cell Surface CXCR4 Density
Because of the key role played by the level of CCR5 and CXCR4 expression at the surface of primary CD4+ T cells in R5 and X4 infection, respectively, we wondered whether these expressions were modified when these cells were cultured in vitro. To address this issue, we followed by quantitative flow cytometry CCR5 and CXCR4 densities at the surface of CD4+ T cell freshly isolated from healthy donors. CCR5 expression was not modified over time (data not shown). By contrast, CXCR4 expression increased drastically in culture. Whereas CXCR4 density measured immediately ex vivo was approximately 2000 molecules per cell, it reached 20,000 molecules per cell after less than 24 hours of in vitro culture and remained high during 2 weeks (data not shown).
CCR5- and CXCR4-binding chemokines are known to induce the internalization of their receptors in vitro.20 The main CCR5-binding chemokines, MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5), are produced by PBMC, particularly by CD8+ T cells and NK cells,21 and are present in the plasma. In infected subjects, their plasma levels range from 5 to 170 pg/mL for MIP-1α, from 3 to 203 pg/mL for MIP-1β, and from 10 to 144 ng/mL for RANTES.22 The only chemokine known to bind to CXCR4, SDF-1 (CXCL12), that is not produced by PBMC but by stromal and endothelial cells,23 is also present in the plasma at concentrations ranging from less than 1 to over 100 ng/mL.24 Logically, in the supernatant cultures, we found the presence of RANTES at a concentration ranging from 250 to 400 pg/mL, but not the presence of SDF-1. For this reason, we hypothesized that the increase in CXCR4 expression we observed in vitro might be the result of the absence of SDF-1-induced CXCR4 internalization. To test this hypothesis, we decided to create a stable cell line able to produce SDF-1. To this end, we transduced 293T cells with HIV-1 vectors harboring the SDF-1 gene. In this way, we obtained a cell line we called 293T/SDF-1, which produced 300 ng/mL of SDF-1. As a negative control, we established concurrently a 293T cell line transduced with the EGFP gene (293T/EGFP cells). When we cocultured PBMC with the 293T/SDF-1 cell line, CD4+ T cell surface CXCR4 density remained at the levels observed in vivo, below 5000 molecules/mL (Fig. 1). On the other hand, in coculture with the negative control 293T/EGFP cells, CD4+ T cells increased their membrane expression of CXCR4 as do CD4+ T cells cultured alone (Fig. 1). By contrast, under the same conditions, CD4+ T cell surface CCR5 densities stayed unmodified (data not shown).
In Culture Conditions Where CD4+ T Cells Express In Vivo Levels of CXCR4 Surface Densities, an X4 Strain Is Less Infective Than an Isogenic R5 Strain
Next, we compared the infectivity of isogenic R5 and X4 strains in this system. To this purpose, we exposed each coculture to the strains Ad8 (R5) and NL4.3 (X4) that differ only partially in their env gene.25 We verified that the concentrations of RANTES and SDF-1 in the various cocultures remained comparable after R5 and X4 infection (data not shown). In both cocultures, CD4+ T cell surface CCR5 densities remained constant (Fig. 2A). In the coculture containing the 293T/SDF-1 cells, as expected, CD4+ T cell surface CXCR4 density remained low, whereas in the coculture containing the 293T/EGFP cells, it increased drastically (Fig. 2B). When we monitored viral production in the cocultures with the negative control cell line 293T/EGFP, we observed that X4 replication was higher than R5 replication (Fig. 2C). The opposite result was obtained when the PBMCs were cocultured with the SDF-1-producing cell line 293T/SDF-1: R5 replication was higher than X4 replication (Fig. 2D).
In this study, we show that culturing primary CD4+ T cells results in a drastic increase in their surface CXCR4 density as a consequence of the absence of the CXCR4 ligand SDF-1 in the culture medium. These results are in keeping with data previously reported by Bermejo et al.15 This drawback may be circumvented by coculturing the lymphocytes with SDF-1-producing cells. Under these conditions, an X4 strain was less fit for infection than an isogenic R5 strain. These results emphasize the link between cell surface CXCR4 density and infectability by X4 strains, a link that has been previously reported for thymocytes and T cells.15,16,26 Our conclusion that R5 strains are more replicative than X4 strains is at odd with the studies of other groups.7,8 Yet, in these studies, coreceptor expression was not checked. We think that all previous in vitro studies on X4 infections must be re-examined by the light of our present observation, and in the future, careful monitoring of coreceptor expression will need to be carried out for an in vitro system to be validated.
Now, why are X4 strains less fit for replication than R5 strains? Various reasons may be proposed, including the following. First, physiological CD4+ T cell surface CCR5 density, which ranges, in CCR5-expressing subjects, from 4000 to over 20,000 molecules/cell18 is higher than CD4+ T cell surface CXCR4 density, which remains most of the time below 3000 molecules/cell.17 Moreover, on CCR5+CXCR4+ cells, this high expression might give an advantage to CCR5 in its competition with CXCR4 for association with CD4 during viral entry.27 Second, CCR5 but not CXCR4 has been reported to be constitutively associated with the CD4 receptor at the surface of the target T cells.28 The fact that CD4 and CCR5 are already physically linked when the virion binds to the first receptor, whereas CD4-bound X4 gp120 has to recruit CXCR4 coreceptors, might give an advantage to R5 strains. Finally, the affinity of R5 gp120 for CCR5 has been reported to be higher than that of X4 gp120 for CXCR4.29,30
Our observation that in conditions mimicking the in vivo situation, CXCR4-using strains are less infectious than CCR5-using strains might help to explain the preponderance of the latter in infected subjects inasmuch as there seems to be some degree of competition between both types of strains in vivo. In support of this notion is the observation that the treatment with a CCR5 antagonist of macaques coinfected with an R5 SIV and an X4 SHIV strain resulted in an increase in the X4 SHIV viral load.31 Likewise, in some infected subjects harboring X4 HIV-1 strains below the level of detection, administration of the CCR5 antagonist maraviroc induced a rise in X4 HIV-1 RNA plasma level.32 Thus, initially, the better aptitude of R5 strains to productively infect CD4+ T cells could give them an advantage that is strengthened by the fact that the presence of a large amount of R5 virions inhibits the development of X4 virions. This situation might be modified if for some reason cell surface CXCR4 expression happens to be increased. The report that individuals infected with X4 strains overexpress CXCR4 at the surface of their CD4+ T cells17 is in keeping with this model.
Ms. Fiser was supported by ANRS and SIDACTION.
1. Richman DD, Bozzette SA. The impact of the syncitium-inducing phenotype of human immunodeficiency virus on disease progression. J Infect Dis
2. Veazey R, Lackner A. The mucosal immune system and HIV-1 infection. AIDS Rev
3. Bou-Habib DC, Roderiquez G, Oravecz T, et al. Cryptic nature of envelope V3 region epitopes protects monocytotropic human immunodeficiency virus type 1 from antibody neutralization. J Virol
4. Harouse JM, Buckner C, Gettie A, et al. CD8+ T cell-mediated CXC chemokine receptor 4-simian/human immunodeficiency virus suppression in dually infected rhesus macaques. Proc Natl Acad Sci USA
5. Fenyö EM, Albert J, Asjö B. Replicative capacity, cytopathic effect and cell tropism of HIV. AIDS
6. Kaneshima H, Su L, Bonyhadi ML, et al. Rapid-high, syncitium-inducing isolates of human immunodeficiency virus type 1 induce cytopathicity in the human thymus of the SCID-hu mouse. J Virol
7. Roy AM, Schweighardt B, Eckstein LA, et al. Enhanced replication of R5 HIV-1 over X4 HIV-1 in CD4(+)CCR5(+)CXCR4(+) T cells. J Acquir Immune Defic Syndr
8. Schweighardt B, Roy A-M, Meiklejohn DA, et al. R5 human immunodeficiency virus type 1 (HIV-1) replicates more efficiently in primary CD4+
T-cell cultures than X4 HIV-1. J Virol
9. Vicenzi E, Bordignon PP, Biswas P, et al. Envelope-dependent restriction of human immunodeficiency virus type 1 spreading in CD4+
T lymphocytes: R5 but not X4 viruses replicate in the absence of T-cell receptor restimulation. J Virol
10. Pastore C, Ramos A, Mosier DE. Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching. J Virol
11. Markham RB, Schwartz DH, Templeton A, et al. Selective transmission of human immunodeficiency virus type 1 variants to SCID mice reconstituted with human peripheral blood mononuclear cells. J Virol
12. Picchio GR, Gulizia RJ, Wehrly K, et al. The cell tropism of human immunodeficiency virus type 1 determines the kinetics of plasma viremia in SCID mice reconstituted with human peripheral blood leukocytes. J Virol
13. ten Haaft P, Murthy K, Salas M, et al. Differences in early virus loads with different phenotypic variants of HIV-1 and SIV(cpz) in chimpanzees. AIDS
14. Wu L, Paxton WA, Kassam N, et al. CCR5 levels and expression pattern correlate with infectability by macrophage-tropic HIV-1, in vitro
. J Exp Med
15. Bermejo M, Martin-Serrano J, Oberlin E, et al. Activation of blood T lymphocytes down-regulates CXCR4 expression and interferes with propagation of X4 strains. Eur J Immunol
16. Moonis M, Lee B, Bailer RT, et al. CCR5 and CXCR4 expression correlated with X4 and R5 HIV-1 infection yet sustained replication in TH1 and TH2 cells. AIDS Res Hum Retroviruses
17. Lin Y-L, Portales P, Baillat V, et al. CXCR4 overexpression during the course of HIV-1 infection correlates with the emergence of X4 strains. J Acquir Immune Defic Syndr
18. Reynes J, Portales P, Segondy M, et al. CD4+
T cell surface CCR5 density as a determining factor of viral load in HIV-1-infected individuals. J Infect Dis
19. Naldini L, Blomer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science
20. Amara A, Le Gall S, Schwartz O, et al. HIV coreceptor downregulation as antiviral principle: SDF-1α-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J Exp Med
21. Cocchi F, De Vico AL, Garzino-Demo A, et al. Identification of RANTES, MIP-1α, and MIP-1β as the major HIV-suppressive factors produced by CD8+ T cells. Science
22. Polo S, Veglia F, Malnati M, et al. Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection. AIDS
23. Kucia M, Reca R, Miekus K, et al. Trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the SDF-1-CXCR4 axis. Stem Cells
24. Derdeyn CA, Costello C, Kilby JM, et al. Correlation between circulating stromal cell-derived factor 1 levels and CD4+
cell count in human immunodeficiency virus type 1-infected individuals. AIDS Res Hum Retroviruses
25. Englund G, Theodore TS, Freed EO, et al. Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1. J Virol
26. Zaitseva M, Lee S, Rabin RL, et al. CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection. J Immunol
27. Lee S, Lapham CK, Chen H, et al. Coreceptor competition for association with CD4 may change the susceptibility of human cells with T-tropic and macrophage-tropic isolates of human immunodeficiency virus type 1. J Virol
28. Xiao X, Wu L, Stantchev TS, et al. Constitutive cell surface association between CD4 and CCR5. Proc Natl Acad Sci USA
29. Doranz BJ, Baik SS, Doms RW. Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events. J Virol
30. Hoffman TL, Canziani G, Rucker J, et al. A biosensor assay for studying ligand-membrane interactions: binding of antibodies and HIV-1 env to chemokine receptor. Proc Natl Acad Sci USA
31. Wolinsky SM, Veazy RS, Kunstman KJ, et al. Effect of a CCR5 inhibitor on viral loads in macaques dual-infected with R5 and X4 primate immunodeficiency viruses. Virology
32. Deeks SG. Challenges of developing R5 inhibitors in antiretroviral naive HIV-infected patients. Lancet