As explained above, passages of the six HIV-1 strains occurred at relatively slow replication rates due to the low availability of CCR5 coreceptor. At the same time, similar cultures were passaged in the presence of the reverse transcriptase inhibitors, ZDV or 3TC, and the CCR5 antagonist TAK-779 at suboptimal concentrations, applying similar pressure on the virus but on different target genes. The gain of CXCR4 use by the three isolates described above was delayed with ZDV, 3TC and TAK-779 (Fig. 2). In both CI3 and CI5, CXCR4-using viruses emerged earlier with TAK-779 compared to the cultures with ZDV or 3TC. (Fig. 2a, c and Table 2). For CI3, emergence of CXCR4-using variants in the presence of TAK-779 (CI3TAK-779) was delayed for 15 passages (49 days) compared to the untreated control (CI3C), whereas ZDV delayed it (CI3ZDV) for 21 passages (70 days). The CI5 strain cultured with TAK-779 (CI5TAK-779) switched coreceptor at 5–9 passages (17–59 days) after the CI5 without drug (CI5C), depending on the experiment. Coreceptor switch variants of CI5 in the presence of ZDV (CI5ZDV) could not be detected even 18 or 33 passages (63 or 115 days) after their detection in the control cultures.
A parallel culture of each strain was maintained with AMD3100 (1 μg/ml). AMD3100 prevented the emergence of CXCR4-using viruses in the cultures of the three clinical isolates that switched in the absence of drug pressure.
Selection of the R5X4 phenotype could also be induced with the monoclonal antibody (mAb) anti-CCR5 2D7 and RANTES (Fig. 2d). The switch of coreceptor use was delayed if compared to the untreated culture, but was noticed earlier with all CCR5 agents when compared to cultures growing at a similar replication rate (in the presence of ZDV or 3TC).
The sensitivity to ZDV, AMD3100 and TAK-779 of each parental virus and all the viruses obtained after the passages was determined in PBMC. EC50 values are shown in Table 2. As expected, almost all the viruses that gained CXCR4 usage were less sensitive to TAK-779. The control CI5 virus (control CI5, CI5C), of R5X4 phenotype, was 30-fold less sensitive to TAK-779 compared to the parental CI5 (EC50 0.003 μg/ml and 0.0001 μg/ml, respectively). Similarly, the EC50 of TAK-779 for CI5TAK-779 and CI5RANTES increased 30-fold and 100-fold for the CI52D7 strain. Comparable results were obtained with the CI3 virus, the switched variants were 40-fold (CI3C) 90-fold (CI3ZDV) and 60-fold (CI3TAK-779) less sensitive to TAK-779. Nevertheless, the R5X4 variant CI4ZDV was as sensitive as the CI4 parental isolate. Concerning the AMD3100 inhibition, parental isolates were totally resistant, but the R5X4 variants gained some sensitivity. However, an EC50 value for AMD3100 could not be calculated, except in two cases, for CI3C (0.03 μg/ml) and for CI4C (0.1 μg/ml). Drug sensitivity in primary cells is prone to higher variation in experimental error and variation in virus titre may explain the 10-fold increase in the AMD3100, ZDV and 3TC CI5 passaged strains.
A number of publications have suggested that CCR5 drug resistance may emerge in the absence of coreceptor switch [28–32]. HIV-1 may become resistant to vicriviroc [28,44] or maraviroc  by utilizing an inhibitor-bound form of the receptor and this has been shown as a preferential mode to circumvent the anti-HIV activity of CCR5 drugs in the absence of coreceptor switch. Coreceptor phenotype testing from the phase II maraviroc trial showed that circulating virus remained CCR5 tropic in 60/62 patients, indicating that X4 variants were not rapidly selected despite CCR5-specific drug pressure . Conversely, we show that in cell culture, HIV-1 strains may switch to CXCR4 use faster with selective pressure on CCR5 use (TAK-779, RANTES or 2D7) than with ZDV, suggesting a preferential selection of X4 virus as a mode of drug resistance in some of the HIV strains tested.
The use of CCR5-targeting drugs requires the prior knowledge of the viral tropism in a given patient. Determination for HIV-1 coreceptor usage is complex and only few methods exist [46,47] that may not be sensitive enough to detect minor X4 or R5/X4 populations [45–50], selection of which would be favoured by a CCR5 antagonist. For instance, X4 variants in two patients on treatment with maraviroc appeared to emerge by outgrowth of a pretreatment CXCR4-using reservoir .
Our observation that a clonal R5 virus may gain CXCR4 use, which also occurred in the presence of CCR5 compounds, shows that minor X4 populations may evolve from the mutants generated within the first 2 weeks from a purely R5 virus. Furthermore, if a minor X4 population is present at the initiation of cell culture, it is intuitive to think that X4 emergence will occur at a similar time/rate in the absence or presence of CCR5 drugs and this did not occur. It is also relevant to bear in mind that for any given compound, the selection of a resistant virus applies for a preexisting minor mutant population with a selective advantage in the presence of the drug. Generation of the mutant virus depends on the intrinsic mutation rate and the replicative capacity of the virus. Therefore, whether the drug-resistant virus (i.e., a CXCR4-using strain) was present at day zero or was generated during cell culture, the emergence of the mutant is independent of the drug that, if present, selects for the drug-resistant virus.
Our results are in line with a recent report of the maraviroc phase III trial concluding that more patients on maraviroc had a change in tropism to dual tropic/mix (R5/X4) or X4 at time of failure than in the placebo control group , underscoring the propensity of CXCR4-using virus to emerge under CCR5 drug pressure. Upon discontinuation of treatment, R5 virus may repopulate and conform the dominating phenotype, suggesting that X4 variants may only have an increased viral fitness in the presence of CCR5 drug pressure.
The X4 variants that emerged in our cultures gained sensitivity to AMD3100 and lost it to TAK-779. However, changes in susceptibility to both compounds were partial, reflecting the retained capacity of the virus to use CCR5 or CXCR4. Notably, one virus strain expanded coreceptor capacity while retaining total sensitivity to TAK-779 in PBMC. Coreceptor switch intermediates with lower affinity to CCR5 and higher sensitivity to CCR5 inhibitors have been recently described . These results were generated in U87-CD4 cells that exclusively express CCR5, allowing evaluation of changes in sensitivity due to changes in the affinity of the virus for the receptor. Reduced or loss of sensitivity to TAK-779 of HIV-1 stains in PBMC cultures, i.e., expressing both CCR5 and CXCR4, probably reflects increased representation of CXCR4-using variants within the virus population that grow out in the presence of drug, and not a correlative measurement of CCR5 binding. Dualtropic viruses exhibit considerable variations in their efficiency to use CXCR4 and CCR5 as coreceptor , and consequently, in their susceptibility to CXCR4 and CCR5 entry inhibitors. HIV-1 strains such as 89.6, defined as dualtropic through coreceptor assays, may be completely blocked by AMD3100 in PBMC and ex vivo lymphoid tissue . Tests for coreceptor use and drug sensitivity in cells expressing both coreceptors  may not always be in agreement, highlighting the necessity of multiple determinations to clearly assess coreceptor preference by HIV-1.
In this cell culture model not all strains were able to switch coreceptor preference, which may reflect an intrinsic capacity of some isolates to switch to X4 or retain the R5 phenotype. It is enticing to suggest the importance of evaluating a significant number of isolates in order to validate if cell culture assays could be used to measure the propensity of a clinical isolate to switch or expand coreceptor preference prior to or after the initiation of a CCR5 drug-containing regimen. It will also be important to determine by clonal analysis if the emerging X4 phenotype is generated by a mixture of R5 and emerging X4 variants, or are dual-tropic (R5X4) viruses.
It becomes clear that cell culture conditions and choice of virus isolate are of utmost importance to induce a coreceptor change in cell culture. This observation can be extended to the development of resistance to CCR5 inhibitors in the absence of coreceptor switch. The virus strains that did not switch coreceptor preference did not become resistant to TAK-779 at the time that cell cultures were stopped (data not shown for CI1, CI2 and BaL). Resistance is commonly developed in cells expressing detectable CCR5 levels and by gradually increasing the compound concentration until relatively high levels are reached [29–31]. The cell type, time in culture and the concentration of HIV inhibitors together with the specific HIV-1 strain that is being selected are factors that affect the outcome of in vitro resistance development.
We thank the NIH AIDS Reference Reagent Program for reagents. This work was supported in part by the Spanish MEC project BFU2006-00966, FIS PI060624 and Red de Investigación sobre el SIDA and the European TRIoH Consortium (LSHB-CT-2003-503480). G. Moncunill is recipient of a scholarship from Generalitat de Catalunya.
1. Berger EA, Doms RW, Fenyo EM, Korber BT, Littman DR, Moore JP, et al
. A new classification for HIV-1. Nature 1998; 391:240.
2. Menendez-Arias L, Este JA. HIV-resistance to viral entry inhibitors. Curr Pharm Des 2004; 10:1845–1860.
3. Bjorndal A, Deng H, Jansson M, Fiore JR, Colognesi C, Karlsson A, et al
. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 1997; 71:7478–7487.
4. Carrillo A, Ratner L. Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants. J Virol 1996; 70:1301–1309.
5. Chan SY, Speck RF, Power C, Gaffen SL, Chesebro B, Goldsmith MA. V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1. J Virol 1999; 73:2350–2358.
6. Cocchi F, DeVico AL, Garzino-Demo A, Cara A, Gallo RC, Lusso P. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nat Med 1996; 2:1244–1247.
7. Hwang SS, Boyle TJ, Lyerly HK, Cullen BR. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 1991; 253:71–74.
8. Pastore C, Nedellec R, Ramos A, Pontow S, Ratner L, Mosier DE. Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J Virol 2006; 80:750–758.
9. Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR. Change in coreceptor use coreceptor use correlates with disease progression in HIV-1–infected individuals. J Exp Med 1997; 185:621–628.
10. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al
. Identification of a major co-receptor for primary isolates of HIV-1. Nature 1996; 381:661–666.
11. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, et al
. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 1996; 86:367–377.
12. Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, et al
. Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS. Ann Intern Med 1993; 118:681–688.
13. Richman DD, Bozzette SA. The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progression. J Infect Dis 1994; 169:968–974.
14. Este JA, Telenti A. HIV entry inhibitors. Lancet 2007; 370:81–88.
15. Baba M, Nishimura O, Kanzaki N, Okamoto M, Sawada H, Iizawa Y, et al
. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci USA 1999; 96:5698–5703.
16. Dorr P, Westby M, Dobbs S, Griffin P, Irvine B, Macartney M, et al
. Maraviroc (UK-427 857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum antihuman immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 2005; 49:4721–4732.
17. Willey S, Peters PJ, Sullivan WM, Dorr P, Perros M, Clapham PR. Inhibition of CCR5-mediated infection by diverse R5 and R5X4 HIV and SIV isolates using novel small molecule inhibitors of CCR5: effects of viral diversity, target cell and receptor density. Antiviral Res 2005; 68:96–108.
18. Strizki JM, Tremblay C, Xu S, Wojcik L, Wagner N, Gonsiorek W, et al
. Discovery and characterization of vicriviroc (SCH 417690), a CCR5 antagonist with potent activity against human immunodeficiency virus type 1. Antimicrob Agents Chemother 2005; 49:4911–4919.
19. Donzella GA, Schols D, Lin SW, Este JA, Nagashima KA, Maddon PJ, et al
. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med 1998; 4:72–77.
20. Este JA. Virus entry as a target for anti-HIV intervention. Curr Med Chem 2003; 10:1617–1632.
21. Este JA, Telenti A. HIV entry inhibitors. Lancet 2007; 370:81–88.
22. Gulick RM, Su Z, Flexner C, Hughes MD, Skolnik PR, Wilkin TJ, et al
. Phase 2 study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV-1-infected, treatment-experienced patients: AIDS Clinical Trials Group 5211. J Infect Dis 2007; 196:304–312.
23. Lalezari JG, DeJesus E, Lampiris H, Gulick R, Saag M, Bridgway C, et al
. Efficacy and safety of maraviroc plus optimized background therapy in viremic ART-experienced patients infected with CCR5-tropic HIV-1: 24-week results of a Phase 2b/3 study in the US and Canada. Fourteenth Conference on Retroviruses and Opportunistic Infections. Los Angeles, CA, February 2007; 2007 [abstract 104bLB].
24. Nelson M, Konourina I, Lazzarin A, Clumeck N, Horban A, Tawadrous M. Efficacy and safety of maraviroc plus optimized background therapy in viremic, ART-experienced patients infected with CCR5-tropic HIV-1 in Europe, Australia, and North America: 24-Week Results. Fourteenth Conference on Retroviruses and Opportunistic Infections. Los Angeles, CA, February 2007; 2007 [abstract 104bLB].
25. Clotet B. CCR5 inhibitors: promising yet challenging. J Infect Dis 2007; 196:178–180.
26. Moore JP, Kitchen SG, Pugach P, Zack JA. The CCR5 and CXCR4 coreceptors–central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. AIDS Res Hum Retroviruses 2004; 20:111–126.
27. Nishikawa M, Takashima K, Nishi T, Furuta RA, Kanzaki N, Yamamoto Y, et al
. Analysis of binding sites for the new small-molecule CCR5 antagonist TAK-220 on human CCR5. Antimicrob Agents Chemother 2005; 49:4708–4715.
28. Kuhmann SE, Pugach P, Kunstman KJ, Taylor J, Stanfield RL, Snyder A, et al
. Genetic and phenotypic analyses of human immunodeficiency virus type 1 escape from a small-molecule CCR5 inhibitor. J Virol 2004; 78:2790–2807.
29. Marozsan AJ, Kuhmann SE, Morgan T, Herrera C, Rivera-Troche E, Xu S, et al
. Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D). Virology 2005; 338:182–199.
30. Trkola A, Kuhmann SE, Strizki JM, Maxwell E, Ketas T, Morgan T, et al
. HIV-1 escape from a small molecule, CCR5-specific entry inhibitor does not involve CXCR4 use. Proc Natl Acad Sci USA 2002; 99:395–400.
31. Baba M, Miyake H, Wang X, Okamoto M, Takashima K. Isolation and characterization of human immunodeficiency virus type 1 resistant to the small-molecule CCR5 antagonist TAK-652. Antimicrob Agents Chemother 2007; 51:707–715.
32. Westby M, Smith-Burchnell C, Mori J, Lewis M, Mosley M, Stockdale M, et al
. Reduced maximal inhibition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. J Virol 2007; 81:2359–2371.
33. Este JA, Cabrera C, Blanco J, Gutierrez A, Bridger G, Henson G, et al
. Shift of clinical human immunodeficiency virus type 1 isolates from X4 to R5 and prevention of emergence of the syncytium-inducing phenotype by blockade of CXCR4. J Virol 1999; 73:5577–5585.
34. Pauls E, Senserrich J, Clotet B, Este JA. Inhibition of HIV-1 replication by RNA interference of p53 expression. J Leukoc Biol 2006; 80:659–667.
35. de Jong JJ, Goudsmit J, Keulen W, Klaver B, Krone W, Tersmette M, et al
. Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity. J Virol 1992; 66:757–765.
36. NIAID and NIH. NIAID and NIH Virology Manual for HIV Laboratories. Washington DC: National Institute of Allergy and Infectious Diseases; Publication NIH-97-3828 1997.
37. Moncunill G, Negredo E, Bosch L, Vilarrasa J, Witvrouw M, Llano A, et al
. Evaluation of the anti-HIV activity of statins. AIDS 2005; 19:1697–1700.
38. Bosch B, Clotet-Codina I, Blanco J, Pauls E, Coma G, Cedeno S, et al
. Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody. Antiviral Res 2006; 69:173–180.
39. Martinez MA, Gutierrez A, Armand-Ugon M, Blanco J, Parera M, Gomez J, et al
. Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication. AIDS 2002; 16:2385–2390.
40. Armand-Ugón M, Clotet-Codina I, Tintori C, Manetti F, Clotet B, Botta M, et al
. The anti-HIV activity of ADS-J1 targets the HIV-1 gp120. Virology 2005; 343:141–149.
41. Pastore C, Ramos A, Mosier DE. Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching. J Virol 2004; 78:7565–7574.
42. Fouchier RA, Groenink M, Kootstra NA, Tersmette M, Huisman HG, Miedema F, et al
. Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule. J Virol 1992; 66:3183–3187.
43. Pollakis G, Kang S, Kliphuis A, Chalaby MI, Goudsmit J, Paxton WA. N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization. J Biol Chem 2001; 276:13433–13441.
44. Pugach P, Marosan A, Ketas T, Landes E, Mooore J, Kuhmann S. HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology 2007; 361:212–228.
45. Westby M, Lewis M, Whitcomb J, Youle M, Pozniak AL, James IT, et al
. Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir. J Virol 2006; 80:4909–4920.
46. Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wrin T, et al
. Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism. Antimicrob Agents Chemother 2007; 51:566–575.
47. Coakley E, Petropoulos CJ, Whitcomb JM. Assessing chemokine co-receptor usage in HIV. Curr Opin Infect Dis 2005; 18:9–15.
48. Wilkin TJ, Su Z, Kuritzkes DR, Hughes M, Flexner C, Gross R, et al
. HIV type 1 chemokine coreceptor use among antiretroviral-experienced patients screened for a clinical trial of a CCR5 inhibitor: AIDS Clinical Trial Group A5211. Clin Infect Dis 2007; 44:591–595.
49. Jensen MA, van 't Wout AB. Predicting HIV-1 coreceptor usage with sequence analysis. AIDS Rev 2003; 5:104–112.
50. Fernandez G, Llano A, Esgleas M, Clotet B, Este JA, Martinez MA. Purifying selection of CCR5-tropic human immunodeficiency virus type 1 variants in AIDS subjects that have developed syncytium-inducing, CXCR4-tropic viruses. J Gen Virol 2006; 87:1285–1294.
51. Pastore C, Nedellec R, Ramos A, Hartley O, Miamidian JL, Reeves JD, et al
. Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching. J Virol 2007; 81:8165–8179.
52. Reeves JD, Gallo SA, Ahmad N, Miamidian JL, Harvey PE, Sharron M, et al
. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc Natl Acad Sci USA 2002; 99:16249–16254.
53. Yi Y, Shaheen F, Collman RG. Preferential use of CXCR4 by R5X4 human immunodeficiency virus type 1 isolates for infection of primary lymphocytes. J Virol 2005; 79:1480–1486.
54. Jensen MA, Li FS, van 't Wout AB, Nickle DC, Shriner D, He HX, et al
. Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 env V3 loop sequences. J Virol 2003; 77:13376–13388.
55. Hoffman NG, Seillier-Moiseiwitsch F, Ahn J, Walker JM, Swanstrom R. Variability in the human immunodeficiency virus type 1 gp120 Env protein linked to phenotype-associated changes in the V3 loop. J Virol 2002; 76:3852–3864.
56. LaRosa GJ, Davide JP, Weinhold K, Waterbury JA, Profy AT, Lewis JA, et al
. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 1990; 249:932–935.
57. Voulgaropoulou F, Tan B, Soares M, Hahn B, Ratner L. Distinct human immunodeficiency virus strains in the bone marrow are associated with the development of thrombocytopenia. J Virol 1999; 73:3497–3504.
58. Troyer RM, Collins KR, Abraha A, Fraundorf E, Moore DM, Krizan RW, et al
. Changes in human immunodeficiency virus type 1 fitness and genetic diversity during disease progression. J Virol 2005; 79:9006–9018.
59. Shankarappa R, Margolick JB, Gange SJ, Rodrigo AG, Upchurch D, Farzadegan H, et al
. Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J Virol 1999; 73:10489–10502.
60. Holuigue S, Ketas T, Moore J. Co-receptor switched and CCR5 inhibitor-resistant HIV-1 variants are generated in response to different selection pressures in vitro. Fourteenth Conference on Retroviruses and Opportunistic Infections. Los Angeles, CA, February 2007; 2007 [abstract 164].