Dual-tropic HIV type 1 isolates vary dramatically in their utilization of CCR5 and CXCR4 coreceptors
Toma, Jonathan; Whitcomb, Jeannette M; Petropoulos, Christos J; Huang, Wei
Monogram Biosciences, South San Francisco, California, USA.
Received 15 January, 2010
Revised 5 May, 2010
Accepted 19 May, 2010
Correspondence to Dr Wei Huang, Monogram Biosciences, 345 Oyster Point Boulevard, South San Francisco, CA 94080, USA. Tel: +1 650 866 7429; fax: +1 650 624 4132; e-mail: email@example.com
Objective(s): Dual HIV-1 utilizes cellular CCR5 and CXCR4 coreceptors to enter host cells. Recent studies indicate that the ability of these viruses to use both coreceptors varies significantly in cell lines expressing CXCR4 or CCR5; however, it is not clear whether differences in coreceptor mediated infection in vitro reflect infection of primary cells in vivo.
Methods: We evaluated coreceptor usage of dual envelope clones from patient viruses using a single-cycle pseudovirus assay conducted in cell lines and a replication-competent assay performed using peripheral blood mononuclear cells. Dual envelope clones were selected and classified into three groups, R5>X4, R5≈X4, and X4>R5, based on their ability to mediate entry by using CXCR4 and CCR5 in a pseudovirus assay.
Results: We observed a high degree of concordance between measurements of coreceptor-mediated entry in pseudovirus and peripheral blood mononuclear cell assays. R5>X4 viruses were efficiently inhibited by a CCR5 antagonist, but not a CXCR4 antagonist, whereas X4>R5 viruses were efficiently inhibited by a CXCR4 antagonist, but not a CCR5 antagonist. R5≈X4 viruses were not inhibited, or only partially inhibited, by either a CCR5 or a CXCR4 antagonist alone.
Conclusions: These observations indicate that measurements of coreceptor use determined using pseudoviruses and coreceptor-expressing cell lines are generally concordant with the results obtained using replication-competent assays and peripheral blood mononuclear cell. This suggests that a considerable fraction of dual viruses preferentially infect either CCR5 or CXCR4 target cells in vivo. The clinical implications of preferential coreceptor utilization by dual viruses, that is, HIV-1 pathogenesis and response to coreceptor antagonists, require additional studies.
The HIV type 1 (HIV-1) envelope glycoproteins (Env) mediate HIV entry. Phenotypic coreceptor usage is typically determined in vitro by assessing the ability of a virus to infect CD4+ cell lines that express either CXCR4 or CCR5 [1,2], or based on syncytia induction in MT2 cells that express CD4 and CXCR4 . Viruses that exclusively utilize the CCR5 coreceptor for entry are defined as R5; those that exclusively use the CXCR4 coreceptor are defined as X4; viruses are classified as dual if they are able to use both CCR5 and CXCR4 . R5 viruses dominate the early stages of HIV infection whereas X4 and dual viruses (CXCR4-using viruses) frequently, but not always, arise later in the disease [5–7] and their emergence is associated with a poor clinical prognosis [8–10]. Widespread and intensive efforts to exploit coreceptor binding as an antiviral target highlight the importance of improving our understanding of HIV coreceptor use. Maraviroc (MVC; Pfizer, UK), a small molecule CCR5 antagonist, was recently approved by the US Food and Drug Administration for use in patients with R5 virus . Another CCR5 antagonist, vicriviroc (Schering-Plough, New Jersey, USA) and a CCR5-specific humanized monoclonal antibody, PRO 140 (Progenics, New York, USA) are currently being evaluated in phase III and phase IIb trials, respectively [12,13]. Several CXCR4 antagonists, including AMD3100 and AMD11070 (AnorMED, Langley, Canada), have also demonstrated potent antiviral activity against X4 virus [14–16]; however, the development of CXCR4 antagonists has trailed CCR5 antagonists owing to concerns over delivery and toxicity . In contrast to the potent and complete inhibition of X4 and R5 viruses by CXCR4 and CCR5 antagonists, respectively, the inhibition of dual viruses by either CXCR4 or CCR5 antagonists has not been fully characterized. We have reported that pseudoviruses produced using cloned dual envelope (env) sequences exhibit different levels of infectivity in U87 cell lines that were engineered to express CD4 and CCR5 or CXCR4 [18–21], whether these observed differences in coreceptor use in cell lines reflects the infectivity of dual viruses in primary cells has not been established.
In this study we characterized 32 dual env clones from 12 HIV-1-infected patients using a single-cycle pseudovirus assay in cell lines and replication-competent assay in primary cells. These dual env clones were obtained from the patient samples submitted to Monogram for coreceptor tropism testing and selected to capture the broad range of infectivity we have observed in the Trofile assay (Table 1). Based upon the amount of luciferase activity (i.e., infection) detected in U87/CD4/CCR5 and U87/CD4/CXCR4 target cells, we assigned each env clone to one of three coreceptor tropim classifications: 11 clones with higher infectivity in CCR5+ target cells [11 720–1 655 718 relative light units (RLU)] compared to CXCR4+ target cells (762–17 092 RLU) were classified as dual R5>X4, seven clones with higher infectivity in CXCR4+ target cells (71 954–811 378 RLU) compared to CCR5+ target cells (513–28 427 RLU) were defined as dual X4>R5, and 14 clones with similar infectivity in both CCR5+ (5552–1 429 209 RLU) and CXCR4+ target cells (6471–1 601 696 RLU) were defined as dual R5≈X4.
The ability of dual Env to mediate virus entry was further characterized by evaluating the infectivity of each dual pseudovirus in the presence of a CCR5 antagonist (Merck, New Jersey, USA) in U87/CD4/CCR5 cells or a CXCR4 antagonist AMD3100 in U87/CD4/CXCR4 cells. Dual pseudoviruses were further evaluated for their ability to infect cells expressing both CCR5 and CXCR4 (U87/CD4/CCR5/CXCR4) in the presence of either a CCR5 antagonist or a CXCR4 antagonist (Fig. 1). Irrespective of the group of dual env clones examined, complete or near complete inhibition of viral infection was observed when U87/CD4/CCR5 cells were infected in the presence of a CCR5 antagonist, or when U87/CD4/CXCR4 cells were infected in the presence of a CXCR4 antagonist (Fig. 1a, b and c). The inhibition of CCR5 or CXCR4 mediated entry of dual viruses is coreceptor specific. However, when U87/CD4/CCR5/CXCR4 target cells were used, dual (R5>X4) clones were effectively inhibited (mean 98%) by the CCR5 antagonist, but not by the CXCR4 antagonist (mean 0%) (Fig. 1d). Reciprocally, dual (X4>R5) clones were effectively inhibited (mean 94.1%) by the CXCR4 antagonist, but not by the CCR5 antagonist (mean 7.3%) (Fig. 1f). Infection by dual (R5≈X4) clones was not inhibited well by either the CCR5 antagonist (mean 31.0%) or the CXCR4 antagonist (mean 10.6%) (Fig. 1e).
Next, we evaluated the infectivity of replication competent dual viruses in primary lymphocytes (peripheral blood mononuclear cells, PBMC). A subset (n = 22, all clones are indicated in Table 1) of the env sequences that were evaluated in the pseudovirus assay were transferred into an infectious molecular clone of HIV-1 by using restriction sites created upstream and downstream of the env region of NL43. Viral stocks were generated by transfection of HEK 293 cells; 5 ng p24 antigen of virus was used to infect phytohemaglutinin stimulated PBMC at 2 × 105 cells per well in triplicate. The pooled PBMCs used for the assay were obtained from four HIV-negative individuals. Inoculates were removed the following day and cells were replenished with fresh medium. Viruses were collected at day 7 postinoculation and assessed for p24 production using an enzyme-linked immunosorbent assay. To assess inhibition, coreceptor antagonists were maintained throughout the course of infection; AMD3100 (0.5 μg/ml), CCR5 antagonist (0.674 μg/ml). Virus replication was determined by averaging the p24 production of the triplicates per each sample. The percent inhibition of infection was determined by comparing p24 production in the presence and absence of drugs. PBMC infections in the presence of either the CCR5 inhibitor or the CXCR4 inhibitor, or both inhibitors are shown in Fig. 1. Infection by dual (R5>X4) clones was effectively inhibited by the CCR5 antagonist (mean 98.7%), but not by the CXCR4 antagonist (mean 3.7%) (Fig. 1g). Reciprocally, infection by dual (X4>R5) clones was effectively inhibited by the CXCR4 antagonist (mean 98.9%), but not by the CCR5 antagonist (mean 14.7%) (Fig. 1i). Dual (R5≈X4) clones were not effectively inhibited by either the CCR5 antagonist (mean 10.6%) or the CXCR4 antagonist (mean 40.1%) (Fig. 1h). However, infection was completely inhibited when both CCR5 and CXCR4 antagonist were present (mean 98.7%) (Fig. 1g, h and i). Notably, our observations using replication-competent viruses in a PBMC assay were consistent with our observations using env pseudoviruses in U87 cell lines.
On the basis of the results we have obtained from single-cycle assays in cell lines and multicycle assays in primary cell cultures, we conclude that dual viruses vary broadly in their ability to use CXCR4 and CCR5 for infection. Certain dual viruses effectively use both the CCR5 and CXCR4 coreceptors whereas others preferentially use CCR5 or CXCR4. Emergence of CXCR4-using viruses during HIV infection is strongly associated with disease progression and consequently is thought to play an important role in virus pathogenesis [8–10]. It is possible that the differences in CCR5 and CXCR4 use of plasma-derived dual viruses that we have observed in this study may represent patient CXCR4-using variants at different stages of env evolution. Given the fact that CXCR4 is expressed in many more CD4+ cells in humans (including thymocytes, hematopoietic progenitor cells, naive T cells and monocytes) than CCR5 (mostly found on memory T cells and macrophages) [22,23], the acquisition of CXCR4 use in late infection may provide viruses with access to larger target cell populations, especially T cells, which in turn may explain the association between the emergence of CXCR4-using viruses and the decline of CD4+ cells in some patients [24,25]. The relative ability of dual viruses to utilize CXCR4 may have a critical impact on coreceptor switching and disease progression. Furthermore, our group and others have documented several cases of dual and X4 virus transmissions in newly infected adults and infants, suggesting that both horizontal and vertical transmission is also a potential source of CXCR4-using virus [20,21,26]. It is important to note that although dual viruses can exhibit the ability to use both coreceptors efficiently in vitro, changes in the availability of target cell populations in vivo may create conditions that favor the rapid evolution of viruses that use one coreceptor over the other.
We observed a striking association between coreceptor utilization and susceptibility to specific coreceptor antagonists. Dual (R5>X4) clones that display efficient use of CCR5, but not CXCR4, were effectively inhibited by a CCR5 antagonist, but not a CXCR4 antagonist, when evaluated in both single-cycle assays that use engineered cell lines and multicycle assays that use primary cells with both coreceptors (CCR5 and CXCR4). Conversely, dual (X4>R5) clones that efficiently use CXCR4, but not CCR5, were inhibited by a CXCR4 antagonist, but not by a CCR5 antagonist in both assay systems. Notably, dual (R5≈X4) clones that efficiently use both CXCR4 and CCR5 were incompletely inhibited by either the CCR5 or the CXCR4 antagonist in both assay systems. The results of this study suggest that the efficiency of CXCR4 and CCR5 use by dual viruses may impact responses to CCR5 and CXCR4 coreceptor antagonists in vivo and provide explanations for the observations seen in clinical trials, in which some dual viruses were not inhibited by either CXCR4  or CCR5  antagonists, whereas other dual viruses were inhibited by a CCR5 antagonist  or a CXCR4 antagonist . It is important to appreciate that the inhibition of viral infection by coreceptor antagonists in patients is more complex than with the individual virus clones studied here. Virus populations composed of mixtures of R5, dual, and/or X4 variants often exist in patients. In a phase I/II study, the CXCR4 antagonist AMD3100 successfully suppressed X4 virus populations, but not R5 nor dual/mixed virus populations . Clonal analysis of the dual/mixed viral populations demonstrated that AMD3100 was unable to inhibit dual variants that exhibited efficient CCR5 use , which is consistent with our findings reported here. However, AMD3100 appeared to suppress the replication of dual variants that utilize CXCR4 efficiently when variants were present as minor subpopulations amongst a larger population of R5 variants . We assert that the most likely explanation for these observations is that dual viruses are unable to effectively compete with R5 viruses when their replication is restricted to CCR5 cell populations. Similar results were recently obtained by examining the composition of dual/mixed virus populations that were inadvertently exposed to MVC in phase IIb/III clinical trials. In several cases, MVC was capable of suppressing virus subpopulations of dual viruses that utilized CXCR4 inefficiently, but not virus subpopulations within the same patient that utilized CXCR4 efficiently . Finally, we and others have shown that minor subpopulations of dual viruses that use CXCR4 efficiently can emerge and predominate when CCR5-mediated entry is blocked by MVC [28–31].
We would like to acknowledge the Monogram Biosciences Clinical Reference Lab for their assistance in performing the phenotypic tropism tests described and Cynthia Sedik for editorial assistance. This work was supported in part by Small Business Innovation Research grant R44-AI-048990.
1. Trouplin V, Salvatori F, Cappello F, Obry V, Brelot A, Heveker N, et al
. Determination of coreceptor usage of human immunodeficiency virus type 1 from patient plasma samples by using a recombinant phenotypic assay. J Virol 2001; 75:251–259.
2. 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.
3. Schuitemaker H, Koot M, Kootstra NA, Dercksen MW, de Goede RE, van Steenwijk RP, et al
. Biological phenotype of human immunodeficiency virus type 1 clones at different stages of infection: progression of disease is associated with a shift from monocytotropic to T-cell-tropic virus population. J Virol 1992; 66:1354–1360.
4. Berger EA, Doms RW, Fenyo EM, Korber BT, Littman DR, Moore JP, et al
. A new classification for HIV-1. Nature 1998; 391:240.
5. Berger EA, Murphy PM, Farber JM. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 1999; 17:657–700.
6. 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.
7. Melby T, Despirito M, Demasi R, Heilek-Snyder G, Greenberg ML, Graham N. HIV-1 coreceptor use in triple-class treatment-experienced patients: baseline prevalence, correlates, and relationship to enfuvirtide response. J Infect Dis 2006; 194:238–246.
8. Daar ES, Kesler KL, Petropoulos CJ, Huang W, Bates M, Lail AE, et al
. Baseline HIV type 1 coreceptor tropism predicts disease progression. Clin Infect Dis 2007; 45:643–649.
9. 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.
10. Brumme ZL, Goodrich J, Mayer HB, Brumme CJ, Henrick BM, Wynhoven B, et al
. Molecular and clinical epidemiology of CXCR4-using HIV-1 in a large population of antiretroviral-naive individuals. J Infect Dis 2005; 192:466–474.
11. Gulick RM, Lalezari J, Goodrich J, Clumeck N, DeJesus E, Horban A, et al
. Maraviroc for previously treated patients with R5 HIV-1 infection. N Engl J Med 2008; 359:1429–1441.
12. 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.
13. Jacobson JM, Saag MS, Thompson MA, Fischl MA, Liporace R, Reichman RC, et al
. Antiviral activity of single-dose PRO 140, a CCR5 monoclonal antibody, in HIV-infected adults. J Infect Dis 2008; 198:1345–1352.
14. 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.
15. Bridger GJ, Skerlj RT, Padmanabhan S, Martellucci SA, Henson GW, Struyf S, et al
. Synthesis and structure-activity relationships of phenylenebis(methylene)-linked bis-azamacrocycles that inhibit HIV-1 and HIV-2 replication by antagonism of the chemokine receptor CXCR4. J Med Chem 1999; 42:3971–3981.
16. Moyle G, DeJesus E, Boffito M, Wong R, Coakley E, Gibney C, et al. CXCR4 antagonism: proof of activity with AMD11070
. In: 14th Conference on Retroviruses and Opportunistic Infections
; 2007; Los Angeles, California, USA.
17. Hendrix CW, Collier AC, Lederman MM, Schols D, Pollard RB, Brown S, et al
. Safety, pharmacokinetics, and antiviral activity of AMD3100, a selective CXCR4 receptor inhibitor, in HIV-1 infection. J Acquir Immune Defic Syndr 2004; 37:1253–1262.
18. Huang W, Eshleman SH, Toma J, Fransen S, Stawiski E, Paxinos EE, et al
. Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations. J Virol 2007; 81:7885–7893.
19. Huang W, Toma J, Fransen S, Stawiski E, Reeves JD, Whitcomb JM, et al
. Coreceptor tropism can be influenced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein. J Virol 2008; 82:5584–5593.
20. Huang W, Toma J, Stawiski E, Fransen S, Wrin T, Parkin N, et al
. Characterization of human immunodeficiency virus type 1 populations containing CXCR4-using variants from recently infected individuals. AIDS Res Hum Retroviruses 2009; 25:795–802.
21. Huang W, Eshleman SH, Toma J, Stawiski E, Whitcomb JM, Jackson JB, et al
. Vertical transmission of X4-tropic and dual-tropic HIV-1 in five Ugandan mother-infant pairs. AIDS 2009; 23:1903–1908.
22. Berkowitz RD, Beckerman KP, Schall TJ, McCune JM. CXCR4 and CCR5 expression delineates targets for HIV-1 disruption of T cell differentiation. J Immunol 1998; 161:3702–3710.
23. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR. The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci U S A 1997; 94:1925–1930.
24. Grivel JC, Penn ML, Eckstein DA, Schramm B, Speck RF, Abbey NW, et al
. Human immunodeficiency virus type 1 coreceptor preferences determine target T-cell depletion and cellular tropism in human lymphoid tissue. J Virol 2000; 74:5347–5351.
25. Jekle A, Keppler OT, De Clercq E, Schols D, Weinstein M, Goldsmith MA. In vivo evolution of human immunodeficiency virus type 1 toward increased pathogenicity through CXCR4-mediated killing of uninfected CD4 T cells. J Virol 2003; 77:5846–5854.
26. Markowitz M, Mohri H, Mehandru S, Shet A, Berry L, Kalyanaraman R, et al
. Infection with multidrug resistant, dual-tropic HIV-1 and rapid progression to AIDS: a case report. Lancet 2005; 365:1031–1038.
27. Fransen S, Bridger G, Whitcomb JM, Toma J, Stawiski E, Parkin N, et al
. Suppression of dualtropic human immunodeficiency virus type 1 by the CXCR4 antagonist AMD3100 is associated with efficiency of CXCR4 use and baseline virus composition. Antimicrob Agents Chemother 2008; 52:2608–2615.
28. 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.
29. Lewis M, Mori J, Simpson P, Whitcomb J, Li X, Robertson D, Westby M. Changes in V3 loop sequence associated with failure of maraviroc treatment in patients enrolled in the MOTIVATE 1 and 2 trials
. In: 15th Conference on Retroviruses and Opportunistic Infections
; 3–6 February 2008; Boston, Massachusetts, USA.
30. Lewis M, Simpson P, Fransen S, Huang W, Whitcomb J, Mosley M, et al
. CXCR4-using virus detected in patients receiving maraviroc in the Phase III studies MOTIVATE 1 and 2 originates from a preexisting minority of CXCR4-using virus. Antivir Ther 2007; 12:S65. Abstract 56.
31. Archer J, Braverman MS, Taillon BE, Desany B, James I, Harrigan PR, et al
. Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing. AIDS 2009; 23:1209–1218.
This article has been cited 3 time(s).
Plos OneInhibition of Dual/Mixed Tropic HIV-1 Isolates by CCR5-Inhibitors in Primary Lymphocytes and MacrophagesPlos One
Frontiers in Bioscience-LandmarkConfronting JC virus and Homo sapiens biological signaturesFrontiers in Bioscience-Landmark
International Journal of Infectious DiseasesPrevalence of R5 strains in multi-treated HIV subjects and impact of new regimens including maraviroc in a selected group of patients with CCR5-tropic HIV-1 infectionInternational Journal of Infectious Diseases
CCR5; CXCR4; coreceptor antagonists; coreceptor tropism; dual; HIV; R5; X4
© 2010 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.
Readers Of this Article Also Read