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.
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