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J Virol. Sep 2010; 84(17): 8777–8789.
Published online Jun 23, 2010. doi:  10.1128/JVI.00333-10
PMCID: PMC2919036

Distinct Molecular Pathways to X4 Tropism for a V3-Truncated Human Immunodeficiency Virus Type 1 Lead to Differential Coreceptor Interactions and Sensitivity to a CXCR4 Antagonist[down-pointing small open triangle]

Abstract

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.

For human immunodeficiency virus type I (HIV-1) to enter a target cell, the gp120 subunit of the viral envelope glycoprotein (Env) must engage CD4 and a coreceptor on the cell surface. Although numerous coreceptors have been identified in vitro, the two most important coreceptors in vivo are the CCR5 (3, 11, 19, 22, 24) and CXCR4 (27) chemokine receptors. HIV-1 variants that can use only CCR5 (R5 viruses) are critical for HIV-1 transmission and predominate during the early stages of infection (86, 90). The importance of CCR5 for HIV-1 transmission is underscored by the fact that individuals bearing a homozygous 32-bp deletion in the CCR5 gene (ccr5-Δ32) are largely resistant to HIV-1 infection (15, 49, 84). Although R5 viruses typically persist into late disease stages, viruses that can use CXCR4, either alone (X4 viruses) or in addition to CCR5 (R5X4 viruses), emerge in approximately 50% of individuals infected with subtype B or D viruses (12, 39, 44). Although not required for disease progression, the appearance of X4 and/or R5X4 viruses is associated with a more rapid depletion of CD4+ cells in peripheral blood and faster progression to AIDS (12, 44, 77, 86). However, it remains unclear whether these viruses are a cause or a consequence of accelerated CD4+ T cell decline (57). The emergence of CXCR4-using viruses has also complicated the use of small-molecule CCR5 antagonists as anti-HIV-therapeutics as these compounds can select for the outgrowth of X4 or R5X4 escape variants (93).

Following triggering by CD4, gp120 binds to a coreceptor via two principal interactions: (i) the bridging sheet, a four-stranded antiparallel beta sheet that connects the inner and outer domains of gp120, together with the base of the V3 loop, engages the coreceptor N terminus (NT); and (ii) more distal regions of V3 interact with the coreceptor extracellular loops (ECLs) (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Although both the NT and ECL interactions are important for coreceptor binding and entry, their relative contributions vary among different HIV-1 strains (23). For example, V3 interactions with the ECLs, particularly ECL2, serve a dominant role in CXCR4 utilization (7, 21, 50, 63, 72), while R5 viruses exhibit a more variable use of CCR5 domains, with the NT interaction being particularly important (4, 6, 20, 67, 83). Although V3 is the primary determinant of coreceptor preference (34), it is unclear how specificity for CCR5 and/or CXCR4 is determined, and, in particular, it is unknown how X4 tropism is acquired. Several reports have shown that the emergence of X4 tropism correlates with the acquisition of positively charged residues in the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 75), raising the possibility that these mutations directly or indirectly mediate interactions with negatively charged residues in the CXCR4 ECLs. However, Env domains outside V3, including V1/V2 (9, 32, 45, 46, 61, 64, 65, 80, 95) and even gp41 (40), can also contribute to coreceptor switching, and it is unclear mechanistically or structurally how X4 tropism is determined.

We previously derived a replication-competent variant of the R5X4 HIV-1 clone R3A that contained a markedly truncated V3 loop (47). This Env was generated by introducing a mutation termed ΔV3(9,9), which deleted the distal 15 amino acids of V3. The ΔV3(9,9) mutation selectively ablated X4 tropism but left R5 tropism intact, consistent with the view that an interaction between the distal half of V3 and the ECLs is critical for CXCR4 usage (7, 21, 43, 50, 59, 60, 63, 72). This V3-truncated virus provided a unique opportunity to address whether CXCR4 utilization could be regained on a background in which this critical V3-ECL interaction had been ablated and, if so, by what mechanism. Here, we characterize two novel X4 variants of R3A ΔV3(9,9) derived by adapting this virus to replicate in CXCR4+ CCR5 SupT1 cells. We show that R3A ΔV3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 variant, designated ΔV3-X4A, acquired changes in the V3 remnant that reestablished an interaction with the CXCR4 ECLs; the other, ΔV3-X4B, acquired changes outside V3 that engendered interactions with the CXCR4 NT. These divergent evolutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ΔV3-X4A showing increased sensitivity relative to R3A and with ΔV3-X4B becoming completely resistant. These findings demonstrate the contributions that interactions with distinct coreceptor regions have in mediating tropism and drug sensitivity and illustrate how HIV's remarkable evolutionary plasticity in adapting to selection pressures can be exploited to better understand its biological potential.

MATERIALS AND METHODS

Cells.

The human SupT1 T lymphoblastoid cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 2 mM penicillin-streptomycin (RPMI-complete). SupT1 cells that stably express human CCR5 (SupCCR5) (52) were maintained in RPMI-complete medium with 300 ng/ml puromycin. A single cell clone of SupT1 cells, termed Sup-Zfn/X4, was generated using zinc finger nucleases (ZFN) (Sangamo) (56, 66, 85) targeting the CXCR4 gene (18; also J. Wang et al., unpublished data). Sup-Zfn/X4 cells were engineered to stably express CCR5 (Sup-Zfn/X4R5+) or CXCR4 (Sup-Zfn/X4+) by transduction with a CCR5- or CXCR4-containing pELNS replication-defective lentiviral vectors, generated as previously described (76). Following transduction, coreceptor-positive cells were isolated using fluorescence-activated cell sorting (FACS) and the anti-CCR5 antibody 2D7 or the anti-CXCR4 antibody 12G5. Note that Sup-Zfn/X4, Sup-Zfn/X4R5+, and Sup-Zfn/X4+ were previously referred to as SupX4, SupX4R5+, and SupX4+, respectively (18). The Japanese quail fibrosarcoma cell line QT6 and the human embryonic kidney cell line 293T were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, and 2 mM penicillin-streptomycin.

Adaptation of R3A ΔV3(9,9) to SupT1 cells.

The generation and adaptation of clade B HIV-1 R3A containing the ΔV3(9,9) mutation, which has a deletion all but the first and last nine amino acids of the V3 loop (not counting the disulfide-bonded cysteines) and includes a Gly-Ala-Gly linker, has been described previously (47). A replication-competent virus bearing the R3A ΔV3(9,9) env on an NL4-3 backbone derived by serial propagation in SupR5R cells, which expressed CCR5 and DC-SIGN-R, retained R5 tropism but was unable to use CXCR4 (47). To adapt this Env to reacquire CXCR4 use, we inoculated a 1:10 mix of SupCCR5 and SupT1 cells with the uncloned, adapted R3A ΔV3(9,9) viral swarm from which the TA1 Env clone was derived (47). Infection was monitored by immunofluorescence microscopy (IFA) using an anti-p24Gag monoclonal antibody (25.4; kindly provided by Jan McClure, University of Washington). A spreading infection was established, and virus-containing supernatants were serially passaged in 1:10 mixes of SupCCR5 and SupT1 cells until infection spread to >10% of the cells, at which point virus-containing supernatants were serially passaged in uninfected SupT1 cells.

Env cloning, plasmid construction, and mutagenesis.

Plasmid pHSPG-R3A, containing the HIV-1 R3A envelope, and plasmid pHSPG-TA1, containing the adapted R3A ΔV3(9,9) env clone TA1, have been described previously (47, 55). To isolate adapted env clones from infected SupT1 cultures, genomic DNA was prepared using a QIAamp DNA minikit (Qiagen) according to the manufacturer's instructions, and env sequences were PCR amplified using HotStar Taq (Invitrogen) and primers that flank the env region. PCR products were then cloned using TOPO TA into pCR2.1 (Invitrogen) and screened for env inserts using restriction analysis and DNA sequencing. Clones chosen for further evaluation were digested with EcoRI and XhoI and ligated to the pHSPG-R3A expression construct and the pNL4-3 HIV-1 genome construct. The identities of the recombinant clones were confirmed using restriction analysis and DNA sequencing. Mutant env genes in pHSPG were created using a QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's protocol. The identities of the mutations were confirmed by DNA sequencing. Selected mutant env genes were digested with EcoRI and XhoI and ligated to the pNL4-3 HIV-1 genome construct to generate recombinant replication-competent viruses. Expression constructs containing CD4, CCR5, and CXCR4 cDNAs and the reporter plasmid encoding luciferase under the control of a T7 promoter have been described previously (81). Expression constructs containing the CXCR4/CXCR2 chimeras have been described previously (21).

Cell-cell fusion assay.

Cell-cell fusion assays were performed as previously described (25, 81, 82). Briefly, effector QT6 cells were generated by infecting cells with the recombinant vaccinia strain VTF1.1 expressing T7 polymerase (2) at a multiplicity of infection of 10 for 1 h at 37°C and then transfecting cells for 5 h with the appropriate env expression vector using the standard calcium phosphate method. Following transfection, effector cells were incubated overnight at 32°C in the presence of rifampin at a concentration of 100 μg/ml. Target QT6 cells were generated by transfection with the desired receptor expression vectors and a T7-luciferase reporter construct by the standard calcium phosphate method for 5 h, followed by overnight expression at 37°C. Effector cells were then added to target cells in the presence of 100 μg/ml rifampin and 100 nM cytosine arabinoside, and cell-cell fusion was assessed 7 to 8 h later by lysing cells with 0.5% Triton X-100—phosphate-buffered saline, adding luciferase substrate (Promega), and quantifying luciferase activity with a Thermo LabSystems Luminoskan Ascent luminometer. Background fusion levels with cells expressing only CD4 were determined and subtracted out prior to data normalization. For AMD3100 inhibition experiments, serial dilutions of AMD3100 (obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) were added to target cells at the time of addition of effector cells, and inhibition of fusion was measured as the reduction in luciferase activity relative to activity in the untreated control.

Replication-competent infection assays.

To generate molecularly cloned viruses, 293T cells were transfected with recombinant pNL4-3 constructs containing selected env genes for 5 h by the standard calcium phosphate method. Cell supernatants were collected 48 h posttransfection and stored at −80°C. Virus concentrations were determined by an enzyme-linked immunosorbent assay for the viral p24 antigen (Perkin-Elmer). SupT1, Sup-Zfn/X4, Sup-Zfn/X4R5+, and Sup-Zfn/X4+ cells were inoculated with equivalent amounts of p24-containing virus. Following an overnight incubation at 37°C, cells were washed in RPMI medium supplemented with 5% FBS to remove excess virus, and viral replication was monitored by measuring viral reverse transcriptase (RT) activity in the culture supernatants. For AMD3100 inhibition experiments, target cells were incubated with various concentrations of AMD3100 at 37°C for 1 h prior to the addition of virus, and AMD3100 was maintained in the culture at the desired concentration throughout the course of the infection.

Flow cytometry.

SupT1 cells were washed and then preincubated in the absence or presence of various concentrations of AMD3100 in FACS buffer for 1 h on ice. Cells were then stained with 4 μg/ml of the anti-CXCR4 monoclonal antibody 12G5 (26) for 30 min in the presence of drug. Cells were washed, and antibody binding was detected with an affinity-purified fluorescein isothiocyanate (FITC)-conjugated secondary antibody and analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer and CellQuest software (Becton Dickinson). Untreated control cells were stained with a primary isotype control antibody to determine background fluorescence levels.

RESULTS

Adaptation of R3A ΔV3(9,9) to infect SupT1 cells.

We previously described the derivation of an R5-tropic, V3-truncated Env (47) from the dual tropic HIV-1 R3A (54). This Env contained a mutation, termed ΔV3(9,9), that introduced a 15-amino-acid deletion in the distal half of V3, leaving only the first and last 9 amino acids (not counting the disulfide-bonded cysteines) connected by a Gly-Ala-Gly linker. As previously described, in cell-cell fusion assays, the ΔV3(9,9) mutation resulted in a selective loss of X4 tropism with CCR5 usage being maintained, albeit at reduced levels (Fig. (Fig.1).1). When an NL4-3 virus bearing the R3A ΔV3(9,9) env was adapted for replication in SupR5R cells, which expressed both CXCR4 and CCR5, it maintained a truncated V3 loop and remained exclusively R5 tropic. A functional env clone, termed TA1, derived from this viral swarm was similarly unable to use CXCR4 for entry, consistent with the view that distal regions of V3 are essential for X4 tropism (47). To determine if viruses containing the ΔV3(9,9) truncation could reacquire X4 tropism, we inoculated a 1:10 mix of SupCCR5 (CXCR4+ CCR5+) and SupT1 cells (CXCR4+ CCR5), respectively, with the adapted R3A ΔV3(9,9) swarm from which TA1 was cloned (Fig. (Fig.1).1). Virus-containing supernatants were serially passaged until >10% of the mixed culture was positive for p24Gag by immunofluorescence microscopy (not shown) and then serially passaged on SupT1 cells alone. This adaptation scheme was carried out twice, yielding two viruses that could infect SupT1 cells with rapid kinetics. The first virus, designated ΔV3-X4A, was passaged 11 times in the SupCCR5-SupT1 mix, followed by 20 passages in SupT1; the second virus, designated ΔV3-X4B, was passaged seven times in the SupCCR5-SupT1 mix followed by 10 passages in SupT1 (Fig. (Fig.11).

FIG. 1.
Scheme summarizing the derivation of the ΔV3-X4A and ΔV3-X4B viruses. Mutagenesis and in vitro adaptation steps are indicated in italics. Introduction of the ΔV3(9,9) mutation in HIV-1 R3A and adaptation to SupR5R cells have been ...

ΔV3-X4A and ΔV3-X4B Envs acquired CXCR4 use.

To determine if the ΔV3-X4A and ΔV3-X4B viruses had reacquired X4 tropism, env clones were PCR amplified from genomic DNA of infected SupT1 cultures and evaluated in a cell-cell fusion assay with QT6 target cells coexpressing CD4 and either CCR5 or CXCR4 (Fig. (Fig.22 A). As previously reported (47), while parental R3A Env could use both CCR5 and CXCR4, TA1 could induce fusion only with CCR5-expressing cells at a level 50% of R3A. In contrast, for ΔV3-X4A clones, all four tested Envs were able to fuse using CXCR4 at levels of 30 to 60% of R3A but used CCR5 poorly with fusion levels of <5% of R3A. For ΔV3-X4B Envs, all four tested clones could use CXCR4 at levels of 39 to 64% of R3A, but unlike the ΔV3-X4A Envs they retained R5 tropism with fusion levels of 32 to 50%. To determine if ΔV3-X4A and ΔV3-X4B env clones could confer the ability to replicate in SupT1 to a replication-competent virus, we generated recombinant NL4-3 viruses containing the ΔV3-X4A.c5 or ΔV3-X4B.c7 env clones and compared their growth to that of TA1 and R3A. As expected, dual-tropic R3A replicated in SupT1 cells with rapid kinetics, reaching peak RT levels (>1.0 × 105 cpm) by day 3, while purely R5-tropic TA1 virus did not replicate in SupT1 and showed only background RT levels up to 20 days postinoculation (Fig. (Fig.2B).2B). As shown in Fig. Fig.33 B, TA1 could replicate in CCR5+ SupT1 cells. In contrast, ΔV3-X4A.c5 and ΔV3-X4B.c7 viruses both replicated with rapid kinetics in SupT1, with peak RT levels (1.3 × 105 and 1.1 × 105 cpm, respectively) by day 6 (Fig. (Fig.2B).2B). Similar to R3A, infection of SupT1 by ΔV3-X4A.c5 and ΔV3-X4B.c7 viruses was highly cytopathic, with extensive syncytia formation and cell killing (not shown).

FIG. 2.
Function of ΔV3-X4A and ΔV3-X4B env clones. (A) Coreceptor use in a cell-cell fusion assay for ΔV3-X4A and ΔV3-X4B env clones. Percent fusion was calculated by using luciferase activity normalized to R3A fusion with QT6 ...
FIG. 3.
Virus growth using CXCR4-negative SupT1 cells to assess coreceptor use. Growth curves for recombinant NL4-3 viruses containing the R3A (A), TA1 (B), ΔV3-X4A.c5 (C), or ΔV3-X4B.c7 (D) env are shown in parental SupT1, Sup-Zfn/X4 ...

To confirm that the ability of ΔV3-X4A.c5 and ΔV3-X4B.c7 viruses to infect SupT1 resulted from their reacquiring X4 tropism, we used a novel CXCR4-negative variant of SupT1 (Sup-Zfn/X4), generated using zinc-finger nuclease (ZFN) technology to disrupt the CXCR4 alleles (reference 18; also Wang et al., unpublished) (Fig. (Fig.3).3). ZFN technology has also been used to disrupt the CCR5 gene in human T-cell lines and primary lymphocytes, rendering them resistant to infection by R5-tropic HIV-1 strains (56, 66). Sup-Zfn/X4 cells were also engineered to stably express CCR5 (Sup-Zfn/X4R5+) or to reexpress CXCR4 (Sup-Zfn/X4+). As shown in Fig. Fig.3A,3A, R3A could replicate in wild-type SupT1 cells but not in Sup-Zfn/X4 cells, which lacked both CXCR4 and CCR5. R3A could also infect Sup-Zfn/X4+ and Sup-Zfn/X4R5+ cells, demonstrating that these ZFN-treated cells remained fully competent for X4- and R5-mediated infection when they expressed CXCR4 or CCR5, respectively (Fig. (Fig.3A).3A). As expected, TA1 replicated in Sup-Zfn/X4R5+ cells but not SupT1, Sup-Zfn/X4, or Sup-Zfn/X4+ cells, which all lacked CCR5 (Fig. (Fig.3B).3B). In contrast, ΔV3-X4A.c5 and ΔV3-X4B.c7 viruses were able to replicate in SupT1 and Sup-Zfn/X4+ cells but not in Sup-Zfn/X4 cells (Fig. 3C and D). Surprisingly, ΔV3-X4B.c7 was unable to replicate in Sup-Zfn/X4R5+ cells, despite the fact that this Env could use CCR5 in cell-cell fusion assays (Fig. (Fig.2A).2A). Nonetheless, these findings clearly demonstrated that both ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs regained X4 tropism during adaption in SupT1 cells.

Mutations in the ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs.

We previously reported that, during adaptation to SupR5R cells, TA1 maintained the ΔV3(9,9) truncation and acquired several mutations in Env compared to the parental R3A sequence, including a deletion of residues 185 to 188 in V1/V2 (designated 185-188Del), R254K in C2, an A to V change in the G-A-G linker in V3, T342A in C3, and A509V at the gp41 N terminus (47) (Fig. (Fig.4).4). When the SupT1-adapted ΔV3-X4A.c5 and ΔV3-X4B.c7 env clones were sequenced, several additional changes were seen compared to the sequence of TA1. The ΔV3-X4A.c5 env acquired 10 mutations in gp120 and 2 in gp41 (Fig. (Fig.44 and Table Table1,1, which also gives HXBc2 numbering). Most striking among these mutations was a 7-amino-acid insertion in the V3 loop (RGRKGVG) that contained three positively charged residues, increasing the V3 length from 23 to 30 amino acids and increasing its net positive charge to +9. This insertion likely resulted from a duplication of the preceding sequence NTRKGVG, followed by point mutations (i.e., N to R and T to G). Six of the other ΔV3-X4A.c5 gp120 mutations resulted in the loss of five putative N-linked glycosylation sites: T305I in V3, T358I in C3, and N392S/T394I, T398A, and T408I in V4. In addition, ΔV3-X4A.c5 contained mutations M98I, M163R, and M296I in gp120 and A579V and T815I in gp41.

FIG. 4.
Alignment of Env sequences for R3A, TA1, ΔV3-X4A.c5, and ΔV3-X4B.c7. Locations of gp120 variable domains (V1/V2, V3, V4, and V5) are indicated. Putative N-linked glycosylation sites are indicated by black dots. Clone TA1 from the adapted ...
TABLE 1.
Amino acid mutations in ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs

The ΔV3-X4B.c7 env acquired 18 mutations (14 in gp120 and 4 in gp41) of which only three (T358I, T398A, and T408I in gp120) were shared with ΔV3-X4A.c5. Although ΔV3-X4B.c7 contained T305A in V3, ablating the same N-linked glycosylation site that was lost in ΔV3-X4A.c5, it maintained the ΔV3(9,9) truncation and did not develop a V3 insertion (Fig. (Fig.44 and Table Table1).1). Similar mutations between ΔV3-X4B.c7 and ΔV3-X4A.c5 included the loss of five of the same predicted N-linked glycosylation sites, three via identical mutations (T358I in C3 and T398A and T408I in V4) and two via distinct mutations (T305A in V3 and N340S in C3). In addition, ΔV3-X4B.c7 lost a glycosylation site in V1/V2 (N128D), acquired two positively charged residues in regions within the bridging sheet (Q205R in the V1/V2 stem and S435R in C4), and lacked the deletion of residues 185 to 188 in V1/V2, leaving an R3A N-linked glycan intact. It also contained four additional point mutations in gp120 (M146V, L177P, V273I, and T461N) and four point mutations in gp41 (A538T, A659S, M684I, and R693K).

Determinants of CXCR4 use for ΔV3-X4A.c5 Env.

Because the V3 loop is the principal determinant of coreceptor use (34), we hypothesized that the RGRKGVG insertion in the ΔV3-X4A.c5 V3 loop was primarily responsible for conferring X4 tropism to this Env. Indeed, when this sequence was inserted into the TA1 Env (designated TA1 ΔV3-X4A V3Ins) and its coreceptor use was evaluated in a cell-cell fusion assay, it was able to use CXCR4 at a level of 69% of ΔV3-X4A.c5 (Fig. (Fig.55 A). The addition of ΔV3-X4A V3Ins also led to a reduction in CCR5 use to 24% of that of TA1 (Fig. (Fig.5B).5B). An NL4-3 virus bearing the TA1 ΔV3-X4A V3Ins Env replicated in SupT1 cells although its kinetics were delayed compared with replication of ΔV3-X4A.c5 (see Fig. S1 in the supplemental material). Interestingly, the env genes PCR amplified from these infected SupT1 cells at peak virus replication all had acquired the T305I mutation in V3 (data not shown), a change that was present in the ΔV3-X4A.c5 Env clone. Thus, although the V3 insertion was a principal determinant for ΔV3-X4A.c5's X4 tropism, the loss of the N-linked glycan in V3 further contributed to CXCR4 use.

FIG. 5.
ΔV3-X4A determinants for CXCR4 use in cell-cell fusion. Fusion for the TA1 env containing the ΔV3-X4A.c5 7-amino-acid V3 loop insertion with CD4+ CXCR4+(A) or CD4+ CCR5+ (B) QT6 cells. Percent fusion was ...

Determinants of CXCR4 use for ΔV3-X4B.c7.

In contrast to ΔV3-X4A.c5, the ΔV3-X4B.c7 Env maintained a truncated V3 loop, indicating that mutations outside V3 likely conferred CXCR4 utilization to this Env. To identify determinants of X4 tropism, we first changed individual mutations in ΔV3-X4Bc.7 back to the TA1 amino acid sequence; however, no single reversion was found to diminish CXCR4 fusion (data not shown). We next introduced into TA1 ΔV3-X4B.c7 either mutations that ablated N-linked glycosylation sites (N128D, T305A, N340S, T358I, and T398A) or a charge change (Q205R and S425R) individually or in various combinations (Fig. (Fig.66 A and C). In addition, residues 185 to 188, deleted in TA1 but present in ΔV3-X4B.c7, were restored in TA1 (185-188Ins), reintroducing an N-linked glycan in V1/V2. We did not evaluate the T408I mutation because, although not present in TA1, this change was present in purely R5-tropic Envs in the culture from which TA1 was derived (data not shown) and was not considered to be contributory to X4 tropism. Three mutations, N128D, 185-188Ins, and T305A, conferred a small but significant increase in CXCR4-dependent fusion (9 to 12% of ΔV3-X4B.c7; P < 0.03, Student's t test) (Fig. (Fig.6A).6A). Although no significant increase was seen when any two of these mutations were combined, when all three were added, CXCR4-mediated fusion increased significantly to 42% of ΔV3-X4B.c7 (P < 0.01). However, no additional single mutation from ΔV3-X4B.c7 when added to the TA1 N128D/185-188Ins/T305A triple mutant resulted in any further increase in CXCR4-mediated fusion (data not shown). To confirm the importance of these three mutations to CXCR4 use, N128D and T305A were removed and 185-188Del was reintroduced into ΔV3-X4B.c7 in combinations (Fig. (Fig.6B).6B). When any two of these changes were made in ΔV3-X4B.c7, CXCR4-dependent fusion decreased 36 to 63%, while combining all three changes reduced fusion to 17% of ΔV3-X4B.c7. Although N128D, 185-188Ins, and T305A were critical for CXCR4 utilization in cell-cell fusion assays, when introduced into TA1 these changes failed to confer infectivity on SupT1 cells, indicating that full use of CXCR4 in the context of a viral infection assay still required contributions from additional Env mutations (data not shown).

FIG. 6.
ΔV3-X4B determinants for CXCR4 use in cell-cell fusion. Fusion for the TA1 env containing ΔV3-X4B.c7 mutations alone and in combination with CD4+ CXCR4+(A) or CD4+ CCR5+ (C) QT6 cells and for the ΔV3-X4B.c7 ...

We also evaluated the effects of these same changes on CCR5 utilization. When introduced into TA1, most of the individual ΔV3-X4B.c7 mutations either had no effect or increased CCR5-mediated fusion (Fig. (Fig.6C).6C). However, T305A, which removed an N-linked glycan from V3, ablated CCR5 use when added alone or in combination with N128D and/or 185-188Ins. Similarly, when this glycan was restored in ΔV3-X4B.c7 alone (not shown) or in association with D128N and/or 185-188Del, CCR5-dependent fusion increased 5- to 6-fold (Fig. (Fig.6C).6C). Thus, the loss of this N-linked glycan in V3 not only contributed to increased X4 tropism but also negatively affected R5 tropism. Reduced CCR5 use in association with the loss of this glycan has been reported for other HIV-1 strains (68, 69).

AMD3100 sensitivity of ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs.

Although ΔV3-X4A.c5 and ΔV3-X4B.c7 both gained the ability to use CXCR4, distinct mutations in each clone were involved, suggesting that these Envs could be interacting with CXCR4 differently. To evaluate this possibility, we assessed their sensitivity to the CXCR4 antagonist AMD3100. This small-molecule CXCR4 antagonist cross-links membrane-proximal aspartic acid residues in the CXCR4 extracellular loops (16, 31, 35) and likely inhibits interactions with the HIV-1 V3 loop by an allosteric mechanism, altering the repertoire of ECL conformations to prevent gp120 binding (16, 91). We first assessed the AMD3100 sensitivity of R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 Envs in a cell-cell fusion assay with CD4+ CXCR4+ target cells (Fig. (Fig.77 A). As shown previously (60), R3A was inhibitable by AMD3100, with a 50% inhibitory concentration (IC50) of 314 nM. In comparison, ΔV3-X4A.c5 was nearly 25-fold more sensitive, with an IC50 of 12.3 nM. In contrast, ΔV3-X4B.c7 was completely resistant to AMD3100, even at concentrations as high as 1,000 nM. To evaluate AMD3100 sensitivity in the context of viral infection, SupT1 cells were inoculated with NL4-3 viruses bearing the R3A, ΔV3-X4A.c5, or ΔV3-X4B.c7 Envs in the presence of various concentrations of AMD3100 (Fig. 7B to D). R3A replication was inhibitable, showing delayed replication at 1,000 nM and complete inhibition at 10,000 nM (Fig. (Fig.7B).7B). In agreement with results from the cell-cell fusion assay, ΔV3-X4A.c5 was markedly more sensitive to AMD3100, with complete inhibition observed at concentrations of ≥100 nM. ΔV3-X4B.C7 was again completely resistant to AMD3100, with no differences in replication kinetics or peak virus production at concentrations as high as 10,000 nM. Flow cytometric analysis using 12G5, an anti-CXCR4 monoclonal antibody whose binding is inhibited by AMD3100, showed that 10,000 nM AMD3100 completely inhibited 12G5 binding to SupT1 cells, demonstrating that this concentration was saturating (see Fig. S2 in the supplemental material). Because replication of ΔV3-X4B.c7 in SupT1 is dependent on CXCR4 (Fig. (Fig.3D),3D), these results clearly indicate that ΔV3-X4B.c7 is able to use AMD3100-bound CXCR4. Thus, although ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs both acquired the ability to use CXCR4, they exhibited distinct and profound differences in their sensitivity to inhibition by this CXCR4 antagonist.

FIG. 7.
Sensitivity to the CXCR4 antagonist AMD3100. (A) Inhibition of R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 by the indicated concentrations of AMD3100 in a cell-cell fusion assay. Percent fusion was calculated by using luciferase activity normalized ...

Use of CXCR2/CXCR4 chimeras by ΔV3-X4A.c5 and ΔV3-X4B.c7 Envs.

Because AMD3100 is thought to inhibit the interaction of V3 with the CXCR4 ECLs, the sensitivity of ΔV3-X4A.c5 to AMD3100 suggested that an interaction with the ECLs was critical for its entry. Conversely, the complete resistance of ΔV3-X4B.c7 to AMD3100 suggested that this Env functioned independently or with less dependence on the ECLs and, instead, was more dependent on the CXCR4 NT or could utilize the ECLs even when bound by drug. To distinguish between these possibilities, chimeric coreceptors generated by swapping domains between CXCR4 and the nonpermissive coreceptor CXCR2 (21) were used in a cell-cell fusion assay (Fig. (Fig.88 A). One chimera, designated 4222, contained the CXCR4 N terminus (NT) grafted onto the CXCR2 ECLs, while the reciprocal chimera, designated 2444, contained the CXCR2 NT grafted onto the CXCR4 ECLs. Both parental R3A and ΔV3-X4A.c5 Envs could induce fusion with target cells that coexpressed CD4 with either CXCR4 or 2444 but not with 4222 or CXCR2, indicating that both principally interacted with the CXCR4 ECLs and were less dependent on the CXCR4 NT. In contrast, ΔV3-X4B.c7 could induce fusion with target cells that coexpressed CD4 and 4222 to a level comparable to its use of CXCR4, suggesting that this Env had acquired a stronger interaction with the CXCR4 NT. However, although ΔV3-X4B.c7 showed some background use of CXCR2 (6% of its CXCR4-mediated fusion), this Env could still induce fusion with 2444-expressing target cells (58% of CXCR4-dependent fusion), suggesting that it also had the ability to interact with the CXCR4 ECLs. To evaluate this possibility, the AMD3100 sensitivities of R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 were determined using the 2444 chimera (Fig. (Fig.8B).8B). As expected, R3A and ΔV3-X4A.c5 were inhibited by AMD3100 (1,000 nM) on both CXCR4- and 2444-expressing target cells. However, while ΔV3-X4B.c7 fusion on CXCR4 was again completely resistant to AMD3100, fusion with the 2444 chimera was sensitive to AMD3100, with an 80% reduction at 1,000 nM. Thus, although ΔV3-X4B.c7 could utilize the CXCR4 NT (i.e., on the 4222 chimera) in an AMD3100-resistant manner, this Env, even with its truncated V3, still retained some capacity for an AMD3100-inhibitable interaction, most likely with the CXCR4 ECLs. However, because ΔV3-X4B.c7 fusion on wild-type CXCR4 was resistant to AMD3100, the NT interaction was likely dominant.

FIG. 8.
CXCR4 determinants for ΔV3-X4A and ΔV3-X4B fusion. (A) Use of CXCR4/CXCR2 coreceptor chimeras (21) in a cell-cell fusion assay for R3A, ΔV3-X4A.c5, and ΔV3-X4B.c7 env clones. The 4222 chimera contained a CXCR4 NT domain ...

DISCUSSION

The HIV-1 Env engages CCR5 and CXCR4 through complex and likely cooperative interactions involving (i) the base of V3 and the bridging sheet domain with the coreceptor NT and (ii) an association of distal portions of V3 with the coreceptor ECLs (13, 14, 36-38, 43, 59, 60, 78, 79, 88). Viruses that initiate infection use only CCR5 and, as a correlate of progressing immunodeficiency, can acquire the ability to use CXCR4 (12, 44, 77, 86, 90). Although the V3 loop largely determines coreceptor specificity and is a critical determinant for this R5-to-X4 coreceptor switch in vivo, the structural basis for coreceptor specificity and the evolution of X4 tropism are unknown. Themes for CXCR4 use have included the acquisition of positively charged amino acids within the V3 stem (17, 29, 87), particularly at positions 11, 24, and 25 (8, 17, 28, 29, 42, 77), the loss of an N-linked glycan at the V3 base (68, 69), and greater V3 exposure, as determined by binding of anti-V3 antibodies (51). However, regions outside V3 have also been implicated in CXCR4 utilization (9, 10, 32, 40, 45, 46, 61, 64, 65, 95), and the mechanisms that underlie how X4 tropism can be acquired are unclear.

In contrast to the V1/V2 and V4 loops, the V3 loop is highly conserved in length, with most HIV-1 V3 loops containing 34 to 35 amino acids, suggesting that there are strong evolutionary pressures to maintain this length for coreceptor engagement (38). In this study, we developed a novel model for evaluating X4 tropism using a recently described HIV-1 variant, termed R3A ΔV3(9,9), that was adapted to replicate in vitro with a 15-amino-acid deletion of the distal half of its V3 loop (47). This ΔV3(9,9) mutation, when introduced into the dual-tropic HIV-1 R3A Env, ablated X4 but not R5 tropism, increased dependence for entry on the CCR5 NT, and conferred resistance to small-molecule CCR5 antagonists that interact with the ECLs to prevent V3 binding (47). Recent evidence has indicated that in the face of a weakened CCR5 interaction, this virus also became more dependent on CD4 for entry (1). The availability of a replication-competent virus with only a 23-amino-acid V3 loop provided a unique opportunity to explore determinants and mechanisms whereby X4 tropism could be acquired on a background in which V3's contribution to CXCR4 use had been eliminated. When this virus was twice adapted to replicate on CXCR4+ CCR5 SupT1 cells, two X4-tropic HIV-1 Envs were derived, ΔV3-X4A.c5 and ΔV3-X4B.c7, each of which exhibited a distinct evolutionary pathway for regaining X4 tropism. While ΔV3-X4A.c5 acquired CXCR4 use through a 7-amino-acid insertion in V3, ΔV3-X4B.c7 maintained a truncated V3 and, instead, with the exception of a lost glycosylation site, acquired mutations that were outside V3. These divergent pathways led to differences in how these Envs interacted with CXCR4, as evidenced by their differential sensitivity to AMD3100 and their use of CXCR4/CXCR2 chimeric coreceptors that isolated interactions with the CXCR4 NT and ECLs.

For ΔV3-X4A.c5, an RGRKGVG insertion in V3 was sufficient to confer CXCR4 use to the R5-tropic TA1 Env in cell-cell fusion assays and to enable a virus containing this insertion to infect SupT1 cells. Although the additional loss of the N-linked glycan in the V3 base at position 305 correlated with more rapid replication in SupT1, these findings suggested that the ability of ΔV3-X4A.c5 V3 to engage CXCR4 ECLs had been restored. Indeed, ΔV3-X4A.c5 could use the 2444 chimera that contained only the CXCR4 ECLs but not the reciprocal 4222 chimera that contained only the CXCR4 NT. However, compared to parental R3A, ΔV3-X4A.c5 exhibited a 25-fold increase in sensitivity to AMD3100, which alters the conformation of the CXCR4 ECL (16, 31, 35), likely indicating that its interaction with the ECLs is suboptimal and thus more easily inhibited. Although we did not formally investigate the determinants within this insert that conferred CXCR4 usage, ΔV3-X4A.c5 still has a V3 loop that is much shorter than that of typical HIV-1s (i.e., 30 amino acids), and it may be compromised in binding to CXCR4. We have previously shown that R3A containing a 2-amino-acid deletion in its V3 base, which selectively ablated R5 but not X4 tropism, also became highly sensitive to AMD3100 (60), suggesting that the CXCR4 interaction for even a slightly shortened V3 becomes impaired. In addition, although three positively charged residues were present in the V3 insert, their positioning may not have been ideal for mediating a full interaction with the CXCR4 ECLs.

For ΔV3-X4B.c7, determinants for X4 tropism were located outside the distal region of V3 and included the loss of N-linked glycosylation sites in V1/V2 (N128D) and the V3 base (T305A) and the restoration of 4 amino acids and an N-linked glycan in V1/V2 that had been lost during the derivation of TA1 (185-188Del). However, even in combination, these three changes conferred only 40% of ΔV3-X4B.c7's ability to use CXCR4 to TA1 in cell-cell fusion assays, and a virus containing the TA1 Env with only these changes was unable to replicate in SupT1 cells, indicating that cooperative and complex interactions with additional mutations are likely involved. In striking contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 could induce fusion with the 4222 chimera, indicating increased dependence on the NT rather than the ECLs. Consistent with this view, its use of wild-type CXCR4 was completely resistant to saturating concentrations of AMD3100, which targets the CXCR4 ECLs. Although the structural basis for this observation is unclear, it is possible that loss of the N-linked glycan at position 305 could have increased exposure of the surface formed by the base of V3 and the bridging sheet that, at least for CCR5, has been shown to engage sulfated tyrosine residues in the coreceptor NT (37) and may play a role in engaging the CXCR4 NT as well. In addition, for one HIV-1, V1/V2 was sufficient to impart AMD3100 resistance (33) in conjunction with an increased dependence on the CXCR4 NT (J. Harrison and R. Doms, personal communication), suggesting that changes in this loop, perhaps involving N-linked glycans and quaternary interactions within the Env trimer (92), could facilitate, directly or indirectly, an NT interaction. Intriguingly, ΔV3-X4B.c7 was also able to use the 2444 chimera in an AMD3100-inhibitable fashion, though at reduced levels (Fig. (Fig.8B),8B), showing that despite a truncated V3, it still could interact with the CXCR4 ECLs in the context of an inefficient NT interaction. Thus, although in a viral infection assay the ΔV3-X4B.c7 env was completely AMD3100 resistant, an AMD3100-sensitive component could be detected, likely reflecting a persisting, albeit weak, interaction with the CXCR4 ECLs. We previously showed that a 4-amino-acid deletion of residues 9 to 12 in the HIV-1/R3A V3 stem ablated CXCR4 use, suggesting that this region directly interacts with the CXCR4 ECLs, while smaller deletions within this 4-amino-acid domain only partially reduced CXCR4 use (60). Because ΔV3-X4B.c7 still contains residues 9 and 10, it is possible that these residues contribute to this interaction.

In our attempt to define determinants for the ΔV3-X4B.c7 env gene's X4 tropism, differences were observed in the results of cell-cell fusion and viral infection assays. Although no single mutation was sufficient, three mutations (N129D, 185-188Ins, and T305A) could confer ~40% of the CXCR4-mediated fusion activity of ΔV3-X4B.c7 to the TA1 env; however, this activity was not sufficient to enable a virus to replicate on CXCR4+ SupT1 cells. We along with others have reported that HIV and simian immunodeficiency virus (SIV) Envs frequently can use coreceptors in cell-cell fusion but do not replicate in cells expressing the same coreceptors (26, 33, 47, 48, 59, 60, 62, 71, 73, 74). This discrepancy likely results from overexpression of Env and receptors in the context of a cell-cell fusion assay, where low-efficiency interactions can be detected that are insufficient to mediate infection when these molecules are expressed at more physiologic levels. In addition, Env mutations that affect trimer stability, viral assembly, or replication are likely to have a greater impact on viral infection than on cell-cell fusion. Nonetheless, although we failed to find determinants that were sufficient to mediate X4-dependent infection for ΔV3-X4B.c7, our results clearly show that in contrast to the ΔV3-X4A.c5 env, ΔV3-X4B.c7 acquired CXCR4 use via mutations outside V3.

Unlike for CCR5, where HIV-1 can, depending on the isolate, interact with multiple coreceptor domains (4, 6, 20, 67, 83), for CXCR4 an interaction of V3 with the ECLs, particularly the second ECL, has been viewed as critical for coreceptor engagement (7, 21, 50, 63, 72). However, our findings for ΔV3-X4B.c7 highlight the extent to which the gp120-coreceptor NT interaction can also impact the development of X4 tropism. Although modestly successful, efforts to predict Env coreceptor specificities based solely on V3 sequence are not always correct, particularly with predictions of coreceptor use for R5X4 and non-clade B Envs (30, 41, 42, 53, 75). Limitations in this approach could result from the differential contribution of the gp120-NT interaction to coreceptor use, with non-V3 changes (9, 10, 32, 40, 45, 46, 48, 61, 64, 65, 95) imparting greater dependence on the CXCR4 NT. Notably, in acquiring X4 tropism primarily through an NT interaction, ΔV3-X4B.c7 lost the ability to use CCR5 in viral infection assays, indicating that at least some specificity for CXCR4 engagement can be conferred through this interaction.

The current generation of small-molecule antagonists for CCR5 and CXCR4 inhibit HIV entry by interacting with membrane-proximal residues within the coreceptor ECLs and limiting the repertoire of ECL conformations that permit interactions with distal regions of V3 (16, 91). For CCR5 inhibitors, in vitro and in vivo derived viral resistance has been associated with changes in and outside V3 that enable viruses either to increase their affinity for CCR5 or, more commonly, to acquire the ability to use drug-bound receptors (5, 58, 70, 89, 94). TA1, which was adapted to replicate with a V3 truncation (47), and an HIV-1 with a smaller 4-amino-acid deletion in the V3 stem (59) were also shown to use drug-bound CCR5 for entry. Although a number of mechanisms are possible, an emerging theme in these studies has been an increased dependence of drug-resistant Envs on the CCR5 NT rather than the ECLs (5, 47, 59, 89). Here, our data on the acquisition of CXCR4 tropism via two distinct pathways, one of which imparted complete AMD3100 resistance, clearly indicate that preferential use of a coreceptor NT can be a more general theme for resistance to small-molecule coreceptor antagonists. While this virus was shown to have an AMD3100-inhibitable interaction with the ECLs (i.e., by its use of a 2444 chimera) (Fig. (Fig.8),8), its NT interaction was clearly dominant and capable of conferring complete AMD3100 resistance to a replication-competent virus. Although the interaction of the coreceptor NT with the V3 base and bridging sheet has not previously been targeted for pharmacologic intervention, it is possible that Envs such as ΔV3-X4B.c7 and/or analogous drug-resistant R5-tropic Envs could be useful in isolating this interaction for assays that can identify new classes of entry inhibitors.

In summary, using an HIV-1 that contained a V3 truncation that ablated its ability to use CXCR4, we demonstrate that X4 tropism could be reacquired through two distinct mechanisms: one involving an insertion that increased the length and net positive charge of V3, reestablished a V3 interaction with the CXCR4 ECLs, and conferred marked sensitivity to AMD3100 and one that principally involved changes outside V3 that increased the efficiency of gp120's interaction with the CXCR4 NT and conferred complete resistance to AMD3100. Our findings highlight the extent to which these two interactions not only determine tropism but also powerfully modulate sensitivity to coreceptor antagonists and perhaps interactions with other host factors. While the V3-ECL and gp120-NT interactions cooperate to mediate viral entry, it is likely that a balance between these interactions also provides evolutionary flexibility that enables the virus to adapt to a number of selection pressures in vitro and in vivo. Moreover, the ability to select for viruses that isolate these interactions may be useful in further structure/function studies and in developing new approaches to screen for novel pharmacologic inhibitors of HIV entry.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank Max Richardson and James Riley for the pELNS lentiviral vector. We also thank Nathaniel Wang, Jianbin Wang, Michael Holmes, Philip Gregory, Edward Rebar, Jeffrey Miller, Lei Zhang, Sarah Hinkley, and colleagues at Sangamo BioSciences for reagents and helpful discussions during the generation of CXCR4-negative SupT1 cells. The CXCR4/CXCR2 chimeric coreceptor constructs were graciously provided by Robert Doms. We also thank Robert Doms and Ronald Collman for helpful discussions. Technological support for p24 assays was provided by the Viral and Molecular Core of the Penn Center for AIDS Research.

This work was supported by grants from the National Institutes of Health, AI-49784 (to J.A.H.) and T32 AI-07632 (to G.Q.D.P.), and a Bill & Melinda Gates Foundation Grand Challenges Program grant (grant 37874; G. M. Shaw, principal investigator).

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 June 2010.

Supplemental material for this article may be found at http://jvi.asm.org/.

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