• No results found

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


Academic year: 2019

Share "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"


Loading.... (view fulltext now)

Full text


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,

termedV3-X4A.c5 andV3-X4B.c7, which took distinct molecular pathways for this change. TheV3-X4A.c5

Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestab-lishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the

CXCR4 antagonist AMD3100. In contrast, theV3-X4B.c7 Env maintained the V3 truncation but acquired

mutations outside V3 that were critical for X4 tropism. In contrast toV3-X4A.c5,V3-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 interac-tions with the CXCR4 ECLs, and/or (ii) mutainterac-tions outside V3 that enhance interacinterac-tions 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 glycopro-tein (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 recep-tors. 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-⌬32) are largely resistant to HIV-1 infection (15, 49, 84). Al-though 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 ap-pearance 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 dif-ferent 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 un-clear 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 muta-tions directly or indirectly mediate interacmuta-tions 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) * Corresponding author. Mailing address: Department of Medicine,

Hematology-Oncology Division, University of Pennsylvania, 356 Bio-medical Research Building II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: (215) 898-0261. Fax: (215) 573-7356. E-mail: hoxie @mail.med.upenn.edu.

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

Published ahead of print on 23 June 2010.


on November 8, 2019 by guest



and even gp41 (40), can also contribute to coreceptor switch-ing, 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 trun-cated V3 loop (47). This Env was generated by introducing a mutation termed⌬V3(9,9), which deleted the distal 15 amino acids of V3. The ⌬V3(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 back-ground 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⌬V3(9,9) derived by adapting this virus to replicate in CXCR4⫹CCR5⫺ SupT1 cells. We show that R3A⌬V3(9,9) could indeed reacquire X4 tropism but through two markedly different mechanisms. One X4 vari-ant, designated ⌬V3-X4A, acquired changes in the V3 rem-nant that reestablished an interaction with the CXCR4 ECLs; the other,⌬V3-X4B, acquired changes outside V3 that engen-dered interactions with the CXCR4 NT. These divergent evo-lutionary pathways led to profound differences in sensitivity to the CXCR4 antagonist AMD3100, with ⌬V3-X4A showing increased sensitivity relative to R3A and with⌬V3-X4B be-coming 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.


Cells.The human SupT1 T lymphoblastoid cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glu-tamine, and 2 mM penicillin-streptomycin (RPMI-complete). SupT1 cells that stably express human CCR5 (SupCCR5) (52) were maintained in RPMI-com-plete 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/X4⫺R5⫹) 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/X4⫺R5⫹, and Sup-Zfn/X4⫹ were previously referred to as SupX4⫺, SupX4⫺R5⫹, 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 R3AV3(9,9) to SupT1 cells.The generation and adaptation of clade B HIV-1 R3A containing the⌬V3(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 previ-ously (47). A replication-competent virus bearing the R3A⌬V3(9,9)envon 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⌬V3(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 an-tibody (25.4; kindly provided by Jan McClure, University of Washington). A spreading infection was established, and virus-containing supernatants were

se-rially 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⌬V3(9,9)envclone TA1, have been described previously (47, 55). To isolate adaptedenvclones from infected SupT1 cultures, genomic DNA was prepared using a QIAamp DNA minikit (Qiagen) according to the manufactur-er’s instructions, and envsequences were PCR amplified using HotStar Taq (Invitrogen) and primers that flank theenvregion. PCR products were then cloned using TOPO TA into pCR2.1 (Invitrogen) and screened forenvinserts using restriction analysis and DNA sequencing. Clones chosen for further eval-uation 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. Mutantenvgenes 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 mutantenvgenes 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 appropriateenvexpression 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␮g/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␮g/ml rifampin and 100 nM cytosine arabi-noside, 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 (Pro-mega), and quantifying luciferase activity with a Thermo LabSystems Luminos-kan 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 vi-ruses, 293T cells were transfected with recombinant pNL4-3 constructs contain-ing selectedenvgenes 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/X4⫺R5⫹, and Sup-Zfn/X4⫹cells were inoculated with equivalent amounts of p24-contain-ing virus. Followp24-contain-ing 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 ab-sence or preab-sence of various concentrations of AMD3100 in FACS buffer for 1 h on ice. Cells were then stained with 4␮g/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.

Nucleotide sequence accession numbers.The⌬V3-X4A.c5 and⌬V3-X4B.c7 envsequences have been deposited in the GenBank database under accession numbers GQ994937 and GQ994936, respectively. The parental R3A, R3A

⌬V3(9,9), and TA1envsequences have GenBank accession numbers AY608577, EF613596, and EF613594, respectively.

on November 8, 2019 by guest




Adaptation of R3AV3(9,9) to infect SupT1 cells.We

pre-viously 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⌬V3(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 ⌬V3(9,9) mutation resulted in a selective loss of X4 tropism with CCR5 usage being maintained, albeit at reduced levels (Fig. 1). When an NL4-3 virus bearing the R3A⌬V3(9,9) envwas adapted for replication in SupR5R cells, which expressed both CXCR4 and CCR5, it maintained a truncated V3 loop and remained ex-clusively R5 tropic. A functionalenvclone, termed TA1, de-rived 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 contain-ing the⌬V3(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⌬V3(9,9) swarm from which TA1 was cloned (Fig. 1). Virus-containing supernatants were serially passaged until

⬎10% of the mixed culture was positive for p24Gagby immu-nofluorescence microscopy (not shown) and then serially pas-saged on SupT1 cells alone. This adaptation scheme was car-ried out twice, yielding two viruses that could infect SupT1 cells with rapid kinetics. The first virus, designated⌬V3-X4A, was passaged 11 times in the SupCCR5-SupT1 mix, followed by 20 passages in SupT1; the second virus, designated⌬ V3-X4B, was passaged seven times in the SupCCR5-SupT1 mix followed by 10 passages in SupT1 (Fig. 1).

V3-X4A andV3-X4B Envs acquired CXCR4 use.To

de-termine if the⌬V3-X4A and⌬V3-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. 2A). 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⌬V3-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⌬V3-X4B Envs, all four tested clones could use CXCR4 at levels of 39 to 64% of R3A, but unlike the⌬V3-X4A Envs they retained R5 tropism with fusion levels of 32 to 50%. To de-termine if⌬V3-X4A and⌬V3-X4Benvclones could confer the ability to replicate in SupT1 to a replication-competent virus, we generated recombinant NL4-3 viruses containing the⌬ V3-X4A.c5 or⌬V3-X4B.c7envclones and compared their growth to that of TA1 and R3A. As expected, dual-tropic R3A repli-cated in SupT1 cells with rapid kinetics, reaching peak RT levels (⬎1.0⫻105cpm) 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. 2B). As shown in Fig. 3B, TA1 could replicate in CCR5⫹SupT1 cells. In con-trast, ⌬V3-X4A.c5 and ⌬V3-X4B.c7 viruses both replicated with rapid kinetics in SupT1, with peak RT levels (1.3⫻105 and 1.1⫻105cpm, respectively) by day 6 (Fig. 2B). Similar to R3A, infection of SupT1 by ⌬V3-X4A.c5 and ⌬V3-X4B.c7 viruses was highly cytopathic, with extensive syncytia formation and cell killing (not shown).

FIG. 1. Scheme summarizing the derivation of the⌬V3-X4A and


⌬V3-X4B viruses. Mutagenesis andin vitroadaptation steps are indi-cated in italics. Introduction of the⌬V3(9,9) mutation in HIV-1 R3A and adaptation to SupR5R cells have been described previously (47) (see Materials and Methods). The adapted R3A⌬V3(9,9) viral swarm was serially passaged in a 1:10 mix of SupCCR5 and SupT1 cells, respectively, until infection of greater than 10% of cells (by IFA) occurred. Virus was then serially passaged in SupT1 cells. This adap-tation scheme was carried out twice, generating two viral swarms, designated⌬V3-X4A and⌬V3-X4B.

FIG. 2. Function of⌬V3-X4A and⌬V3-X4Benvclones. (A) Co-receptor use in a cell-cell fusion assay for⌬V3-X4A and⌬V3-X4Benv

clones. Percent fusion was calculated by using luciferase activity nor-malized to R3A fusion with QT6 cells coexpressing CD4 with CCR5 or CXCR4. The means of three experiments plus the standard error of the means are shown. (B) Growth curves for recombinant NL4-3 viruses containing the R3A, TA1,⌬V3-X4A.c5, or⌬V3-X4B.c7envin CD4⫹CXCR4⫹CCR5⫺SupT1 cells. RT activity in culture superna-tants was measured at the indicated time points. Results from a rep-resentative experiment are shown.

on November 8, 2019 by guest



To confirm that the ability of⌬V3-X4A.c5 and⌬V3-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. 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/X4⫺R5⫹) or to reexpress CXCR4 (Sup-Zfn/X4⫹). As shown in Fig. 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/X4⫺R5⫹cells, demon-strating that these ZFN-treated cells remained fully competent for X4- and R5-mediated infection when they expressed CXCR4 or CCR5, respectively (Fig. 3A). As expected, TA1 replicated in Sup-Zfn/X4⫺R5⫹cells but not SupT1, Sup-Zfn/ X4⫺, or Sup-Zfn/X4⫹cells, which all lacked CCR5 (Fig. 3B). In contrast,⌬V3-X4A.c5 and⌬V3-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,⌬V3-X4B.c7 was un-able to replicate in Sup-Zfn/X4⫺R5⫹cells, despite the fact that this Env could use CCR5 in cell-cell fusion assays (Fig. 2A). Nonetheless, these findings clearly demonstrated that both

⌬V3-X4A.c5 and⌬V3-X4B.c7 Envs regained X4 tropism dur-ing adaption in SupT1 cells.

Mutations in theV3-X4A.c5 andV3-X4B.c7 Envs. We

previously reported that, during adaptation to SupR5R cells, TA1 maintained the⌬V3(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 (desig-nated 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. 4). When the SupT1-adapted⌬V3-X4A.c5 and ⌬V3-X4B.c7 env clones were sequenced, several addi-tional changes were seen compared to the sequence of TA1. The⌬V3-X4A.c5envacquired 10 mutations in gp120 and 2 in gp41 (Fig. 4 and Table 1, which also gives HXBc2 numbering). Most striking among these mutations was a 7-amino-acid in-sertion 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 preced-ing sequence NTRKGVG, followed by point mutations (i.e., N to R and T to G). Six of the other⌬V3-X4A.c5 gp120 muta-tions 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,⌬V3-X4A.c5 contained mutations M98I, M163R, and M296I in gp120 and A579V and T815I in gp41.

The ⌬V3-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 ⌬V3-X4A.c5. Although ⌬ V3-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),⌬V3-X4A.c5 (C), or⌬V3-X4B.c7 (D)envare shown in parental SupT1, Sup-Zfn/X4⫺, Sup-Zfn/X4⫺R5⫹, and Sup-Zfn/X4⫹ cells. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

on November 8, 2019 by guest



X4B.c7 contained T305A in V3, ablating the same N-linked glycosylation site that was lost in⌬V3-X4A.c5, it maintained the ⌬V3(9,9) truncation and did not develop a V3 insertion (Fig. 4 and Table 1). Similar mutations between⌬V3-X4B.c7 and ⌬V3-X4A.c5 included the loss of five of the same pre-dicted N-linked glycosylation sites, three via identical muta-tions (T358I in C3 and T398A and T408I in V4) and two via distinct mutations (T305A in V3 and N340S in C3). In addi-tion,⌬V3-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, leav-ing 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 forV3-X4A.c5 Env.Because

the V3 loop is the principal determinant of coreceptor use (34), we hypothesized that the RGRKGVG insertion in the


⌬V3-X4A.c5 V3 loop was primarily responsible for conferring X4 tropism to this Env. Indeed, when this sequence was in-serted into the TA1 Env (designated TA1⌬V3-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⌬V3-X4A.c5 (Fig. 5A). The addition of⌬V3-X4A V3Ins also led to a re-duction in CCR5 use to 24% of that of TA1 (Fig. 5B). An NL4-3 virus bearing the TA1⌬V3-X4A V3Ins Env replicated in SupT1 cells although its kinetics were delayed compared with replication of⌬V3-X4A.c5 (see Fig. S1 in the supplemen-tal material). Interestingly, theenvgenes PCR amplified from these infected SupT1 cells at peak virus replication all had acquired the T305I mutation in V3 (data not shown), a change FIG. 4. Alignment of Env sequences for R3A, TA1,⌬V3-X4A.c5, and⌬V3-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 R3A⌬V3(9,9) swarm contained the original⌬V3(9,9) mutation with an Ala-to-Val mutation in the Gly-Ala-Gly linker, a deletion of residues 185 to 188 in V1/V2, and three other point mutations in Env. Clone⌬V3-X4A.c5 contained a 7-amino-acid insertion in the truncated V3 loop and 12 other point mutations in Env, compared with the TA1 sequence. Clone⌬V3-X4B.c7 maintained the⌬V3(9,9) truncation in V3 but did not contain a deletion in V1/V2 and contained 18 other point mutations in Env compared with TA1 sequence.

on November 8, 2019 by guest



that was present in the⌬V3-X4A.c5 Env clone. Thus, although the V3 insertion was a principal determinant for⌬V3-X4A.c5’s X4 tropism, the loss of the N-linked glycan in V3 further contributed to CXCR4 use.

Determinants of CXCR4 use forV3-X4B.c7.In contrast to


⌬V3-X4A.c5, the⌬V3-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⌬V3-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⌬V3-X4B.c7 either mu-tations 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. 6A and C). In addition, residues 185 to 188, deleted in TA1 but present in⌬V3-X4B.c7, were restored in TA1 (185-188Ins), reintroducing an N-linked glycan in V1/V2. We did not eval-uate 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 signif-icant increase in CXCR4-dependent fusion (9 to 12% of⌬ V3-X4B.c7; P ⬍ 0.03, Student’s t test) (Fig. 6A). Although no significant increase was seen when any two of these mutations FIG. 5.⌬V3-X4A determinants for CXCR4 use in cell-cell fusion.


Fusion for the TA1envcontaining the⌬V3-X4A.c5 7-amino-acid V3 loop insertion with CD4⫹ CXCR4⫹(A) or CD4⫹ CCR5⫹ (B) QT6 cells. Percent fusion was calculated by using luciferase activity normal-ized to⌬V3-X4A.c5 fusion with CD4⫹CXCR4⫹targets (A) or to TA1 fusion with CD4⫹CCR5⫹targets (B). The means of three experiments plus standard error of the means are shown.

TABLE 1. Amino acid mutations in⌬V3-X4A.c5 and⌬V3-X4B.c7 Envs

Protein Domaina

R3A no. HxB no.


R3A TA1 ⌬V3-X4A.c5 ⌬V3-X4B.c7

gp120 C1 98 100 M - I

-V/V2 128 130 N - - D

146 147 M - - V

163 165 M - R

-177 179 L - - P

185–188 187⫹3 TTKN .... ....

----C2 205 203 Q - - R

254 252 R K -

-273 271 V - - I

296 294 M - I

-V3 305 303 T - I A

308–322 306–321 RVTLGPGRVYYTTGQ ....GVG... ....GVGRGRKGVG. ....GVG...

C3 340 339 N - - S

342 341 T A -

-356 355 N - - S

358 358 T - I I

V4 392 392 N - - S

394 394 T - - I

398 398 T - A A

408 413 T - I I

C4 435 440 S - - R

V5 461 466 T - - N

gcp41 Ecto 509 512 A V V V

538 541 A - - T

579 582 A - V

-659 662 A - - S

MSD 684 687 M - - I

693 696 R - - K

CT 815 818 T - I


C, constant domain; V, variable domain; Ecto, ectodomain; MSD, membrane-spanning domain; CT, cytoplasmic tail. b

Amino acid changes in gp120 and gp41 are shown for the indicated Env clones relative to the parental R3A. Dashes indicate identical residues, and dots indicate deletions.

on November 8, 2019 by guest



were combined, when all three were added, CXCR4-mediated fusion increased significantly to 42% of ⌬V3-X4B.c7 (P

0.01). However, no additional single mutation from ⌬ V3-X4B.c7 when added to the TA1 N128D/185-188Ins/T305A tri-ple mutant resulted in any further increase in CXCR4-medi-ated 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⌬V3-X4B.c7 in combinations (Fig. 6B). When any two of these changes were made in ⌬V3-X4B.c7, CXCR4-dependent fusion de-creased 36 to 63%, while combining all three changes reduced fusion to 17% of⌬V3-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). We also evaluated the effects of these same changes on CCR5 utilization. When introduced into TA1, most of the individual⌬V3-X4B.c7 mutations either had no effect or in-creased CCR5-mediated fusion (Fig. 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⌬V3-X4B.c7 alone (not shown) or in association with D128N and/or 185-188Del, CCR5-dependent fusion increased 5- to 6-fold (Fig. 6C). Thus, the loss of this N-linked glycan in V3 not only contributed to increased X4 tropism but also negatively

af-fected 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 ofV3-X4A.c5 andV3-X4B.c7 Envs.

Although⌬V3-X4A.c5 and⌬V3-X4B.c7 both gained the abil-ity to use CXCR4, distinct mutations in each clone were in-volved, 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 rep-ertoire of ECL conformations to prevent gp120 binding (16, 91). We first assessed the AMD3100 sensitivity of R3A,⌬ V3-X4A.c5, and⌬V3-X4B.c7 Envs in a cell-cell fusion assay with CD4⫹CXCR4⫹ target cells (Fig. 7A). As shown previously (60), R3A was inhibitable by AMD3100, with a 50% inhibitory concentration (IC50) of 314 nM. In comparison,⌬V3-X4A.c5 was nearly 25-fold more sensitive, with an IC50of 12.3 nM. In contrast,⌬V3-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,


⌬V3-X4A.c5, or⌬V3-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. 7B). In agreement with FIG. 6.⌬V3-X4B determinants for CXCR4 use in cell-cell fusion. Fusion for the TA1envcontaining⌬V3-X4B.c7 mutations alone and in combination with CD4⫹ CXCR4⫹(A) or CD4⫹ CCR5⫹ (C) QT6 cells and for the ⌬V3-X4B.c7env with mutations reverted back to the corresponding TA1 residues with CD4⫹CXCR4⫹(B) or CD4⫹CCR5⫹(D) QT6 cells. Percent fusion was calculated by using luciferase activity normalized to⌬V3-X4B.c7 fusion with CD4⫹CXCR4⫹targets (A and B) or to TA1 fusion with CD4⫹CCR5⫹targets (C and D). The means of three experiments plus standard error of the means are shown.

on November 8, 2019 by guest



results from the cell-cell fusion assay,⌬V3-X4A.c5 was mark-edly more sensitive to AMD3100, with complete inhibition observed at concentrations of ⱖ100 nM. ⌬V3-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⌬V3-X4B.c7 in SupT1 is de-pendent on CXCR4 (Fig. 3D), these results clearly indicate that ⌬V3-X4B.c7 is able to use AMD3100-bound CXCR4. Thus, although⌬V3-X4A.c5 and ⌬V3-X4B.c7 Envs both ac-quired the ability to use CXCR4, they exhibited distinct and profound differences in their sensitivity to inhibition by this CXCR4 antagonist.

Use of CXCR2/CXCR4 chimeras byV3-X4A.c5 and

V3-X4B.c7 Envs. Because AMD3100 is thought to inhibit the

interaction of V3 with the CXCR4 ECLs, the sensitivity of

⌬V3-X4A.c5 to AMD3100 suggested that an interaction with the ECLs was critical for its entry. Conversely, the complete resistance of ⌬V3-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 distin-guish between these possibilities, chimeric coreceptors

gener-ated by swapping domains between CXCR4 and the nonper-missive coreceptor CXCR2 (21) were used in a cell-cell fusion assay (Fig. 8A). 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⌬V3-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 inter-acted with the CXCR4 ECLs and were less dependent on the CXCR4 NT. In contrast,⌬V3-X4B.c7 could induce fusion with target cells that coexpressed CD4 and 4222 to a level compa-rable to its use of CXCR4, suggesting that this Env had acquired a stronger interaction with the CXCR4 NT. How-ever, although⌬V3-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,⌬V3-X4A.c5, and⌬V3-X4B.c7 were determined using the 2444 chimera (Fig. 8B). As expected, R3A and ⌬V3-X4A.c5 were inhib-ited by AMD3100 (1,000 nM) on both CXCR4- and 2444-expressing target cells. However, while⌬V3-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⌬ V3-FIG. 7. Sensitivity to the CXCR4 antagonist AMD3100. (A) Inhibition of R3A,⌬V3-X4A.c5, and⌬V3-X4B.c7 by the indicated concentrations of AMD3100 in a cell-cell fusion assay. Percent fusion was calculated by using luciferase activity normalized to fusion with CD4⫹CXCR4⫹targets in the absence of drug for eachenv. The means of three experiments plus standard error of the means are shown. (B to D) Growth curves for recombinant NL4-3 viruses containing the R3A (B),⌬V3-X4A.c5 (C), or⌬V3-X4B.c7 (D)envin the presence of the indicated concentrations of AMD3100. RT activity in culture supernatants was measured at the indicated time points. Results from a representative experiment are shown.

on November 8, 2019 by guest



X4B.c7 could utilize the CXCR4 NT (i.e., on the 4222 chi-mera) 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 ⌬V3-X4B.c7 fusion on wild-type CXCR4 was resistant to AMD3100, the NT inter-action was likely dominant.


The HIV-1 Env engages CCR5 and CXCR4 through com-plex 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 core-ceptor 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 pos-itively 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

HIV-1 R3A Env, ablated X4 but not R5 tropism, increased dependence for entry on the CCR5 NT, and conferred resis-tance 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, ⌬V3-X4A.c5 and⌬ V3-X4B.c7, each of which exhibited a distinct evolutionary path-way for regaining X4 tropism. While ⌬V3-X4A.c5 acquired CXCR4 use through a 7-amino-acid insertion in V3, ⌬ V3-X4B.c7 maintained a truncated V3 and, instead, with the ex-ception 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 interac-tions with the CXCR4 NT and ECLs.

For⌬V3-X4A.c5, an RGRKGVG insertion in V3 was suf-ficient 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⌬V3-X4A.c5 V3 to engage CXCR4 ECLs had been restored. Indeed,⌬V3-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,⌬V3-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,


⌬V3-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 com-promised 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 FIG. 8. CXCR4 determinants for⌬V3-X4A and⌬V3-X4B fusion.

(A) Use of CXCR4/CXCR2 coreceptor chimeras (21) in a cell-cell fusion assay for R3A,⌬V3-X4A.c5, and⌬V3-X4B.c7envclones. The 4222 chimera contained a CXCR4 NT domain with the CXCR2 ECLs, and the 2444 chimera contained a CXCR2 NT with the CXCR4 ECLs. Percent fusion was calculated by using luciferase activity normalized to fusion with CD4⫹CXCR4⫹QT6 cells for eachenv. (B) Use of CXCR4 and 2444 in the absence or presence of 1,000 nM AMD3100 in a cell-cell fusion assay for R3A, ⌬V3-X4A.c5, and ⌬V3-X4B.c7 env

clones. Percent fusion was calculated by using luciferase activity nor-malized to fusion on each coreceptor in the absence of drug. The means of three experiments plus standard error of the means are


on November 8, 2019 by guest



present in the V3 insert, their positioning may not have been ideal for mediating a full interaction with the CXCR4 ECLs. For⌬V3-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 ⌬V3-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 interac-tions with additional mutainterac-tions are likely involved. In striking contrast to⌬V3-X4A.c5,⌬V3-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 con-centrations 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 indi-rectly, an NT interaction. Intriguingly,⌬V3-X4B.c7 was also able to use the 2444 chimera in an AMD3100-inhibitable fash-ion, though at reduced levels (Fig. 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 ⌬V3-X4B.c7 env was completely AMD3100 resistant, an AMD3100-sensitive component could be detected, likely reflecting a persisting, albeit weak, interac-tion 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⌬V3-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⌬V3-X4B.c7

envgene’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 fu-sion activity of ⌬V3-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 fre-quently can use coreceptors in cell-cell fusion but do not rep-licate 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 mu-tations 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 deter-minants that were sufficient to mediate X4-dependent infec-tion for⌬V3-X4B.c7, our results clearly show that in contrast to the⌬V3-X4A.c5env,⌬V3-X4B.c7 acquired CXCR4 use via mutations outside V3.

Unlike for CCR5, where HIV-1 can, depending on the iso-late, interact with multiple coreceptor domains (4, 6, 20, 67, 83), for CXCR4 an interaction of V3 with the ECLs, particu-larly the second ECL, has been viewed as critical for corecep-tor engagement (7, 21, 50, 63, 72). However, our findings for

⌬V3-X4B.c7 highlight the extent to which the gp120-corecep-tor 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). Limita-tions in this approach could result from the differential contri-bution 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,

⌬V3-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 mem-brane-proximal residues within the coreceptor ECLs and lim-iting the repertoire of ECL conformations that permit inter-actions with distal regions of V3 (16, 91). For CCR5 inhibitors,

in vitroandin vivoderived 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 tro-pism via two distinct pathways, one of which imparted com-plete 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. 8), its NT interaction was clearly dominant and capable of conferring complete AMD3100 resistance to a replication-competent vi-rus. 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

⌬V3-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:

on November 8, 2019 by guest



pressuresin vitroandin 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.


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).


1.Agrawal-Gamse, C., F. H. Lee, B. Haggarty, A. P. Jordan, Y. Yi, B. Lee, R. G. Collman, J. A. Hoxie, R. W. Doms, and M. M. Laakso.2009. Adaptive mutations in a human immunodeficiency virus type 1 envelope protein with a truncated V3 loop restore function by improving interactions with CD4. J. Virol.83:11005–11015.

2.Alexander, W. A., B. Moss, and T. R. Fuerst.1992. Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and theEscherichia coli lacrepressor. J. Virol.66:2934–2942. 3.Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M.

Murphy, and E. A. Berger.1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ receptor as a fusion cofactor for macrophage-tropic HIV-1. Science272: 1955–1958.

4.Atchison, R. E., J. Gosling, F. S. Monteclaro, C. Franci, L. Digilio, I. F. Charo, and M. A. Goldsmith.1996. Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines. Science274: 1924–1926.

5.Berro, R., R. W. Sanders, M. Lu, P. J. Klasse, and J. P. Moore.2009. Two HIV-1 variants resistant to small molecule CCR5 inhibitors differ in how they use CCR5 for entry. PLoS Pathog.5:e1000548.

6.Bieniasz, P. D., R. A. Fridell, I. Aramori, S. S. Ferguson, M. G. Caron, and B. R. Cullen.1997. HIV-1-induced cell fusion is mediated by multiple re-gions within both the viral envelope and the CCR-5 co-receptor. EMBO J. 16:2599–2609.

7.Brelot, A., N. Heveker, O. Pleskoff, N. Sol, and M. Alizon.1997. Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity. J. Virol.71:4744–4751.

8.Cardozo, T., T. Kimura, S. Philpott, B. Weiser, H. Burger, and S. Zolla-Pazner.2007. Structural basis for coreceptor selectivity by the HIV type 1 V3 loop. AIDS Res. Hum. Retroviruses23:415–426.

9.Carrillo, A., and L. Ratner.1996. Cooperative effects of the human immu-nodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells. J. Virol.70:1310–1316.

10.Cho, M. W., M. K. Lee, M. C. Carney, J. F. Berson, R. W. Doms, and M. A. Martin.1998. Identification of determinants on a dualtropic human immu-nodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4. J. Virol.72:2509–2515.

11.Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu,

AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Sci-ence273:1856–1862.

16.De Clercq, E.2003. The bicyclam AMD3100 story. Nat. Rev. Drug Discov. 2:581–587.

17.De Jong, J. J., A. De Ronde, W. Keulen, M. Tersmette, and J. Goudsmit. 1992. Minimal requirements for the human immunodeficiency virus type 1 V3 domain to support the syncytium-inducing phenotype: analysis by single amino acid substitution. J. Virol.66:6777–6780.

18.Del Prete, G. Q., B. Haggarty, G. J. Leslie, A. P. Jordan, J. Romano, N. Wang, J. Wang, M. C. Holmes, D. C. Montefiori, and J. A. Hoxie.2009. Der-ivation and characterization of a simian immunodeficiency virus SIVmac239 variant with tropism for CXCR4. J. Virol.83:9911–9922.

19.Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, D. R. Littman, and N. R. Landau.1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature381:661–666.

20.Doranz, B. J., Z. H. Lu, J. Rucker, T. Y. Zhang, M. Sharron, Y. H. Cen, Z. X. Wang, H. H. Guo, J. G. Du, M. A. Accavitti, R. W. Doms, and S. C. Peiper. 1997. Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1. J. Virol.71:6305–6314.

21.Doranz, B. J., M. J. Orsini, J. D. Turner, T. L. Hoffman, J. F. Berson, J. A. Hoxie, S. C. Peiper, L. F. Brass, and R. W. Doms.1999. Identification of CXCR4 domains that support coreceptor and chemokine receptor functions. J. Virol.73:2752–2761.

22.Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms.1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell85:1149–1158.

23.Dragic, T.2001. An overview of the determinants of CCR5 and CXCR4 co-receptor function. J. Gen. Virol.82:1807–1814.

24.Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Na-gashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton.1996. HIV-1 entry into CD4⫹cells is mediated by the chemokine receptor CC-CKR-5. Nature381:667–673.

25.Edinger, A. L., and R. W. Doms.1999. A cell-cell fusion assay to monitor HIV-1 Env interactions with chemokine receptors. Methods Mol. Med. 17:41–49.

26.Endres, M. J., P. R. Clapham, M. Marsh, M. Ahuja, J. D. Turner, A. McKnight, J. F. Thomas, B. Stoebenau-Haggarty, S. Choe, P. J. Vance, T. N. Wells, C. A. Power, S. S. Sutterwala, R. W. Doms, N. R. Landau, and J. A. Hoxie.1996. CD4-independent infection by HIV-2 is mediated by fusin/ CXCR4. Cell87:745–756.

27.Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger.1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science272:872–877.

28.Fouchier, R. A., M. Brouwer, S. M. Broersen, and H. Schuitemaker.1995. Simple determination of human immunodeficiency virus type 1 syncytium-inducing V3 genotype by PCR. J. Clin. Microbiol.33:906–911.

29.Fouchier, R. A., M. Groenink, N. A. Kootstra, M. Tersmette, H. G. Huisman, F. Miedema, and H. Schuitemaker.1992. Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule. J. Virol.66:3183–3187.

30.Garrido, C., V. Roulet, N. Chueca, E. Poveda, A. Aguilera, K. Skrabal, N. Zahonero, S. Carlos, F. Garcia, J. L. Faudon, V. Soriano, and C. de Men-doza.2008. Evaluation of eight different bioinformatics tools to predict viral tropism in different human immunodeficiency virus type 1 subtypes. J. Clin. Microbiol.46:887–891.

31.Gerlach, L. O., R. T. Skerlj, G. J. Bridger, and T. W. Schwartz.2001. Molecular interactions of cyclam and bicyclam non-peptide antagonists with the CXCR4 chemokine receptor. J. Biol. Chem.276:14153–14160. 32.Groenink, M., R. A. Fouchier, S. Broersen, C. H. Baker, M. Koot, A. B. van’t

Wout, H. G. Huisman, F. Miedema, M. Tersmette, and H. Schuitemaker.

on November 8, 2019 by guest



1993. Relation of phenotype evolution of HIV-1 to envelope V2 configura-tion. Science260:1513–1516.

33.Harrison, J. E., J. B. Lynch, L. J. Sierra, L. A. Blackburn, N. Ray, R. G. Collman, and R. W. Doms.2008. Baseline resistance of primary human immunodeficiency virus type 1 strains to the CXCR4 inhibitor AMD3100. J. Virol.82:11695–11704.

34.Hartley, O., P. J. Klasse, Q. J. Sattentau, and J. P. Moore.2005. V3: HIV’s switch-hitter. AIDS Res. Hum. Retroviruses21:171–189.

35.Hatse, S., K. Princen, L. O. Gerlach, G. Bridger, G. Henson, E. De Clercq, T. W. Schwartz, and D. Schols.2001. Mutation of Asp(171) and Asp(262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100. Mol. Pharmacol.60:164–173.

36.Hu, Q., K. B. Napier, J. O. Trent, Z. Wang, S. Taylor, G. E. Griffin, S. C. Peiper, and R. J. Shattock.2005. Restricted variable residues in the C-terminal segment of HIV-1 V3 loop regulate the molecular anatomy of CCR5 utilization. J. Mol. Biol.350:699–712.

37.Huang, C. C., S. N. Lam, P. Acharya, M. Tang, S. H. Xiang, S. S. Hussan, R. L. Stanfield, J. Robinson, J. Sodroski, I. A. Wilson, R. Wyatt, C. A. Bewley, and P. D. Kwong.2007. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science317:1930– 1934.

38.Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L. Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and P. D. Kwong.2005. Structure of a V3-containing HIV-1 gp120 core. Science 310:1025–1028.

39.Huang, W., S. H. Eshleman, J. Toma, S. Fransen, E. Stawiski, E. E. Paxinos, J. M. Whitcomb, A. M. Young, D. Donnell, F. Mmiro, P. Musoke, L. A. Guay, J. B. Jackson, N. T. Parkin, and C. J. Petropoulos.2007. Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations. J. Vi-rol.81:7885–7893.

40.Huang, W., J. Toma, S. Fransen, E. Stawiski, J. D. Reeves, J. M. Whitcomb, N. Parkin, and C. J. Petropoulos.2008. Coreceptor tropism can be influ-enced by amino acid substitutions in the gp41 transmembrane subunit of human immunodeficiency virus type 1 envelope protein. J. Virol.82:5584– 5593.

41.Jensen, M. A., M. Coetzer, A. B. van’t Wout, L. Morris, and J. I. Mullins. 2006. A reliable phenotype predictor for human immunodeficiency virus type 1 subtype C based on envelope V3 sequences. J. Virol.80:4698–4704. 42.Jensen, M. A., F. S. Li, A. B. van’t Wout, D. C. Nickle, D. Shriner, H. X. He,

S. McLaughlin, R. Shankarappa, J. B. Margolick, and J. I. Mullins.2003. Improved coreceptor usage prediction and genotypic monitoring of R5-to-X4 transition by motif analysis of human immunodeficiency virus type 1 envV3 loop sequences. J. Virol.77:13376–13388.

43.Kajumo, F., D. A. Thompson, Y. Guo, and T. Dragic.2000. Entry of R5X4 and X4 human immunodeficiency virus type 1 strains is mediated by nega-tively charged and tyrosine residues in the amino-terminal domain and the second extracellular loop of CXCR4. Virology271:240–247.

44.Karlsson, A., K. Parsmyr, E. Sandstrom, E. M. Fenyo, and J. Albert.1994. MT-2 cell tropism as prognostic marker for disease progression in human immunodeficiency virus type 1 infection. J. Clin. Microbiol.32:364–370. 45.Koito, A., G. Harrowe, J. A. Levy, and C. Cheng-Mayer.1994. Functional

role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization. J. Virol.68:2253–2259.

46.Koito, A., L. Stamatatos, and C. Cheng-Mayer.1995. Small amino acid sequence changes within the V2 domain can affect the function of a T-cell line-tropic human immunodeficiency virus type 1 envelope gp120. Virology 206:878–884.

47.Laakso, M. M., F. H. Lee, B. Haggarty, C. Agrawal, K. M. Nolan, M. Biscone, J. Romano, A. P. Jordan, G. J. Leslie, E. G. Meissner, L. Su, J. A. Hoxie, and R. W. Doms.2007. V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies. PLoS Pathog.3:e117.

48.Lin, G., A. Bertolotti-Ciarlet, B. Haggarty, J. Romano, K. M. Nolan, G. J. Leslie, A. P. Jordan, C. C. Huang, P. D. Kwong, R. W. Doms, and J. A. Hoxie. 2007. Replication competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistant to chemokine receptor antagonists. J. Virol.81:9956–9966.

49.Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E. MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau.1996. Homozy-gous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell86:367–377.

50.Lu, Z., J. F. Berson, Y. Chen, J. D. Turner, T. Zhang, M. Sharron, M. H. Jenks, Z. Wang, J. Kim, J. Rucker, J. A. Hoxie, S. C. Peiper, and R. W. Doms. 1997. Evolution of HIV-1 coreceptor usage through interactions with distinct CCR5 and CXCR4 domains. Proc. Natl. Acad. Sci. U. S. A.94:6426–6431. 51.Lusso, P., P. L. Earl, F. Sironi, F. Santoro, C. Ripamonti, G. Scarlatti, R. Longhi, E. A. Berger, and S. E. Burastero.2005. Cryptic nature of a con-served, CD4-inducible V3 loop neutralization epitope in the native envelope

glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains. J. Virol.79:6957–6968. 52.Means, R. E., T. Matthews, J. A. Hoxie, M. H. Malim, T. Kodama, and R. C.

Desrosiers.2001. Ability of the V3 loop of simian immunodeficiency virus to serve as a target for antibody-mediated neutralization: correlation of neu-tralization sensitivity, growth in macrophages, and decreased dependence on CD4. J. Virol.75:3903–3915.

53.Mefford, M. E., P. R. Gorry, K. Kunstman, S. M. Wolinsky, and D. Gabuzda. 2008. Bioinformatic prediction programs underestimate the frequency of CXCR4 usage by R5X4 HIV type 1 in brain and other tissues. AIDS Res. Hum. Retroviruses24:1215–1220.

54.Meissner, E. G., V. M. Coffield, and L. Su.2005. Thymic pathogenicity of an HIV-1 envelope is associated with increased CXCR4 binding efficiency and V5-gp41-dependent activity, but not V1/V2-associated CD4 binding effi-ciency and viral entry. Virology336:184–197.

55.Meissner, E. G., K. M. Duus, F. Gao, X. F. Yu, and L. Su.2004. Character-ization of a thymus-tropic HIV-1 isolate from a rapid progressor: role of the envelope. Virology328:74–88.

56.Miller, J. C., M. C. Holmes, J. Wang, D. Y. Guschin, Y. L. Lee, I. Rupniewski, C. M. Beausejour, A. J. Waite, N. S. Wang, K. A. Kim, P. D. Gregory, C. O. Pabo, and E. J. Rebar.2007. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol.25:778–785.

57.Moore, J. P., S. G. Kitchen, P. Pugach, and J. A. Zack.2004. The CCR5 and CXCR4 coreceptors—central to understanding the transmission and patho-genesis of human immunodeficiency virus type 1 infection. AIDS Res. Hum. Retroviruses20:111–126.

58.Moore, J. P., and D. R. Kuritzkes.2009. A piece de resistance: how HIV-1 escapes small molecule CCR5 inhibitors. Curr. Opin. HIV AIDS4:118–124. 59.Nolan, K. M., G. Q. Del Prete, A. P. Jordan, B. Haggarty, J. Romano, G. J. Leslie, and J. A. Hoxie.2009. Characterization of a human immunodefi-ciency virus type 1 V3 deletion mutation that confers resistance to CCR5 inhibitors and the ability to use aplaviroc-bound receptor. J. Virol.83:3798– 3809.

60.Nolan, K. M., A. P. Jordan, and J. A. Hoxie.2008. Effects of partial deletions within the human immunodeficiency virus type 1 V3 loop on coreceptor tropism and sensitivity to entry inhibitors. J. Virol.82:664–673.

61.Ogert, R. A., M. K. Lee, W. Ross, A. Buckler-White, M. A. Martin, and M. W. Cho.2001. N-linked glycosylation sites adjacent to and within the V1/V2 and the V3 loops of dualtropic human immunodeficiency virus type 1 isolate DH12 gp120 affect coreceptor usage and cellular tropism. J. Virol.75:5998– 6006.

62.Otto, C., B. A. Puffer, S. Po¨hlmann, R. W. Doms, and F. Kirchhoff.2003. Mutations in the C3 region of human and simian immunodeficiency virus envelope have differential effects on viral infectivity, replication, and CD4-dependency. Virology315:292–302.

63.Parolin, C., A. Borsetti, H. Choe, M. Farzan, P. Kolchinsky, M. Heesen, Q. Ma, C. Gerard, G. Palu, M. E. Dorf, T. Springer, and J. Sodroski.1998. Use of murine CXCR-4 as a second receptor by some T-cell-tropic human im-munodeficiency viruses. J. Virol.72:1652–1656.

64.Pastore, C., R. Nedellec, A. Ramos, S. Pontow, L. Ratner, and D. E. Mosier. 2006. Human immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J. Vi-rol.80:750–758.

65.Pastore, C., A. Ramos, and D. E. Mosier.2004. Intrinsic obstacles to human immunodeficiency virus type 1 coreceptor switching. J. Virol.78:7565–7574. 66.Perez, E. E., J. Wang, J. C. Miller, Y. Jouvenot, K. A. Kim, O. Liu, N. Wang, G. Lee, V. V. Bartsevich, Y. L. Lee, D. Y. Guschin, I. Rupniewski, A. J. Waite, C. Carpenito, R. G. Carroll, J. S. Orange, F. D. Urnov, E. J. Rebar, D. Ando, P. D. Gregory, J. L. Riley, M. C. Holmes, and C. H. June.2008. Establish-ment of HIV-1 resistance in CD4⫹T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol.26:808–816.

67.Picard, L., G. Simmons, C. A. Power, A. Meyer, R. A. Weiss, and P. R. Clapham.1997. Multiple extracellular domains of CCR-5 contribute to hu-man immunodeficiency virus type 1 entry and fusion. J. Virol.71:5003–5011. 68.Pollakis, G., S. Kang, A. Kliphuis, M. I. Chalaby, J. Goudsmit, and W. A. Paxton. 2001. N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor uti-lization. J. Biol. Chem.276:13433–13441.

69.Polzer, S., M. T. Dittmar, H. Schmitz, and M. Schreiber.2002. The N-linked glycan g15 within the V3 loop of the HIV-1 external glycoprotein gp120 affects coreceptor usage, cellular tropism, and neutralization. Virology304: 70–80.

70.Pugach, P., A. J. Marozsan, T. J. Ketas, E. L. Landes, J. P. Moore, and S. E. Kuhmann.2007. HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry. Virology361:212–228. 71.Reeves, J. D., S. A. Gallo, N. Ahmad, J. L. Miamidian, P. E. Harvey, M.

Sharron, S. Pohlmann, J. N. Sfakianos, C. A. Derdeyn, R. Blumenthal, E. Hunter, and R. W. Doms.2002. Sensitivity of HIV-1 to entry inhibitors correlates with envelope/coreceptor affinity, receptor density, and fusion kinetics. Proc. Natl. Acad. Sci. U. S. A.99:16249–16254.

72.Reeves, J. D., N. Heveker, A. Brelot, M. Alizon, P. R. Clapham, and L. Picard.1998. The second extracellular loop of CXCR4 is involved in

on November 8, 2019 by guest



by rhesus TRIM5␣. J. Virol.82:11117–11128.

77.Richman, D. D., and S. A. Bozzette.1994. The impact of the syncytium-inducing phenotype of human immunodeficiency virus on disease progres-sion. J. Infect. Dis.169:968–974.

78.Rizzuto, C., and J. Sodroski.2000. Fine definition of a conserved CCR5-binding region on the human immunodeficiency virus type 1 glycoprotein 120. AIDS Res. Hum. Retroviruses16:741–749.

79.Rizzuto, C. D., R. Wyatt, N. Hernandez-Ramos, Y. Sun, P. D. Kwong, W. A. Hendrickson, and J. Sodroski.1998. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science280:1949–1953. 80.Ross, T. M., and B. R. Cullen.1998. The ability of HIV type 1 to use CCR-3

as a coreceptor is controlled by envelope V1/V2 sequences acting in con-junction with a CCR-5 tropic V3 loop. Proc. Natl. Acad. Sci. U. S. A. 95:7682–7686.

81.Rucker, J., B. J. Doranz, A. L. Edinger, D. Long, J. F. Berson, and R. W. Doms.1997. Cell-cell fusion assay to study role of chemokine receptors in human immunodeficiency virus type 1 entry. Methods Enzymol.288:118– 133.

82.Rucker, J., A. L. Edinger, M. Sharron, M. Samson, B. Lee, J. F. Berson, Y. Yi, B. Margulies, R. G. Collman, B. J. Doranz, M. Parmentier, and R. W. Doms.1997. Utilization of chemokine receptors, orphan receptors, and her-pesvirus-encoded receptors by diverse human and simian immunodeficiency viruses. J. Virol.71:8999–9007.

83.Rucker, J., M. Samson, B. J. Doranz, F. Libert, J. F. Berson, Y. Yi, R. J. Smyth, R. G. Collman, C. C. Broder, G. Vassart, R. W. Doms, and M. Parmentier.1996. Regions in beta-chemokine receptors CCR5 and CCR2b that determine HIV-1 cofactor specificity. Cell87:437–446.

84.Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C. Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth, R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier.1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant al-leles of the CCR-5 chemokine receptor gene. Nature382:722–725.

and T. H. Lee.2003. Effect of amino acid substitution of the V3 and bridging sheet residues in human immunodeficiency virus type 1 subtype C gp120 on CCR5 utilization. J. Virol.77:3832–3837.

89.Tilton, J. C., H. Amrine-Madsen, J. L. Miamidian, K. M. Kitrinos, J. Pfaff, J. F. Demarest, N. Ray, J. L. Jeffrey, C. C. Labranche, and R. W. Doms.2010. HIV type 1 from a patient with baseline resistance to CCR5 antagonists uses drug-bound receptor for entry. AIDS Res. Hum. Retroviruses26:13–24. 90.van’t Wout, A. B., N. A. Kootstra, G. A. Mulder-Kampinga, N. Albrecht-van

Lent, H. J. Scherpbier, J. Veenstra, K. Boer, R. A. Coutinho, F. Miedema, and H. Schuitemaker.1994. Macrophage-tropic variants initiate human im-munodeficiency virus type 1 infection after sexual, parenteral, and vertical transmission. J. Clin. Invest.94:2060–2067.

91.Watson, C., S. Jenkinson, W. Kazmierski, and T. Kenakin.2005. The CCR5 receptor-based mechanism of action of 873140, a potent allosteric noncom-petitive HIV entry inhibitor. Mol. Pharmacol.67:1268–1282.

92.Wei, X., J. M. Decker, S. Wang, H. Hui, J. C. Kappes, X. Wu, J. F. Salazar-Gonzalez, M. G. Salazar, J. M. Kilby, M. S. Saag, N. L. Komarova, M. A. Nowak, B. H. Hahn, P. D. Kwong, and G. M. Shaw.2003. Antibody neutral-ization and escape by HIV-1. Nature422:307–312.

93.Westby, M., M. Lewis, J. Whitcomb, M. Youle, A. L. Pozniak, I. T. James, T. M. Jenkins, M. Perros, and E. van der Ryst.2006. 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. 80:4909–4920.

94.Westby, M., C. Smith-Burchnell, J. Mori, M. Lewis, M. Mosley, M. Stock-dale, P. Dorr, G. Ciaramella, and M. Perros.2007. Reduced maximal inhi-bition in phenotypic susceptibility assays indicates that viral strains resistant to the CCR5 antagonist maraviroc utilize inhibitor-bound receptor for entry. J. Virol.81:2359–2371.

95.Yoshimura, K., S. Matsushita, A. Hayashi, and K. Takatsuki.1996. Rela-tionship of HIV-1 envelope V2 and V3 sequences of the primary isolates to the viral phenotype. Microbiol. Immunol.40:277–287.

on November 8, 2019 by guest



FIG. 2. Function of �clones. Percent fusion was calculated by using luciferase activity nor-malized to R3A fusion with QT6 cells coexpressing CD4 with CCR5 orCXCR4
FIG. 4. Alignment of Env sequences for R3A, TA1, �compared with the TA1 sequence. CloneV5) are indicated
TABLE 1. Amino acid mutations in �V3-X4A.c5 and �V3-X4B.c7 Envs
FIG. 6. �corresponding TA1 residues with CD4combination with CD4normalized toV3-X4B determinants for CXCR4 use in cell-cell fusion


Related documents

non emergency group 44 cases out of 48 patients were having abdominal pain

When assayed at 12 weeks after infection, spleens of LP-BM5 MuLV-infected perforin knock- outs had frequencies of cells producing infectious ecotropic and MCF viruses comparable

All regions of the ORF2 and ORF3 proteins containing immunoreactive epitopes in both the Burmese and Mexican strains of HEV were included in the artificial mosaic protein (Fig..

1 To study the prevalence of mitral annular calcification in chronic kidney disease patients compared to people with normal kidney function.. To find out various parameters

Stable BHK cell lines derived using the cloned variant vectors (lanes S1, S2, SF2A, SF1B, and SF2C) and naive BHK cells electroporated with parental vector RNAs (lanes SINBV and

Topo I preferen- tially binds to fully formed double hexamers (Fig. 3), strongly indicating that topo I associates with the initiation complex after the T-antigen double hexamer

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to

risk stratification of patients with acute ischemic heart disease.. These