Abstract
An increasingly large number of antiviral agents that prevent entry of human immunodeficiency virus (HIV) into cells are in preclinical and clinical development. The envelope (Env) protein of HIV is the major viral determinant that affects sensitivity to these compounds. To understand how changes in Env can impact entry inhibitor sensitivity, we introduced six mutations into the conserved coreceptor binding site of the R5 HIV-1 strain YU-2 and measured the effect of these changes on CD4 and coreceptor binding, membrane fusion levels and rates, virus infection, and sensitivity to the fusion inhibitors enfuvirtide (T-20) and T-1249, the CCR5 inhibitor TAK-779, and an antibody to CD4. The mutations had little effect on CD4 binding but reduced CCR5 binding to various extents. In general, reductions in coreceptor binding efficiency resulted in slower fusion kinetics and increased sensitivity to TAK-779 and enfuvirtide. In addition, low CCR5 binding usually reduced overall fusion and infection levels. However, one mutation adjacent to the bridging sheet β21 strand, P438A, had little effect on fusion activity, fusion rate, infectivity, or sensitivity to enfuvirtide or T-1249 despite causing a marked reduction in CCR5 binding and a significant increase in TAK-779 sensitivity. Thus, our findings indicate that changes in the coreceptor binding site of Env can modulate its fusion activity, infectivity, and entry inhibitor sensitivity by multiple mechanisms and suggest that reductions in coreceptor binding do not always result in prolonged fusion kinetics and increased sensitivity to enfuvirtide.
Human immunodeficiency virus type 1 (HIV-1) strains that are resistant to existing reverse transcriptase and protease inhibitors are becoming more common and account for a growing fraction of new infections in North America and Europe (27, 37). The development of a new class of antiviral agents that prevent entry of HIV into cells is a promising prospect for therapy, as viruses resistant to reverse transcriptase and protease inhibitors remain sensitive to these compounds (38, 39). Indeed, addition of the recently licensed entry inhibitor enfuvirtide (also called T-20 and Fuzeon) to an optimized background regimen of reverse transcriptase and protease inhibitors results in an average 10-fold reduction in viral load, which in many cases is sustained over a prolonged time period (32-34). Entry inhibitors described to date block binding of the viral envelope (Env) protein to CD4, binding of Env to the coreceptor, or the membrane fusion reaction itself (20, 38). However, Env is the most variable HIV protein, so the use of entry inhibitors may be complicated by significant variability in the sensitivity of diverse HIV-1 strains to these drugs (31). Characterizing the viral and host determinants that impact entry inhibitor sensitivity may provide information that can be used to guide the clinical application of entry inhibitors.
The HIV-1 entry process involves binding of the trimeric Env protein to CD4 and a coreceptor, either CCR5 or CXCR4 (6, 19). CD4 binding by the gp120 subunit of Env induces conformational changes that enable subsequent binding to a coreceptor (6, 19). These changes include the exposure of a conserved region in gp120 that, in conjunction with the V3 loop of gp120, mediates coreceptor binding (12, 13, 30, 48, 52, 56). Mutations in the coreceptor-binding site of gp120 have been shown to modulate the affinity of gp120 for CCR5 and CXCR4 (5, 47, 48), although the effects of these mutations on membrane fusion activity have not been investigated in detail (42, 53). Coreceptor inhibitors that bind to either CCR5 or CXCR4 have been described, with several showing efficacy in early clinical trials (A. L. Pozniak, G. Fätkenheuer, M. Johnson, I. M. Hoepelman, J. Rockstroh, F. Goebel, S. Abel, I. James, M. Rosario, C. Medhurst, J. Sullivan, M. Youle, and E. Van der Ryst, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother., 2003, abstr. H-443; J. Reynes, R. Rouzie, T. Kanouni, V. Baillat, B. Baroudy, A. Keung, C. Hogan, M. Markowitz, and M. Laughlin, Abstr. 9th Conf. Retrovir. Opportunistic Infect., 2002, abstr. 1).
gp120-coreceptor interactions induce structural alterations in the membrane-spanning gp41 subunit of Env that lead to membrane fusion (20). Fusion is thought to be induced by insertion of the fusion peptide at the N terminus of gp41 into the host cell membrane, after which this region is brought into close proximity to the transmembrane domain of gp41 via the formation of a coiled-coil structure composed of two helical domains in the ectodomain of gp41 (9, 55). The fusion inhibitor enfuvirtide, a peptide based on the sequence of the second helical region (HR2) in gp41, blocks formation of the coiled coil and thus prevents membrane fusion (11). Mutations in the first helical region (HR1) of gp41, selected for both in vitro and in vivo, can affect viral sensitivity to enfuvirtide, presumably by altering the affinity of enfuvirtide for HR1 (46, 54).
Differences in how Env engages its receptors can influence entry inhibitor sensitivity. We have shown that changes in the V3 loop of gp120 that reduce coreceptor affinity enhance the sensitivity of virus to coreceptor inhibitors and enfuvirtide (42). Reduced affinity for coreceptor can delay fusion kinetics, increasing the length of time that the binding site for enfuvirtide is exposed. We have also described a single-amino-acid change in the conserved coreceptor binding site that reduces gp120 affinity for CCR5 and increases viral sensitivity to enfuvirtide and a CCR5 inhibitor (42). Still, given the great variation in the sensitivity of primary virus strains to coreceptor antagonists and enfuvirtide, there are likely other mechanisms that could make viruses more or less sensitive to enfuvirtide and other entry inhibitors.
To investigate this, we introduced a series of amino acid changes in the coreceptor binding site of gp120 and examined the effects of these mutations on CD4 and CCR5 binding, membrane fusion levels, membrane fusion kinetics, viral infectivity, and sensitivity to several entry inhibitors. Our results show that reductions in CCR5 binding correlated with increased sensitivity to the CCR5 ligand TAK-779. There was also a correlation between fusion extent, fusion kinetics, susceptibility to soluble CD4 (sCD4)-induced fusion triggering, enfuvirtide sensitivity, and infectivity. In general, reductions in CCR5 binding delay fusion kinetics and enhance sensitivity to entry inhibitors that act downstream of CD4 binding. Reduced coreceptor affinity can also reduce fusion levels and viral infectivity. However, there is not a strict correlation between affinity and entry inhibitor sensitivity; we describe a point mutation, adjacent to the β21 strand of the bridging sheet, which significantly reduced gp120-CCR5 affinity without obviously affecting fusion kinetics, fusion or infection levels, or sensitivity to enfuvirtide. This mutant was susceptible to soluble CD4-induced fusion, in contrast to all the other mutants besides one that exhibited wild-type CCR5 binding. Our results indicate that the complex interactions between the highly variable Env protein and its receptors can modulate fusion activity and entry inhibitor sensitivity via multiple mechanisms.
MATERIALS AND METHODS
Cells.
293T, NP2 (57), NP2/CD4 (51), QT6, U87/CD4/CCR5 (7, 16), T-REx/CCR5 (42), and JC53 CD4/CCR5+ HeLa (40) cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U of penicillin, and 100 μg of streptomycin per ml. In addition, 1 mg of G418 per ml was used to maintain CD4 expression in NP2/CD4 cells, 0.3 mg of G418 plus 1 μg of puromycin per ml was used to maintain CD4 and CCR5 expression in U87/CD4/CCR5 cells, and 200 μg of zeocin plus 5 μg of blasticidin per ml was used to maintain CCR5 and Tet repressor genes in T-REx/CCR5 cells. High-level CCR5 expression was induced in T-REx/CCR5 cells by addition of 10 ng of doxycycline (Sigma) per ml to the culture medium.
The T-cell line CEMSS-R5 (25), stably expressing CCR5, was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U of penicillin, and 100 μg of streptomycin per ml (RPMI/10/PS). Peripheral blood mononuclear cells (PBMCs), cultured in RPMI/20/PS, were stimulated for 3 days with phytohemagglutinin (PHA; 5 μg/ml) and then cultured with interleukin-2 (IL-2; 50 U/ml) for 2 to 3 days prior to infection. CD8+ T cells were depleted from PBMCs with MACS colloidal superparamagnetic MicroBeads conjugated to monoclonal mouse anti-human CD8 antibodies (Miltenyi Biotech), according to the manufacturer's instructions.
Plasmids.
YU-2 Env was cloned into the MluI and PspOM1 sites of a modified pCI expression construct (Promega), modified to contain hepatitis B virus PRE to enable high-level, rev-independent Env expression (8, 42), to generate a gp160 expression construct that contained both T7 and cytomegalovirus promoters. T202G, K421D, I423S, P437A, P438A, and G441V amino acid changes (numbering according to HXB2) were introduced into the coreceptor-binding site of YU-2 Env with specific oligonucleotides and the Quikchange site-directed mutagenesis kit (Stratagene), according to the manufacturer's instructions. Stop codons were introduced at the gp120/gp41 cleavage junctions of YU-2 and mutant YU-2 gp160 expression constructs, with specific oligonucleotides and the Quikchange site directed mutagenesis kit, to generate gp120 expression constructs. StuI-SalI restriction fragments of YU-2 coreceptor binding site mutant Envs, with the exception of T202G, were subcloned into a molecular clone of YU-2 (pJD364; kindly provided by Jeff Dvorin) to generate coreceptor binding site mutant YU-2 proviral clones. A T202G Env mutant proviral clone was generated by concurrent site-directed mutagenesis of T202G, with specific oligonucleotides, and PCR amplification of a fragment encompassing the NcoI-StuI restriction sites in the vpr and env genes, respectively, of pJD364. An NcoI-StuI restriction fragment of this PCR product was then subcloned into pJD364.
The β-lactamase gene was cloned into the KpnI and XhoI sites of the pcDNA3.1+ expression plasmid (kindly provided by Mike Miller, Merck & Co.). The pGEM2 T7-luc expression plasmid was obtained from Promega. The pNL-luc (10, 14), CD4 (23), CXCR4 (43), CCR5 (43) and Env expression constructs have been described previously.
Env receptor binding assays.
gp120s were produced from 293T cells that were calcium phosphate transfected with gp120 expression constructs and infected with vTF1.1 vaccinia virus encoding T7 polymerase (1) to drive expression from the T7 promoter. Cell culture supernatants were harvested 24 h posttransfection, and gp120 concentrations were determined by enzyme-linked immunosorbent assay (ELISA) as previously described (44) with the exception that gp120 was detected with an HIV-1 Env-specific rabbit serum and a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody (Amersham Life Science) followed by TMB substrate (KPL).
The receptor binding efficiencies of YU-2 and YU-2 mutant gp120s were determined with a cell surface binding assay in which bound protein is detected by immunostaining and flow cytometry analysis as previously described (42, 44). CD4-negative T-REx/CCR5 cells, induced to express a high level of CCR5, were used to determine Env-CCR5 binding efficiency in the absence and presence of 5 μg of sCD4 per ml to induce coreceptor binding site exposure. NP2 and NP2/CD4 cells were used to determine Env-CD4 binding efficiency. Bound gp120 was detected with an HIV Env-specific rabbit serum and a phycoerythrin-conjugated anti-rabbit immunoglobulin antibody (Pharmingen).
Env expression analysis.
Total Env expression and gp120/gp160 processing levels of YU-2 and YU-2 mutant gp160 expression constructs were compared by Western blot analyses. QT6 cells were transfected with the Env expression plasmid and infected with vTF1.1 to drive Env expression from the T7 promoter. Cells were harvested 24 h posttransfection, and clarified lysates were assayed for gp160 and gp120 content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting followed by detection with an HIV Env-specific rabbit serum, a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody (Amersham), and Supersignal Femto substrate (Pierce). Blots were imaged on a Fujifilm LAS-1000 Plus luminescent image analyzer, and relative Env expression levels were compared with Fujifilm Science Lab 98 Image Gauge software.
The cell surface Env expression levels of YU-2 and YU-2 mutants were compared by immunostaining and flow cytometry analysis of QT6 cells transfected or infected with vTF1.1, as described above. Env was detected with an HIV Env-specific rabbit serum and a phycoerythrin-conjugated anti-rabbit immunoglobulin antibody (Pharmingen).
Cell-cell fusion assay.
QT6 effector cells, transfected with Env expression plasmids and infected with a vaccinia virus encoding T7 polymerase (vTF1.1) (1), were added to QT6 target cells cotransfected with a luciferase reporter construct under the control of a T7 promoter (T7-luc), CD4 and CCR5 expression plasmids. Cell-cell fusion, resulting from a functional interaction between the Env-expressing effector cells and receptor-expressing target cells, was detected by assaying for T7 polymerase-driven luciferase expression within the linear range of the assay. This assay has been described in detail previously (49).
Env fusion kinetics.
The fusion kinetics of YU-2 wild-type and mutant Envs were determined in a β-lactamase reporter cell-cell fusion assay, based on that recently described by Lineberger et al. (36). QT6 effector cells, cotransfected with Env and β-lactamase expression constructs and infected with vTF1.1, were added to CD4/CCR5+ HeLa target cells labeled with CCF2-AM as previously described (36). Cell-cell fusion was detected by assaying for a shift from green to blue fluorescence, indicating β-lactamase cleavage of CCF2. Fluorescence was quantitated with a CytoFluor Series 4000 Fluorescence multiwell plate reader (PerSeptive Biosystems), and the results are expressed as the ratio of blue to green fluorescence obtained with Env-transfected effectors minus the ratio of background blue and green fluorescence obtained with empty-vector-transfected cells.
Virus infection assays.
Luciferase reporter pseudotype viruses bearing YU-2 and YU-2 mutant Envs were generated by cotransfection of 293T cells with the gp160 and pNL-luc expression constructs as previously described (10, 14). Pseudotype viruses, normalized for p24 content, were used to infect U87/CD4/CCR5 cells, and infection was analyzed by assaying for luciferase expression 3 days postinfection.
YU-2 and YU-2 mutant replication-competent viruses, generated by transfection of proviral constructs into 293T cells, were normalized for p24 content and used to infect CEMSS-R5 cells and CD8+-T-cell-depleted PBMCs. Input virus was removed by extensive washing, and culture supernatants were harvested at various times postinfection for p24 analysis of viral replication.
Fusion inhibition assays.
The enfuvirtide, T-1249, TAK-779 and CD4-specific monoclonal antibody sensitivity of Env-mediated fusion was determined by fusing cells in the presence of different concentration of inhibitors as previously described (45).
RESULTS
Coreceptor binding site mutagenesis of YU-2.
The bridging sheet and structurally adjacent regions of gp120 have been shown to play a role in mediating binding to coreceptors, with mutations in this highly conserved domain affecting the affinity with which gp120 binds to CCR5 (47, 48). In addition, we found that a single-amino-acid change (K421D), immediately adjacent to the β20 strand of the bridging sheet, not only reduced the affinity with which gp120 bound to CCR5 but increased Env sensitivity to enfuvirtide by approximately 30-fold (42). To investigate the effect of mutations in this highly conserved region on receptor binding, membrane fusion, infection of primary cells, and sensitivity to entry inhibitors more fully, we produced a panel of six point mutants in the context of gp120 expression constructs in order to measure receptor binding, gp160 expression constructs in order to quantitatively measure membrane fusion and Env-mediated infection, and proviral constructs in order to measure the effects of these mutations on virus infection and replication. Mutations T202G and I423S reside in the β3 and β20 strands, respectively, of the bridging sheet domain, K421D lies immediately adjacent to the β20 strand of the bridging sheet, and mutations P437A, P438A, and G441V lie adjacent to the β21 strand of the bridging sheet (Fig. 1). The effects of these mutations on the CCR5 binding ability of modified YU-2 gp120, with a truncated N terminus and lacking the V1 and V2 loops, has been described previously (47, 48).
FIG. 1.
Mutation sites on YU-2 gp120. Space-filling model of YU-2 gp120, with the conserved bridging sheet domain and two-domain sCD4 in ribbon format. Mutated residues are indicated, as are the V1/V2 and V3 loop stems. All of the mutations lie within or adjacent to the bridging sheet strands. The model was rendered with RasMol 2.7.1 from protein databank file 1G9N; YU-2 gp120 envelope glycoprotein complexed with CD4 and induced neutralizing antibody 17b (29).
Impact of YU-2 gp120 mutations on CD4 and CCR5 binding.
Supernatants containing YU-2 and YU-2 mutant gp120s, secreted from transiently transfected 293T cells, were quantitated for gp120 content by ELISA. All gp120s were secreted from cells with similar efficiency. Supernatants containing equal amounts of gp120, as judged by ELISA analysis, were incubated with NP2 or NP2/CD4 cells; the cells were then washed, and bound gp120 was detected by flow cytometry. The amount of gp120 used for this assay was empirically adjusted to within the linear range of the assay so that a correlation between gp120 concentration and binding could be measured. We found that the six YU-2 mutants bound to CD4 similarly to the wild-type protein (Fig. 2A), consistent with previous results in which these mutations were examined in the context of YU-2 gp120 core proteins with 82 residues deleted from the N terminus and lacking the V1 and V2 loops (YU-2 Δ82ΔV1V2) (47, 48).
FIG. 2.
Receptor binding efficiencies of YU-2 mutant Envs. The CD4 and CCR5 binding efficiencies of YU-2 and YU-2 mutant Envs were determined by immunostaining and flow cytometry analysis of gp120 binding to the surface of (A) NP2 and NP2/CD4 cells and (B) TREx/CCR5 cells in the presence and absence of sCD4. Results represent the average and standard error of the mean of three independent experiments.
Based on previous studies and the structure of gp120, we anticipated that the mutations introduced into intact YU-2 gp120 would impact binding to CCR5 (30, 42, 47, 48). To address this, equal amounts of each gp120 protein were incubated with sCD4 to trigger the conformational changes needed for CCR5 binding. The gp120-sCD4 complexes were then incubated with T-REx/CCR5 cells that had been induced to express high levels of CCR5. The binding assay was also performed in the absence of sCD4 as a specificity control. After binding, the cells were washed, and bound gp120 was detected by flow cytometry. Once again, the amount of gp120 was adjusted to within the linear range of this binding assay. The YU-2 mutants exhibited three different CCR5 binding phenotypes. We found that P437A bound to CCR5 as efficiently as wild-type YU-2, T202G and I423S bound with approximately 25% of wild-type efficiency, while K421D, P438A, and G441V either did not bind to CCR5 or did so at a level below the limit of detection of this equilibrium binding assay (Fig. 2B). The relative CCR5 binding efficiencies of these mutants correlated well with those reported by Rizzuto et al., using YU-2 Δ82ΔV1V2 proteins (47, 48), though our binding efficiencies were lower. This may be due to different assay sensitivities or the presence of the N-terminal 82 residues, and V1/V2 loops in our intact gp120 constructs may reduce CCR5 binding efficiency due to structural constraints, without hindering relative CD4 binding efficiencies.
Impact of YU-2 mutations on membrane fusion.
While the effects of mutations within the coreceptor binding site region on receptor binding have been assessed, most studies have not examined the effects of these mutations on Env function. To study relationships between mutations in this region, their effects on receptor binding as well as Env function in cell-cell fusion assays, we first confirmed that each of the six YU-2 mutants was expressed and processed efficiently in the context of full-length Env. Cells expressing each protein were assayed for cell surface Env expression by flow cytometry or lysed and analyzed by Western blotting for the presence of gp160 and gp120. We found that all of these proteins, which differ from each other by only a single amino acid, were expressed and processed at similar levels, consistent with the fact that each gp120 protein was efficiently secreted from cells (Fig. 3 and data not shown).
FIG. 3.
Expression analysis of YU-2 mutant Envs. Env expression was analyzed in QT6 cells transfected with Env expression plasmids and infected with a vaccinia virus encoding T7 polymerase to drive expression. Total Env expression was determined by SDS-PAGE and Western blot analysis of cell lysates, followed by detection of Env with a specific rabbit serum and a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody. Expression was quantitated with Fujifilm Science Lab 98 Image Gauge software to analyze chemiluminescent signals imaged on a Fujifilm LAS-1000 Plus luminescent image analyzer. Serial threefold dilutions of cell lysates ensured that detection was within the linear range of the assay. Results represent the average and standard error of the mean of four independent experiments. Cell surface Env expression was detected by immunostaining and flow cytometry analysis. Results represent the mean fluorescence intensity of a representative experiment, expressed as a percentage of the mean intensity obtained with YU-2.
To measure the membrane fusion activity of each Env, we used two different cell-cell fusion assays in which cells expressing Env are mixed with reporter cells expressing CD4 and CCR5. The first assay measures relative levels of fusion, while the second measures fusion rates. With the first assay, only two of the YU-2 mutants mediated efficient cell-cell fusion (Fig. 4A; note the log scale). The P438A Env fused as efficiently as wild-type YU-2, while P437A mediated membrane fusion was reduced by only twofold (Fig. 4A). Fusion mediated by the T202G and G441V Envs was reduced approximately fivefold relative to that with the wild type, while fusion mediated by K421D and I423S was reduced more than 10-fold (Fig. 4A). No fusion was observed with any Env on target cells expressing the CD4 receptor only (data not shown).
FIG. 4.
Fusion and infection efficiencies of YU-2 Env mutants. (A) Relative fusion was determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing the indicated Envs and T7 polymerase and QT6 target cells expressing CD4, CCR5, and a T7 luciferase reporter plasmid. Results are expressed as a percentage of YU-2-mediated fusion and represent the average and standard error of the mean of at least seven independent experiments. (B) Relative luciferase reporter pseudotype virus infection of U87/CD4/CCR5 cells. Envs were pseudotyped onto the surface of a reporter virus core, and virus stocks, normalized by p24 assay, were used to infect U87/CD4/CCR5 cells. Cell lysates were assayed for luciferase activity 3 days postinfection. Results are expressed as a percentage of the luciferase activity in YU-2-infected cells and represent the average and standard error of the mean of at least three independent experiments.
The fact that K421D, P438A, and G441V could mediate fusion demonstrates that these Envs must bind to CCR5, though with affinities below the detection limit of our binding assay. The efficient cell-cell fusion mediated by P438A was surprising, since we could not detect binding of the gp120 derived from this Env to CCR5. Taken together, these results indicate that reduced gp120-CCR5 binding usually results in reduced fusion activity. However, there is not a perfect correlation between relative CCR5 binding and relative fusion levels, as G441V and especially P438A fused more efficiently than might have been expected from their undetectable gp120-CCR5 binding profiles. It is possible that P438A and G441V reduce CCR5 affinity in the context of monomeric gp120s but not CCR5 avidity in the context of the intact Env trimers. However, as described below, we found that P438A and G441V were more sensitive to inhibition by a CCR5 ligand, as was K421D, suggesting that these mutations do in fact reduce coreceptor avidity in the context of the trimeric Env protein.
Effects of YU-2 mutations on fusion kinetics.
We employed a second type of cell-cell fusion assay that makes it possible to measure the rate as well as extent of membrane fusion (34). In this assay, cells expressing CD4 and CCR5 are loaded with the fluorescent dye CCF2-AM, after which they are mixed with cells expressing the desired Env protein as well as β-lactamase. Upon fusion, β-lactamase is introduced into the cytoplasm of the target cell, where it cleaves CCF2, resulting in a change in emission wavelength that can be quantitated in a fluorometer. When this assay was used and the extent of fusion was determined after 80 min, the relative overall fusion levels for the YU-2 mutants compared to that of YU-2 were higher than observed with the luciferase assay, though the overall pattern remained the same (Fig. 5A). Thus, P438A fused as efficiently as the wild type, P437A also mediated efficient fusion, and T202G and G441V were the next most efficient Envs, while K421D and I423S fused about fourfold less efficiently than wild-type YU-2 (Fig. 5A). Assay-based differences in relative fusion levels of YU-2 and mutant Envs may be accounted for by differences in target cell receptor expression levels, which were higher in the β-lactamase fusion assay. Additionally, the ratio of Env-expressing effector cells to target cells was higher in the β-lactamase fusion assay.
FIG. 5.
Kinetics of YU-2 Env mutant fusion. The efficiency and kinetics of YU-2 Env-mediated cell-cell fusion were determined in a β-lactamase reporter assay with QT6 effector cells expressing the indicated Env, a β-lactamase reporter plasmid and T7 polymerase, and CD4/CCR5+ HeLa target cells loaded with the green fluorescent dye CCF2-AM. Upon cell-cell fusion, β-lactamase cleavage of the CCF2 substrate results in a change from green to blue fluorescent emission that was quantitated in a fluorometer. (A) Fusion expressed as a ratio of blue to green fluorescence. Points represent the average ± standard deviation of triplicate wells from an individual experiment. The data are representative of at least three independent experiments. (B) Fusion (ratio of blue to green fluorescence) expressed as a percentage of maximum fusion for each Env to aid comparison of relative kinetic profiles.
The rate of Env-mediated membrane fusion can influence viral sensitivity to certain types of entry inhibitors (42). Since the rate of membrane fusion is dependent in part on Env-receptor affinity, we tested the panel of YU-2 mutants in the β-lactamase fusion assay to see if reduced CCR5 binding caused by the various point mutations affected membrane fusion kinetics as well as extents. We found a good correlation between the rate and extent of fusion (Fig. 5A and B). Mutants P437A and P438A caused cell-cell fusion at rates nearly indistinguishable from that of the wild type, with a time to half-maximal fusion (t1/2) of approximately 22 min (Fig. 5B). Mutants T202G and G441V, which caused an intermediate amount of fusion, exhibited delayed fusion kinetics, with a t1/2 of approximately 32 min, while I423S and K421D exhibited fusion kinetics that were delayed by approximately twofold from that of YU-2, with a t1/2 of approximately 39 and 44 min, respectively (Fig. 5B). Thus, under the conditions used here, point mutations in the coreceptor binding site that reduced fusion activity also reduced the rate of membrane fusion. These results also showed that P438A, despite exhibiting reduced binding to CCR5, fused not only as efficiently as YU-2, but also as quickly (Fig. 5A and B).
sCD4-induced triggering of YU-2 Env mutant fusion.
sCD4 can be used to induce fusion of some HIV Envs with CD4-negative coreceptor-positive cells. We examined whether sCD4 could induce fusion of cells expressing mutant YU-2 Env proteins with CD4−/CCR5+ cells. In this assay, Env is not tethered to the host cell membrane through interactions with CD4, and under these conditions an Env with low affinity for CCR5 would not be expected to fuse with CD4-negative cells unless CCR5 binding rapidly and efficiently triggered the conformational changes needed for membrane fusion. Therefore, we added increasing amounts of sCD4 to cells expressing the different Env proteins and measured fusion with CD4−/CCR5+ cells. Wild-type YU-2 was triggered most efficiently by sCD4 (Fig. 6). sCD4 triggering of P438A was as efficient as or better than that by P437A at higher sCD4 concentrations (Fig. 6). P437A fusion was consistently reduced at 25 μg/ml compared to 1 or 5 μg of sCD4 per ml (Fig. 6). In contrast, sCD4 did not induce fusion by K421D or G441V, which, like P438A, exhibited no detectable CCR5 binding, or even T202G or I423S Envs, which did bind detectably to CCR5 (Fig. 6). Taken together, the results of these fusion experiments show that there is a good but imperfect correlation between gp120-CCR5 binding efficiency and membrane fusion activity and that some mutations can reduce coreceptor binding without obviously affecting membrane fusion activity, perhaps by making Env more sensitive to receptor triggering.
FIG. 6.
sCD4 triggering of YU-2 mutant Env fusion. sCD4-induced fusion was determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing Env and T7 polymerase and QT6 target cells expressing a T7 luciferase reporter plasmid and CCR5 receptor only. The indicated amount of sCD4 was added to target and effector cells, and luciferase activity was measured 7 h later. Fusion is expressed as a percentage of that observed with YU-2 on target cells expressing a T7 luciferase reporter plasmid and both cellular CD4 and CCR5. Results represent the average and standard error of the mean of four independent experiments.
Sensitivity to entry inhibitors.
Having introduced mutations into conserved regions of YU-2 that reduced CCR5 binding and in some cases altered fusion efficiency and kinetics, we next determined the impact of these changes on the sensitivity of each Env to a CD4-specific antibody, a small-molecule inhibitor of CCR5 binding (TAK-779) (2), and the fusion inhibitors enfuvirtide and T-1249. None of the gp120 mutations significantly affected the ability of a monoclonal antibody to CD4 to prevent membrane fusion (Fig. 7A), consistent with our finding that the mutations had little impact on gp120-CD4 binding (Fig. 2A). In contrast, several of the point mutations greatly affected the ability of TAK-779 to block membrane fusion (Fig. 7B). The three mutations that reduced CCR5 binding efficiency to below the detection limits of our binding assay (K421D, P438A, and G441V) had the greatest impact on TAK-779 sensitivity, with the K421D and G441V mutants being exquisitely sensitive to TAK-779 (approximately 60- and 30-fold more sensitive than YU-2, respectively) and P438A reducing the amount of TAK-779 needed to achieve 50% inhibition from approximately 8,700 nM to approximately 2,600 nM (Fig. 7B). The two point mutations that reduced binding to CCR5 by approximately fivefold (T202G and I423S) reduced TAK-779 sensitivity less than twofold, while P437A had little effect on TAK-779 sensitivity (Fig. 7B), just as it had no apparent effect on CCR5 binding (Fig. 2B). Thus, there was a good correlation between the effects of mutations on CCR5 binding and their effects on TAK-779 sensitivity, as we have noted previously (42).
FIG. 7.
Entry inhibitor sensitivity of YU-2 Env mutants. Fifty percent inhibition (IC50) values for fusion inhibition of YU-2 mutants by (A) a CD4-specific monoclonal antibody, (B) TAK-779, (C) enfuvirtide, and (D) T-1249, determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing Env and T7 polymerase and QT6 target cells expressing CD4, CCR5 and a T7 luciferase reporter plasmid. The concentration of each inhibitor needed to reduce fusion activity by 50% was determined in at least three independent experiments, and results represent average values and the standard error of the mean.
Fusion inhibition by enfuvirtide did not correlate as well with gp120-CCR5 binding, though there was a good a correlation between enfuvirtide sensitivity, fusion levels, and fusion kinetics (Fig. 7C, 2B, 4A, and 5). The rank order of the various YU-2 Env mutants for fusion kinetics (YU-2 ≥ P438A ≥ P437A > G441V ≥ T202G > I423S > K421D; Fig. 5B) was the same as their rank order for enfuvirtide sensitivity (Fig. 7C). From previous studies with V3 chimeric and nonisogenic Envs (42; J. Reeves, B. Puffer, N. Ahmad, C. Derdeyn, M. Sharron, T. Edwards, D. Carlin, P. Harvey, T. Pierson, E. Hunter, and R. Doms, Abstr. 9th Conf. Retrovir. Opportunistic Infect., 2002, abstr. 82), absolute fusion levels are not indicative of relative enfuvirtide sensitivity. For example, two Envs that fuse to a similar extent can exhibit enfuvirtide sensitivities that vary by greater than 10-fold. When experiments were performed with the fusion inhibitor T-1249, which is about a log more potent than enfuvirtide, considerably less variability in drug sensitivity was observed between YU-2 and YU-2 mutants, which may be a consequence of T-1249 potency (Fig. 7D). Since none of the mutations in gp120 directly affect the binding site in gp41 for enfuvirtide, we conclude that their effects on enfuvirtide sensitivity are largely due to differences in fusion kinetics, with slower fusion kinetics resulting in prolonged exposure of the enfuvirtide binding site.
Impact of YU-2 mutations on virus infection.
To investigate if the fusogenicity of YU-2 mutants correlated with their infection and replication profiles, we first produced luciferase reporter viruses by pseudotyping each Env protein onto HIV cores from an env-defective reporter virus that expresses the luciferase gene in place of nef (10, 14). The resulting virus stocks were normalized by p24 ELISA and used to infect U87 cells stably expressing CD4 and CCR5. The results of these experiments were similar to those obtained with the cell fusion assays, though in all cases the YU-2 mutants functioned relatively less well than wild-type YU-2 in infection than in cell-cell fusion (Fig. 4B). Thus, P438A mediated efficient virus infection; infection mediated by P437A was reduced by approximately 4-fold, T202G and G441V infection was reduced approximately 25-fold, while infection mediated by K421D and I423S was reduced by at least 10-fold (Fig. 4B).
We also introduced five of the various mutations into a YU-2 provirus so that their impact on viral replication in a CCR5-expressing T-cell line (CEMSS-R5; Fig. 8A) and CD8+ T-cell-depleted PBMC cultures (Fig. 8B) could be determined. For these assays, either 1 ng (top panels) or 10 ng (bottom panels) of each virus stock was used, with similar results. The replication efficiency of the YU-2 mutants was comparable to their relative fusion and single-round pseudotype infection assay results. As for the pseudotype infections, the replication efficiency of the mutant Envs was affected to a greater extent than in cell-cell fusion assays. In CEMSS-R5 cells, with 10 ng of p24-normalized virus/105 cells, P438A replicated to about the same level as YU-2; P437A and T202G peak p24 levels were reduced approximately 5-fold, G441V peak p24 levels were reduced approximately 15-fold, and K421D replicated poorly, with peak p24 levels reduced by approximately 200-fold (Fig. 8A, bottom panel). Even though overall infection levels were different in CEMSS-R5 cells, the replication kinetics of mutant and wild-type viruses were similar, with peak p24 values occurring 5 days postinfection (Fig. 8A). In CD8+-T-cell-depleted PBMCs, with 10 ng of p24-normalized virus/105 cells, P438A replicated to a similar extent as YU-2, P437A and T202G peak p24 levels were reduced approximately 3-fold, while K421D and G441V peak p24 levels were reduced by approximately 10-fold (Fig. 8B, bottom panel). With YU-2 and P438A, infection of PBMCs reached or neared plateau p24 levels 10 days postinfection, whereas p24 values with the other mutant viruses generally peaked at an earlier time point and then decayed, which likely indicates a reduced capacity to establish a spreading infection (Fig. 8B).
FIG. 8.
Replication efficiency of YU-2 mutant viruses. Growth curves of replication-competent YU-2 mutant viruses in the (A) CEMSS-R5 T-cell line and (B) CD8+-T-cell-depleted PBMC cultures, with 1 or 10 ng of p24-normalized input virus/105 cells as indicated. Input virus was removed by extensive washing, and viral replication was evaluated by analyzing culture supernatants for p24 production at the indicated days postinfection. Results represent average p24 values plus standard deviation of the results for triplicate wells from representative experiments.
DISCUSSION
The HIV-1 Env protein mediates virus entry by sequentially binding CD4 and either CCR5 or CXCR4, which trigger conformational changes in Env that lead to membrane fusion (20). The types of receptors engaged by Env and how Env engages those receptors are factors that can influence viral tropism and pathogenesis. Thus, viruses that evolve from using CCR5 to using CXCR4 appear to be less adept at establishing infections in new hosts but are associated with accelerated disease progression in those with established infections (15), while decreased dependence on CD4 levels has been linked to macrophage tropism in the simian immunodeficiency virus system and neurotropism for both simian immunodeficiency virus and HIV (4, 22, 26, 41). Theoretically, how well an Env binds to a coreceptor and how Env responds to receptor binding could also influence tropism and pathogenesis. For example, some Envs could be more easily triggered than others, either requiring fewer receptor-binding events to elicit membrane fusion inducing conformational changes or undergoing conformational changes more quickly. These factors are also likely to influence sensitivity to different entry inhibitors. Given the impressive variability of Env and its ability to respond to selective pressures, it is therefore perhaps not surprising that primary HIV-1 strains can differ considerably in their sensitivity to entry inhibitors (31). Identifying the mechanisms that account for this variability could make the administration of these new compounds more efficient as they move forward in clinical development.
Sensitivity to entry inhibitors can be directly influenced by changes in Env that impact inhibitor binding sites and indirectly by changes that affect how Env engages and responds to receptor binding events. Changes in the HR1 domain of the gp41 subunit, which comprises the binding site for the fusion inhibitor enfuvirtide, can affect virus sensitivity to this newly approved drug both in vitro and in vivo (46, 54). Alternatively, changes in gp120 that affect coreceptor binding can also affect enfuvirtide sensitivity even though this compound binds to gp41 (17, 18, 42). We have found that reduced coreceptor binding efficiency can slow fusion kinetics, leading to prolonged exposure of the enfuvirtide binding site and increased sensitivity to this fusion inhibitor. With multiple domains in Env playing roles in receptor binding and the ensuing conformational changes, it is likely that multiple regions in Env can affect sensitivity to different classes of entry inhibitors.
In this study, we investigated how changes in one functionally important region in Env, the conserved coreceptor binding site in gp120, can impact sensitivity to several different entry inhibitors. The V3 loop of gp120 is primarily responsible for mediating coreceptor specificity, determining whether CCR5, CXCR4, or both coreceptors can be used by a given virus strain (12, 13, 52, 56). Recent modeling data suggest that the V3 loop of gp120 mimics a region of the chemokine ligands for CCR5 or CXCR4 (50) and interacts with the extracellular loops of the coreceptor. In contrast, the coreceptor binding site, at least for CCR5, appears to interact with the receptor's N-terminal domain. The precise role of the coreceptor binding site in virus entry is not clear. While mutations introduced into this highly conserved region often result in reduced affinity of gp120 for CCR5 or CXCR4 (5, 47, 48), the effects of mutations in this region on virus infection are largely undescribed. Because of its role in modulating coreceptor affinity and its highly conserved nature, we introduced mutations into this region to assess not only how they affect coreceptor binding, but how this in turn impacts Env function in both fusion and infection assays.
In good agreement with work by Rizzuto et al. (47, 48), we found that mutations in the coreceptor binding site reduced the binding efficiency of gp120 for CCR5. Monomeric gp120-receptor binding affinities can differ from Env trimer-receptor binding affinities, but it is likely that coreceptor binding site mutations reduce the binding efficiency of both native Env trimers and monomeric gp120s for CCR5, since we found a good correlation between CCR5 binding and sensitivity to the CCR5 inhibitor TAK-779. Since TAK-779 does not appear to efficiently downregulate CCR5, its primary mode of action is likely to occupy at least a portion of the gp120 binding site on this coreceptor, making it more likely that mutations that increase Env affinity for CCR5 will decrease sensitivity to TAK-779, while mutations that decrease coreceptor affinity will enhance sensitivity to TAK-779. Thus, we conclude that the single-amino-acid changes studied here that reduced CCR5 binding also reduced CCR5 affinity in the context of intact Env trimers.
We observed a partial correlation between coreceptor binding efficiency and absolute fusion levels, with a notable exception discussed below (P438A). While only a few different Env proteins have been examined carefully for coreceptor binding affinity, those that have been studied show that relatively broad ranges of affinities are compatible with efficient virus infection. gp120 proteins tend to bind to CCR5 with affinities between 4 and 15 nM (21, 56). For CXCR4, laboratory-adapted HIV-1 strains and primary R5X4 isolates bind more weakly, with Kds of between 200 and 500 nM (3, 28). Whether relatively poor affinity for CXCR4 will prove to be a general feature of X4 HIV-1 strains is not known, though high-affinity interactions with CXCR4 are possible, as evidenced by HIV-2 VCP and HIV-2 ROD/B (35). Interestingly, these viruses can infect CXCR4-positive cells independently of CD4 (24, 43), due perhaps in part to their high affinity for this coreceptor.
While we did not directly measure the affinity with which the various YU-2 gp120 proteins interacted with CCR5, we can conclude that those which bound below the limit of detection in the equilibrium binding assay did so with affinities of >100 nM. Since these viruses were also >3- to approximately 60-fold more sensitive to TAK-779, it is apparent that a number of single-amino-acid changes in gp120 reduced CCR5 affinity by 10-fold or more. This reduction in binding efficiency was accompanied by reduced fusion activity, with the exception of P438A. It is interesting, however, that I423S and T202G bound detectably to CCR5, indicating that they bind to this coreceptor with higher affinity than most X4 HIV strains bind to CXCR4. Despite this, both I423S and T202G caused membrane fusion at reduced levels. There appears to be a paradox between X4 and R5 virus strains with regard to coreceptor affinity and membrane fusion efficiency, with lower affinities being more compatible with efficient membrane fusion via CXCR4 than via CCR5. It will be interesting to determine if CXCR4 binding triggers conformational changes in Env either more efficiently or more quickly than CCR5 binding, which might allow for tolerance of poorer affinities.
Our results show that there was a correlation between fusion extent, fusion kinetics, susceptibility to sCD4-induced fusion, enfuvirtide sensitivity, and infectivity. In general, we found that reductions in coreceptor binding efficiency correlated with reduced fusion efficiency and infectivity, slower fusion kinetics, and enhanced sensitivity to entry inhibitors that target CCR5 binding or fusion. However, the correlation was not perfect, as we found that P438A reduced gp120-CCR5 binding to below the detection limit of our equilibrium binding assay but resulted in an Env that was essentially wild type with regard to fusion kinetics, fusion extent, infectivity, and replication kinetics. The P438A mutant was more sensitive to TAK-779, consistent with a reduction in coreceptor affinity. However, it was essentially wild type with regard to enfuvirtide sensitivity. The mechanism by which P438A Env mediates fusion with nearly wild-type efficiency and kinetics despite reduced CCR5 affinity is not clear. Perhaps the P438A mutation affects the affinity of monomeric gp120 for CCR5 to a greater extent than it does in the trimeric Env protein. Alternatively, the P438A Env may also enhance the efficiency or kinetics with which receptor binding triggers conformational changes that lead to membrane fusion. Indeed, P438A was the only mutant besides P437A that could be triggered by sCD4 to fuse with CD4− CCR5+ cells. Interestingly, of all the mutants that we examined, P438A was the only one that exhibited wild-type or increased binding to a CD4 binding site monoclonal antibody, a CCR5 binding site monoclonal antibody, and a monoclonal antibody that binds to a CD4-induced coreceptor-binding site epitope, in the context of a YU-2 core gp120 protein (47, 48). This may indicate that the P438A mutation results in an Env with a more receptor-accessible conformation, which in turn may result in more efficient receptor triggering.
In summary, by examining a panel of Env mutants containing single-amino-acid changes in and around the bridging sheet region of gp120 that constitutes a coreceptor-binding domain, we have established that coreceptor affinity is an underlying phenotypic property that correlates with sensitivity to the CCR5 inhibitor TAK-779 and partly with enfuvirtide sensitivity. Enfuvirtide sensitivity correlated well with the extent and kinetics of YU-2 Env mutant-mediated fusion. Additionally, we identified a point mutant that exhibits fusion, infection, and enfuvirtide and T-1249 sensitivity profiles similar to those of wild-type Env but exhibits a relatively low CCR5 affinity. These results indicate that a number of mechanisms are likely to contribute to the differential sensitivity of primary viruses to entry inhibitors and show that properties other than coreceptor affinity can modulate fusion, infection, and entry inhibitor sensitivity.
Acknowledgments
We acknowledge Aimee Kessler for technical support with p24 assays and DNA preps, the University of Pennsylvania CFAR for p24 assays, and Karin Strecker for phlebotomy procedures. We also thank Jeff Dvorin for plasmid pJD364, Mike Miller for the β-lactamase expression plasmid, Trimeris for enfuvirtide and T-1249, Jim Hoxie for a CD4-specific monoclonal antibody, Emily Platt and David Kabat for CD4/CCR5+ HeLa cells, Hiroo Hoshino for NP2 and NP2/CD4 cells, and the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, for TAK-779.
This work was supported by NIH grant R01 40880 and grants from the Burroughs Wellcome Foundation and the Elizabeth Glaser Pediatric AIDS Foundation. J.D.R. is supported by grant 106437-34-RFGN from the American Foundation for AIDS Research.
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