Membrane binding properties of 10E8-3R.

Following the approach described in our previous works (24, 25) (see schematics displayed in Fig. 2A), virus-like (VL) vesicle flotation experiments were first used to establish the effect of the 3R mutations on Fab 10E8 partitioning from water into membranes (Fig. 2B). Anti-gp120 antibody PG9, used as a control for the absence of an interaction with membranes, and 10E8-WT showed similar flotation profiles after sucrose centrifugation, with most input antibody being recovered in pellets (i.e., from nonfloating fractions). In sharp contrast, under the same experimental conditions, 67% of 10E8-3R was found cofloating with vesicles (rhodamine [Rho] emission spectra in Fig. 2B, bottom). The antibody signal was detected mainly in the nonfloating fractions when the same experiment was run in the absence of vesicles. To ensure that the observed membrane binding phenomenon was dependent on the specific location of the 3R mutation, three Arg residues were instead placed into solvent-exposed positions S153R, S193R, and S199R of the 10E8 constant-domain light chain. The resultant 10E8-3R(C) mutant was found in the pellet fractions after centrifugation. Thus, the minimal amount of Fab 10E8-3R(C) detected in association with vesicles followed the pattern of parental 10E8-WT. These results indicate that light chain 3R mutation S30R/N52R/S67R specifically endows the 10E8 antibody with the capacity to partition into VL vesicles.

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FIG 2

Partitioning of Fabs into membranes measured by a vesicle flotation assay. (A) Diagram illustrating the method. Fabs (1.5 μM) were incubated with Rho-PE-labeled VL LUVs (1.5 mM) and subsequently deposited onto a stepwise sucrose gradient (time zero [t₀]). After ultracentrifugation (t₁), four different fractions were collected based on their densities. (B) Partitioning of Fab 10E8 and its derivatives into VL LUVs. The presence of vesicles and Fab in the different fractions was probed by rhodamine emission and Western blotting, respectively. VL LUVs (emission spectra at the bottom right) were found in the third and fourth fractions (i.e., floated fractions). An additional fraction, employing SDS, was collected to recover the material attached to the surface of the tube. Analyzed samples include (from top to bottom) PG9, an anti-gp120 antibody used as a negative control; 10E8-WT; 10E8-3R; 10E8-3R(−LUVs), a 10E8-R3 sample centrifuged in the absence of VL vesicles; and 10E8-3R(C), a control Fab containing a 3R mutation within the constant domain. The values displayed on the right correspond to the percentages of antibody found cofloating with vesicles, calculated by densitometry.

Assays using fluorescently labeled Fabs were additionally performed to prove the membrane binding capacity of 10E8-3R in an intact system (i.e., without physical separation of the vesicle-bound specimens). Figure 3A displays confocal fluorescence microscopy data for GUVs incubated with Fab. To carry out these experiments, GUVs were stained with 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) (emission rendered in green), and Fabs were labeled with the Abberior Star Red probe (KK114) (rendered in red), the latter of which was conjugated with heavy-chain residue C216_HC of the constant domain. In accordance with the spontaneous partitioning of the 3R mutant into bare membranes, VL GUVs incubated with the KK114-10E8-3R mutant were simultaneously stained with Laurdan and KK114. In contrast, no KK114 staining was detected in GUVs incubated with KK114-10E8-WT.

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FIG 3

Partitioning into membranes measured by fluorescence techniques. (A) Partitioning of fluorescently labeled Fabs KK114-10E8-WT and KK114-10E8-3R into VL GUVs. Micrographs display confocal images of VL GUVs at the equatorial plane. The lipid bilayer was labeled with Laurdan, and bound antibody was imaged following fluorescence emission of KK114 (green and red, respectively). The micrographs of both samples were rendered with equal contrast and brightness to best appreciate the difference in emission intensity. Bars, 2.5 μm. (B) Diagram illustrating the spectroscopic changes, namely, the increase in the emission intensity and maximum shift, that occur after transferring the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to the CDRH3 tip of Fabs from the solvent to the membrane (3, 24, 25). The thiol-reactive fluorescence probe used was N,N’-dimethyl-N-(iodoacetyl)-N’-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine (IANBD). (C) Binding of Fab 10E8 to VL LUVs monitored by changes in NBD fluorescence. Emission spectra of NBD-10E8-WT and NBD-10E8-3R were measured in solution (gray solid line) or in the presence of increasing concentrations of VL vesicles (black solid and dotted lines). Prior to spectrum collection, NBD-labeled Fabs (0.5 μM) were incubated with different concentrations of VL LUVs, as indicated. a.u., arbitrary units.

Membrane binding by 10E8-3R was also assessed by using Fabs labeled with the molecular sensor 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) (Fig. 3B). This approach to probe protein-membrane interactions was described in our previous studies (3, 24, 25) and is based on monitoring changes in fluorescence emission that occur upon transferring the NBD probe from an aqueous solvent to the less polar environment of the lipid bilayer. The changes in NBD emission, i.e., the increase in intensity and blue shift of the emission maximum wavelength, are exemplified by the model spectra displayed in Fig. 3B (left). A shift of the emission maximum wavelength and an increase of the fluorescence intensity were observed for samples incubated with NBD-10E8-3R, which is consistent with a tendency of the 3R mutant to associate spontaneously with VL membranes (Fig. 3C, bottom right). In contrast, incubation of Fab NBD-10E8-WT Fab with VL large unilamellar vesicles (LUVs) did not induce appreciable changes in the emitted fluorescence (Fig. 3C, bottom left).

To confirm the contribution of electrostatic interactions to the observed partitioning of Fab 10E8-3R Fab, we next quantified the antibody-membrane interaction by spectroscopic titration of NBD-labeled Fab against vesicles that combined 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) lipids at different molar ratios (26, 27) (Fig. 4). The experimental values, adjusted to equation 1, allowed the estimation of a mole fraction partition coefficient (K_x) of 0.2 × 10⁷ for DOPC-DOPS (50:50) vesicles, a value that falls in the range of those observed for peripheral membrane proteins such as phospholipase C-ζ or the juxtamembrane domain of ErbB1 (28, 29). Moreover, the K_x values decreased upon the reduction of the content of the negatively charged lipid DOPS (Fig. 4A). Consistently, the linear plot of partitioning free energies (ΔG_obs) as a function of the surface potential (Ψ₀) displayed a positive slope (Fig. 4B), following the pattern expected for antibody-membrane interactions being driven by electrostatic forces (30, 31). In conclusion, 10E8-3R followed a membrane binding mechanism that resembled a peripheral membrane interaction, as was described previously for Fab 4E10 (25) (see below).

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FIG 4

Binding of 10E8-3R to DOPC:DOPS LUVs monitored by changes in NBD fluorescence. (A) Titration curves obtained by plotting the fraction of NBD-10E8-3R bound as a function of the concentration of lipid accessible on DOPC:DOPS vesicles (half the total lipid concentration) that contained 50, 25, or 10 mol% of DOPS (data depicted in blue, red, and green, respectively). Each symbol on the plot represents an average of data from three independent experiments (± standard deviations if larger than the symbol). The molar fraction partition coefficients, K_x, displayed were calculated from the best fit of equation 1 to the data (curves). (B) Plot of the free energy of partitioning versus the membrane surface potential in the above-described lipid vesicles, estimated according to equations 2 and 3, respectively. The positive slope of the linear regression is consistent with the favorable contribution of electrostatic interactions to membrane partitioning.

Effects on the biological function of 10E8 imparted by the 3R mutation.

Having determined that the 3R mutations enabled the binding of 10E8 to lipid vesicles, we tested their effect on polyreactivity and neutralizing activity (Fig. 5 and ​and6).6). We first assessed the propensity of 10E8 and the cognate 3R mutant to bind to HEp-2 cells, which have been used previously for determining antibody polyreactivity (8, 32). We found that, in contrast to the positive control, 4E10 IgG, neither 10E8-WT nor 10E8-3R bound to HEp-2 epithelial cells (Fig. 5A). We also compared the polyreactivities of 10E8-WT and 10E8-3R in a standard ELISA (8, 33) against various lipids, DNAs, as well as the unrelated proteins ovalbumin (OVA) and bovine serum albumin (BSA). By these assays, neither 10E8-WT nor 10E8-3R showed significant polyreactivity, even at high concentrations, with the exception of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) binding, in which case 10E8 and the 3R mutant showed weak binding signals similar to that of the 4E10 IgG control (Fig. 5B).

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FIG 5

Analysis of 10E8-3R polyreactivity. (A) Immunofluorescence staining experiment 1 using 10E8-3R against HEp-2 cells. Fabs 10E8-WT and 10E8-3R were used at a concentration of 25 μg/ml. 4E10 IgG was used as a positive control. Images shown are at a ×200 magnification. (B) ELISA binding of 10E8-3R against various antigens. Fab PV04 and 4E10 IgG were used as a negative control and a positive control, respectively. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; mAbs, monoclonal antibodies.

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FIG 6

Neutralization of HIV-1 by 10E8-3R. Fabs 10E8-WT and 10E8-3R were tested in a neutralization assay using TZM-bl target cells and a cross-clade panel of the following HIV-1 isolates: 94UG103 (A), 92RW020 (B), 92BR020 (C), IAVI C22 (D), 16055 (E), and 92TH021 (F).

We next tested the effect of the 3R mutations on the neutralization capacity of 10E8 against a panel of six HIV-1 isolates used previously as an indicator of cross-clade neutralization breadth (34). As illustrated by the data displayed in Fig. 6, 10E8-3R was notably more potent against all six isolates than was 10E8-WT (5- to 35-fold decrease in the 50% inhibitory concentration [IC₅₀]) (Table 1). Ten additional isolates were tested, and 10E8-3R was also more potent against the majority of these isolates, including simian immunodeficiency virus (SIV) strain TAN2 (Table 1). In total, the 3R mutations increased 10E8 neutralization potency against 14 of 16 isolates, with an average 8.9-fold decrease in the IC₅₀; however, the neutralization potency against two isolates (Q23.17 and CH181.12) did not change, so the effect appears to be somewhat isolate dependent.

10E8-3R antibody engagement of the MPER peptide and HIV-1 Env.

The finding that the 3R mutation did not make 10E8 polyreactive indicates that the differences in the binding and neutralization of 10E8 caused by the mutations are specific. It was shown previously that 10E8 neutralizing activity correlates with its capacity for epitope recognition in a membrane environment (3). Therefore, we sought to establish whether the overall enhanced neutralization observed for 10E8-3R correlated with an improved epitope binding function (Fig. 7 and ​and88).

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FIG 7

Effect of enhanced electrostatic interactions on epitope recognition at the membrane surface. (A) Partitioning of KK114-labeled Fabs 10E8-WT and 10E8-3R into VL GUVs decorated with the MPER(671–693) epitope peptide. Conditions are otherwise as described in the legend of Fig. 3. (B) Changes of fluorescence emission spectra upon incubation of NBD-labeled 10E8-WT or 10E8-3R with vesicles that contained 1.7 μM MPER(671–693) (black and blue lines, respectively). The gray line represents the emission spectra of the NBD-labeled Fab fragments in solution. (C) Increase in the fractional emission and change in the position of the maximum fluorescence emission wavelength in the presence of increasing concentrations of vesicles that contained 1.7 μM peptide (top and bottom, respectively). The initial value of fluorescence (F₀) was determined from the maximum intensity of the labeled Fab fragment in solution, i.e., before the addition of peptide-containing vesicles. Final fluorescence values (F) correspond to the NBD intensity measured after incubation with vesicles. Black and blue symbols correspond to WT and 3R Fabs, respectively. Each data point corresponds to the average of results from three titrations (± standard deviation), as displayed in panel B.

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FIG 8

Binding of 10E8-3R to native Env trimers on virions. (A) HIV-1 virions displaying Comb-mut Env were incubated with Fabs 10E8-WT or 10E8-3R, followed by treatment with mild detergent and the resolution of Env-Fab complexes using BN-PAGE Western blotting. Binding of an antibody to the Env trimer causes it to run more slowly on the gel and the cognate band to shift upwards on the blot. Estimation of the migration of a trimer with one, two, or three Fabs bound (left) is based on gel mobility shifts by control Fabs PGT126 (3 Fabs/trimer), PGT151 (2 Fabs), and PG9 (1 Fab). (B) BN-PAGE gel mobility shift analysis was performed as described above for panel A, except that after Fabs and virus were incubated together for 30 min, the virus was pelleted, and unbound Fab was removed. Thus, the gel mobility shift reflects only binding to the trimer while it was incorporated into the virion membrane. (C) Intensities of bands on BN-PAGE Western blots were quantified by using ImageJ software, and the percentage of the trimer bound by antibody was calculated by comparing the intensities of the unliganded trimer band in the presence and absence of Fab. The results shown are the averages of data from four (standard assay) and two (washout) independent BN-PAGE Western blot assays.

Figure 7A (top) displays confocal images of peptide-containing GUVs that were probed by Laurdan labeling and with Fab KK114-10E8-WT. These GUVs exhibited simultaneous staining of the lipid bilayer by both Laurdan and KK114 probes (green and red, respectively), consistent with the previously reported binding of 10E8 to the MPER peptide inserted into membranes (3). The relatively intense KK114 staining of GUVs observed upon incubation with Fab KK114-10E8-3R suggests that the optimized 3R mutant bound with a higher apparent affinity than the parental antibody to GUV membranes decorated with the epitope peptide (Fig. 7A, bottom). These observations were further supported by experiments performed by using NBD-labeled Fabs (Fig. 7B and ​andC).C). Binding to vesicles containing the MPER peptide resulted in NBD fluorescence changes (Fig. 7B), i.e., increases in emission and maximum blue shift (Fig. 7C), that were overall more prominent in the 10E8-3R variant than in parental Fab 10E8-WT. This observation was again consistent with the more efficient epitope recognition in the membrane milieu by the 3R mutant.

To determine whether the increase in neutralization by 10E8-3R corresponded to an increase in its binding affinity for virion-associated Env trimers, we next turned to a blue native PAGE (BN-PAGE) gel mobility shift assay. Virions were produced from a molecular clone of Comb-mut whose Env protein has been shown to be >95% cleaved and trimeric (35). When a Fab fragment binds to the Env spike, the trimer will run more slowly on BN-PAGE gels, shifting the cognate band upwards on the gel. The 10E8-3R Fab fragment shifted the trimer band at a 5-fold-lower concentration than 10E8-WT, suggesting that the 3R mutations increased the affinity for the trimer (Fig. 8A and ​andC)C) (shifting 50% effective concentrations [EC₅₀s] of 2.3 μg/ml for the WT and 0.47 μg/ml for 3R). To determine whether the membrane contributed to the enhanced binding capacity of the 10E8-3R Fab fragment, we also performed a “washout” version of the gel mobility shift assay, in which virus was pelleted and unbound antibody was removed prior to the addition of detergent. This experiment yielded results similar to those of the standard assay (Fig. 8B and ​andC).C). We conclude that the 3R mutations enhance the binding of 10E8 to the unliganded Env trimer in the virus membrane. We note that neutralization by 10E8 and other MPER antibodies typically involves binding to both unliganded Env and receptor-activated forms of Env, and it is possible that the 3R mutations also affect binding to Env in the latter state, but this was not tested here due to the experimental challenges involved.

Production, characterization, and labeling of Fabs.

Antibody sequences were cloned into plasmid pColaDuet and expressed in the Escherichia coli T7-shuffle strain. Recombinant expression was induced at 18°C overnight with 0.4 mM isopropyl-d-thiogalactopyranoside when the culture reached an optical density (OD) of 0.8. Cells were harvested and centrifuged at 8,000 × g, after which they were resuspended in a buffer containing 50 mM HEPES (pH 7.5), 500 mM NaCl, 35 mM imidazole, DNase (Sigma-Aldrich, St. Louis, MO), and an EDTA-free protease inhibitor mixture (Roche, Madrid, Spain). Cell lysis was performed by using an Avestin Emulsiflex C5 homogenizer. Cell debris was removed by centrifugation, and the supernatant was loaded onto a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (GE Healthcare). Elution was performed with 500 mM imidazole, and the fractions containing the His-tagged proteins were pooled, concentrated, and dialyzed against a solution containing 50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 1 mM dithiothreitol (DTT), and 0.3 mM EDTA in the presence of purified tobacco etch virus protease (50). Fabs were separated from the cleaved peptides containing the His₆ tag by an additional step in a Ni-nitrilotriacetic column. The flowthrough fraction containing the antibody was dialyzed overnight at 4°C against sodium acetate (pH 5.6) supplemented with 10% glycerol and subsequently loaded onto a MonoS ion-exchange chromatography (IEC) column (GE Healthcare). Elution was carried out with a gradient of potassium chloride, and the fractions containing purified Fab were concentrated and dialyzed against a buffer containing 10 mM sodium phosphate (pH 7.5), 150 mM NaCl, and 10% glycerol. For the preparation of mutant Fabs, the KOD-Plus mutagenesis kit (Toyobo, Osaka, Japan) was employed according to the instructions of the manufacturer. Labeling with the polarity-sensitive NBD probe was performed as described previously (51, 52). In brief, a cysteine-substituted Fab derivative (W100b_HCC) was first generated by site-directed mutagenesis and modified with a sulfhydryl-specific iodoacetamide derivative of NBD. Thus, this procedure results in the conservative replacement of the Trp indole ring by the similarly bicyclic nitrobenzoxadiazole ring, which makes comparable changes in polarity scored by the NBD label. For confocal microscopy experiments, the fluorescence probe KK114 was introduced in vitro at position C216_HC according to the same procedure as the one used for the NBD probe.

Lipid vesicle production.

LUVs made from the VL lipid mixture (DOPC, Chol, SM, DOPE, and DOPS at a molar ratio of 14:46:17:16:7) were produced according to the extrusion method (53). To this end, lipid suspensions were subjected to 10 freeze-thaw cycles prior to extrusion 10 times through 2 stacked polycarbonate membranes with a nominal pore size of 0.1 μm (Nuclepore, Inc., Pleasanton, CA, USA). GUVs with the same composition were produced by spontaneous swelling according to procedures described previously (54, 55). Briefly, lipids were mixed in CHCl₃-CH₃OH (9:1), and the organic solvent was removed by desiccation for 1 h. Dried silica beads covered with lipid were collected and transferred to a 7.5-g/liter sucrose buffer to induce the spontaneous swelling of GUVs. For the preparation of peptide-GUV complexes, 0.66 μM peptide was added to the observation dish in an isosmotic buffer containing 10 mM HEPES and 150 mM KCl (pH 7.4), followed by the addition of the formed vesicles.

Vesicle flotation assays.

The partition of the antibody into membranes was examined by vesicle flotation experiments in sucrose gradients according to methods described previously by Yethon et al. (56). In brief, 100 μl of VL LUVs labeled with the lipid rhodamine-PE (Avanti Polar Lipids, Alabaster, AL) was incubated with Fabs at 25°C for 15 min under continuous stirring (800 rpm). The mixture was adjusted to a sucrose concentration of 1.4 M in a final volume of 300 μl and subsequently deposited on a stepwise gradient composed of successive solutions containing 0.8 M (400 μl) and 0.5 M (300 μl) sucrose. The gradient was centrifuged at 4°C at 436,000 × g for 3 h in a TLA120.2 rotor (Beckman Coulter, Brea, CA). After centrifugation, four fractions of 250 μl each were collected. The material that adhered to the centrifugation tubes was extracted by washing the tubes with 250 μl of a solution of 1% (wt/vol) SDS at 100°C. Antibody Fab fragments were detected in SDS-PAGE gels by using Western blotting, and the presence of liposomes was monitored by measuring rhodamine fluorescence. Densitometry of the blotted Fab bands was performed by using ImageJ software, and the percentage of Fab bound to vesicles was calculated from the band intensities measured in the vesicle-floating fractions, relative to the sum of the intensities measured in all fractions.

Confocal microscopy.

Images were acquired on a Leica TCS SP5 II microscope (Leica Microsystems GmbH, Wetzlar, Germany) as described previously (55, 57). Laurdan-stained GUVs were excited at 780 nm by using a 63× water immersion objective (numerical aperture [NA] = 1.2), 512- by 512-pixel images were acquired at 400 Hz per scanning line, and emission was imaged at 435 ± 20 nm. The KK114-labeled Fab fragments were excited at 633 nm by using an HeNe laser, and emission was imaged at 775 ± 125 nm.

Spectroscopic titration.

NBD fluorescence emission spectra were recorded with the excitation wavelength fixed at 470 nm. The emission spectrum of a sample lacking the fluorophore was subtracted from the spectrum of the equivalent sample containing the fluorophore. Partitioning curves were computed from the fractional changes in emitted NBD fluorescence when 150 nM NBD-labeled Fab was titrated with increasing lipid concentrations. The mole fraction partition coefficients, K_x, were determined by fitting the experimental values to a hyperbolic function (58):

where [L] is the concentration of accessible lipid and K is the lipid concentration at which the bound peptide fraction is 0.5. Therefore, K_x = [W]/K, where [W] is the molar concentration of water. The observed free energy of water-membrane partitioning was subsequently calculated according to the following expression:

where R is the ideal gas constant and T is the temperature of the binding reaction. For the estimation of the electrical potential at the membrane surface as a function of the PS content, the following equation was used:

where c is the number of ions per volume and σ is the surface charge density (30). To calculate the latter parameter, a surface area per phospholipid of 69.5 Ų was considered, and net charges of 0 and −1 were assigned to DOPC and DOPS, respectively (59).

HEp-2 cell immunofluorescence assay.

Antibodies (Fabs) were assessed for the ability to stain HIV-1-negative human epithelial HEp-2 (VIRGO ANA/HEp-2) cells on glass slides by indirect-immunofluorescence microscopy. Fabs were diluted to 25 μg/ml, and 10 μl was used for each test, according to the manufacturer's specifications. Fluorescein isothiocyanate (FITC)-conjugated goat anti-human Fab (Jackson) was used as the secondary probe. Slides were photographed by using an Evos fluorescence microscope (Invitrogen). Staining experiment 1 with 10E8-3R and staining experiment 2 with 4E10-3R were performed separately; the intensities of antibody staining can be compared within but not between experiments.

ELISA for antibody polyreactivity.

Ninety-six-well plates were coated with 10 nM cholesterol, DOPS, POPC, or DOPE (Sigma) in 50 μl 100% methanol and left to evaporate overnight at 4°C. Wells were coated with other antigens (500 ng/well in phosphate-buffered saline [PBS]) overnight at 4°C without evaporation. The wells were washed three times with PBS containing 0.05% Tween 20 (PBST) and blocked for 1 h at 37°C with 4% nonfat dry milk (NFDM) in PBS. The wells were washed three times with PBST, Fabs diluted in 0.4% NFDM–PBST were added to the wells, and the wells were incubated for 2 h at 37°C. Following three washes with PBST, a goat anti-human Fab-HRP conjugate (1:500 in 0.4% NFDM–PBST) was added to the wells, and the wells were incubated for 45 min at room temperature (RT). The wells were washed three times with PBST, and the plate was developed by using the 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Pierce) at 37°C. The OD at 450 nm was determined by using a Synergy H1 plate reader (BioTek).

Virus production.

Pseudotyped HIV-1 was generated by the cotransfection of 293T cells with Env plasmid DNA and HIV-1 backbone plasmid pSG3ΔEnv (60) by using 25,000-molecular-weight (25K) polyethyleneimine (PEI), as previously described (61). Envs of HIV isolates JRFL, LAI, and SF162 were obtained from the NIH AIDS Reagent Program; Envs of 92RW020, 94UG103, 92BR020, 92TH021, IAVI C22, and BG505 were obtained from D. Burton (The Scripps Research Institute); Env of 16055 was obtained from R. Wyatt (Scripps); Env of COT6 was obtained from L. Morris (University of Witwatersrand); Env of Q23.17 was obtained from J. Overbaugh (Fred Hutchinson Cancer Research Center); Envs of Du422.01 and CH181.12 were obtained from M. Seaman (Beth Israel Deaconess Medical Center); and Env of TAN2 was obtained from B. Hahn (University of Pennsylvania). Envs were expressed from the pSVIII plasmid; Comb-mut, by exception, was contained in the pcDNA plasmid. Virions for BN-PAGE analysis were produced by transient transfection using the molecular clone pLAI-Comb-mut (35).

Neutralization assays.

Neutralization was determined by using a single-cycle neutralization assay with CD4⁺ CXCR4⁺ CCR5⁺ TZM-bl cells as target cells. Antibodies were added to virus in cell culture medium (Dulbecco's modified Eagle's medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin), and the mixture was incubated for 1 h at 37°C prior to addition to target cells. Following 72 h of incubation at 37°C, cells were lysed, Bright-Glo luciferase reagent (Promega) was added, and luminescence in relative light units (RLUs) was measured by using a Synergy H1 plate reader (BioTek).

This study was supported by the Spanish MINECO (BIO2015-64421-R [MINECO/FEDER UE] to J.L.N.) and the Basque Government (IT838-13 to J.L.N.). Support was also provided by the NIH NIAID (R01 AI114401 and R01 AI098602 to M.B.Z.) and the James B. Pendleton Charitable Trust (M.B.Z.). E.R. and S.I. received predoctoral fellowships from the Basque Government.

We acknowledge valuable technical assistance from Rubén Sanchez-Eugenia and Miguel García-Porras.