Design of Env with loop deletions.
Env variants with variable loop deletions have been generated by us and others for functional, structural, and vaccine studies, but most of these constructs are slightly different and no comparative studies have been performed. It is a priori hard to predict which deletions are preferable in terms of Env folding and function. We have constructed a set of Env variants with different deletions in the V1/V2 and V3 regions in the context of the CXCR4-using LAI isolate, which is very convenient for evolution experiments. Some of these deletions were of novel design, while others were based on published studies to provide a comparison. Two-dimensional schematics and assumed three-dimensional (3-D) structures of some variants are provided in Fig. .
Variants 1 and 2 are of novel design. In contrast to most previously described V1/V2 deletion variants in which the disulfide bonds (between C119 and C205 and/or C126 and C196) are maintained and the V1/V2 region replaced with a Gly-Ala-Gly linker, we replaced the respective cysteines with two adjoining alanines, thus creating a continuous protein backbone (Fig. ). Variants 3 and 4 were derived and recloned from our previous studies of disulfide-stabilized Env constructs with loop deletions (60
). Note that these were derived from the JR-FL strain, and part of the flanking regions between the NdeI and StuI restriction sites that were used for subcloning were also derived from JR-FL (see Materials and Methods). We hypothesized that this sequence variation compared to the other constructs might result in different evolution routes. Variants 5 and 6 were designed to allow for the evolution of an alternative disulfide-bonded architecture of the V1/V2 stem. Variant 5 retained the cysteines at positions 126 and 131, while their counterparts, C157 and C196, were eliminated. We hypothesized that an alternative disulfide bond could be formed between positions 126 and 131 to rearrange the V1/V2 stump. Mutant 6, lacking C196, contained an uneven number of cysteines, which usually is disadvantageous, but we envisaged that it may facilitate new evolution routes by allowing for the addition or removal of a cysteine. In addition, mutant 6 contains an HXB2 V1 region that is 5 amino acids shorter than that of LAI. Some other amino acids in V1 also differ compared to wt LAI (Fig. ).
Variant 8 is a copy of the variant described by Wyatt et al. and was used to crystallize the gp120 core (Fig. ) (14
). After prolonged culturing of this variant in a previous study by Cao et al., an evolved variant was identified with a change in the Gly-Ala-Gly linker region: Asp-Ala-Gly (10
). We reproduced this mutant (variant 9). Building on the results of the study by Cao et al., we hypothesized that the Gly-Ala-Gly linker may not be optimal, and we constructed additional variants with changes in the linker region to test their relative functionality and allow for diverse evolution pathways to improve Env function. We generated variants with the following linker residues: variant 10, Gly-Asp-Gly; variant 11, Asp-Asn-Gly; and variant 12, Gly-Thr-Gly. Note that construct 11 contains an extra site for N-linked glycosylation within the linker region. Variant 14 was also taken from the Wyatt studies (78
Most mutants were constructed such that the bridging sheet between the inner and outer domains, which forms upon CD4 binding and is part of the coreceptor binding domain, remained intact (Fig. ). However, variants 1 and 14 lack β2 and β3, while variants 5 and 6 lack β3.
We also constructed three variants with deletions in the V3 loop (variants 15 to 17; Fig. ). Variant 15 lacks 7 amino acids in the N-terminal part of the V3 loop, while variant 16 lacks 10 amino acids at the C-terminal end. These deletions are combined in variant 17, in which the conserved tip sequences are retained.
Functional characterization of Env with loop deletions.
To assess the activity of the Env variants with loop deletions, we performed single-cycle infection assays with the respective mutants. The variant viruses were produced in SupT1 cells and used to infect TZM-bl reporter cells containing the luciferase gene under the control of the long-terminal-repeat promoter. V1/V2 mutants 1, 3 to 6, and 14 and V3 mutants 15 to 17 did not exhibit any activity in these functional assays (Fig. and results not shown). In contrast, V1/V2 variants 2, 9, and 11 remained relatively efficient in infecting the reporter cells, although not as efficient as wt Env. Variants 8, 10, and 12 displayed a low but reproducible level of infection.
Functional analysis of the deletion variants. TZM-bl reporter cells (confluence of 70 to 80%) were infected with 1.0 ng of mutant or wt virus in the presence of saquinavir in a 96-well plate, and luciferase activity was measured after 48 h.
Interestingly, V1/V2 variants 8 to 12, which are very similar in design with only minor changes in the linker replacing V1/V2, showed considerably different activities in these infection experiments. Mutants 9 and 11 containing the linkers Asp-Ala-Gly and Asp-Asn-Gly, respectively, were more active than variants 8 (Gly-Ala-Gly), 10 (Gly-Asp-Gly), and 12 (Gly-Thr-Gly). The activity of variant 2 shows that the traditional deletion design—that is, the retention of a disulfide bond linked by a small flexible stretch of amino acids—is not necessary. The Cys126-Cys196 disulfide bond is replaced by two adjoining alanines, thus forming a continuous protein backbone. This Cys126-Cys196 disulfide bond is required for virus replication in the context of the wt virus (73
) but apparently not anymore when the V1/V2 domain is deleted.
We also tested these viruses in spreading infections on SupT1 T cells and primary CD4+ T cells (see below). The replication experiments in SupT1 cells mirrored the single-cycle experiments with some qualitative differences. Most of the mutants were not able to replicate in primary CD4+ cells, but some replication was observed for mutants 2, 9, and 11.
Oxidative folding of Env with V1/V2 deleted.
The adverse effects of the V1/V2 deletions on recombinant trimers are ill-defined (11
). Since these effects do not become apparent in monomeric gp120, we do expect them to be rather subtle and act primarily on the trimeric complex. To exclude that the deletions impair early Env biosynthesis (i.e., oxidative folding in the endoplasmic reticulum), we examined the oxidative folding of Env mutants with V1/V2 deleted using a previously developed pulse-chase assay (42
). Env variants were expressed from plasmids under the control of the T7 promoter, using a recombinant vaccinia virus system (25
). The cells were pulse-labeled for 10 min and chased for the indicated times (Fig. ). The chase was stopped by cooling and the addition of alkylating agent to block free sulfhydryl groups. Lysates were immunoprecipitated and glycans were removed for analysis of the disulfide bond formation (42
). The maturation of Env is slow: after 4 h, the majority of wt Env reaches the native state (22
). Another striking feature of Env is the completely posttranslational cleavage of the signal peptide. A mutual relationship exists between Env oxidative folding and signal peptide removal: the lack of removal disturbs oxidative folding, whereas the formation of disulfide bonds is necessary to allow cleavage of the signal peptide (42
Signal peptide cleavage was followed over time by analyzing Env in lysates by reducing SDS-PAGE (Fig. ). Directly after the pulse, a single band appeared (Fig. , lane 1, Ru), representing gp160 with its signal peptide still attached. Cleavage started after ~30 min of chase (Fig. , lane 2, Rc). After 4 h of chase, the majority of the gp160 had lost its signal peptide (Fig. , lane 4). The decrease in signal after a 4-h chase was primarily caused by the cleavage of gp160 into gp120 and gp41 and the subsequent release of gp120 into the culture medium (Fig. , lanes 1 to 2) (47
We subjected the V1/V2 variants to the same assay. Since all variants behaved roughly similarly in these pulse-chase assays, we only show the results obtained with variants 1, 3, and 4, which are representative for all variants. Variants 1, 3, and 4 showed similar kinetics in the cleavage of the signal peptide, notwithstanding the mobility differences caused by loop deletions (Fig. ). The ratio between uncleaved and cleaved Env after 30 min of chase is similar to that of the wt (Fig. ; compare lanes 2, 4, 10, and 14).
To monitor the oxidative folding more directly, we followed the disulfide bond formation by analyzing the samples with nonreducing SDS-PAGE. The formation of disulfide bonds during folding increases the compactness of gp160 and, as a consequence, its electrophoretic mobility (8
). Immediately after the pulse, wt gp160 appeared as a fuzzy band that represents gp160 with different sets of disulfide bonds representing folding intermediates (Fig. , lane 1). With time, the folding intermediates gradually disappeared (Fig. , lanes 2 to 4) in favor of a fast migrating sharp band, which represents native, folded gp160 containing all its disulfide bonds. The mutants showed a similar oxidation pattern, as after 30 min of chase, some molecules reached their native state (Fig. , lanes 6, 10, and 14). Folding intermediates did appear somewhat more diffuse and heterogeneous, but the mobility differences between the mutants and the wt preclude any conclusions. The intensity of the native bands increased over the first hour and then disappeared from the lysate because of the cleavage of gp160 into gp120 and gp41 and the subsequent shedding of gp120 into the medium (Fig. ).
Major defects thus did not appear in the early biosynthesis of the Env variants with V1/V2 deletions, in contrast to the Env variants lacking disulfide bonds essential for oxidative folding (73
). Even the more-drastic deletion variants, 1
, and 14
, did not show significant defects in oxidative folding. We argue that the consequences of V1/V2 deletion only become apparent later, for example, during trimer assembly or maintenance of the trimeric spike.
Evolution of Env with V1/V2 deleted.
Regardless of the causes underlying the adverse effects of V1/V2 deletion, we examined whether the function of the Env constructs with loop deletions could be rescued. For this purpose, we used spontaneous virus evolution, which is likely to select for compensatory second-site changes. SupT1 cultures transfected with each of the molecular clones (three or four independent cultures per variant) were maintained for prolonged times (4½ months) as described in Materials and Methods and monitored for the appearance of faster-replicating variants by visual inspection for the appearance of syncytia and by CA-p24 ELISA. The initial CA-p24 production after the bulk transfection of SupT1 cells roughly mirrored the single-cycle infection experiments described above (data not shown). Viruses were passaged cell free onto uninfected cells when signs of active virus spread were apparent.
Cultures of the V1/V2 mutants 1 and 5 and all of the V3 mutants never produced replicating virus. These deletions, hence, are incompatible with residual Env function, which is needed to allow virus evolution. The V3 viruses were therefore excluded from further experiments. Virus spread was measured in 4/4 cultures of variant 3, 4/4 of variant 4 (of which two were lost during subsequent cell-free passage [see below]), 3/4 of variant 6, and 1/4 of variant 14, suggesting that although the deletions quite severely affected Env function, the virus was able to rescue this function by evolution.
In some cases, we lost replicating viruses during cell-free passage, indicating that although Env function was sufficient for cell-cell spread, some of these variants were defective in virus-cell infection (some cultures of variants 2, 4, 10, and 12). The virus variants differed in their ability to form syncytia and the morphology of syncytia (data not shown). These different properties, which also changed over the course of the evolution experiment, could not be correlated with CA-p24 production and replication kinetics, pointing at mechanistic differences in Env function.
Functional analysis of revertant Envs with V1/V2 deleted.
To confirm that the evolved viruses indeed gained replication capacity, the mutant and adapted viruses were directly compared in an infection experiment. SupT1 cells were infected with equal amounts of virus, and virus spread was monitored by CA-p24 ELISA (Fig. ). Mutant 2 replicated quite efficiently already, and the evolved variants 2A and 2D therefore did not readily display further improvements. Mutants 8 to 12 replicated efficiently as well, but evolution in some cases did improve replication (e.g., 10B, 12B, and 12C). In contrast, variants 8B and 11B lost replication capacity. Mutant 3 was a poorly replicating virus, and the viruses from three out of four cultures (3B to D but not 3A) clearly showed an improvement. Mutant 4 was worse than mutant 3, and both evolved variants were greatly improved, 4C being the best. Mutant 6 was as poor as mutant 4, but all three evolved variants (6A to C) displayed wt-like virus spread. Mutant 14 appeared completely replication defective, but a single evolved variant, 14B, did appear, although it replicated with a 4-day delay compared to that of the wt.
FIG. 4. Replication of mutant and adapted viruses. (A) 400 × 103 SupT1 cells were infected with 100 pg virus, and replication was monitored for 18 days by CA-p24 ELISA. (B) 200 × 103 primary CD4+ T cells were infected with 500 pg virus, (more ...)
We also tested the V1/V2 mutants and evolved variants for replication in primary blood cells (Fig. ). Most mutants were unable to replicate in primary CD4+ cells, but variants 2, 9, and 11 displayed a low level of virus spread. We observed improved replication for some of the evolved variants (variants 6B, 9B, 10B, and 12C), although virus spread was still very poor compared to that of the wt virus. Some variants lost the capacity to replicate in primary T cells (2A, 2D, and 11C), which may indicate specific adaptation to the SupT1 T-cell line with which the evolution experiment was performed. To establish that the evolved viruses had improved their Env function, we also performed single-cycle infection assays with TZM-bl cells (Fig. ). Most variants showed improved infectivity in TZM-bl cells upon evolution (3A to D, 4C, 4D, 6A to C, 8B, 8C, 9B, 10B, and 14D), but some variants became less infectious (2A, 2D, 8A, 9A, 9C, and 11A to C), pointing at SupT1-specific adaptations. In summary, using virus evolution, we obtained several fully replication-competent Env variants with large V1/V2 deletions.
Functional analysis of adapted Envs. TZM-bl reporter cells were infected with 1.0 ng of mutant or wt virus in the presence of saquanavir, and luciferase activity was measured after 48 h.
Genotypic analysis of revertant Envs with V1/V2 deleted.
We next investigated the unlikely scenario that the evolved virus variants had reintroduced sequences at the location of the V1/V2 deletion. The V1/V2 domain of provirus in revertant cultures after 4½ months of evolution was PCR amplified and subjected to gel electrophoresis, and it showed no change in size compared to that of the input mutant viruses (Fig. ). Apparently, V1/V2 sequences cannot be easily rebuilt.
FIG. 6. No restoration of V1/V2 sequences. The V1/V2 domain and surrounding sequences from proviral DNA in evolution cultures were PCR amplified and analyzed by gel electrophoresis. The lengths of the amplified fragments are 460 bp (wt), 253 bp (variant 2), 304 (more ...)
To identify determinants that are responsible for the improvement of Env function, the complete env
genes were PCR amplified and sequenced. The resulting sequences represent the predominant variants in the viral quasispecies. The identified substitutions are given in Table . All of the cultures contained changes to the input mutant sequence except for culture 14B, suggesting that mutations elsewhere in the genome underlie the improved replication capacity (for example, elevated transcription or alternative RNA splicing, resulting in more Env mRNA [54
]). Several interesting observations can be made based on the sequence analysis (see Discussion). A selected set of mutant and adapted Env variants were pursued in follow-up experiments.
Observed amino acid changes in evolved ΔV1/V2 virus variantsa
Neutralization sensitivity of variants with V1/V2 deleted.
To assess the neutralization sensitivity of mutants and revertants with V1/V2 deleted, we performed neutralization assays with a set of MAbs to gp120 and gp41. We chose to perform these experiments with the wt and the 2, 4C, 6B, 9, 9B, 10B, 11, and 11A variants because these viruses efficiently infected TZM-bl cells used in the neutralization experiments and because most of these viruses also infected primary CD4+ T cells. We included the mutant-revertant pairs 9 to 9B and 11 to 11A to investigate whether the neutralization sensitivity changed upon evolution. The inhibition curves are shown in Fig. , and the respective IC50 are given in Table .
FIG. 7. Neutralization sensitivity of mutant and adapted viruses. TZM-bl cells were infected with 1.0 ng virus as described in the legend to Fig. and in Materials and Methods. Virus was preincubated with the indicated amount of MAb for 30 min at (more ...)
Neutralization sensitivity of the ΔV1/V2 variantsa
We first tested the neutralization sensitivity to the 4E10 MAb directed against the membrane proximal region in gp41. We hypothesized that the exposure of the 4E10 epitope would not be affected significantly by the truncation of the V1/V2 domain. However, we found that all variants were more sensitive than the wt virus (IC50 ranging from 1.2 to 2.8 μg/ml versus ~10 μg/ml for the wt). The deletion of the V1/V2 domain in gp120 apparently increased the accessibility of the membrane proximal domain in gp41. Mutant viruses and evolved variants did not show major differences (compare 9 to 9B and 11 to 11A).
The epitope for the glycan-dependent 2G12 MAb is located on the outer face of gp120 (61
). Most viruses exhibited increased sensitivity to 2G12 (IC50
ranging from 0.080 to 0.22 μg/ml versus 6.1 μg/ml for the wt), suggesting that the exposure of the epitope is increased upon the deletion of the V1/V2 domain. As for 4E10, no major differences were apparent for the two mutant-revertant pairs. Revertant 10B was resistant to 2G12 neutralization, consistent with the loss of the 295 glycan which is part of the 2G12 epitope as a result of the T297I substitution (Table ) (61
). Resistance was not complete, since even low 2G12 concentrations resulted in an infection inhibition of 25%. Possibly a 2G12-sensitive subpopulation was present within the viral quasispecies that had not acquired the T297I substitution yet.
Mutants 2, 9, and 11 were highly sensitive to neutralization by CD4-IgG2 used as a surrogate for CD4 (IC50
of 0.0055 to 0.0090 μg/ml) compared to the wt virus (IC50
of 0.58 μg/ml), confirming that the V1/V2 domain is involved in limiting the accessibility of CD4BS. Of the adapted viruses, the 6B variant, which contains most of the V1 sequences, was more resistant to CD4 than the variants with V1/V2 deleted (IC50
of 0.034 μg/ml) but more sensitive than the wt virus. These results suggest that on the native trimer, both V1 and V2 are involved in the shielding of CD4BS. The evolved 9B and 11A variants (IC50
of 0.16 and 0.015 μg/ml, respectively) were more resistant than the original mutants 9 and 11. Their affinities for CD4 may be lower, caused by the substitutions located in or near CD4BS (G458D, F382L, and M434T). Residue 458 is a contact residue for CD4 through main chain interactions (38
), and a G458A substitution indeed causes a dramatic decrease in CD4 binding (51
We next tested the sensitivity to the broadly neutralizing antibody b12 directed to CD4BS (9
). All mutants were highly sensitive to b12 neutralization (IC50
of 0.010 to 0.018 μg/ml for the mutants and 0.77 μg/ml for the wt), but interestingly, complete neutralization was not achieved even at high b12 concentrations (~35% residual infectivity). We have repeatedly observed this phenomenon but do not have an adequate explanation. Most adapted viruses were also highly sensitive to b12 (IC50
of 0.0066 to 0.013 μg/ml) but could be inhibited completely. An exception was the 6B variant, which was similarly sensitive to b12 as to the wt (IC50
of 0.37 μg/ml).
MAb b6 is derived from the same phage library as b12 and also targets CD4BS (56
), but it does not neutralize the wt virus, presumably because its angle of binding to gp120 is incompatible with binding to the Env trimer (51
). Indeed, wt LAI was resistant to b6 neutralization. In contrast, we found that the mutant viruses with V1/V2 deleted were sensitive to b6 neutralization, although b6 was less potent than b12 against these viruses (IC50
of 0.072 to 0.39 μg/ml). The adapted viruses were equally sensitive to b6 neutralization (IC50
of 0.054 to 0.14 μg/ml), except 6B, which was quite resistant (IC50
, 2.13 μg/ml), although not as resistant as the wt virus, suggesting that both V1 and V2 play a role in covering the b6 epitope on trimeric Env.
The 17b MAb is directed to an epitope overlapping the coreceptor binding site. The creation and exposure of the epitope are induced by CD4 binding. The neutralizing potency of 17b is therefore limited (41
). While the wt virus was resistant to 17b neutralization, the mutant viruses were sensitive to 17b (IC50
of 0.24 to 1.7 μg/ml), indicating that V1/V2 deletion increases the exposure of the epitope (78
). Consistent with this, variant 6B, containing most of V1, was resistant to 17b neutralization. The evolved variants 9B, 10B, and 11A were also resistant to 17b neutralization, most likely because the acquisition of the V120E, P417T, and M434T changes located in β2, β19, and β21 of the four-stranded bridging sheet damaged the 17b epitope (38
Improvement of the synthesis and secretion of soluble stabilized Env.
Our goal is to use substitutions selected during evolution experiments to improve the folding and secretion of soluble stabilized Env immunogens. To provide a proof of principle that this is possible, we cloned selected deletion variants into an expression vector for SOS gp140 based on the JR-FL isolate (6
). The PCR fragments from different time points during the evolution of the ΔV1/V2.6 variant (Fig. ) were cloned into an SOS gp140 vector and sequenced. Most clones contained the mutations identified in the population sequence at the respective time points (indicated in the labels of Fig. ), but one clone of culture 6A contained an additional substitution (M104I) (Fig. ). The resulting SOS gp140 constructs were transiently expressed in 293T cells in the presence of abundant furin (6
). The cell lysates and supernatants were subjected to SDS-PAGE and Western blotting to analyze the intracellular and secreted Env (Fig. ). Full-length SOS gp140 was expressed efficiently as reported previously (6
), but the expression of the SOS ΔV1/V2.6 variant was reduced. In contrast, all four evolved variants were efficiently expressed and secreted in the culture supernatant, with some quantitative differences. These results indicate that compensatory changes were selected in the virus evolution experiments that improved the biosynthesis and secretion of stabilized gp140.
FIG. 8. Substitutions improve the folding and secretion of stabilized gp140 constructs. The ΔV1/V2.6 deletion and compensatory changes identified in various evolution cultures were introduced in an expression vector for SOS gp140. The variants were expressed (more ...)
Since we wish to employ evolved variants in recombinant trimer constructs, we also analyzed the oligomerization of the SOS gp140 constructs using native electrophoresis (BN-PAGE; Fig. , bottom panel). The wt gp140 forms a mixture of predominantly dimers and trimers, as does SOS gp140, although the oligomerization of SOS gp140 varies considerably between experiments and can be modulated by additional stabilization (62
). The evolved SOS ΔV1/V2.6 variants showed a similar pattern, indicating that the deletion and compensatory substitutions are compatible with efficient oligomerization.