Here we set out to investigate the effects of replacing the complex glycans on the neutralizing face of HIV-1 Env trimers with oligomannose glycans. We looked at infectivity of the modified trimers, their susceptibility to a deglycosylating enzyme, its effects on infectivity, and the basis for that effect. We also compared the neutralization sensitivity of viruses bearing modified glycans with a mutant virus that eliminates a key glycan of the V3 loop.
We initially observed that GnTI- cells can express functional virus. This finding is mirrored by the observation that soluble gp120 and gp140 trimers produced from GnTI- cells retain an ability to be recognized by sCD4 and CD4i MAbs (32
). It is also consistent with earlier observations that functional virus can be produced in the presence of glycan analogs such as kifunensine and swainsonine (36
) that inhibit stages of glycan processing immediately preceding and following that of GnTI enzyme (Fig. ). Thus, only inhibitors that affect very early stages of glycan maturation, such as NB-DNJ, appear to dramatically impact infectious function (37
). The infectious potential per Env expression in GnTI- cells appeared to be proportional to that of the corresponding virus prepared in parent 293T cells, suggesting that expression in GnTI- cells has no ill effects on Env function (32
). Thus, the GnTI- cell effect of replacing complex glycans with Man5
did not adversely affect the folding and oligomerization of the gp160 precursor into trimers or its processing into gp120/gp41. The lack of effect on oligomerization was expected, considering that this step in Env synthesis occurs in the endoplasmic reticulum and precedes that of ablating GnTI in the cis
-Golgi complex. However, gp160 processing is a very late step in Env synthesis that occurs in the trans
-Golgi network (81
). The proper gp160 processing in GnTI- cells therefore suggests that the complex glycan antennae play little or no role in modifying the oligomer's sensitivity to furin or related proteases that mediate this late maturation step.
Another way to investigate the importance of glycans in HIV-1 infection is to treat virus with glycosidases. However, despite the sensitivity of soluble Env proteins to these enzymes, studies until now have found native Env trimers to be resistant to all but those that target the outer glycan moieties (32
). In contrast to the modest effects of mannosidase (reduced infection) and sialidase (enhanced infection) (76
), here we found that endo H rapidly ablated the infectivity of GnTI- but not the parent virus. In BN-PAGE, GnTI- trimers rapidly dropped in size with endo H treatment, while the parent trimers remained largely endo H resistant. Three possibilities might explain this observation: (i) that N-glycans are directly involved in conformational changes necessary for fusion; (ii) that the removal of glycans exposes Env trimers to proteases and therefore leads to degradation; and (iii) that the removal of glycans exposes hydrophobic protein domains, leading to aggregation. We do not favor the latter two possibilities. Although a significant loss of the GnTI- trimer band staining in BN-PAGE (Fig. ) could suggest degradation, it required prolonged endo H treatment and was much slower than would be expected if this were to account for the rapid loss of infectivity (Fig. ). The retention of binding by various MAbs in Fig. suggests that the loss in trimer staining relates to a slow loss in 2G12 staining, rather than to degradation. We did not observe significant aggregation in our BN-PAGE analysis, although we note that the gentle detergents we used to release Env from the viral membrane may have masked any aggregating effects.
To further understand the basis of the effect of endo H on infectivity, it is useful to consider whether all or only a fraction of glycans are removed. In BN-PAGE, there appeared to be a much slower second phase of digestion after the rapid initial phase of glycan removal. Similarly, endo H treatment of JR-FL gp120 produced from Drosophila melanogaster
cells was previously shown to remove only 90% of the oligomannose carbohydrate (58
). This probably reflects the different sensitivities of the glycans of the neutralizing and silent faces. The former is decorated more sparsely with glycans that are therefore more accessible, while the latter domain exhibits closely packed and therefore enzyme-resistant glycans. In a previous study, the glycans that decorate soluble gp140 trimers produced in GnTI- cells were found to comprise a mixture of Man5-9
), in contrast to the uniform Man5
sugars that decorate most proteins produced in these cells. Therefore, the steric constraints that limit the trimming of oligomannose precursors of the silent domain appear to be unaffected by expression in GnTI- cells. This is perhaps not surprising, considering that mannose trimming is necessary to create the Man5
substrate of the GnTI enzyme (Fig. ). It also explains the difficulty of removing these glycans from native Env trimers, even when they are expressed in GnTI- cells (17
). Overall, our results suggest the loss in infectivity due to endo H treatment is due to removal of glycans from the neutralizing face.
Further BN-PAGE experiments (Fig. ) revealed that endo H did not affect trimer-sCD4 binding or its ability to induce the CD4i and V3 epitopes, suggesting that receptor and coreceptor binding is competent. This is consistent with previous studies in which these ligands were able to recognize deglycosylated forms of gp120 (12
). Thus, the lack of function of endo H-treated trimer appears to stem from a post-CD4/CCR5 binding entry block.
The above observations have been modeled in Fig. . This figure represents our best imagining of the scenario, while acknowledging the many gaps and caveats in the available information from which these images are constructed. HXBc2 gp120 structures (in red) are derived from a b12 complex (126
) and are fit into the cryoelectron tomogram of native unliganded trimers of the BaL isolate (69
). The complex glycans of the neutralizing face of parent trimers (Fig. , orange glycans) are replaced by Man5
in GnTI- trimers (Fig. ). Endo H treatment of these trimers results in the rapid removal of Man5
and other high-mannose glycans from the neutralizing face, leaving behind single GlcNac stumps (Fig. , four orange glycans and one slate blue glycan), while the tightly packed oligomannose moieties on the silent face are relatively endo H resistant and therefore remain intact. Figure shows a heavily depleted trimer that retains its gross architecture, as supported by BN-PAGE (Fig. and ). In one scenario, the lack of neutralizing face glycans lining both receptor binding sites may limit the ability of trimers to transmit the effects of receptor/coreceptor binding by undergoing further conformational changes, perhaps involving gp41. Possibly, the removal of these glycans could affect gp120 dissociation from gp41 and subsequent exposure of the fusion peptide (124
). Previously, NB-DNJ-treated virus Env was also found to be capable of binding to CD4, despite its lack of infectivity (37
). Furthermore, some single glycan mutants can ablate infectivity, for example, the N301 mutant in SF162 was not functional in one study (74
). Thus, receptor-induced conformational changes may depend in part on proper glycan processing, as well as their frank presence, so that they can properly orchestrate post-receptor binding refolding events in fusion.
Our neutralization results suggested that the effect of “lowering the glycan fence” (as modeled by the GnTI- virus) compared to “knocking out a fence post” (as modeled by the N301Q mutant) was relatively subtle: while the N301Q virus was more sensitive to V3, nonneutralizing CD4bs mAbs sCD4, and at least one of the CD4i MAbs, the GnTI- virus was more sensitive only to V3 MAbs and sCD4. Previous studies suggest a largely predictable order in which glycan deletion mutants become neutralization sensitive, with increases in V3 and CD4bs exposure being frequently observed, often together, while increases in CD4i and MPER epitope exposure occur only for exceptionally sensitive mutants (54
). Exposure of CD4i epitopes may require larger holes in the protective shell, provided only by the removal of three glycans in one study (54
) or by the complete removal of glycans from the neutralizing face (Fig. , lane 3). The N301Q mutant falls into the latter “exceptionally sensitive” category, in keeping with the loss of a particularly large glycan (3
). However, the GnTI- virus does not fit into either category, as, despite its V3 sensitivity, it remained resistant to nonneutralizing CD4bs MAbs and other specificities.
Two possibilities might explain the slightly increased sensitivity of the N301Q mutant to CD4i MAbs: (i) an altered steady-state trimer conformation or (ii) slower fusion kinetics that might grant prolonged access to the transition state targeted by these MAbs. The former possibility is supported by the observation of increased sensitivity to MAb 15e. This MAb is expected to block primary receptor binding, and its exposure should therefore stem from an altered ground state conformation. This explanation is also consistent with the lack of change in sensitivity to MPER MAbs or the T-20 peptide, whose epitopes become better exposed during fusion (7
) and therefore might be expected to exhibit a greater potency against Envs that fuse more slowly. It has been suggested that “nonpotent” CD4bs and CD4i MAbs generally fail to neutralize, due to the quaternary constraints of trimers that restrict the conformational reorganization required for binding (57
). This model was generated from observing the unusually high entropic penalties associated with binding of “nonpotent” CD4bs and CD4i MAbs to monomeric gp120, compared to the lower penalty of b12 binding. Applying this model to the present scenario, the replacement of the N301 glycan might alter the steady-state conformation of the trimer or else may relieve the restrictions on conformational change that normally prevent the binding of these MAbs.
The GnTI- virus remained resistant to nonneutralizing CD4bs and CD4i MAbs, suggesting that the glycan stems or “fence posts” affected by the N301Q mutation, but not by GnTI- expression, are important for maintaining trimers in a native conformation in which these specificities remain occluded. Furthermore, in contrast to the decreased CD4 dependency of some glycan point mutants, CD4i epitope exposure (a surrogate for coreceptor binding) on the GnTI- virus was still dependent on sCD4 binding, in keeping with the retention of native conformation.
Although the GnTI- virus does not embody any conformational changes that grant access to otherwise-nonneutralizing ligands, it does allow greater access to those that do recognize the parent trimers. The dramatically increased sensitivity of GnTI- viruses to sCD4 (and to a lesser extent b12) suggests a relief of the steric constraints to accessing the CD4 binding loop in the GnTI- trimers. We have modeled this in Fig. . Here, the yellow area on each red gp120 protomer represents the b12 binding site, which closely approximates the CD4 binding site. For example, comparing the lower panels of Fig. , it appears that the complex carbohydrate at position N276 of the parent gp120, as part of the “fence” that surrounds the receptor binding sites, may impinge on the b12 binding site, and its replacement with the smaller Man5GlcNac2 in Fig. opens up the b12 binding site, ostensibly allowing increased b12/sCD4 access. Positions N397 and N463 may offer a lesser though similar scenario, depending on the angle of entry of b12/sCD4.
Studies with monomeric gp120 suggest that certain mutations can increase the binding of sCD4 but not CD4bs MAbs, while other mutations have the reverse effect (122
). Therefore, the differential sensitivity profiles of N301Q and GnTI- viruses may be a simple reflection of alternative trimer steady-state conformations that generally favor the binding of one or the other of these ligands.
In marked contrast to the GnTI- virus's resistance to nonneutralizing CD4bs and CD4i MAbs, its increased V3 loop MAb sensitivity was almost as dramatic as that of the N301Q mutant. In the absence of a conformational change (as described above), one interpretation is that the large complex antennae of the N301Q mutant protects the V3 loop from MAb binding. An alternative explanation is that V3 loop exposure may be a result of disrupting the global network glycan-glycan hydrogen bonds, whose role may in part be to protect the V3 loop. The fact that we observed a V3-sensitive phenotype with the GnTI- virus without exposure of other conformationally occluded epitopes suggests, however, that increased V3 sensitivity does not require gross changes in trimer conformation.
Perhaps the closest precedent of the present analysis of GnTI- virus neutralization sensitivity is a study of an SIV isolate expressed in the presence of swainsonine. However, the sensitivity of this virus to SIV-infected macaque sera was not significantly affected (76
). The difference compared to the generally increased sensitivity we observed with GnTI- and HIV-1-infected plasmas may relate to the fact that swainsonine inhibits a later step in glycan synthesis: the conversion of hybrid into complex glycans, leaving behind a “higher fence” than expression in GnTI- cells (Fig. ). Alternatively, the difference may be related to the difficulties in detecting increased sensitivity to certain epitopes amid a background of neutralizing activity targeting another epitope(s), as we observed with plasma LTNP2 (Fig. ).
Overall, our neutralization analysis suggests that lowering the glycan fence and knocking out a fence post have markedly different consequences, with the major difference being that only the latter can alter trimer conformation. This is logical, considering that glycans participate in earliest stages of Env folding, so that deletion of one may have a greater consequence on conformation than the ablation of GnTI, which affects a later stage in glycoprotein maturation (67
). Indeed, eliminating glycans can affect gp160 processing and the exposure of distal epitopes, supporting a conformational effect (74
). That N301Q mutation has no effect on CD4bs MAb recognition of monomeric gp120 (54
) further supports the idea that N301 glycan removal does not directly expose the CD4bs. It is possible that the removal of the N301 glycan affects its interaction with the V1V2 loop, whose repositioning may lead to increased exposure of the CD4bs (54
). In comparison, the replacement of complex glycans with Man5
has a more subtle effect on the glycan shell.
From a broader perspective, our results may have some relevance for ongoing attempts to crystallize native Env trimers. The replacement of complex glycans with simpler glycans that can then be removed by glycosidases has been a key tool in facilitating the formation and analysis of diffractable crystals. The ability of endo H-treated trimers to still bind to receptor and coreceptor analogs suggests that the fundamental properties of the trimer remain intact, despite these treatments, and therefore that a structure of such a molecule would largely reflect that of the untreated trimer.
Our findings also have some bearing on attempts to design an Env-based vaccine to elicit broad NAb responses. Further investigation of the effects of glycan modification on the immunogenicity of candidate vaccines is clearly warranted (4
). However, a balance may need to be struck between increasing the exposure of neutralizing epitopes and avoiding the exposure of nonneutralizing epitopes that are not available on the native Env spike. Specifically, GnTI- Env trimers retain the same conformation as their native counterparts and relieve the constraints on sCD4 and possibly b12 binding without exposing nonneutralizing epitopes, with the important exception of the V3 loop. Thus, if GnTI- trimers are considered as immunogens, modifications may need to be engineered to dampen the sensitivity to V3 loop MAbs (88
), so that antibody responses may instead focus on the more-exposed CD4 binding site. Finally, GnTI- viruses may also be useful tools to help us to better understand the specificities of neutralizing MAbs that depend partly or wholly on glycans (45