Alterations in gp120 glycans or the gp41 fusion peptide-proximal region modulate the stability of the human immunodeficiency virus (HIV-1) envelope glycoprotein pretriggered conformation

Abstract

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry into host cells by binding receptors, CD4 and CCR5/CXCR4, and fusing the viral and cell membranes. In infected cells, cleavage of the gp160 Env precursor yields the mature Env trimer, with gp120 exterior and gp41 transmembrane Env subunits. Env cleavage stabilizes the State-1 conformation, which is the major target for broadly neutralizing antibodies, and decreases the spontaneous sampling of more open Env conformations that expose epitopes for poorly neutralizing antibodies. During HIV-1 entry into cells, CD4 binding drives the metastable Env from a pretriggered (State-1) conformation into more “open,” lower-energy states. Here, we report that changes in two dissimilar elements of the HIV-1 Env trimer, namely particular gp120 glycans and the gp41 fusion peptide-proximal region (FPPR), can independently modulate the stability of State 1. Individual deletion of several gp120 glycans destabilized State 1, whereas removal of a V1 glycan resulted in phenotypes indicative of a more stable pretriggered Env conformation. Likewise, some alterations of the gp41 FPPR decreased the level of spontaneous shedding of gp120 from the Env trimer and stabilized the pretriggered State-1 Env conformation. State-1-stabilizing changes were additive and could suppress the phenotypes associated with State-1-destabilizing alterations in Env. Our results support a model in which multiple protein and carbohydrate elements of the HIV-1 Env trimer additively contribute to the stability of the pretriggered (State-1) conformation. The Env modifications identified in this study will assist efforts to characterize the structure and immunogenicity of the metastable State-1 conformation. IMPORTANCE The elicitation of antibodies that neutralize multiple strains of HIV-1 is an elusive goal that has frustrated the development of an effective vaccine. The pretriggered shape of the HIV-1 envelope glycoprotein (Env) spike on the virus surface is the major target for such broadly neutralizing antibodies. The “closed” pretriggered Env shape resists the binding of most antibodies but is unstable and often assumes “open” shapes that elicit ineffective antibodies. We identified particular changes in both the protein and the sugar components of the Env trimer that stabilize the pretriggered shape. Combinations of these changes were even more effective at stabilizing the pretriggered Env than the individual changes. Stabilizing changes in Env could counteract the effect of Env changes that destabilize the pretriggered shape. Locking Env in its pretriggered shape will assist efforts to understand the Env spike on the virus and to incorporate this shape into vaccines.

Membrane HIV-1 envelope glycoproteins stabilized more strongly in a pretriggered conformation than natural virus Envs

“…In that process, we have identified several natural Env amino acid polymorphisms that individually stabilize the pretriggered conformation of the functional HIV-1 membrane Env trimer. 32 , 33 , 110 , 115 Here, we demonstrate that combinations of these changes yield Env variants from diverse HIV-1 strains that are additively stabilized in a pretriggered conformation, some to a degree well beyond that of natural HIV-1 strains. Increased stability of the pretriggered Env conformation correlated with increased trimer subunit association, increased sensitivity to neutralization by conformation-dependent bNAbs, and decreased capacity to mediate membrane fusion and virus entry.…”

“…Compared with their unstabilized Env counterparts, viruses with functional HIV-1 Envs that are stabilized in a pretriggered conformation exhibit relative resistance to soluble CD4 (sCD4), CD4mcs, and exposure to cold (0°C). 32 , 33 , 87 , 90 , 91 , 110 By screening natural Env polymorphisms, we identified several that conferred phenotypes associated with stabilization of the pretriggered HIV-1 AD8 Env. 32 , 110 , 115 We also identified two examples where combinations of individual Env changes enhanced these phenotypes in an additive manner: (1) a set of three changes (Q114E/Q567K/A582T) (herein called Tri) involving interior gp120 and gp41 elements near the trimer axis 32 and (2) a set of three changes (A532V/I535M/L543Q) in the gp41 fusion peptide-proximal region (herein called FPPR) 110 ( Figure S1 ).…”

“…Moreover, Env changes that stabilize sgp140 SOSIP.664 trimers do not necessarily stabilize the pretriggered conformation of membrane Envs. 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 Conversely, several changes that have been found to stabilize the pretriggered conformation of membrane HIV-1 Envs involve Env regions that are heterogeneous or disordered in sgp140 SOSIP.664 trimer structures. 32 , 33 , 109 , 110 , 113 , 114 For these reasons, efforts to stabilize the pretriggered conformation of membrane Envs have proceeded empirically.…”

“… 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 Conversely, several changes that have been found to stabilize the pretriggered conformation of membrane HIV-1 Envs involve Env regions that are heterogeneous or disordered in sgp140 SOSIP.664 trimer structures. 32 , 33 , 109 , 110 , 113 , 114 For these reasons, efforts to stabilize the pretriggered conformation of membrane Envs have proceeded empirically. In that process, we have identified several natural Env amino acid polymorphisms that individually stabilize the pretriggered conformation of the functional HIV-1 membrane Env trimer.…”

Preservation of memory B cell homeostasis in an individual producing broadly neutralising antibodies against HIV-1

“…Indeed, another bnAb with an overlapping epitope, 3BC315, was shown to cause dissociation of HIV-1 Env trimers 45 . Conversely, co-engagement of both gp41 and gp120 subunits by the antibody may restrict conformational rearrangements at the gp41-gp120 interface involved in the opening of the Env structure prior to activation of the fusion machinery 74–77 . Finally, binding at the very base of HIV-1 Env, ELC07 is expected to come in close contact with the viral membrane (Fig S2D).…”