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. 2023 Sep 28;97(9):e0059223.
doi: 10.1128/jvi.00592-23. Epub 2023 Sep 11.

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

Zhiqing Zhang #  1   2 , Qian Wang #  1   2 , Hanh T Nguyen  1   2 , Hung-Ching Chen  3 , Ta-Jung Chiu  3 , Amos B Smith Iii  3 , Joseph G Sodroski  1   2
Affiliations

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

Zhiqing Zhang et al. J Virol. .

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.

Keywords: CD4-mimetic compound; State-1 conformation; cold sensitivity; glycan; glycosylation; mutant; stabilizing mutation; transmembrane glycoprotein; trimer; virus entry.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
HIV-1AD8 Env regions altered in this study. Schematic representations of the HIV-1AD8 Env are shown, with gp120 and gp41 subunits designated. The proteolytic cleavage site [(508)REKR(511)] between gp120 and gp41 is colored red. The gp120 variable regions (V1–V5) and conserved regions (C1–C5) are indicated. The following elements are labeled: SP, signal peptide; FP, fusion peptide; HR1 and HR2, heptad repeat regions 1 and 2; MPER, membrane-proximal external region; TM, transmembrane region; CT, cytoplasmic tail. The N-linked glycosylation sites are depicted above the Env diagram. The sequences in V1 and V2 (A) and the Env region surrounding the gp120-gp41 cleavage site (B) are shown. The HIV-1AD8 Env amino acids are numbered according to standard nomenclature, referring to the prototypic HXB2 sequence (94). In A, the N-linked glycosylation sites in the V1/V2 region, several of which are modified in this study, are colored purple, and are numbered.
Fig 2
Fig 2
Phenotypes of V1/V2 insertion mutants. (A) The sequences of the HIV-1AD8 V1/V2 insertion mutants are shown. The gp120-gp41 cleavage site is designated with a triangle. S, signal peptide; FP, fusion peptide; HR1 and HR2, heptad repeat regions 1 and 2; T, transmembrane region; CT, cytoplasmic tail. The Asn 136 and Asn 141 glycosylation sites are shown. (B) Pseudovirus particles were prepared from transfected 293T cells, lysed, and analyzed by western blotting. The gp160, gp120, and gp41 Envs and the p24 CA (capsid) and p17 MA (matrix) proteins are shown. (C) The inhibition of the infectivity of recombinant viruses pseudotyped by the indicated wild-type (wt) or mutant HIV-1AD8 Envs by the CD4mc BNM-III-170 is shown. (D) Recombinant viruses with wt or mutant Envs were incubated at 0°C for up to 2 days (left panel) or 5 days (right panel), and their infectivity on TZM-bl cells was measured. The infectivity relative to a control virus not incubated at 0°C is reported. (E) The inhibition of the infectivity of recombinant viruses with the wt or mutant Envs by BMS-806 is shown. In C–E, the means and SDs of triplicate measurements are shown. The experiments were repeated with comparable results.
Fig 3
Fig 3
Phenotypes of Env glycosylation mutants. (A) The processing and subunit association of the wt and indicated HIV-1AD8 Env mutants in transfected HOS cells were evaluated. The two upper panels show the cell lysates, and the lower panel shows the cell supernatants. The upper panel shows the total input Envs in the cell lysates. The middle panel shows the Env proteins precipitated by Ni-NTA beads, using the His6 tags at the carboxyl terminus of the gp41 glycoprotein. The lower panel shows the gp120 in the cell supernatants, which was precipitated with Galanthus nivalis lectin (GNL)-beads. (B and D) Recombinant viruses with the indicated wt or mutant Envs were incubated at 0°C for the indicated times, and their infectivity on TZM-bl cells was measured. The infectivity relative to a control virus not incubated at 0°C is reported. (C) Virus particles were prepared from transfected 293T cells, lysed, and analyzed by western blotting. (E and F) Recombinant viruses pseudotyped with the indicated wt or mutant Envs were incubated with the indicated concentrations of BNM-III-170 (E) or the 19b pNAb (F) for 1 h at 37°C. The virus preparations were then incubated with TZM-bl cells, and the infectivity was measured 48 h later. In B and D–F, the means and SDs of triplicate measurements are shown. The experiments were repeated with comparable results.
Fig 4
Fig 4
Viral phenotypes of Envs with combinations of glycosylation changes. (A–D) Recombinant viruses pseudotyped with the indicated Env variants were incubated at 0°C for the period of time shown, and their infectivity on TZM-bl cells was measured. The infectivity relative to a control virus not incubated at 0°C is reported. (E and F) Recombinant viruses pseudotyped with the indicated Envs were incubated with BNM-III-170 (E) or BMS-806 (F) for 1 h at 37°C. Viruses were then incubated with TZM-bl cells for 48 h, and the infectivity was measured. The means and SDs of triplicate measurements are reported. The experiments were repeated with comparable results.
Fig 5
Fig 5
Stability of viral Envs on ice. Virus particles with the indicated Envs were prepared from the supernatants of transfected 293T cells and incubated at 0°C for the period of time shown. The viruses were then pelleted, lysed, and analyzed by western blotting (A). The gp120/gp41 ratio (%) on the virus particles at each time point relative to that on a control virus not incubated at 0°C is reported (B). The results of a typical experiment are shown. The experiment was repeated with comparable results.
Fig 6
Fig 6
Effects of HIV-1AD8 Env FPPR changes on processing, subunit association, and gp120-trimer association. HOS cells transiently expressing the wt HIV-1AD8 Env and the indicated mutants with C-terminal His6 tags were harvested 72 h after transfection. The cells were lysed, and fractions of the cell lysates were reserved as Input samples. The remainder of the cell lysates was incubated at room temperature for 1.5 h with Ni-NTA beads. The cell supernatants were incubated at room temperature for 1.5 h with GNL beads. Total cell lysates (Input), Ni-NTA precipitates, and GNL precipitates were western blotted with a goat anti-gp120 antibody. HOS cells transfected with the empty pcDNA3.1 vector serve as a negative control. The GNL precipitates from the cell supernatants reflect the level of gp120 shed from the cell-associated Env and were used to calculate subunit association. The Ni-NTA precipitates from the cell lysates measure the relative levels of gp160 and gp120 precipitated in the presence of NP-40 detergent and were used to calculate gp120-trimer association. The results of a typical experiment are shown.
Fig 7
Fig 7
Phenotypes of HIV-1AD8 Env variants with changes near the gp120-gp41 cleavage site. Env processing, subunit association, gp120-trimer association, cell-cell fusion, and infectivity are shown for each HIV-1AD8 Env mutant, normalized to those values observed for the wt HIV-1AD8 Env. Cold sensitivity represents the pseudovirus infectivity after 24 h of incubation at 0°C, relative to that of the untreated virus. The 50% inhibitory concentrations (IC50 values) of the CD4-mimetic compound, BNM-III-170, are reported in µM. N/A, not available; ND, not determined. Values are colored according to the key, based on their fold increase or decrease compared to those of the wt HIV-1AD8 Env. The results shown are the means and SDs derived from at least two independent experiments.
Fig 8
Fig 8
Effects of combinations of FPPR changes on HIV-1AD8 Env processing, subunit association, and gp120-trimer association. HOS cells transiently expressing the indicated wt and mutant HIV-1AD8 Env variants were processed as described in the Fig. 6 legend. The results of a typical experiment are shown.
Fig 9
Fig 9
Phenotypes of HIV-1AD8 Env FPPR combination mutants. The phenotypes of Envs with combined changes and selected Env mutants with individual amino acid changes in the FPPR were compared to those of the wild-type HIV-1AD8 Env. Values are colored according to the key, based on their fold increase or decrease compared to those of the wt HIV-1AD8 Env. The results shown are the means and SDs derived from two independent experiments.
Fig 10
Fig 10
Analysis of HIV-1AD8 Env FPPR mutants expressed from an infectious HIV-1 proviral clone. (A) HEK 293T cells were transfected with plasmids containing an infectious NL4-3 provirus with the envs expressing the indicated wild-type (wt) or mutant AD8 Bam Envs. The Bam changes (S752F I756F) restore the natural HIV-1AD8 Env sequence near the AD8/HXBc2 junction and reduce the clipping of the gp41 cytoplasmic tail by the viral protease (90). Seventy-two hours later, the cell supernatants were collected, filtered (0.45 µm), and aliquoted. The virus-containing supernatants were incubated on ice for 0 or 48 h, after which they were centrifuged at 14,000 × g for 1 h at 4°C. The pellets were resuspended in 1× PBS and represent the virion samples. The supernatants were incubated with GNL beads for 1.5 h at room temperature; the GNL precipitates represent the shed gp120. Virion and shed gp120 samples were western blotted with a goat anti-gp120 antibody. (B) The infectivity of the virions with the indicated HIV-1AD8 Env variants without treatment or after 24 and 48 h of incubation on ice was measured on TZM-bl cells. The values are normalized to that of the untreated wt AD8 Bam virions. The fold increases observed for the Env mutants relative to the values for the wt AD8 Bam virus under the same treatment conditions are colored according to the key. The inhibition of virus infectivity after 1 h incubation at 37°C with the CD4mc, BNM-III-170, or antibodies is reported. The 50% inhibitory concentrations (IC50 values) are reported in µM for BNM-III-170 and in µg/mL for the antibodies. Note that all the viruses were resistant to the 2F5 and 4E10 bNAbs against the gp41 membrane-proximal external region (MPER). The results shown are the means and SDs derived from two independent experiments. Inhibition by BNM-III-170 is colored according to the key. (C) Purified virus particles were incubated with a panel of broadly neutralizing antibodies (bNAbs) and poorly neutralizing antibodies (pNAbs) for 1 h at room temperature. The virus-antibody mixture was washed with 1× PBS and centrifuged. The virus-antibody pellet was lysed and precipitated with Protein A-agarose beads for 1 h at 4°C. The beads were washed three times and western blotted with a goat anti-gp120 antibody and the 4E10 anti-gp41 antibody.
Fig 11
Fig 11
Location of Env changes in current Env structural models. (A) A cryo-EM structure of the AE2.1 variant of the HIV-1AD8 Env trimer (110) is shown from the perspective of the target cell (left panel) or as a side view, with the viral membrane at the top of the figure (right panel). Although the detailed structure of the State-1 conformation of the membrane Env trimer is unknown, the structure of the AE2.1 Env trimer, which has been suggested to represent a default intermediate (State-2-like) conformation (110), is informative. Specific glycosylated Asn residues studied herein are indicated. Well-defined, stable densities in the cryo-EM map allowed modeling of the peptide-proximal glycans at Asn 295, Asn 301, and Asn 332, whereas the glycans associated with Asn 130, Asn 136, and Asn 411 were not resolved (110). Removal of the gp120 glycans in cyan resulted in State-1-destabilizing phenotypes. Where resolved, these glycans project into the interprotomer spaces of the Env trimer. Removal of the Asn 136 glycan (orange) resulted in the phenotypes associated with State-1 stabilization. The poorly resolved glycan at Asn 136 presumably projects from the gp120 V1 loop into the solvent. (B) A side view of the AE2.1 variant of the HIV-1AD8 trimer is shown, with the MPER and viral membrane at the top of the figure. The fusion peptide (FP; green), FPPR (blue), and α9 helices (orange) are highlighted. The structures of these regions in all three protomers of the asymmetric AE2.1 Env trimer are shown. In the asymmetric AE2.1 Env trimer, two opening angles between the protomers are >120°, and one opening angle is <120° (110). The FPPR residues that are implicated in State-1 stabilization are shown as blue Corey-Pauling-Koltun (CPK) models. Note that the conformation of the FPPR differs in the two protomers associated with the larger opening angles compared with that in the protomer associated with the smaller opening angle. The as-yet-known structure of the State-1 Env may hypothetically consist of a symmetric trimer. In such a model, the FPPR could contribute to State-1 stabilization and Env trimer symmetry through interactions with the α9 helices and/or MPERs of the adjacent protomers. FPPR interactions with α9 or MPER could also potentially influence the conformations of the adjacent HR1N regions. The HR1N region is helical in the protomers associated with the larger opening angles, whereas HR1N is a random coil in the protomer associated with the smaller opening angle. Thus, variation in the interactions of the FPPR and HR1N regions with other Env elements can potentially influence trimer symmetry.
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