Research Article

Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer

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Science  04 Mar 2016:
Vol. 351, Issue 6277, pp. 1043-1048
DOI: 10.1126/science.aad2450

A more complete look at the HIV-1 envelope

HIV-1 uses its envelope protein (Env), a large glycoprotein present on the viral surface, to enter target cells. Env forms trimers on the viral surface. Structural studies of solubilized Env trimers have provided important insights into viral entry and antibody binding, but soluble trimers lack several important insoluble regions of the native protein. Lee et al. used cryo–electron microscopy to solve the structure of a trimeric Env protein of HIV-1, missing only its cytoplasmic tail, in complex with broadly neutralizing antibodies. A more complete understanding of Env's structure may aid in vaccine design ef orts.

Science, this issue p. 1043

Abstract

The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4+ T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure.

The HIV-1 envelope glycoprotein (Env) houses the receptor binding site and fusion machinery to infect target cells. The intrinsic instability of and glycosylation on Env have made solving a high-resolution structure a daunting task. Low-resolution tomographic reconstructions of Env on the viral surface have described the overall shape of the trimer (1, 2), and more recently, structures of an engineered, soluble clade A BG505 SOSIP.664 trimer have been solved at high resolution (39). The BG505 SOSIP.664 trimer interacts preferentially with broadly neutralizing antibodies (bnAbs) but not with nonneutralizing antibodies (10) and has promising immunogenic properties (11, 12). Although these data suggest that this soluble trimer recapitulates native Env, it is not known what effect the stabilizing mutations or the lack of the membrane proximal external region (MPER) and transmembrane domain (TM) have on the Env structure.

BG505 SOSIP.664 shares a highly similar architecture with wild-type Env

We studied the JR-FL Env strain with the cytoplasmic tail (CT) deleted (hereafter referred to as EnvΔCT). In some isolates, the deletion of the CT has been shown to increase the exposure of nonneutralizing epitopes (13), but the deletion of CT in JR-FL does not abolish the ability of the trimer to fuse and infect (14, 15). Our previously described protocol (16) for extracting the complex formed by JR-FL EnvΔCT and the bnAb PGT151 was modified to make the sample amenable for cryogenic electron microscopy (cryo-EM) (fig. S1, A and B), resulting in a 4.2 Å–resolution reconstruction (Fig. 1, A and B, and figs. S1 to S3). Similar to our negative-stain reconstructions (16), PGT151 Fab bound in an asymmetric manner, with a maximum of two Fabs per trimer. Because the three gp140 interfaces of the Env trimer were nonequivalent, we hereafter refer to them as interfaces 1, 2, and 3 (Fig. 1A).

Fig. 1 Cryo-EM reconstruction of JR-FL EnvΔCT.

(A) Reconstruction of JR-FL EnvΔCT in complex with PGT151 Fab at 4.2 Å resolution, segmented to highlight densities corresponding to gp120 (yellow), gp41 (blue), PGT151 Fab (pink), and the micelle surrounding the MPER and TM domain (gray). The three possible PGT151 binding sites are labeled as interface 1 (unliganded), interface 2, and interface 3. (B) Model of the EnvΔCT ectodomain, colored as in (A). The Fab light and heavy chains are colored in pink and magenta, respectively. Glycans are shown as spheres, with the gp120 and gp41 glycans shown in light and dark green, respectively. (C) Simplified cartoon of gp41. Most of the portion of HR1 that does not have a regular secondary structure in SOSIP trimers (residues 548 to 568) is here revealed to be an α-helix. To distinguish this region from the central HR1 helix (residues 571 to 593), we call these two helices HR1N and HR1C, respectively. The complete HR1 spans residues 534 to 593. The cartoon cylinder and loops are colored according to the sequence shown at the bottom.

The clade B JR-FL EnvΔCT shares a similar topology to BG505 SOSIP.664, despite the lack of stabilizing mutations and the difference in subtypes (68.5% sequence identity) (fig. S4 and fig. S5, A and E). Differences were observed at the trimer apex and the N-terminal region of heptad repeat 1 (HR1N) of gp41. In the JR-FL trimer, the inter-V1/V2 loop region of the trimer apex is more loosely associated than in the unliganded BG505 SOSIP.664 [Protein Data Bank identifier (PDB ID) 4ZMJ] (fig. S5B). This phenomenon is consistent with fluorescence resonance energy transfer studies of viral Env (17), as well as studies of clades B and C SOSIP.664 trimers in which loop mobility and flexibility have been observed (18, 19). Despite this weaker interaction, the V3 loop in all three protomers remains in contact with the base of V2 on the adjacent protomer (figs. S5B and S6A), and therefore it probably confers the majority of the stability at the trimer apex.

In the published BG505 SOSIP.664 structures, HR1N does not adopt a regular secondary structure (49), whereas in our JR-FL EnvΔCT model, this region is helical (fig. S5D). We attribute this difference to the I559P mutation in SOSIP that disrupts the propensity of the HR1 peptide to form an extended and stable α-helix during fusion (20). In contrast, the SOS disulfide bond does not cause any major conformational differences relative to the wild-type structure (fig. S5C). As in the BG505 SOSIP.664 structures (49), most of the C terminus of HR2 is helical until residue 664. However, hydrogen deuterium exchange mass spectrometry (HDXMS) studies (21) have demonstrated that the C-terminal region of HR2 in BG505 SOSIP.664 has a flexible topology. Whereas the C-terminal region of gp41 is observed in our JR-FL EnvΔCT structure, the micelle-embedded MPER and TM just downstream of HR2 were both unresolved (Fig. 1A; fig. S1, D and E; and fig. S2C). Crystal structures of MPER peptide–Fab complexes have also shown that MPER can adopt different conformations (2224).

Model building of newly resolved regions

We used the BG505 SOSIP.664 and PGT151 Fab x-ray structure coordinates [PDB IDs 4TVP (6) and 4NUG (16), respectively] as starting models for building and refinement (figs. S3, S5F, and S6 and table S1). The fusion peptide (FP, residues 512 to 527) and HR1N (548 to 568) (Fig. 1C and fig. S4) regions of gp41 were both resolved in the current structure. The HR1N helix in interfaces 2 and 3 was tilted by ~24° away from the center of the trimer and ~26° to the right in comparison with interface 1 (Fig. 2A), when viewed normal to the threefold axis of the trimer. The HR1N and HR1C form a helix-turn-helix–type motif but contain residues with high helical propensity in the turn region (Fig. 2A and fig. S4). The HR1N region in the unliganded protomer is less well defined than the liganded HR1N helices (fig. S5D), as would be expected for a conformationally variable segment.

Fig. 2 Conformational changes induced by PGT151 binding.

(A) Compared with interface 1 (gray), HR1N in interfaces 2 (blue) and 3 (teal) is shifted about 24° outwards toward the Env surface and 26° toward gp120 of the same protomer (yellow surface). The position of the I559 residue is shown in bright yellow. (B) The FP (teal) is inserted into a hydrophobic pocket formed by the PGT151 CDR loops (CDRH2, magenta; CDRH3, purple; CDRL3, orange). The hydrophobic aromatic residues in these CDRs are shown as sticks.

In the BG505.664 x-ray structure, the N terminus of the FP (residues A512 to A518) is disordered and appears to project into the solvent. Similarly, at interface 1 of the cryo-EM model, residues A512 to L520 are unresolved, but a density adjacent to HR1N could be attributed to the FP (fig. S7A); this suggests that the hydrophobic N terminus of the FP may be inserted into the trimer core in wild-type Env, as observed in another class I viral fusion protein, influenza hemagglutinin (25). At the PGT151 liganded interfaces, there are changes in both the antibody and the FP. The majority of the Fab heavy- and light-chain residues adopt the same conformation as when the Fab is unliganded, except for the heavy-chain complementarity-determining region 3 (CDRH3) of PGT151 (P100a to Y100l), which is substantially different in the two structures (fig. S7B). Upon PGT151 binding, the entire FP is resolved (fig. S7C), where it projects away from the trimer and is sequestered in a pocket formed between the PGT151 Fab CDRH2, CDRH3, and CDRL3 (L, light chain) loops via hydrophobic and backbone interactions (Fig. 2B and fig. S7D). Because the FP is pulled away from the trimer core in both BG505 SOSIP.664 and PGT151-bound Env, which stabilizes the prefusion trimer, this conformation of the FP may counterintuitively contribute to trimer stability.

Newly revealed glycans in the wild-type Env trimer

The Env trimers in our studies contain fully processed native glycans. Similar to the two other cryo-EM structures (5, 9), at least the two core N-acetylglucosamine (GlcNAc) moieties are visible at the majority of glycosylation sites, except in disordered peptide regions such as V1 and V4 (fig. S6, B to D, and table S1). The glycans in the PGT151 epitope are ordered, allowing us to resolve four highly branched glycans at N241 and N448 in gp120 and N611 and N637 in gp41.

Gp120 glycans have been studied extensively, but much less is known about gp41 glycans. In our structure, glycans at positions N625 and N616 were only resolved up to the core GlcNAc residues (table S1). The ordered glycans at positions N611 and N637 that interact with PGT151 were built as complex glycans with N-acetyllactosamine (LacNAc) branching, which is consistent with published glycan array binding data (Fig. 3, A to D, and figs. S8 and S9) (26). The base GlcNAc residues of the N611 and N637 glycans were core-fucosylated (Fig. 3, A and B, and figs. S8 and S9), which is consistent with glycan array data demonstrating fucose-dependent binding differences by some clonal relatives of PGT151 (26) and with binding studies (fig. S10).

Fig. 3 Glycan structures on the Env trimer.

(A) The glycan at position N611 makes extensive contacts with PGT151 Fab. The glycan residues are colored according to the diagram in (C) and (D). (B) As in (A), but for the N637 glycan. (C) The glycans modeled at N611 and (D) N637. Only sugar moieties resolved in the current structure are shown. (E) The N241 and N448 glycans (different shades of green) are in close proximity to the CDRL3 and FWRH3 of PGT151.

The N611 glycan is minimally a tri-antennary glycan, with the two LacNAc units of the mannose(α1–6) [Man(α1–6)] arm resolved. The LacNAc(β1–6) branch packs against the heavy-chain framework region 3 (FRWH3). The LacNAc(β1–2) interacts with a highly conserved CDRH2 region of the PGT151 family and also extends far enough to make potential contacts with the FP of the adjacent gp41 (Fig. 3, A and C; fig. S8; and fig. S12, A and B).

The N637 glycan is a tetra-antennary glycan (Fig. 3, B and D, and fig. S9), and the core trisaccharide interacts along the length of CDRH3 (Fig. 3B). The interactions are probably backbone-mediated, because the PGT151-family CDRH3 sequences in this stretch are variable (fig. S12, A and C). The Man(α1–3) branch projects between the heavy chain and light chain, with the LacNAc(β1–4) unit primarily interacting with CDRH1 and the LacNAc(β1–2) branch interacting with the C terminus of CDRL2. The GlcNAc(β1–6)Man(α1–6) branch interacts with another well-conserved region in CDRL2 (Fig. 3B and fig. S12, A and C).

Previous low-resolution modeling and glycan knockout neutralization assays allowed us to predict that glycans from N262 and N448 in one gp120 protomer, and from N276 in the adjacent gp120 protomer on a BG505 trimer, could interact with the PGT151 Fab (16). In this study, we observed a density for a glycan at N241 (glycosylation site ~96% conserved, not present in BG505), which, along with the N448 glycan, extends toward FWRH3 (Fig. 3E and fig. S11, A and B). The N276 glycan is the least resolved of the four PGT151-proximal gp120 glycans (fig. S11C), suggesting that it makes considerably fewer contacts relative to N448 or N241, although is positioned to restrict accessibility to the PGT151 epitope.

In TZM-bl neutralization assays, the IC50 (half-minimal inhibitory concentration) improved slightly when the N241 or N448 glycan was knocked out in JR-FL pseudovirus (26), corroborating our structural observations. In the same assay, the presence of either the N611 or the N637 glycan alone in gp41 was sufficient for neutralization, albeit with decreased potency (26). In JR-FL, the reduced neutralization potency due to the knockout of N637 was recovered by a second, simultaneous knockout of the N448 glycan. This effect is not observed for BG505 pseudovirus, which naturally lacks the N241 glycan. Furthermore, in BG505 pseudovirus, the N637A glycan knockout does not cause as large a decrease in neutralization potency as in JR-FL (26). We therefore hypothesize that the N448 glycan imposes a steric hurdle for PGT151 binding, but only in the presence of the highly conserved N241 glycan (Fig. 4A). The N241 glycan probably limits the range of motion of the N448 glycan, which in turn limits access to the PGT151 epitope, in a similar manner to steric restriction of epitopes by glycans that has been observed with other bnAbs (2729). It has also been suggested that the presence or absence of one glycan can affect the conformational space that can be occupied by adjacent glycans (30). This type of mechanism illustrates the complex nature of Env surface accessibility and the difficulty of determining complete epitopes outside of high-resolution structures. Lastly, the D2/D3 branch of the very highly conserved N262 oligomannose glycan is in close proximity to the FWRL3 of PGT151; this suggests interactions with the D2 arm, although it is unclear whether this glycan is hindering or enhancing PGT151 binding (fig. S11D).

Fig. 4 The complete PGT151 epitope.

(A) A model of PGT151-glycan interactions. Glycans from up to four different subunits (two from gp120 and two from gp41) of two protomers of the trimer can lock the Fab in its bound form (left). Some of the glycans bind PGT151 with high affinity (black arrows), but there are numerous steric barriers that need to be overcome (red arrows). Glycans N241 and N448 probably have an inhibitory effect on PGT151 binding by influencing the conformations of each other. The lack of the N241 glycan (right) alleviates steric pressure by N448 (blue arrow). Different gp120 subunits are shown in shades of yellow, gp41 subunits in shades of blue, Fab light and heavy chains in pink and magenta, gp120 glycans in green, and gp41 glycans in dark green. (B) When PGT151 is bound to glycans at N611 and N637, HR2 is locked in a bent conformation and therefore cannot undergo conformational changes into the extended postfusion form. Colors are as in (A). (C) PGT151 CDRL1 (yellow) and the N637 glycan fucose (Fuc, dark green) interact with a glutamic acid– and asparagine-rich region of HR1N on the adjacent gp41 (Q551 to N554). The CDRH3 (purple) inserts between HR1N and the FP, and these interactions cap HR1N to lock gp41 in the prefusion conformation. The interacting residues in the Fab and HR1N are shown in orange. Only the core Man(Fuc)GlcNAc2 residues are shown for the N637 glycan for clarity. In (B) and (C), red arrows with crosses show that gp41 regions are blocked from undergoing conformational changes. (D) A measurement of the inter-gp41 distances in PGT151-bound JR-FL (left) compared with the unliganded BG505 trimer (right). The unliganded BG505 trimer measures ~37 Å between the Cα of N628 and N637 (residues shown in yellow) on the adjacent protomer, whereas the distance between the same two residues measures ~35 Å at the PGT151-liganded interfaces. However, the inter-gp41 distance at interface 1 (~44 Å) is ~9 Å farther apart in comparison with the liganded interfaces, indicating that the trimer becomes asymmetric in the PGT151-bound form. The three shades of blue differentiate the three gp41 monomers.

Trimers of different genotypes may contain different glycoforms, especially in gp41 (31), and variation at these sites may be responsible for a neutralization plateau (26). Our model illustrates that the PGT151 epitope extends beyond the gp41 glycans and includes gp120 glycans that may also contribute to incomplete neutralization. Overall, PGT151 is highly glycan-dependent, with up to six glycans in JR-FL (N241, N262, N276, N448, N611, and N637) surrounding the antibody (Fig. 4A).

Mechanism of trimer stabilization

Unlike any other bnAb identified so far, including gp41-gp120 interface antibodies such as 35O22 (fig. S13A) (32), PGT151 stabilizes the metastable prefusion Env for prolonged periods (16). Contact with both N611 and N637 glycans probably prevents the HR2 helix from progressing to the postfusion conformation (Fig. 4B). PGT151 and its clonal relatives all have a long CDRL1 of 16 residues and a conserved N28 (D28 in PGT154 to -158; fig. S12A) at the CDRL1 tip that interacts with a conserved region at the end of HR1N (sequence conservation: Q551 to Q552, 100%; N553, 58%; N554, 99%). Additionally, CDRH3 is wedged between the HR1N and the FP-proximal HR1 region. In this manner, the CDRL1 and CDRH3 cap the end of a short helix to prevent it from extending into a longer helix and thereby thwart the transition to a postfusion conformation (Fig. 4C). PGT151 has an unusual binding stoichiometry of two Fabs per trimer, even though there is no obvious steric barrier to the binding of the third Fab (fig. S13B). Rather, binding of two PGT151 antibodies appears to have an allosteric effect, altering the conformation of the third binding site (Fig. 4D).

10E8-bound conformation of Env

To visualize the MPER, we added MPER-specific bnAb 10E8 to the complex and analyzed the JR-FL EnvΔCT–PGT151 Fab–10E8 Fab complex by means of cryo-EM (fig. S14, A and B). Although 10E8 had some stabilizing effect on the MPER, we still observed flexibility in this region (Fig. 5A and fig. S14C), which is consistent with a previously solved crystal structure of the MPER peptide–10E8 Fab complex (Fig. 5C) (22). Despite variation in the protein conformation and binding occupancies on the trimer, we obtained an 8.8 Å–resolution reconstruction from a subset of the JR-FL EnvΔCT–PGT151 Fab–10E8 Fab complexes (Fig. 5A and fig. S14D). This reconstruction revealed that the center of the trimer at the base is empty, similar to the JR-FL EnvΔCT–PGT151 structure. Again, we did not observe a three-helix bundle formed by the TM, but MPER bound to 10E8 and the HR2-MPER interface could be visualized (Fig. 5B and fig. S15A). The HR2-MPER connectivity was substantially different from the crystal structure and emphasizes the flexibility in this region (Fig. 5C and fig. S15A).

Fig. 5 JR-FL EnvΔCT bound to PGT151 and 10E8.

(A) Cryo-EM reconstruction of JR-FL EnvΔCT (gray) in complex with both PGT151 (pink) and the MPER-binding antibody 10E8 (blue) at 8.8 Å resolution (left). The JR-FL EnvΔCT–PGT151 reconstruction (low pass–filtered to 8.8 Å) is shown on the right for comparison. The reconstructions indicate that when 10E8 is bound, the trimer is lifted off the membrane (red arrow), suggesting a conformational change in the MPER and TM. (B) The Env HR2-MPER connectivity in the 10E8-bound form is modeled into the EM density. (C) A comparison of the position of residues 659 to 670 in the two asymmetric units of the 10E8-bound MPER peptide x-ray model (dark and light gray), superimposed on the complete Env model (teal), in which the primary MPER epitope (residues 671 to 685) is shown in yellow. This N-terminal segment exhibits different conformations in the two asymmetric units. D664 is colored in red as a point of reference. 10E8 is shown as the white surface.

10E8 recognizes Env primarily via CDRH3 contacts with MPER helix 672 to 683, with additional interactions between FWRH3 and the gp41-gp120 interface (Fig. 6, A and B). Moreover, N88 and N625 glycans are positioned to sterically block 10E8 binding. The N88 glycan, built as a ManGlcNAc2 in the 4.2 Å model, clashes with FWRH1, and a complete glycan at N625 would also clash with the 10E8 Fab constant region (Fig. 6, A and B). The N625Q mutation increases maximum neutralization of JR2, and glycoforms affect the degree of neutralization plateauing (33). In the trimer structure, both the N88 and N625 glycans are accessible for glycan processing and have been predicted to be complex (3436). Thus, these glycans could restrict access to the MPER epitope. Most MPER antibodies bind to gp41 fusion intermediates (3739), but although 10E8 potently neutralizes virus after CD4 attachment (33), it also neutralizes the prefusion trimer (22, 32). Other studies have suggested that the CD4-bound form of Env may be lifted from the membrane and would therefore provide greater accessibility to membrane proximal epitopes (32). CD4 binding opens Env via a rotation in gp120, which moves the N88 glycan farther away from the base of the trimer (Fig. 6C). Nuclear magnetic resonance structures of MPER peptides, as well as our EnvΔCT model (fig. S16), suggest that the ground-state MPER epitope is embedded in the membrane (15). Thus, although MPER is difficult to access, MPER antibodies can either bind MPER while membrane-embedded or during some transient exposure when the trimer is lifted off the membrane surface.

Fig. 6 10E8 contact analysis in the context of the Env ectodomain.

(A and B) A model of the 10E8 epitope in the context of the intact Env trimer. The Fab constant region (dark and light gray surface) and the nearby Env gp120 (white) are also shown. On the left in (A) and the middle-lower right in (B) is the gp41 in which the 10E8 Fab makes primary interactions with MPER residues 671 to 685 (yellow). Additional contacts could be made with the HR2 and C-terminal region of the FP in the adjacent gp41 (teal), as well as regions in gp120 (white). These additional contacts to Env within a 4 Å radius of 10E8 are colored red. Many of these interactions are probably mediated by FWRH3 (orange). The model also demonstrates that the N88 (A) and N625 (B) glycans could sterically obstruct 10E8 binding (red circles). The glycans modeled here are ManGlcNAc2 for N88 and GlcNAc for N625 (table S1), but they are expected to be larger in native Env. (C) Glycans at N88 and N625 sterically hinder 10E8 binding to the trimer (left, red arrows). Binding of 10E8 (left, purple) or CD4 (center right) lifts the MPER up from the membrane relative to the ground state (center left). In the CD4-bound conformation, the opening of the trimers results in rotation of the gp120s, moving N88 away from the 10E8 binding site and relieving some steric hindrance (right, blue arrow). Colors are as in Fig. 4A. The red-orange areas in the cartoons on the right indicate the CD4-bound state.

In the negative-stain reconstruction of 10E8 in complex with MPER-containing BG505 SOSIP.683 (fig. S15, B and C), the Fab does not induce an opening of the trimer, as it does when soluble CD4 or CD4-induced antibodies are bound (2). Although the 2D class averages of 10E8-bound BG505 SOSIP.683 trimers display distinguishable Fab densities (fig. S15B), the particles refine poorly in the 3D reconstruction, and the 10E8 Fab binds at a different angle than in EnvΔCT (fig. S15C), which again is consistent with MPER flexibility. Without the TM, however, the Fab approaches from the bottom of the trimer, which would be impossible in the context of the viral membrane. Cross-linking the ectodomain of full-length Env also has no effect on MPER-bnAb binding, despite the reduced CD4 binding (40). Together, these observations indicate that (i) MPER is largely inaccessible on the viral membrane, with the membrane imposing a steric hurdle for the Fab approach angle, and (ii) 10E8 binds a transiently exposed MPER, which is not necessariliy the CD4-bound conformation of Env nor a fusion-transition conformation, in contrast with previous observations (41). The appearance of a gap between the HR2 C terminus and the micelle in our 10E8-bound structure (Fig. 5A) probably represents the optimal display of the membrane-anchored MPER epitope with all additional constraints in place. The epitope of 10E8, and perhaps of other MPER antibodies, is therefore more complex than previously thought, involving elements from multiple gp41 protomers, as well as from gp120.

Discussion

Here we present the cryo-EM reconstruction of a cleaved wild-type JR-FL EnvΔCT trimer in complex with PGT151 Fab at 4.2 Å resolution, demonstrating the structure not only of wild-type Env but also of a type I viral fusion protein with an intact TM, which unexpectedly was found to be flexible. The PGT151 epitope includes the FP and an extensive network of primary and secondary glycan interactions that stabilize the prefusion conformation of the Env trimer. The MPER appears to be sequestered in the detergent micelle in the unliganded state, but it is outside the micelle in the 10E8-bound structure, suggesting a dynamic topology. This property—in combination with steric constraints from gp120, gp41, and glycans at N88 and N625—effectively shields the conserved MPER. Thus far, MPER peptide vaccines, though immunogenic, produce nonneutralizing antibodies, probably because of the lack of the additional constraints provided by the trimer and membrane (42, 43); our model suggests that the minimalistic MPER epitope peptide presentation may not be the most ideal strategy to elicit MPER bnAbs. Overall, our data indicate that Env is a pliable structure in which several of the protein-protein interfaces can be remodeled, making it a difficult moving target for the immune system.

Supplementary Materials

www.sciencemag.org/content/351/6277/1043/suppl/DC1

Materials and Methods

Figs. S1 to S16

Table S1

References (4464)

REFERENCES AND NOTES

Acknowledgments: We thank C. Blattner for the PGT151 and JR-FL EnvΔCT plasmids, J. Torres and N. Overney for helping with plasmid preparations, T. Nieusma for technical assistance, A. Sarkar and L. Kong for advice on glycan modeling, and I. A. Wilson for helpful comments and discussion. The data from this study are tabulated in the main paper and in the supplementary materials. The cryo-EM reconstructions of JR-FL EnvΔCT–PGT151 and JR-FL EnvΔCT–PGT151 Fab–10E8 Fab and the model of JR-FL EnvΔCT–PGT151 have been submitted to the PDB and the Electron Microscopy Data Bank with accession codes PDB-5FUU, EMD-3308, EMD-3309, and EMD-3312. This work was supported by the NIH (grant UM1 AI100663), the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium through the Collaboration for AIDS Vaccine Discovery (grants OPP1084519 and OPP1115782), and the California HIV/AIDS Research Program Dissertation Award (to J.H.L.). This work was partially funded by IAVI with the generous support of the U.S. Agency for International Development (USAID), the Ministry of Foreign Affairs of the Netherlands, and the Bill and Melinda Gates Foundation; a full list of IAVI donors is available at www.iavi.org. The contents of this manuscript are the responsibility of the authors and do not necessarily reflect the views of USAID or the U.S. government. The EM work was conducted at the cryogenic electron microscopy facility at The Scripps Research Institute. This is manuscript number 29175 from the Scripps Research Institute.
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