Subnanometer structures of HIV-1 envelope trimers on aldrithiol-2-inactivated virus particles

Abstract

The HIV-1 envelope glycoprotein (Env) trimer, composed of gp120 and gp41 subunits, mediates viral entry into cells. Recombinant Env trimers have been studied structurally, but characterization of Env embedded in intact virus membranes has been limited to low resolution. Here, we deploy cryo-electron tomography and subtomogram averaging to determine the structures of Env trimers on aldrithiol-2 (AT-2)-inactivated virions in ligand-free, antibody-bound and CD4-bound forms at subnanometer resolution. Tomographic reconstructions document molecular features consistent with high-resolution structures of engineered soluble and detergent-solubilized Env trimers. One of three conformational states previously predicted by smFRET was not observed by cryo-ET, potentially owing to AT-2 inactivation. We did observe Env trimers to open in situ in response to CD4 binding, with an outward movement of gp120-variable loops and an extension of a critical gp41 helix. Overall features of Env trimer embedded in AT-2-treated virions appear well-represented by current engineered trimers.

Extended Data Fig. 1.. Comparison of cryoET maps of the ligand-free HIV-1/SIV-1 Env trimer.

(a) In situ structure of Env trimer on the surface of SIV-1 mac329 virus with truncated cytoplasmic tail (EMD 1246). (b) Structure of Env trimer of SIV-1mneE11S virus (EMD 1216). (c) Structure of Env trimer of HIV-1_BaL virus (EMD 5019). (d) Structure of ligand-free HIV-1_BaL Env trimer (current work).

Extended Data Fig. 2.. CryoET resolution estimations.

(a) Resolution estimation using FSC 0.5 as cutoff value. (b) Local resolution estimation of ligand-free, 10–1074 and 3BNC117 double antibodies, and sCD4–17b bound HIV-1_BaL Env trimer is estimated with Resmap. For each map, two projections are shown: Z plane projection is shown as a top view, Y plane projection is shown as a side view.

Extended Data Fig. 3.. Model fitting.

Env trimers from other two strains, omitting the binding antibodies, are rigid body docked into the cryoET map. (a) 3H109L and 35O22 bound B41 SOSIP.664 (PDB 6MUF) (b) PGT122 and PGV19 bound BG505 NFL.664 (PDB 6B0N). (c) Segmented characteristic helical densities of gp41 subunits in the cryoET map. (d-f) Trimeric HR1C, partial HR1N and HR2 from 4ZMJ fit well into the rod-shape densities.

Extended Data Fig. 4.. Variable loop regions and glycosylation sites on the top of the ligand-free Env trimer map.

(a) Variable loop regions on the top of the Env trimer are differently colored in the map. (b) Potential glycosylation densities at the apical surface of BaL strain virus. Ligand-free monomer of fully glycosylated JR-FLΔCT (PDB 5FUU) was docked in the cryoET map. The glycan moiety was shown as sticks.

Extended Data Fig. 5.. Comparison between the cryoET model for HIV-1_BaL Env and BG505 SOSIP.664 in complex with different antibody combinations.

Each ligand and its binding protomer were projected into central plane and the dihedral angles were measured with UCSF Chimera. (a) CryoET model of 10-1074-3 and BNC117 bound Env. (b) 10–1074 bound BG505 SOSIP-based immunogen RC1 (PDB 6ORN). (c) 3BNC117 bound BG505 SOSIP.664 (PDB 5V8M). (d) 10–1074 and IOMA bound BG505 SOSIP.664 (PDB 5T3Z). All angular values are listed in panel (e).

Extended Data Fig. 6.. 10–1074 and 3BNC117 cannot stabilize State 1 of AT-2 treated HIV-1_BaL Env.

(a, b) 10–1074 and 3BNC117 Fabs exhibit a preference for State 1 of virus Env in the absence of AT2-inactivation (a) Representative fluorescence (top, donor Cy3 in green and acceptor Cy5 in red) and FRET trace (bottom, resulting FRET in blue and hidden Markov model idealization in red) of individual V1V4 labeled ligand-free HIV-1_NL4–3 virus Env. (b) FRET histogram of HIV-1_NL4–3 Env in the presence of broadly neutralization antibodies 10–1074 (50 μg ml⁻¹) and 3BNC117 (50 μg ml⁻¹), overlaid with that of ligand-free HIV-1_NL4–3 Env. (c, d) 10–1074 and 3BNC117 are unable to restore State 1-predominance observed on native virus Env after AT-2 treatment shifted the conformational equilibrium towards State 2. (c, d) experiments as in (a, b) of AT-2 chemically inactivated HIV-1_NL4–3 virus Env, respectively. (e) Quantification of relative state occupancy of HIV-1_NL4–3 virus Env, derived from FRET histograms in (b, d).

Extended Data Fig. 7.. The conformational effect of AT-2 lies in the ectodomain.

FRET histograms of HIV-1_JR-FL Env without cytoplasmic tail under conditions: ligand-free and untreated (a); ligand-free Env after AT-2 treatment (b); and in the additional presence of 10–1074 (50 μg ml⁻¹) and 3BNC117 (50 μg ml⁻¹) (c). (d) Quantification of the corresponding relative state occupancies.

Figure 1.. In situ cryoET structure of ligand-free HIV-1 Env trimer.

(a) A slice through a representative tomogram of AT-2-treated HIV-1 virons. The scale bar is 100 nm. (b) A zoom-in view of one virion. Note that Env trimers on virus surface are readily visible. The scale bar is 50 nm. (c, d) Two perpendicular slices of a sub-tomogram average of the HIV-1 Env trimer. The scale bar is 5 nm. (e) Segmentation of the Env trimer is shown in front and top views, respectively. The gp120 subunit is colored in light blue, the gp41 ectodomain in orange, the MPER in purple, and the membrane in gray. (f) Fitted model of gp120 (blue) and gp41(orange) subunits from the crystal structure of ligand-free BG505 SOSIP.664 (PDB 4ZMJ).

Figure 2.. CryoET structure of HIV-1 Env with antibodies 10–1074 and 3BNC117 at sub-nanometer resolution reveals domain features of in situ trimer.

(a) A slice through a representative reconstruction of HIV-1 viruses in complex with 10–1074 and 3BNC117 Fabs. The scale bar is 100 nm. (b) A zoom-in view of one virion with Env trimers. The scale bar is 50 nm. (c) A central slice of a sub-tomogram average of the Env trimer bound with 10–1074 and 3BNC117. The scale bar is 5 nm. (d) A segmentation of the antibody-bound Env trimer shows 10–1074 (green), 3BNC117 (cyan), gp120 (blue) and gp41 (orange) subunits. (e) Enlarged views of gp41-interactive region of gp120 and gp41 (upper panel) with residue-level model fitting in the density map (Bottom panel). (f, g) Side and bottom views of gp41 at two different contour levels (0.156 and 0.121), respectively. The gp41 model from PGT151-bound JR-FL Env trimer (PDB 5FUU) fitted into the segmented maps, respectively (bottom); gp41 colored in orange, with the C-terminal region of the HR1-N highlighted in watermelon red.

Figure 3.. AT-2-treatment increases the prevalence of State 2 HIV-1 Env on virus.

(a, b, c) AT-2 treatment shifts the State 1-predominance of the conformational landscape of ligand-free HIV-1_NL4–3 (a: left panel), HIV-1_BG505 (b: left panel), and HIV-1_JR-FL (c: left panel) towards State 2. FRET histograms of ligand-free (left panels) and AT-2 treated (middle panels), and the corresponding quantification of relative conformational states occupancy (right panels) for neutralization-sensitive lab-adapted isolate HIV-1_NL4–3 (a), and neutralization-resistant primary isolates HIV-1_BG505 (b) as well as HIV-1_JR-FL (c), respectively. FRET histograms compiled by a number (n) of individual FRET traces represent mean ± s.e.m., from three independent groups of smFRET traces. FRET histograms overview conformational profiles of native Env trimers in the context of intact viruses, while bar-graphs quantify the relative occupancy for each state.

Figure 4.. In situ cryoET structure at 9.7 Å resolution of sCD4_D1D2- and 17b-bound Env trimer.

(a) A slice through a representative tomogram shows multiple virions. The scale bar is 100 nm. (b) A zoom-in view of one virion with Env spikes. The scale bar is 50 nm. (c, d) Sub-tomogram average shows sCD4_D1D2- and 17b-bound Env trimer from side and top views, respectively. The scale bar is 10 nm. (e) Segmentation of the Env trimer shows sCD4_D1D2 (purple), 17b (green), gp120 (blue) and gp41(orange) subunits in both side and top views. (f) Atomic models of gp120, gp41, 17b and sCD4 fit well into the map (PDB 5VN3). (g) V1V2 loop fitted from sCD4(D1D2), 21C-bound, and 8ANC195-bound B41 SOSIP.664 (PDB 6EDU).

Figure 5.. The central helical density of gp41 in situ resembles that observed in high-resolution SOSIP structures.

(a) gp41 subunit of ligand-free BaL HIV-1 Env. (b) gp41 subunit of 10–1074 and 3BNC117 bound Env. (c) sCD4–17b bound BaL HIV-1 Env. (d) sCD4–17b bound B41 SOSIP.664 (EMD 8713) and (e) sCD4-E51 bound BG505 SOSIP.664 (EMD 20605). The high-resolution single particle cryoEM maps were low-pass filtered to 10Å. Segments of gp41 are depicted as below, in the cryoET maps, HR1-N and fusion peptide (FP) are colored in red, HR1-C of gp41 in orange; for both reported EM maps, HR1-N densities are colored in pink, HR1-C are colored in yellow. The base portion of all the gp41s are colored in orange. (f) Each corresponding atomic structure of HR1-C helices was fitted into the maps that were viewed from the top and side views.

Figure 6.. sCD4- and 17b-bound Env trimer adopts a more open conformation in situ in the HR1-N and HR2 regions of gp41.

(a) One copy of HR1-N and adjacent HR2 are segmented in sCD4–17b bound Env map. HR1-N is colored in red and HR2 colored in orange. (b, c, d, e) Segmentation of HR1-N and HR2 from four different open-state structures, respectively. (b) sCD4–17b bound B41 SOSIP.664 (EMD 8713). (c) sCD4–17b-8ANC195 bound BG505 SOSIP.664 (EMD 7516). (d, e) Two classes of sCD4-E51 bound BG505 SOSIP.664 (EMD 20605 and EMD 20607). (f, g, h, i) Each of the four atomic models are docked into the cryoET map, respectively. HR1-N and HR2 of gp41 subunits are highlighted. HR1-N and HR2 are colored in green (f, g), in purple (h) and in magenta (i, j). (f) sCD4–17b bound B41 SOSIP.664 structure (PDB 5VN3). (g) A zoom view of panel f. (h) sCD4–17b-8ANC195 structure (PDB 6CM3). (i, j) Two structures of E51-sCD4 bound BG505 SOSIP.664 (PDB 6U0L and 6U0N).