Cryo-electron tomography of trimeric SIV Env. (A) Slice (2.4 nm thick) through a reconstructed, cryo-electron tomogram of SIVmac239 virions incubated with sCD4 (30 μM). Env spikes on the surface of the viral membrane as well as the inner viral core are visible. Bar, 100 nm. (B) Schematic illustrating relationship between the 3D structure of the sCD4-Env complex and the slices through the tomographic reconstruction shown in C, with gp120 in red, gp41 in blue, and viral membrane in gray. (C) Slices through the tomographic reconstruction of sCD4-bound SIVmac239 Env with successive rounds of refinement shown in each row. The first column on the left shows a side-projection view through the density map before refinement (first row) and through the 12 successive rounds of refinement. The second and successive columns show slices every 4.1 Å starting at the base (density corresponding to the transmembrane glycoprotein) through Env to the apex (density corresponding to gp120 and sCD4) in the last column on the right. sCD4 density can be visualized from the second iteration onwards (third row from the top) as punctate dots in the slices at the apex of the spike.

Comparison of trimeric SIV Env molecular architecture in native and sCD4-bound states. (A to C) Consecutive slices illustrating the density of the map at the apex of the spike in native SIVmneE11S Env (A), sCD4-bound SIVmneE11S Env (B), and sCD4-bound SIVmac239 Env (C). The densities from gp120 and sCD4 are clearly delineated. (D and E) Density map of SIVmneE11S Env displayed as an isosurface (blue and gray represent Env and viral membrane, respectively) in native (D) (58) and in sCD4-bound (E) conformations. (F) Superposition of density map from sCD4-Env complex on SIVmneE11S (shown in mesh) with positive difference density map (yellow isosurface) obtained by subtracting the native density map from the sCD4-bound density map. (G to I) Native, sCD4-bound, and superposition of liganded density maps with difference density maps for trimeric SIVmac239 Env, as in D to F for SIVmneE11S. Although both positive and difference densities were calculated for the respective maps, the positive difference density overwhelms the negative difference density peaks and are not visible at the threshold displayed. The mass contained within the displayed difference density threshold was estimated by procedures implemented in the EMAN software (39) and is ∼78 kDa, roughly matching the approximate molecular mass of 3 sCD4 molecules (24). No difference in structure was observed when sCD4 incubation was carried out at 37°C instead of 4°C.

Coordinate fits of gp120 and sCD4 into native and sCD4-bound SIV Env maps. (A and B) Perspective view of native SIVmneE11S (A) and SIVmac239 (B) Env fit with gp120 coordinates (1GC1.pdb) (58). Env is displayed as a transparent blue isosurface, with gp120 coordinates shown as gray space-filling model with V1/V2, V3, and sCD4-binding site shown in red, green, and yellow, respectively. Red spheres indicate the locations of the V1/V2 loops missing from the coordinates. (C and D) Rigid body fitting with X-ray coordinate subset containing gp120 and sCD4 coordinates (PDB ID:1GC1) into sCD4-bound SIVmneE11S (C) and SIVmac239 (D) Env density maps. Env and gp120 are displayed as in A and B, with sCD4 shown in yellow ribbons. (E and F) Top views of the density maps and coordinate fits shown as in C and D for sCD4-bound SIVmne E11S (E) and SIVmac239 (F) Env, respectively. The small opening at the apex of the trimer can be visualized by the displacement in the estimated portions of the V1/V2 loops (red spheres) compared to the native state.

Molecular architecture and coordinate fits of sCD4-bound SIV CP-MAC Env. (A and B) Native (A) (58) and sCD4-bound (B) SIV CP-MAC Env density map obtained from 3D classification and averaging rendered as a blue isosurface with gray lipid bilayer. (C) Greatest positive difference density (yellow isosurface) when subtracting the native SIV CP-MAC Env (A) density map from that of sCD4-bound trimeric Env (B) density map, shown here as a blue mesh. (D) Fit of gp120 subset of 1GC1 coordinates into native SIV CP-MAC Env density map. The gp120 protomers are rotated outward compared to the conformation observed for native SIVmac239 and SIVmneE11S Env trimers. (E) Fit of gp120 and sCD4 components of 1GC1 coordinates into the sCD4-bound SIV CP-MAC Env density map. Coordinates of gp120 are displayed as in Fig. 3, with sCD4 shown as yellow ribbons.

Molecular architecture of CCR5-binding site antibody (7D3) complexes on SIV CP-MAC Env with fitted gp120 coordinates. (A and B) Perspective views of 7D3-bound (red ribbons) SIV CP-MAC Env shown in blue transparent isosurface rendering in the absence (A) (58) and presence (B) of sCD4 (yellow ribbons). Insets show the same averaged maps as in the main figure panel, but without the fitted coordinates. Coordinate fits of a subset of gp120 X-ray coordinates from 1GC1 (A) or gp120 and sCD4 (B) rendered as in Fig. 3. Residues highlighted in magenta and blue indicate gp120 residues involved in coreceptor binding (47) and gp120 residues which have complete sequence conservation between HIV-1 and SIV, respectively. The 17b component of 1GC1 coordinates were used to model the 7D3 Fab fragment (red ribbons), since no crystal structure is available for 7D3 Fab, and it was then fitted into difference density calculated by subtracting the native SIV CP-MAC Env density from that of the antibody-bound map (see Materials and Methods for details).

Comparison of the binding site footprint for coreceptor binding site (CoRbs) and V3-loop targeting antibodies. (A) Top view of trimeric SIV CP-MAC Env complexed with coreceptor binding site antibody 7D3 in the presence of bound sCD4. (B) Top view of trimeric HIV-1 BaL Env complexed with coreceptor binding site antibody 17b in the presence of bound sCD4 (38). Fitted X-ray coordinates for the coreceptor binding site antibodies 7D3 and 17b are shown as red (A) and blue (B) ribbons, respectively. As in Fig. 5, the X-ray coordinates from 17b Fab were used to represent 7D3 for coordinate fitting (see Materials and Methods for details). (C) Magnified top view showing locations of fitted coordinates for gp120, sCD4, and 7D3 (red ribbons) in the SIV Env map shown in A, superimposed with the location of 17b (blue ribbons) based on the corresponding ternary complex with HIV-1 gp120 and sCD4. (D and E) Visualizing locations of V3-loop and coreceptor binding site antibodies in the context of SIV CP-MAC/sCD4/7D3 ternary complex. To better understand how the binding of 7D3 to SIV CP-MAC gp120 compares with the binding of comparable antibodies directed against HIV-1 gp120, we compared the locations of these CD4i (CD4-induced) antibodies after placing them at the apex of the spike by using the complexed V3 peptide as a structural marker for alignment, as suggested in reference 32. Perspective (D) and top (E) views of the sCD4/7D3-bound density map (mesh) superposed with predicted locations of various V3-loop reactive antibodies. Positions were derived by first fitting gp120 X-ray coordinates (1GC1 with 17b shown in blue) without V3 into the density map, aligning the coordinates with V3-loop containing gp120 coordinates (2B4C with X5 shown in brown) and subsequent superposition of V3-loop peptides of V3-binding antibody complexes (59.1, 447-52D, 50.1, 83.1, and 58.2 in orange, green, yellow, purple and pink van der Waals surfaces, respectively).

Top and schematic views of trimeric SIV envelope glycoproteins in native and sCD4-bound states. (A) Top views of the density maps (shown as a transparent blue isosurface) with fitted gp120 coordinates for SIVmne, sCD4-bound SIVmne, SIV CP-MAC, and sCD4-bound CP-MAC, shown from left to right. The red spheres indicate the likely locations of the V1/V2 loops, which are not present in the X-ray coordinates. (B) Schematic as in A, representing the essentially closed (first two) and open (last two) states of trimeric Env without and with sCD4 bound, showing gp120, gp41, and sCD4 shown in red, cyan, and yellow, respectively.