Visualization of retroviral envelope spikes in complex with the V3 loop antibody 447-52D on intact viruses by cryo-electron tomography

Abstract

The gp120 portion of the envelope spike on human immunodeficiency virus type 1 (HIV-1) plays a critical role in viral entry into host cells and is a key target for the humoral immune response, and yet many structural details remain elusive. We have used cryoelectron tomography to visualize the binding of the broadly neutralizing monoclonal antibody (MAb) 447-52D to intact envelope spikes on virions of HIV-1 MN strain. Antibody 447-52D has previously been shown to bind to the tip of the V3 loop. Our results show antibody arms radiating from the sides of the gp120 protomers at a range of angles and place the antibody-bound V3 loop in an orientation that differs from that predicted by most current models but consistent with the idea that antibody binding dislodges the V3 loop from its location in the Env spike, making it flexible and disordered. These data reveal information on the position of the V3 loop and its relative flexibility and suggest that 447-52D neutralizes HIV-1 MN by capturing the V3 loop, blocking its interaction with the coreceptor and altering the structure of the envelope spike.

Importance: Antibody neutralization is one of the primary ways that the body fights infection with HIV. Because HIV is a highly mutable virus, the body must constantly produce new antibodies to counter new strains of HIV that the body itself is producing. Consequently, antibodies capable of neutralizing multiple HIV strains are comparatively few. An improved understanding of the mechanism of antibody neutralization might advance the development of immunogens. Most neutralizing antibodies target the Env glycoprotein spikes found on the virus surface. The broadly neutralizing antibody 447-52D targets the highly conserved β-turn of variable loop 3 (V3) of gp120. The importance of V3 lies in its contribution to the coreceptor binding site on the target cell. We show here that 447-52D binding to V3 converts the Env conformation from closed to open and makes the V3 loop highly flexible, implying disruption of coreceptor binding and attachment to the target cell.

FIG 1

(A) Ribbon representation of the crystal structure of Fab 447D MAb from PDB 1Q1J (58). The heavy chain is colored cornflower blue and the light chain is colored yellow, with the V3 MN peptide in red. The two views are approximately perpendicular to the pseudo 2-fold axis. (B) View of a plausible spike atomic model showing the rough location of the V3 loop. The 3-fold axis of the spike is approximately vertical in the picture. The coloring scheme is as follows: gp120 residues 90 to 124, green; residues 198 to 396, orange; and residues 410 to 492, yellow. The intact V3 loop, residues 298 to 327, is navy blue, and the GPGR loop at the tip of V3 is red.

FIG 2

Images of antibody labeled HIV-1 MN virions. (A) A slice through the x-y plane of one of the HIV-1 MN cryo-electron tomograms. The spikes are significantly fewer compared to the short-tailed SIV (it has more spikes on its membrane surface due to truncation of the cytoplasmic tail) but still visible on the viral surface. Parts of the cores within some of the virions are also visible but the AZT treatment significantly disrupts the core of the virion. Scale bar, 100 nm. (B to E) Representative slices through individual virions following binning. The density range displayed is the mean ± 3σ. Binning (averaging the densities of a 2×2×2 cube into a single voxel) enhances the signal-to-noise ratio, thereby improving the visibility of the spikes and bound antibody. The tomograms were then reinterpolated back to the original voxel size to reduce pixelation. Panels B and D come from virions marked with white arrows in the lower-magnification view shown in panel A. The binding can be seen clearly in some of the spikes (white arrows) on the viral membrane. Antibodies sometimes extend up relative to the membrane plane and sometimes parallel to the membrane plane.

FIG 3

Density maps from the one-arm classification of HIV-1 MN Env spike with bound 447-52D antibody. In the left-hand column is a view down the 3-fold axis of the spike. The numbers in the top row of column A label the spike arms, thereby defining each arm as to whether classification was carried out, arm 1, or not, arms 2 and 3. The central column is a view obtained by rotating the spike average by 45° about the horizontal axis and away from the observer. The right-hand column is a view tangential to the membrane plane. Each map is superimposed on the symmetrized average of the unliganded control spike arm at 50% transparency. The contour threshold used for the unliganded control spike is the same for all figures that show it. (A) The class average having the least density due to bound MAb, which was used for making the symmetrized control spike. (B to G) The six class averages showing density due to bound antibody. (H) Average of classes 1 to 6 weighted according to the number of class members. The coloring scheme is as follows: class 0, orange; class 1, brick; class 2, yellow; class 3, cyan; class 4, red; class 5 magenta; class 6, green; and the average of 1 to 6, dodger blue. These images can also be seen as an aligned, animated, and annotated sequence in Movie S2 in the supplemental material.

FIG 4

(A) Superimposed difference maps of all antibody-bound class averages with the symmetrized, antibody-free average. From left to right top view; 45° oblique view; side view tangential to the membrane. The numbers in panel A label the spike arms, thereby defining each arm by whether classification was carried out, arm 1, or not, arms 2 and 3. Images of the individual difference maps overlaid on the control spike can be visualized in Movie S3 in the supplemental material. (B) Intersection of the vectors through the quasi 2-fold axis of the Fab atomic model fitted by NMFF to the class averages with the symmetrized, antibody-free spike envelope. These four class averages are the only ones for which the vector intersected. Each of the four vectors intersects the envelope approximately at the location of the base of the V3 loop. (C) Comparison of difference maps from HIV/447D Env spikes with the soluble, partially deglycosylated spike trimer KNH1144 SOSIP 664G with antibody PGT128 bound (colored goldenrod), EMDB code emd-1970 (89). Images of the individual difference maps overlaid on emd-1970 can be seen in Movie S5 in the supplemental material. (D) Comparison of the difference maps for the 447-52D class averages with the 3-D reconstruction of PGT135 in complex with the BG505 SOSIP.664 gp140 trimer (90) colored olive (EMDB code emd-2331). Images of the individual difference maps overlaid with emd-2331 can be seen in Movie S6 in the supplemental material.

FIG 5

Crystal structure of PGT128 Fab in complex with an engineered glycosylated gp120 outer domain with a miniV3 loop (eODmV3), PDB 3TYG (1), aligned onto the gp120 spike pseudoatomic model from the present study. The 3TYG crystal structure is shown in orange-red, and the gp120 pseudoatomic model from the present work shown in yellow. The PGT128 Fab is shown in blue and cyan. The symmetrized average Env spike from the present study is provided as a point of reference. Panels A and B show the crystal structure as it would be positioned when fitted in the PGT128 image reconstruction (emd-1970), followed by alignment of emd-1970 to our control spike reconstruction. Panels C and D are after alignment on the gp120 atomic model used in the present study. The alignment was done manually using CHIMERA. The major movement requires a small outward radial movement and a small downward axial movement as indicated by the narrow black lines. There is also an ∼15° clockwise rotation along an axis roughly perpendicular to the radial direction of the spike arm. Panels E and F have the added global average of the antibody bound subvolumes from the single arm classification shown in dodger blue.