Sequence of the HXBc2 gp120 core and residue-by-residue fractional variation. The fraction of non-HXBc2 sequences at each residue for HIV-1 group M (blue column), clade B (green column), and clade C (red column) are plotted along the primary sequence of the HXBc2 core of gp120. Variable loops V1-V2 and V3 were replaced with Gly-Ala-Gly tripeptides, and V4 was deleted because of unreliable alignment. Sequence numbering follows the HXBc2 convention. Symbols under the HXBc2 sequence in green and orange depict gp120 contacts with b12 and CD4, respectively. Open circles, gp120 main-chain contacts; open circles with rays, gp120 side chain contacts; filled circles, gp120 main- and side-chain contacts. The CD4-binding loop, which is the prominent b12 and CD4 contact surface on gp120, is highlighted with a red box.

Effect of amino acid variants within the CD4-binding loop on b12 binding to monomeric gp120 (A, C, and E) and viral neutralization sensitivity to b12 (B, D, and F). ELISA binding curves show monoclonal antibody (Ab) 17b binding in black, as a control for the capture of wild-type (WT) and mutated gp120, and b12 binding in red. Solid symbols are WT gp120, and open symbols are the indicated point mutants. Control 17b binding was performed in the presence of sCD4. OD450, optical density at 450 nm.

Effect of mutations outside the b12 epitope such as Δ197, Δ301, and gp41 (569 and 675) on b12 binding to monomeric gp120 (A, C, and E) and on viral neutralization sensitivity to b12 (B, D, and F). ELISA binding curves show monoclonal antibody (Ab) 17b binding in black as a control for the capture of wild-type (WT) and mutated gp120 and b12 binding in red. Solid symbols are WT gp120, and open symbols are the indicated point mutants. OD450, optical density at 450 nm. (G) Illustration of the altered conformation of the Env spike by mutations outside the b12 epitope. Glycans at 197 and 301 appear to restrict the quaternary flexibility of gp120 within the viral Env spike, thus limiting the Env movement required for the exposure of the b12 epitope. Glycan removal at these sites likely alters the quaternary restriction of conformational changes, and this appears to allow for increased accessibility to the b12 contact surface. A similar effect may occur with alterations in gp41 residues 569 and 675.

HIV-1 gp120 sequence variation displayed in dendrogram format and mapped on the three-dimensional structure of the core of gp120. (A) Maximum-likelihood trees for HIV-1 group M (left), clade B (middle), and clade C (right). A value of 0.1 indicates the distance scale. The 19 clade B and 18 clade C reference viruses are highlighted with blue and red squares, respectively. (B) Sequence variation from HXBc2 in the context of the b12 epitope and gp120 core structure. The top row, from left to right, shows the molecular surface of the HXBc2 core gp120 in blue, with the b12 contact surface highlighted in green. Subsequent images display protein sequence variability from HXBc2 for group M, clade B, and clade C strains. Variability is displayed utilizing a white-to-red color gradient, which corresponds to 0 to 100% variation from the HXBc2 sequence. Hence, a 100% value for a specific site among clade C viruses indicates that all clade C viruses contained an amino acid that is different from that found in the HXBc2 isolate. Note that although the intrinsic variation within group M is greater than that of clade C, the average variation of clade C from the HXBc2 isolate is greater. To aid visualization, the borders of the b12 epitope are highlighted with a green outline. Some regions of the gp120 core display high variation (bright red), while the b12 epitope is relatively conserved except in the CD4-binding loop, where there is substantial variation. The middle row shows ribbon representations corresponding to the top-row surface representation. The dash boxes highlight the CD4-binding loop. The bottom row is a close-up, with Cα atoms of residues of the CD4-binding loop displayed in spheres and selectively marked according to the HXBc2 residue numbers. Note the level of variation (in red) near residues 364, 369, and 373.

Amino acid variants within the CD4-binding loop in the context of b12- and CD4-bound structures of the gp120 core. (A) Structure-based computational models display likely side-chain rotomers for natural variants 364H, 369L, and 373 M (shown with carbon atoms in magenta), which are predicted to be incompatible with b12 binding. These models are displayed in the context of the b12-gp120 core structure, shown in stick representation, with gp120 residues shown with blue carbon atoms and b12 residues with green carbon atoms. Potential clashes are highlighted in gold. These clashes may occur with other gp120 residues within the b12-bound conformation and/or with b12 residues such as Asn31, Tyr98, and Trp100. (B) The locations of residues 364, 369, and 373 (highlighted in purple) in CD4-bound (left) and b12-bound (right) structures of the gp120 core are shown in both ribbon diagrams (top row) and close-up surface representations (bottom row) of the CD4-binding loop. The CD4 and b12 contact sites are colored yellow and green, respectively. Boxed areas in the top row are magnified in the indicated insets. In the bottom row, portions of CD4 (orange) and b12 complementarity-determining regions (CDRs) CDR-H1, CDR-H2, and CDR-H3 (orange) are shown as a ribbon diagram, and portions of the borders of their contact sites on the CD4-binding loop of gp120 are marked with red lines. CD4 interacts with only one side of the CD4-binding loop, while the CDRs of b12 grab on both sides of the CD4-binding loop. Residues 364, 369, and 373, with naturally occurring mutations, reside outside of the conserved CD4-binding site (yellow) but are contained within the b12 contact surface (green). Although residue 364 appears to be outside the b12 epitope in the viewing angle shown in the middle bottom panel, its side chain actually falls within the b12 contact area on the other side of the CD4-binding loop, as shown in the bottom right panel.

Statistical analysis of b12 binding and neutralization. (A) Comparison of b12 binding to gp120 from b12 neutralization-sensitive (b12^S) and -resistant (b12^R) viruses from clade B and C (left). The middle and right panels show the linear regression of b12 binding of gp120s and the neutralization of Env pseudoviruses from clade B and C. (B) Comparison of sCD4 neutralization sensitivity between b12^S and b12^R viruses from clade B and C (left). The middle and right panels show the linear regression of sCD4 and b12 neutralization activity against Env pseudoviruses from clade B and C. (C) Comparisons of b12 binding to clade B and C gp120 (left), b12 (middle), and sCD4 (right) neutralization activity against clade B and C Env pseudoviruses. The horizontal bars show geometric means. P values were derived by the Mann-Whitney test. The b12 and sCD4 neutralization IC₅₀s used in these plots were converted from micrograms/milliliter to nanomolars using a molecular mass of 148 kDa for b12 and 46 kDa for sCD4.