Structure/Function Studies Involving the V3 Region of the HIV-1 Envelope Delineate Multiple Factors That Affect Neutralization Sensitivity

Abstract

Antibodies (Abs) specific for the V3 loop of the HIV-1 gp120 envelope neutralize most tier 1 and many tier 2 viruses and are present in essentially all HIV-infected individuals as well as immunized humans and animals. Vaccine-induced V3 Abs are associated with reduced HIV infection rates in humans and affect the nature of transmitted viruses in infected vaccinees, despite the fact that V3 is often occluded in the envelope trimer. Here, we link structural and experimental data showing how conformational alterations of the envelope trimer render viruses exceptionally sensitive to V3 Abs. The experiments interrogated the neutralization sensitivity of pseudoviruses with single amino acid mutations in various regions of gp120 that were predicted to alter packing of the V3 loop in the Env trimer. The results indicate that the V3 loop is metastable in the envelope trimer on the virion surface, flickering between states in which V3 is either occluded or available for binding to chemokine receptors (leading to infection) and to V3 Abs (leading to virus neutralization). The spring-loaded V3 in the envelope trimer is easily released by disruption of the stability of the V3 pocket in the unliganded trimer or disruption of favorable V3/pocket interactions. Formation of the V3 pocket requires appropriate positioning of the V1V2 domain, which is, in turn, dependent on the conformation of the bridging sheet and on the stability of the V1V2 B-C strand-connecting loop.

Importance: The levels of antibodies to the third variable region (V3) of the HIV envelope protein correlate with reduced HIV infection rates. Previous studies showed that V3 is often occluded, as it sits in a pocket of the envelope trimer on the surface of virions; however, the trimer is flexible, allowing occluded portions of the envelope (like V3) to flicker into an exposed position that binds antibodies. Here we provide a systematic interrogation of mechanisms by which single amino acid changes in various regions of gp120 (i) render viruses sensitive to neutralization by V3 antibodies, (ii) result in altered packing of the V3 loop, and (iii) activate an open conformation that exposes V3 to the effects of V3 Abs. Taken together, these and previous studies explain how V3 antibodies can protect against HIV-1 infection and why they should be one of the targets of vaccine-induced antibodies.

FIG 1

The V3 loop in the context of the gp120 trimer. (a) Overview of the V3 loop environment. The crown of the V3 hairpin (stick representation) is docked in a pocket formed by the V1V2 loop from the same protomer (green), the gp120 core with its V1V2 stem from the same protomer (magenta), as well as the V1V2 stem from the adjacent gp120 protomer (V1V2′; orange). (b to d) Close-ups of the interactions between the V3 crown and other domains. The residues indicated by arrows were mutated in the studies described here and are denoted by space-filling transparent spheres. The structures are reoriented as appropriate to best depict the key features of the respective interfaces. (b) Residue N197 in the β3 strand of the V1V2′ stem (indicated by an arrow) in the adjacent protomer interacts with V3. The first N-acetylglucosamine residue of the glycan is also visible and is marked N197gl. Residues F159 and M161 (indicated by arrows) interact with V1V2 of the same protomer (c), and residue L125 in the V1V2 stem in the gp120 core (indicated by an arrow) interacts with the V3 of the same protomer (d). Images were generated from the structure with PDB accession number 4TVP (24).

FIG 2

Overview of mutations made in gp120 to assess the impact on exposure of V3. (Top) Linear diagram (90) showing the five constant regions (C1 to C5) and five variable regions (V1 to V5) of gp120 with the amino acids in each region that were mutated individually in this study. Putative glycosylation sites are shown by the symbols above the bar. (Bottom) A two-dimensional depiction of gp120 with mutated residues shown by stick representation. As negative controls, pseudoviruses carrying the V2 mutation V182Q and the C4 mutation N448Q or T450A were constructed but are not shown here. The image was generated from the structure with PDB accession number 4TVP (24).

FIG 3

Effects of mutations on neutralization sensitivity of pseudoviruses carrying the WT gp120 of clade B strain JR-FL.JB or Env with mutations at position 197 or 184. (A) Neutralization of JR-FL.JB WT and three mutants with mutations at position 197 by anti-V3 MAb 1-79, soluble CD4IgG2, and antiparvovirus MAb 1418. Dotted lines, neutralization of the WT pseudovirus; solid lines, neutralization of the JR-FL mutants. (B) IC₅₀ neutralization values (in micrograms per milliliter) for anti-V3 MAbs against pseudoviruses carrying the envelope of WT clade B strain JR-FL or JR-FL mutants with substitutions at position 197 or 184. Pseudoviruses were also tested with positive and negative controls (CD4IgG2 and MAb 1418, respectively). The TZM.bl cell neutralization assay was used as described above; all experiments were performed in triplicate, and data are shown as the average of all data points from two experiments. Color coding uses red for values of <0.5 μg/ml, orange for values of 0.5 to 5.0 μg/ml, yellow for values of 5.1 to 25 μg/ml, and gray for values of 25 to >50 μg/ml.

FIG 4

The bridging sheet is an antiparallel β sheet in the structure of the monomer but has a mixed parallel/antiparallel conformation in the SOSIP trimer. (Left) The V2 stem residue A204 and C4 residues M434 and I424 form a buried hydrophobic core in the mixed β sheet of the SOSIP trimer (24). (Right) In contrast, in the structure of the gp120 monomer, there is an antiparallel β-sheet conformation and the three residues (A204, M434, and I424) separate, and two of the side chains become exposed (modified from the structure with PDB accession number 4TVP) (24). Thus, disruption of the trimer core would be expected to alter the packing of the V1V2 stem and/or shift the equilibrium toward the antiparallel configuration of the monomer gp120 bridging sheet structures, with downstream effects on the V1 and V2 loops as well as their interaction with V3.

FIG 5

The glycan at N301 restricts the mobility of the V3 loop, sharply increasing the buried conformation. A histogram of RMSDs from the in-pocket state for 500 lowest-energy conformations which was generated by 300 flexibility simulations of the V3 stem and crown in the presence (blue bars) or absence (red bars) of the glycan at position 301 is shown. RMSDs near zero correspond to the V3 buried (in-pocket) conformation, while high RMSDs indicate the released state. Estimated error bars represent the square root of the frequencies (Poisson statistics).

FIG 6

Simulations of the lowest-energy conformations of V3 in the presence and absence of the Man9 glycan attached to N301. The proportions of released and in-pocket conformations of the V3 loops are shown for the 50 lowest-energy V3 conformations (100 Monte Carlo simulations) in the presence of the glycan at N301 (A) and for the 50 lowest-energy V3 conformations (100 Monte Carlo simulations) in the absence of the glycan at N301 (B).

FIG 7

The glycan at N160 can stabilize the flexible elements of V2. Representative conformations from flexibility simulations of the loop that connects the B and C strands of V2 in the presence (upper) and absence (lower) of the glycan at position 160 are shown. The glycan reduces the mobility of the flexible loop.

FIG 8

Effect of mutations in the CD4 binding site on neutralization sensitivity. (A) Neutralization sensitivity of the JR-FL N280E mutant to V3 MAb 2219. Solid line, the JR-FL N280E mutant; dashed line, the JR-FL wild type. x axis, % neutrallization. (B) Neutralization of the JR-FL G458D mutant to V3 MAb 2219. The lines are as described in the legend to panel A. x axis, % neutralization. (C) IC₅₀ neutralization values for JR-FL and YU2 WT and mutant pseudoviruses tested with human anti-V3 MAbs. The colors are as described in the legend to Fig. 3 but with the addition of blue boxes, which denote increased resistance to neutralization by CD4IgG2. ND, not done; *, data from reference .