Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

AbstractHere, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions

Partial Enzymatic Deglycosylation Preserves the Structure of Cleaved Recombinant HIV-1 Envelope Glycoprotein Trimers

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The gly-can-depleted Env trimers should be useful for structural and immunogenicity studie

Broadly Neutralizing Antibody PGT121 Allosterically Modulates CD4 Binding via Recognition of the HIV-1 gp120 V3 Base and Multiple Surrounding Glycans

<div><p>New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC₅₀ less than 0.05 µg ml⁻¹. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664) reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.</p> </div

X-ray crystallography statistics.

*<p>Values in parentheses are for the highest resolution shell.</p>†<p>R_sym = Σ|I-<i>|/Σ<i>, where I is the observed intensity, and <i> is the average intensity of multiple observations of symmetry related reflections.</i></i></i></p><i><i><i>‡<p>R = Σhkl∥Fobs|−|Fcalc∥/Σhkl|Fobs|.</p>§<p>R_free calculated from 5% of the reflections excluded from refinement.</p></i></i></i

Thermodynamic parameters of PGT 121 antibodies and CD4 binding to gp120 and SOSIP.664 trimers measured by isothermal titration calorimetry.

%<p>Reported values are averages from at least two independent measurements and associated errors are approximately 10% of the average. Representative isotherms can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s006" target="_blank">Figs. S6</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s007" target="_blank">S7</a>.</p>*<p>The binding isotherms do not allow to accurately determining these binding parameters.</p>#<p>The change in Gibbs free energy (ΔG) was determined using the relationship: ΔG_binding = RTlnK_d<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-deAzevedo1" target="_blank">[75]</a>.</p>&<p>The stoichiometry of binding (N) is directly affected by errors in protein concentration measurements, sample impurity and glycan heterogeneity on gp120.</p

Structure and sequence characterization of antibodies of the PGT121 family.

<p>A) Superimposition of the PGT121 (green), PGT122 (cyan) and PGT123 (magenta) Fab crystal structures determined at 2.8 Å, 1.8 Å and 2.5 Å resolutions, respectively. Only the variable domains are shown with secondary structure rendering. The three CDRs of the light and heavy chains are labeled and colored in different shades of blue and red to orange, respectively. The 24-residue HCDR3 loop divides the paratope into two faces: an open face composed from HCDR1, HCDR2 and the base of HCDR3 and a more elongated face from LCDR1, LCDR3 and the tip of HCDR3. B) Sequence conservation between antibodies of the PGT121 family mapped on the PGT122 crystal structure, which is rendered as a surface representation. Identical residues in the three antibodies are colored green while divergent sequences are represented in white. Two regions of clustered sequence identity are predicted to play an important role for antigen recognition (conserved faces 1 and 2). These figures were generated using UCSF Chimera <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-Pettersen1" target="_blank">[70]</a>. C) Sequence alignment among antibodies of the PGT121 family with the consensus sequence shown above and non-conserved residues shown below for each antibody. The six light chain (LC) and heavy chain (HC) CDRs and Framework Regions (FR) are indicated based on Kabat alignment. This figure was generated using Jalview <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342-Waterhouse1" target="_blank">[72]</a>.</p

Alanine-scanning mutagenesis of the PGT121, PGT122 and PGT123 paratopes.

<p>A) Surface residues predicted to play a role in mediating antigen recognition were identified from the crystal structures, subsequently mutated to alanine, and the resulting IgG mutants were tested for their ability to neutralize HIV-1 JR-CSF pseudoviruses. The fold increase in the neutralization IC₅₀ compared to WT IgG is reported in the table by color code: green, <2; yellow, 2–5; orange, 5–9; and red, >9. Mapping these results on the PGT121, PGT122 and PGT123 crystal structures allowed identification of a region crucial for mediating neutralization near the elongated face, whereas side chains of residues in the open face appear to play a more secondary role in HIV neutralization. Residues that show an increase in the neutralization IC₅₀ of >9 (red) compared to WT IgG upon mutation to alanine are labeled on the structures. B) The PGT121 open face accommodates a biantennary glycan from a symmetry-related Fab molecule in the crystal (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s002" target="_blank">Figs. S2</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003342#ppat.1003342.s003" target="_blank">S3</a>). PGT121 is rendered as a gray surface, whereas the glycan is shown as magenta sticks. The blue mesh is the 2Fo-Fc electron density map countered at a 1.2 sigma level around the glycan moiety. The figure was generated using Pymol.</p