Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab) and single chain variable fragment (ScFv) antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes

Mass estimations by sedimentation velocity analysis.

<p>Mass estimations by sedimentation velocity analysis.</p

Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles

<div><p>The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a <i>K</i>_d of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.</p></div

Sedimentation velocity absorbance <i>c(s)</i> distributions for various gp41 analogues at 20°C in the presence of an excess of DPC micelles.

<p>(A-C) Data collected in 20 mM sodium phosphate, pH 7 and 150 mM NaCl, except for 17–194 which was recorded in the absence of NaCl. Values derived for constructs A through I at pH 7 are listed numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.t001" target="_blank">Table 1</a>. SV data for 17–194 at pH 4.2 was acquired in 50 mM sodium acetate with 25 mM KCl and 10 mM DPC (panel A, blue) and 17–172 at pH 4 and 5 (panel D: green and orange, respectively) in 50 mM sodium acetate with 10 mM DPC. Protein concentrations are as follows: 17–194 (pH 4.2), ~10 μM; 17–194 (pH 7), 1–2 μM; all others, 10–20 μM. The trimer/monomer distribution was unchanged in the absence or presence of 150 mM NaCl at pH 6 (panel D, red dotted trace). Slight variations in the sedimentation coefficients for the 17–172 and 17–172 ^Cys-MTSL trimer arise from differences in the isotopic labeling: ²H ¹³C ¹⁵N-labeled 17–172 complexed with ¹H-DPC as compared to ²H-labeled 17-172^Cys-MTSL complexed with ²H-DPC.</p

Mutations Proximal to Sites of Autoproteolysis and the α‑Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV‑1 Protease

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1–3 comprising residues 30–37, 60–67, and 88–95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10–35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated <i>in vitro</i> using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR^PR20‑3) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR^PR20‑3 because of the cumulative effect of eight noncluster mutations present in TFR-PR20^PR‑12. Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function

DEER EPR measurements with MTSL labels in various positions of the 17–172 construct.

<p>Positions of labels are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.g001" target="_blank">Fig 1</a>. Results of the DeerAnalysis2015 Tikhonov Regularization fits [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref048" target="_blank">48</a>] of the background corrected data acquired at pH 7 and pH 4 in the presence of ~2-fold excess of DPC micelles. The regularization parameter, α was determined by examination of the relevant L-curves (α = 10 in all cases except for the ^N-Cys17-172 data where α = 1000 was the best choice). Plots of the raw data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s006" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s007" target="_blank">S7</a> Figs. Previously published data indicating the fraying of the C-HR region by the addition of C34 to the 6HB formed by the construct 35–144 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a>) in 10 mM Tris-HCL, pH 7.6, and 150 mM NaCl (without DPC) is superimposed (orange, panel F) for comparison with 17-172^C-Cys in the presence of DPC at pH 4. The blue trace depicts the distance distribution of 35–144 trimer in the absence of C34 and DPC at pH 7. P(<i>r</i>) distribution calculated using the MMM software package for 17-172^L661C labeled with MTSL is superimposed for comparison with the experimental data in panel G (red trace).</p

Binding isotherm for the interaction of Z13e1 with 17–172.

<p>The peaks in red indicate the heat released after each addition of 17–172 into the antibody solution. Traces in blue indicate control titrations using either just the antibody in the cell titrated with the buffer (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 2 mM DPC) shown in B (black) or the same buffer titrated with the antigen shown in A and B (blue). (C) The data were best fit using a single binding constant to calculate the thermodynamic parameters.</p

Sedimentation velocity absorbance <i>c(s)</i> distributions for MTSL-labeled 17–172.

<p>Experiments were carried out using 50 μM ²H-MTSL labeled, 17-172^L661C (blue) and 17-172^C-Cys (red) in 50 mM sodium acetate, pH 4, and 31% nondeuterated glycerol in the presence of ~2-fold excess of DPC micelles. Samples were prepared similar to those used for DEER at a concentration of 50 μM with the above buffer in H₂O instead of D₂O. 17–172 construct which is mainly a monomer in 50 mM sodium acetate at pH 4 and DPC, but in the absence of glycerol, is shown as control (black, rescaled to fit c(<i>s</i>) axis).</p

Amino acid sequence, domain organization and structure representations of HIV-1 gp41.

<p>(A) Numbering of residues corresponds to their positions in the HIV-1 Env sequence. Gp41 spans residues 512 to 856 (in red) of gp160. (B) Abbreviations are as follows: FP, fusion peptide; FPPR, fusion peptide proximal region; N-HR, N-heptad repeat; IL, immune-dominant linker; C-HR, C-heptad repeat; MPER, membrane proximal external region; TM, transmembrane region; and CT, intraviral C-terminal domain, respectively. For ease of designating the various constructs used in this study, gp41 is renumbered from 1–345 (in black). The longest sequence used in this study spans residues 17 to 194 corresponding to Env numbering 528 to 705. Except for 35-144^IL, all constructs have their IL region (residues 69 through 116) replaced with the L6 spacer (SGGRGG) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a>). They are designated based on the amino acid sequence they encompass. When not substituted with the L6 spacer, the construct is designated with an IL (in superscript) following the designation. The various constructs used are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.s001" target="_blank">S1 Fig</a> (C). The 6HB spanning A533 to R580 (FPPR/N-HR) and M629-N677 (C-HR/MPER) is modeled from pdb entries 1SZT [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref014" target="_blank">14</a>] and 2X7R [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160597#pone.0160597.ref017" target="_blank">17</a>]. Terminal residues are indicated in red on the ribbon. Positions substituted with cysteines for MTSL-labeling (chemistry shown in D) are R633, L661, G527 and L684. These positions are indicated also in red on the ribbon (in C) and the sequence with a ball and stick (in A). The epitopes recognized by the antibodies 2F5, 4E10 and Z13e1 are shown in (A).</p