Radar graphs illustrating the data on the global cross-HIV-1-strain conservation neutralization epitopes targeted by mAbs.

<p>The mAb is indicated in black bold letters above its graph. Each radar graph is a polygon, each point of which is representative of each subtype and each axis of which is proportional to the fraction of that subtype in the whole set of circulating worldwide strains. Thus the point labeled “C” is subtype C and its axis from the center of the polygon is as long as all the other axes combined, because subtype C represents half (50%) of the worldwide population of viruses. The percentage of each subtype that contains the neutralization epitope targeted by the mAb is plotted on each axis. Connecting each one of these plotted points traces out an inner polygon colored red that is visually representative of the population of circulating viruses that contain the sequence motif for the neutralization epitope targeted by that mAb. Thus, if most of the area enclosed by the polygon is colored red, most of the circulating HIV-1 viruses in the human population worldwide contain the epitope targeted by the indicated mAb in their sequences.</p

Sequence Motifs for Epitopes Targeted by Individual anti-V3 mAbs Derived from 3D Structures of mAbs:V3 Complexes.

<p>Infecting Donor HIV Subtype indicates the subtype of the virus infecting the patient from which the mAb was isolated. V3 loop numbering is as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015994#s2" target="_blank">Methods</a>. “x” in the sequence motifs indicates that any amino acid may occupy that position.</p

Fraction of HIV-1 Pseudoviruses of five subtypes Neutralized by anti-V3 mABs.

<p>Summary of neutralization breadth of individual mAbs from previously published experiments. The column headings for each of the subtypes displays the subtype name and the number of HIV-1 pseudoviruses of that subtype tested for neutralization by the indicated anti-V3 mAbs. Each cell shows the percentage of tested HIV-1 pseudoviruses which are neutralized by the indicated mAb of that row. The <i>in vitro</i> neutralization data from Hioe et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015994#pone.0015994-Hioe1" target="_blank">[35]</a> was summarized in this table.</p

mAb 3074, 2557, and 268-D bound to V3 loop peptides.

<p>(A) Structure of mAb 3074 (grey molecular surface) bound to a peptide (red ribbon and blue stick depiction) with the same sequence as the crown of the V3 loop of subtype AG isolate VI191. (B) Structure of mAb 2557 (grey molecular surface) bound to a peptide (red ribbon and blue stick depiction) with the same sequence as the crown of the V3 loop of subtype B isolate NY5. (C) Structure of mAb 268-D (grey molecular surface) bound to a peptide (red ribbon and blue stick depiction) with the same sequence as the crown of the V3 loop of subtype B isolate MN. The side-chains of the V3 peptide that are buried in the molecular surface of the mAb are colored blue and labeled.</p

Fraction of Circulating HIV Strains Worldwide That Contain the Sequence Motifs for anti-V3 mAb Epitopes.

<p>The column headings for each of the subtypes displays the subtype name and, in parentheses, the percentage of the whole set of worldwide circulating viruses that belong to that subtype. Each cell shows the percentage of worldwide viruses, all of that subtype, that contain the sequence motif targeted by the indicated mAb of that row: thus “11%” for 3074 and subtype A indicates that 11% of the 12% of worldwide viruses that are subtype A contain the 3074 targeted epitope. Each cell in the Total column indicates the percentage of worldwide circulating viruses that contain the signature motif of the epitope targeted by the indicated mAb of that row.</p

Results of the <i>in silico</i> modeling of amino acid substitutions in V3 loop epitopes.

<p>For each mAb epitope resolved crystallographically, <i>in silico</i> substitutions were made to the structure of the complex, the perturbation of the change was minimized conformationally and the change in energy of the complex was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015994#s2" target="_blank">Methods</a> (right column). Only amino acid substitutions occurring naturally in the viral population in more than 4% at each position were considered in the study. Red colored values indicate energetically silent or non-disruptive substitutions that retained a similar mAb contact and which were then added to the motif describing the epitope. *One change resulted in energetic improvement but a conformational change such that contact with the mAb was lost, thus not contributing to the motif.</p

HIV’s Nef Interacts with β-Catenin of the Wnt Signaling Pathway in HEK293 Cells

<div><p>The Wnt signaling pathway is implicated in major physiologic cellular functions, such as proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Proliferation and migration are important responses of T-cells, which are major cellular targets of HIV infection. Using an informatics screen, we identified a previously unsuspected interaction between HIV’s Nef protein and β-catenin, a key component of the Wnt pathway. A segment in Nef contains identical amino acids at key positions and structurally mimics the β-catenin binding sites on endogenous β-catenin ligands. The interaction between Nef and β-catenin was confirmed <i>in vitro</i> and in a co-immunoprecipitation from HEK293 cells. Moreover, the introduction of Nef into HEK293 cells specifically inhibited a Wnt pathway reporter. </p> </div

Nef inhibits Tcf –luciferase reporter activity.

<p>A. Promoter negative control: HEK293 cells were transfected with the TopFlash or the FopFlash reporters and with 50 ng Nef encoding plasmid (WT-Nef, the indicated mutants or empty vector) and incubated with Wnt-conditioned media (Wnt-CM) or control media that contain no Wnt. The TopFlash plasmid has TCF binding sites that are mutated in the FopFlash plasmid. The sites in FopFlash do not interact with TCF and therefore FopFlash serves as a negative control. Renilla luciferase was used for normalization purposes. Data shown here represent two experiments, each done in triplicate. WT-Nef significantly (P<0.00001, t-test) inhibits TCF reporter activity as compared to empty vector and also compared to the β-catenin motif mutants, D186A-Nef and F191A-Nef (P<0.00001, t-test) when co-transfected with TopFlash. B. The firefly/renilla values from panel A of this figure were used to calculate the TopFlash/FopFlash ratios of each condition. The resultant TopFlash/FopFlash ratios were then used to calculate the percent inhibition by WT-Nef and the indicated mutants on transcription in cells stimulated with Wnt.</p

Nef interacts with β-catenin.

<p>A. Interaction of Nef and β-catenin <i>in </i><i>vitro</i>. Purified recombinant WT-GST-Nef (or indicated D186A/F191A mutants), His- β-catenin and nickel beads mixed in physiological buffer, washed and eluted (See Methods). Top panel: immunoblot using mouse anti- β -catenin antibody (Ab) for detection. Middle panel: immunoblot using mouse anti-GST Ab for detection of Nef. Bottom panel: immunoblot using mouse anti-GST Ab for detection of Nef in total <i>E. coli</i> expression extract (input). This experiment was repeated three times and one representative immunoblot is shown here. B. Interaction of Nef and endogenous β-catenin in cells. HEK293 cells expressing wild-type (WT), the indicated mutants of Nef or empty vector (EV) by transfection are lysed and co-immunoprecipitated using a mouse anti- β-catenin Ab. Uppermost panel: immunoblot of immunoprecipitate (IP) using mouse anti- β-catenin Ab for detection. 2^nd to top panel: immunoblot of IP using anti-TCF4 Ab for detection. 3^rd to top panel: immunoblot of IP using mouse anti-Nef Ab for detection. Lowest panel: immunoblot of the total cell lysate prior to IP (input) using mouse anti-Nef Ab showing level of Nef (or mutant Nef) expression. This experiment was repeated three times and one representative immunoblot is shown here. </p

β-catenin ligands and the identification of Nef as a novel ligand.

<p>A. Upper panel: superimposition of known ligands of β-catenin as bound to β-catenin. β-catenin is shown in white, TCF3 is shown in green, ICAT is shown in yellow, LEF is shown in magenta, E-cadherin is shown in black, APC20 mer is shown in orange and APC15mer is shown in blue. Lower panel: same as the upper panel, however, β-catenin is not displayed. The region where the ligands converge to the same conformation is marked with a black line and titled “structural convergence” in the diagram. The two aspartates of TCF3 at the margins of the convergence are shown in x-stick representation. This region was used as the structural alignment of the ligands for the purpose of deriving the motif illustrated in panel B. B. Flow chart of the steps to identify Nef as a candidate β-catenin ligand. The chart begins with a structurally determined multiple alignment that was used to derive the β-catenin binding pattern motif. The most important residues for binding to β-catenin are colored in red. In grey, residues that were excluded from the motif as they don’t occupy the same position in 3D bound to the receptor (arginine from ICAT and lysine from APC 15 mer). Next, a Prosite-style sequence pattern was constructed to reflect the 3D structural pattern of compatibility of each residue at each specific location in the β-catenin ligand. For example, position 4 in the central region exhibits an isoleucine in some ligands and a leucine or methionine in others in contact with a hydrophobic patch on β-catenin. Thus, any of these three side chains may occupy this position and this portion of the pattern was defined as “[ILM]” to reflect this characteristic. The derived pattern then served as an input for the “MyHits” website that identified the above motif within the Nef, nucleocapsid and the MGF proteins.</p