Analysis of Sam68-binding SH3 domains <i>in vivo</i> by FRET-analysis.

<p>Expression constructs for CFP-tagged Sam68 and YFP-tagged SH3 domains were used to co-transfect 293T cells as indicated. 48 h post transfection, cells were harvested for flow cytometric analysis. Direct protein interaction <i>in vivo</i> was assayed by determining FRET from CFP to YFP by exciting CFP at 405 nm and measuring fluorescence with filters 450/50 (CFP only) vs. 585/42 (CFP + YFP-FRET-signal). (A) Representative diagrams showing the shift of cell populations as a result of FRET. Based on the negative control (CFP, or CFP-Sam68, and YFP on separate plasmids) and the positive control (CFP-YFP-fusion protein on one plasmid) two gates were defined, enclosing cells that do not exhibit FRET (R2, red), or that do exhibit FRET (R3, green), which is manifest by a shift to the left (i.e. lower CFP emission) and simultaneously to the top (i.e. higher YFP-emission). The degree of this shift depends on the FRET-efficiency. (B) FRET signals for all domains assayed. Results are shown as mean ± standard deviation from three independent experiments.</p

Interaction of Sam68 PxxP peptide mutants with SH3 domains.

<p>W  =  wildtype, M1 to M7  =  mutants, n/a  =  not applicable.</p><p>Compilation of phage-ELISA results indicating changes in binding of the SH3 domains to the PxxP-peptide-mutants compared to the wildtype peptide (++), classified as affinity reduced (+) or interaction abolished (0).</p><p>Core residues of the PxxP motifs are underlined; introduction of alanine point mutations is highlighted by a black background.</p

Secondary structure prediction of wildtype and mutant Sam68.

<p>(A) Schematic representation of Sam68 domains and positions of proline-rich motifs. The three motifs not binding to SH3 domains are enclosed in brackets. RG  =  arginine glycine rich region, NK  =  N-terminal of KH domain, KH  =  hnRNP K homology domain, CK  =  C-terminal of KH domain, YY  =  tyrosine rich region, NLS  =  nuclear localization sequence (B) Prediction of secondary structure by JPred, white bars: helical regions, black bars: extended regions. (C) Prediction of intrinsically disordered regions by IUpred.</p

Sam68-binding SH3 domains as identified by bio-panning of recombinant Sam68 against the human SH3-proteome phage-display library.

a<p>Numbers refer to supplementary table from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540-Krkkinen1" target="_blank">[31]</a>.</p>b<p>SH3 domains already reported as Sam68-binders, compare suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540.s004" target="_blank">Table S2</a>.</p>c<p>For a complete listing see suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038540#pone.0038540.s005" target="_blank">Table S3</a>.</p

Interaction of Sam68-PxxP-mutants with SH3 domains.

<p>Expression constructs for CFP-tagged Sam68-mutants defective in either any one of the SH3-interacting PxxP-motifs (Sam68ΔP0, −ΔP3, −ΔP4, −ΔP5), or defective in all (Sam68ΔP0345), were cotransfected with YFP-tagged SH3 domains from Yes, Fyn, p85α, or OSF, or wildtype-Sam68 into 293T-cells and analyzed for direct interaction <i>in vivo</i> by performing FRET-analysis as in Fig. 2. Results are shown as mean ± standard deviation from three independent experiments. Significant reduction (p<0.05 in Student’s T-test) of the signal as compared to wildtype is marked with an asterisk.</p

Identification of Sam68-PxxP-motifs responsible for SH3-domain-binding.

<p>Peptides corresponding to the seven PxxP-motifs (P0 to P6) of Sam68 were purified as GST-fusions and analyzed for interaction with the indicated SH3 domains by phage-ELISA. Results are expressed semi-quantitatively as half-maximal binding occuring at <10¹¹ (+++), 10¹¹−10¹² (++), >10¹² cfu/ml (+).</p

Affinity analysis of 447-52D antibody to individual members of the Env/V3 library.

<p>HEK293T cells were separately transduced at low MOI (0.1) by one of the pQL9 derived lentiviral particles expressing the indicated chimeric Env/V3-variant, respectively. 48 h post transduction, cells were stained with MAb 447-52D and an APC-labeled secondary antibody. The mean values of two independent experiments corrected for equal GFP expression are shown. <b>A</b> A time-course with 447-52D antibody (10 µg/mL) was performed to record the incubation-time needed for equilibrium binding. FACS data are expressed as the MFI for every member of the model library at the different incubation times, respectively. <b>B</b> 447-52D antibody concentrations were serially diluted and incubated on infected cells at equilibrium incubation-time (1 h at 4°C) to obtain a concentration dependent binding profile. <b>C</b> Cell populations stained with 0.1 µg/mL 447-52D antibodies are depicted separately in order to highlight the differential binding at this concentration. <b>D</b> Dissociation constants (K_D) were calculated from the antibody-titration curves shown in (B) based on the µM concentrations derived from the 447-52D molecular weight and therefore expressed as Kd (µM). Data points were fitted by nonlinear least squares regression (One site; Fit total and nonspecific binding, Graphpad Prism 5), and the resulting K_d as well as R² values are listed (#, non-calculable).</p

Schematic overview of the FACS-panning procedure.

<p>HEK293T cells are transfected with a plasmid mix comprising (i) a lentiviral vector plasmid (pQL9/11) encoding the Env/V3 model library, (ii) a packaging construct (pTNpack) as well as (iii) pVSV-G to render released particles transduction competent (1). Linkage between geno- and phenotype from the initially “unlinked” virus library is achieved by low MOI infection of new cells (2). Antibodies (e.g. bnMAb) are applied to bind cells expressing the various envelopes, respectively (3). Envelope expressing cells displaying the highest MFI to the bnMAb and an internal control (GFP) are selected by a FACS-sorting procedure (4). Cells are collected and lysed prior to amplifying the envelope genes from genomic DNA (5). The recovered envelope genes are cloned into pQL9/11 and analyzed by sequencing and realtime-PCR (6). Fresh cells are transfected (together with pTN pack and pVSV-G) to produce new virus for Low MOI infection of fresh cells, thus entering a new cycle of selection.</p

Influence of the modified constructs on Gag- and Pol-specific CD8⁺ T cell immune responses.

<p>BALB/c mice (n = 6 per group) were i.m. inoculated with equimolar amounts of the indicated plasmid DNA constructs (80 µg of ^ΔMGPN, ^MGPN, ^ΔMG^FSPN, ^MG^FSPN; 57 µg of ^MGag; 68 µg of PN). After 12 days, spleen cells were isolated and tested for specific cellular immune responses by measuring IFNγ production after stimulation with Gag (black) or Pol (grey) specific peptides. IFNγ production was determined by FACS analysis after intracellular staining of IFNγ. Cell culture medium served as a negative control. (A) Influence of budding competence and the functional frameshift on Gag- and Pol-specific CD8⁺ T cell immune responses. (B) Impact of separating the Gag and PolNef reading frames on Gag- and Pol-specific CD8⁺ T cell responses. Data shown are representative of two experiments.</p

Enrichment of Env/V3-variant MN.^a

a<p>Values obtained by qPCR.</p>b<p>Calculations were made according to the formula <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#pone.0109196-Khare1" target="_blank">[72]</a>.</p><p># cycle of panning.</p><p>Enrichment of Env/V3-variant MN.^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109196#nt101" target="_blank">a</a>}</p