Figure 1.
Putative complexes formed by Nef Acidic Cluster with TRAF2 and TRAF6.
Upper panels: sequence alignment between Nef AC and TRAF2 (A) or TRAF6 (B) binding sites in known TRAF interactors. The conserved residues within the consensus sequence are in black background whereas the residues present in Nef AC as well as in TRAF2 and TRAF6 interactors are in gray background. Mismatched Ala in Nef compared to TRAF6 consensus in (B) is in gray. TRAFs interactors sequences are as in [22], [23]. Lower panels: schematic representation of the modelled complexes formed by Nef AC with TRAF2 (A) and TRAF6 (B). Nef AC-TRAF2 complex has been modelled using the three-dimensional structure of the TRAF2/4-1BB complex (PDB code: 1D0J) [22] as a template. Nef AC backbone is shown in green, TRAF2 backbone in gold. Nef AC-TRAF6 complex has been modelled using the three-dimensional structure of the TRAF6/RANK complex (PDB code 1LB5) [23]. Nef AC backbone is shown in green, TRAF6 backbone in magenta.
Figure 2.
Acidic Cluster Nef mutant characterization.
(A) Circular dichroism spectroscopy analysis of myr+ wild type recNef protein (black line) and myr+ 4EA recNef mutant (red line) revealed similar secondary structure content from both proteins. (B) GST-HCKSH3 pull down assays with recombinant Nef protein showed binding to both wild type Nef and the 4EA mutant in a comparable manner (lanes 7–9), supporting the structural integrity of the protein. For input control, recNef proteins were similarly incubated alone (lanes 1–3) or in presence of GST beads (lanes 4–6). (C, D) Wild type Nef and 4EA mutants are internalized by MDMs. Cells were treated with 300 ng/ml of the corresponding recombinant proteins labelled with AlexaFluor488 green dye. For CM (C), MDMs were cultured on glass bottom dishes and treated for 30′ wt or 4EA myr+ recNefSF2-Alexa488, then counterstained with PKH26 red fluorescent dye, fixed and analysed. Left panels: merged images of stained cells. Right panels: Differential Interference Contrast (DIC) images. (D) For CFM analysis cells were treated for the indicated times with wild type (left panel) or 4EA (right panel) recNef proteins. Gray plot: untreated cells; sky blue: 30′: light blue: 2 h; blue: 6 h; dark blue 24 h.
Figure 3.
Nef induces NF-κB signalling in an Acidic Cluster dependent manner.
Seven days old human primary MDMs were treated for 30′ with 100 ng/ml of myr+ wild type (WT) recNef, the protein lacking the first 44 aminoacids (ΔN-Term), mutated in the myristoylation site (G2A) or myr+ recNef 4EA mutant. Total cell protein extracts (50 µg) were analyzed by Western Blot as reported in Materials and Methods section using specific anti phospho-IKKα/IKKβ and anti IKKα/IKKβ (A) or with anti IκB-α (B) antibodies. In both (A) and (B) β-tubulin steady-steate expression level was used as an internal loading control. Results shown represent one out three different donors.
Figure 4.
Dose-response analysis of IL-6 and TNFα mRNA.
Primary human MDMs were treated for 2 h with the indicted doses of myr+ wild type recNef (dark line, empty circles) or myr+ 4EA recNef mutant (gray line, empty squares). Cells were processed as reported in the Materials and Methods section. Upper panel: IL-6 mRNA levels; lower panel: TNFα mRNA levels. Results in figure and in Table I represent two out of four independent experiments.
Table 1.
RT-PCR analysis of cytokines and chemokines induced in MDMs following recNef treatment.
Figure 5.
Nef-dependent activation of IRF-3 and synthesis of IFNβ requires the integrity of the Acidic Cluster.
(A) MDMs were treated with myr+ wt as well as with ΔN-Term, G2A or myr+ 4EA recNef (100 ng/ml for 30′). Total cell protein extracts (30 µg) were analyzed by Western Blot using anti-IRF3 specific antibodies. IRF-3 activation was visualized as accumulation of the slower migrating form of the protein corresponding to the iperphosphorylated form and the decrease of the faster migrating band. β-tubulin steady-steate expression level was used as an internal loading control. (B) MDMs were treated for 2 h with myr+ wt as well as with ΔN-Term, G2A or myr+ 4EA recNef (100 ng/ml, 2 h). Cell were processed, total RNA was isolated and Real Time PCR was performed as described in Material and Methods section. Results are expressed as fold of induction using the levels of IFNβ mRNA expression in untreated cells as reference. (C) Supernatants collected from MDMs treated for 2 h with twofold dilution of myr+ wt (black line, empty circles), 4EA (gray line, empty squares) or 100 ng/ml of heat-inactivated myr+ wt recNef (black triangle), were tested for the induction of the antiviral state on A549 indicator cells. Results are expressed as International Units (IU) of IFN/ ml. Results were obtained from four independent healthy donors.
Figure 6.
Tyrosine phosphorylation of STAT1, STAT2 and STAT3 requires the Nef Acidic Cluster.
MDMs were treated for 2 h with 100 ng/ml of myr+ wt as well as with ΔN-Term, G2A or myr+ 4EA recNef. Total cell protein extracts (50 µg) were analyzed by Western Blot using specific anti phosphotyrosine-STAT1, anti phosphotyrosine-STAT2 and anti phosphotyrosine-STAT3 antibodies. Steady-state expression levels of STAT1, STAT2 and STAT3 were evaluated using corresponding specific antibodies whereas β-tubulin steady-steate expression level was used as an internal loading control. Results were obtained from four independent healthy donors.
Figure 7.
Nef interacts in vitro with TRAF2.
Recombinant GST-Nef proteins (WT or 4EA mutants) were incubated with THP-1 total cellular lysates. Pull-down assay was performed as reported in Materials and Methods section and samples were analyzed on 9% SDS-PAGE. Western Blot was performed using anti-TRAF2 (Upper panel) or anti-Hck (Middle panel) specific antibodies. As gel loading control, samples were analyzed on SDS-PAGE and proteins were detected by Ponceau S staining (Lower panel). Band intensities were quantified using ImageJ v1.32J and data were normalizing using Hck signals as calibrator. Mean intensity as well as standard deviation was calculated using data collected from three independent experiments and statistical analysis performed using InStat3 and ANOVA test.
Figure 8.
Both TRAF2 and TRAF6 silencing interfere with Nef-induced STAT1 and STAT2 tyrosine phosphorylation.
THP-1 cells were transfected with specific TRAF2, TRAF6 or unrelated (i.e. for HPV16 E7) siRNA as described in Material and Methods section. Cells were then incubated with recNef (200 ng/ml) for 6 h. Total cell protein extracts (50 µg) were analyzed by 7% SDS-PAGE and Western Blot was performed using specific anti phosphotyrosine-STAT1 and anti phosphotyrosine-STAT2 antibodies. STAT1 and STAT2 steady-state expression levels were checked using the corresponding specific antibodies. Anti-TRAF2 and anti-TRAF6 antibodies were used to verify the specificity of silencing, ruling out off-target effects. β-tubulin steady-state expression level was used as an internal loading control. Results reported in figure represent one out of three independent silencing experiments.
Table 2.
Oligonucleotides used to perform RT-PCR.