Nef-treated microglial cells release iNOS-dependent neurotoxic factors.

<p>(<b>A</b>) BV-2 cells were treated for 48 h with myr⁺Nef_SF2 (200 ng/ml) in the presence (light gray bars) or absence (dark gray bars) of L-NAME at 10 μM concentration. Supernatants were collected and NO₂^- content was measured using the Griess colorimetric assay. (<b>B</b>) iNOS expression was evaluated on total cell lysates by Western Blot using specific anti-iNOS antibodies (upper panel) and anti-β-tubulin antibodies as an internal loading control (lower panel). (<b>C</b>) NB41A3 cells were incubated for 24 h with supernatants from (<b>A</b>). (<b>D</b>) NB41A3 cells were treated for 24 h with myr⁺Nef_SF2 (200 ng/ml) or with LPS (100 ng/ml). Black line: No L-Name treatment; Gray line: L-Name (10 μM). (<b>E</b>) NB41A3 cells were incubated for 24h with conditioned supernatants derived from untreated BV-2 cells or treated for 48 h with 200 ng/ml of wild type (WT), G2A, 4EA’, 4EA” and ΔN-terminal deleted myr⁺Nef_SF2 proteins. In (<b>C)</b> to (<b>E)</b>, cell viability was measured by flow cytometry using Propidium Iodide (PI) dye exclusion and data were expressed as percentage of PI positive cells. One out of three independent experiments is shown.</p

Nef-dependent iNOS induction requires NF-κB.

<p>(<b>A</b>) BV-2 cells were pretreated for 1 h with BMS-345541 (5 μM) and then incubated for 2, 4 and 6 h with myr⁺Nef_SF2 (100 ng/ml) in presence of BMS-345541. Total mRNAs were isolated and analyzed for iNOS mRNA expression as described in the materials and methods section. Empty circles: untreated cells (Ctr); gray empty squares: BMS-345541; filled circles: recNef_SF2; gray filled squares: BMS-345541 plus recNef_SF2. (<b>B</b>) Cells were stimulated as in (<b>A</b>) for 4, 6 and 8 h, then lysed and total cell lysates analyzed by Western Blot for iNOS expression. β-Tubulin was used as an internal loading control. (<b>C</b>) BV-2 cells were pre-treated with BMS-345541 (1 or 2.5 μM), then stimulated for 24 h with the indicated amount of myristoylated recNef_SF2 in presence of BMS-345541. Supernatants were collected and NO₂^- content evaluated. Black bars: no inhibitor; dark gray bars: BMS-345541 at 1 μM concentration; light gray bars: BMS-345541 at 2.5 μM concentration. Ctr: control cells. Data shown were from one of three independent experiments.</p

Nef synergizes with IFNβ in iNOS production.

<p>(<b>A</b>) BV-2 cells were treated for 6 h with myr⁺Nef_SF2 (100 ng/ml), IFNβ (200 IU/ml) or a combination of both. iNOS mRNA was measured by real time RT-PCR as reported in the materials and methods section. (<b>B</b>) Cells were incubated for 24 h with the indicated dose of myr⁺Nef_SF2 with or without IFNβ (50 IU/ml). Cells were also incubated with heat-inactivated recNef_SF2 (inactiv. recNef, 500 ng/ml) or, as control, with LPS or pre-heated LPS (500 ng/ml each). Total cellular lysates were analyzed by Western Blot for iNOS expression. β-Tubulin expression was used as an internal loading control. (<b>C</b>) Cells were treated like in (<b>B</b>) and NO₂^- content in supernatants was measured using the Griess colorimetric assay. Empty diamond: IFNβ; filled circles: myr⁺Nef_SF2; gray empty circles: heat-inactivated myr⁺Nef_SF2; filled squares: recNef_SF2 plus IFNβ; gray empty squares: heat-inactivated myr⁺Nef_SF2 plus IFNβ.</p

Nef treatment of microglial cells induces iNOS expression.

<p>BV-2 cells (<b>A</b>) or purified primary murine microglial cells (<b>B</b>) were treated for the indicated time with myr⁺Nef_SF2 (100 ng/ml in <b>A</b>, 200 ng/ml in <b>B</b>, closed circles). Total cellular RNAs were isolated and real time RT-PCR analysis was performed as reported in the materials and methods section. Results were expressed using the 2^-ΔΔCT method using basal mRNA level in untreated cells (Ctr, open circles) at T = 0 as a calibrator and GAPDH level as an internal loading control. (<b>C</b>, <b>D</b>) BV-2 cells were treated for 24 h with the indicated amounts of myr⁺Nef_SF2, heat inactivated myr⁺Nef_SF2 (Inactiv. recNef), LPS or heat treated LPS. Total cell lysates were analyzed by Western Blot for iNOS (<b>C</b>, upper panel) levels and, as internal loading control, β-Tubulin expression (<b>C</b>, lower panel). (<b>D</b>) NO₂^- content in the supernatants was quantified using the Griess colorimetric assay as reported in the materials and methods section. Dark gray bars: native myr⁺Nef or LPS. Light gray bars: heat pre-treated (Inactiv.) myr⁺Nef or heat pre-treated LPS. Results from one of five independent experiments are shown. (<b>E</b>) Cell phenotyping by flow cytometry of human monocytes and M1 macrophages obtained as described in materials and methods. According to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130189#pone.0130189.ref077" target="_blank">77</a>], human monocytes were CD14⁺/CD163⁺/CD86⁺ whereas M1 macrophages were CD14^-/CD163^-/CD86^bright. (<b>F</b>) M1 human macrophages were left untreated or treated for 24, 48 and 72 h with IFNγ, 100 ng/ml wild type myr⁺Nef_SF2 plus IFNγ, heat pre-treated myr⁺Nef_SF2 and IFNγ or 100 ng/ml LPS plus IFNγ. Total cellular extracts were analyzed by Western Blot to evaluate iNOS expression using specific antibodies as described in materials and methods. β-tubulin expression as internal loading control. Blots are representative of two independent experiments.</p

Nef-induced IFNβ production concurs to Nef-dependent iNOS induction.

<p>(<b>A</b>) BV-2 cells were treated for the indicated time with myr⁺Nef_SF2 (100 ng/ml) in the presence of anti-IFNβ neutralizing antibodies as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130189#pone.0130189.ref036" target="_blank">36</a>]. iNOS mRNA was analyzed as reported in materials and methods section. Dark gray bars: 4 h treatment; light gray bars: 8 h treatment. (<b>B</b>) Cells were treated as in (<b>A</b>) for the indicated time and iNOS expression evaluated by Western Blot. β-Tubulin expression was used as an internal loading control. (<b>C</b>) Cells were treated as in (<b>A</b>) for 24 h, supernatants were then collected and NO₂^- content measured using the Griess colorimetric assay.</p

Nef myristoylation and the acidic cluster are essential to induce iNOS.

<p>BV-2 cells were left untreated or incubated with 100 ng/ml wild type (WT), G2A, two different preparations of the EEEE→AAAA mutant (4EA’ and 4EA”) and ΔN-terminal deleted myr⁺Nef_SF2 proteins. (<b>A</b>) iNOS mRNA was measured by real time RT-PCR using total RNA isolated from cells treated for 6 h. (<b>B</b>) Total cellular extracts obtained from cells treated for 24h were analyzed by Western Blot using anti-iNOS antibodies, whereas β-tubulin expression levels were used as an internal loading control. (<b>C</b>) Supernatants collected from cells treated as in (<b>B</b>) were measured for NO₂^- content using the Griess colorimetric assay. One out of three independent experiments is shown.</p

Nef treatment induces STATs tyrosine phosphorylation, I-B degradation and IRF-1 expression in BV-2 microglial cells.

<p>(<b>A</b>) BV-2 cells were left untreated or incubated with 100 ng/ml wild type myristoylated Nef derived from HIV-1 SF2 strain (myr⁺Nef_SF2) for different times (left panel) or for 5 h with different amounts of myr⁺Nef_SF2 (right panel). Total cellular extracts were analyzed by Western Blot to evaluate STAT-1, -2 and -3 tyrosine phosphorylation or protein expression using specific antibodies as described in materials and methods. (<b>B</b>, <b>C)</b> Cells were incubated for the indicated time with myr⁺Nef_SF2 (100 ng/ml) or, as a positive control, with LPS (500 ng/ml) for 30’ in (<b>B</b>) and 6 h in (<b>C</b>). Whole cell lysates were analyzed by Western Blot for I-κB-α (<b>B</b>) or IRF-1 (<b>C</b>) expression. β-tubulin expression was used as an internal loading control. Results reported in the figure are from four independent experiments.</p

Graphic summary of the ability of each compound to activate HIV within each cell model: (A) primary CD4 T cell models and patient cell outgrowth assay (QVOA), and (B) J-Lat T cell line clones.

<p>Each compound and concentration tested is listed on the X-axis. In the primary CD4 cell models, each compound was tested using cells from 2, 3 or 4 different donors and in duplicate or triplicate with cells from each donor (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003834#s4" target="_blank">Methods</a> Section for details). For the QVOA, results from the limiting dilution cultures from 3 patients were pooled to calculate one common IUPM (infectious units per million cells) value which was then normalized to that obtained with PHA. With the J-Lat clones, experiments were performed in triplicate. Asterisks represent “not done”.</p

List of stimuli used in this study and their corresponding signaling pathways.

<p>List of stimuli used in this study and their corresponding signaling pathways.</p

Heatmap visualization of the ability of each compound to activate HIV within each model when excluding (A) and including (B) data from the QVOA model.

<p>A reduced set of compounds was analyzed in (<b>B</b>) since not every compound was run at every concentration in the QVOA. The clustergram at the left of each heatmap reflects the relationships between compounds based on their ability to activate HIV across compounds. Since cells in all models responded to PHA with high strength, ranking was normalized within each model to the response to PHA at 10 µg/mL and, therefore, all models display in the heatmap the same relative responsiveness to this treatment. The clustergram at the top of each heatmap reflects the relationship between each model based on their response to compounds. Clustergrams were created by calculating Euclidean distances and then clustering distances using the average linkage method. The numbers at the nodes of clusters are AU p-values where 95% represents a <i>p</i>-value cut-off of 0.05 and only values 95% or greater are depicted. Red cells in the heatmaps reflect HIV activation whereas blue or blank cells indicate that the compound did not effectively activate HIV.</p