Comparison of pDC IFNα production triggered by mature versus immature DENV particles.

<p>(<b>A–E</b>) The impact of mutations in prM (substitutions R88A, K90A and R91A) on the levels of intracellular DENV RNA (<b>A</b>), extracellular DENV RNA (<b>B</b>) infectious virus production <b>(C</b>), transmission of E GP from cells harboring the WT and mutant DENV genomes to pDCs (<b>D</b>) and quantification of IFNα secretion by pDCs (<b>E</b>) analyzed as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004434#ppat-1004434-g006" target="_blank">Figure 6</a>. Results of 3 independent experiments in triplicates are expressed as percentages relative to the WT DENV genome (error bars represent means ± SD). (<b>F–I</b>) The impact of a furin inhibitor, at the indicated concentrations, on the prM∶E ratios in extracellular viral supernatants detected by ELISA (<b>F</b>), levels of intracellular DENV RNA (<b>G</b>), extracellular DENV RNA and infectious virus production (<b>H</b>), and quantification of IFNα secretion by pDCs triggered by co-cultured with DENV infected cells or incubation with TLR7 agonist (R848 at 50 ng/mL) (<b>I</b>). Results are expressed relative to the untreated cells (-), set at 100 (error bars represent the means ± SD, 3 independent experiments in triplicates). (<b>J–M</b>) The impact of furin overexpression, or not (-), in 293T cells infected by DENV at different MOIs, as indicated. The panels are displayed as in (<b>F–I</b>), with extracellular prM∶E ratios (<b>J</b>), intracellular DENV RNA (<b>K</b>), extracellular DENV RNA and infectious virus production (<b>L</b>), and quantification of IFNα secretion by co-cultured pDCs (<b>M</b>). Results are expressed relative to 293T cells infected at a MOI of 1 in absence of Furin overexpression (indicated as 293T-1), set to 100 (error bars represent the means ± SD, 3 independent experiments in triplicates). Arrows indicate results below the detection limit of assay as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004434#ppat-1004434-g006" target="_blank">Figure 6</a>. NA; Not Applicable.</p

Time course analysis of pDC IFNα production triggered by DENV infected cells.

<p>(<b>A</b>) Quantification of IFNα in the supernatants of PBMCs and pDCs isolated from two different donors (#1 and #2, as indicated) and co-cultured with DENV infected Huh7.5.1 cells (DENV cells) or treated with 100 µl of supernatant from the latter cells (DENV SN, viral titers ≈3×10⁶ and 1×10⁶ ffu/ml for experiments with Donor #1 and #2, respectively) for the indicated time of incubation. Uninfected Huh-7.5.1 cells are referred to as (cont) cells. Error bars represent the means ± SD, n = 3. Arrows indicate results below the limit of detection of the IFNα ELISA assay (<i>i.e.</i> 12.5 pg/ml). (<b>B</b>) Quantification of IFNα-positive cells at the indicated incubation times of PBMCs or pDCs isolated from two different donors (#1 and #2, as indicated) and co-cultured with DENV infected Huh7.5.1 cells (DENV cells) or treated with 100 µl of supernatant from the latter cells (DENV SN, viral titers of ≈3×10⁶ ffu/ml). The pDC surface marker CD123 and intracellular IFNα staining were assessed by FACS analysis. Results are expressed as the percentages of IFNα positive cells in CD123 positive- and negative-populations.</p

Viral functions required for pDC IFNα production triggered by DENV infected cells.

<p>The impact of mutations in NS5 (Rep^−/−, substitutions G81A, G83A and G85A), capsid (ΔV51–54, deletion V51-to-L54) and E (substitutions D215A, H244A or P217A) on the levels of intracellular DENV RNA (<b>A</b>), infectious virus production (<b>B</b>), extracellular DENV RNA (<b>C</b>) analyzed at the indicated times post-transfection of WT and mutant DENV genomes and quantification of IFNα secretion by pDCs co-cultured with cells harboring the WT and mutant DENV genomes (<b>D</b>), control cells (-). GE; genome equivalent. In D, cells harvested at 24 hours post-transfection were co-cultured with pDCs for 20 hours. Results of 3 independent experiments in triplicates are expressed as percentages relative to the WT DENV genome. Error bars represent the means ± SD, paired Student's t test, *p<0.05, **p<0.01. Triangles indicate the mutants (<i>i.e.</i>, Rep−/− and ΔV51–54) that belong to a separated group, statistically different from the WT and the other mutants of the panel in regards to the intracellular DENV GE levels at 48 hours post-transfection using a non-parametrical analysis (ANOVA). Arrows indicate results below the limit of detection of RT-qPCR: 20 intracellular DENV GE/µg total RNA and 30 extracellular DENV GE/ml; infectious titers: 20 ffu/ml and IFNα ELISA: 12.5 pg/ml. (<b>E</b>) DENV E GPs are transferred from cells harboring the WT and mutant DENV genomes to pDCs in co-cultures for 8 hours. Results are expressed as the percentages of DiI stained pDCs that contain E GP dot(s) detected by immunostaining and analyzed by confocal microscopy. Representative pictures are displayed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004434#ppat.1004434.s008" target="_blank">Figure S8</a>. Similar results were obtained in 3 independent experiments and ≈20 pDCs surrounded by E GP positive/DiI negative cells were observed per experimental condition.</p

Accumulation of actin network and clustering of DENV surface protein at the cell contacts.

<p>(<b>A–E</b>) Imaging of the actin network in co-cultures of pDCs and DENV infected Huh7.5.1 cells (8 hour-incubation). (<b>A</b>) Confocal microscopy analysis of actin stained with CF488A-conjugated phalloidin (green), IFNα detected by immunostaining (purple) and nuclei stained with Hoechst (blue). (<b>B–E</b>) Magnification of yellow-boxed pDC/DENV infected cell contact, shown in (A). (<b>B</b>) 3D reconstitution. (<b>C</b>) Confocal microscopy analysis of actin projected on the phase contrast imaging. (<b>D</b>) Z-axis projection corresponding to the blue line, shown in (A). (<b>E</b>) Color-coded image to represent fluorescence intensity with gradient indicating the intensity from weak (blue) to strong signal (white). Similar results were obtained in 3 independent experiments. Star mark (*): pDCs and hash mark (#): Huh7.5.1 cells. (<b>F–K</b>) Imaging of DENV E glycoprotein (E GP) clustering at cell contacts between pDC and DENV infected cells (co-cultures for 8 hours). (<b>F</b>) Confocal microscopy analysis of with actin (green), pDCs stained with DiI membrane dye (red), E GP (purple, immunodetection without permeabilization step) and nuclei (blue). (<b>G</b>) Confocal microscopy analysis of E GP-nuclei projected on the phase contrast imaging. (<b>H–K</b>) Magnification of yellow-boxed cell contacts, shown in (F). (<b>H</b>) Actin-E GP detection. (<b>I</b>) E GP projected on phase contrast. (<b>J</b>) Z-axis projection corresponding to the blue line in (F). (<b>K</b>) 3D reconstitution. Similar results were obtained in 3 independent experiments. (<b>L–Q</b>) Imaging of DENV prM clustering at cell contacts between pDC and DENV infected cell in co-culture for 8 hours. (<b>L</b>) Confocal microscopy analysis of prM (green, immunodetection without permeabilization step), DiI-stained pDC (red) and nuclei (blue). (<b>M</b>) Confocal microscopy analysis of prM-nuclei projected on the phase contrast imaging. (<b>N–Q</b>) Magnification of yellow-boxed cell contacts, shown in (L). (<b>O</b>) prM staining projected on phase contrast. (<b>P</b>) Z-axis projection corresponding to the blue line in (L). (<b>Q</b>) 3D reconstitution. Results are representative of 3 independent experiments.</p

pDC IFNα secretion is triggered by DENV infected primary cells and is not DENV-restricted.

<p>(<b>A–B</b>) Quantification of IFNα in supernatants of pDCs co-cultured with infected monocyte-derived macrophages (mo-M, DENV) (<b>A</b>) or with infected monocyte-derived dendritic cells (mo-DCs, DENV) (<b>B</b>) or treated with their supernatant (SN). Error bars represent the means ± SD. Results are representative of 2 independent experiments. Insets, time course analysis of intracellular DENV genome equivalent (GE) levels in mo-M (<b>A</b>) and mo-DC (<b>B</b>) post-infection with a MOI of 3. Results are expressed as DENV GE/µg total RNA (means ± SD, n = 3). (<b>C</b>) Quantification of IFNα in supernatants of pDCs co-cultured with infected cells (INF cells) or treated with 100 µl of supernatant from the latter cells (INF SN). Cont cells, uninfected parental cells. Arrows indicate results below the limit of detection of the IFNα ELISA (<i>i.e.</i>, 12.5 pg/ml). Error bars represent the means ± SD (n, independent experiments, in duplicate: Huh7.5.1 cells/DENV, n = 8; BHK-21 cells/DENV, n = 3; Hela cells/DENV, n = 2; 293T cells/DENV, n = 5; Vero cells/DENV, n = 2; 293T cells/WNV, n = 5). Because of the limited numbers of isolated pDCs, each experiment comprises a part of the panel of cell lines, thus DENV infected Huh7.5.1 cells and their supernatants were included in each independent experiment (n = 8) and served as a reference to compare and collate results of independent experiments. Parallel determination of intracellular DENV RNA levels, expressed as DENV GE/µg total RNA (<b>D</b>) and infectious virus production, expressed as ffu/ml (<b>E</b>).</p

Recognition and pDC antiviral signaling triggered by DENV infected cells.

<p>(<b>A</b>) DENV RNA is transferred from infected cells to pDCs. Upper panels, projections of confocal microscopy analysis of DENV RNA (green) detected by FISH in DENV infected Huh-7.5.1 cells (DENV cells/no pDC) and in pDCs after 5 hours of co-culture with uninfected cells (cont cells/pDCs) or with DENV infected cells (DENV cells/pDCs), pDC DiI dye (red), IFNα protein (white) and nuclei (blue). Lower panels, consecutive Z-axis sections and 3D reconstitutions with magnification of the yellow-boxed pDC. Cell contours on the DENV RNA panels labeled with dotted lines surrounding DiI staining. Yellow arrows; DENV RNA dots inside pDC, *; pDC and #; Huh7.5.1 cells. Summary table, percentages of IFNα-positive- or negative-pDCs (green colored lines) that contain DENV RNA after co-culture with DENV cells, control cells or DENV cells with FISH procedure performed in absence of DENV probe (no probe), as controls. Similar results were obtained in 3 independent experiments, # cells; numbers of cells included in the analysis. (<b>B</b>) Quantification of IFNα in the supernatants of pDCs that were preincubated, or not, with TLR7 antagonist (IRS661, 0.35 µM), as indicated, then co-cultured with DENV infected Huh7.5.1 cells (DENV cells) or stimulated by TLR7 agonist (R848, 50 ng/mL) or TLR9 agonist (ODN2216, 0.1 µM) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004434#ppat.1004434-Kawai1" target="_blank">[1]</a>. Results are expressed as the percentages of IFNα produced in the absence of TLR7 antagonist (means ± SD, n = 4, paired Student's t test: *, p<0.01). (<b>C</b>) Quantification of TNFα and IL6 in supernatants of pDCs co-cultured with DENV cells, treated with DENV SN (viral titers ≈2×10⁶ ffu/ml) or TLR7 agonist, set-up as in (B) (means ± SD, n = 2 for IL6, n = 3 for TNFα). Arrows indicate results below the limit of detection (TNFα and IL6, 8 and 25 pg/ml, respectively). (<b>D</b>) Left panel, quantification of surface CD83 and CD86 on CD123-gated cells by FACS analysis at the indicated times, set-up as in (B), Right panel, parallel quantification of supernatant IFNα (means ± SD, n = 3).</p

pDCs robustly produce IFNα in response to DENV infected cells.

<p>(<b>A</b>) Quantification of IFNα in the supernatants of PBMCs, pDC-depleted PBMCs and isolated pDCs (Responders) co-cultured with DENV infected Huh7.5.1 cells (DENV cells) or treated with 100 µl of supernatants from the latter (DENV SN), as indicated (Inducers). Viral titers of DENV SN ≈2.5×10⁶ foci forming units (ffu)/ml. pDC depletion/enrichment was performed using an anti-BDCA-4 antibody. Cell #; number of co-cultured responder cells. Cont cells; uninfected Huh-7.5.1 cells. Arrows indicate results below the detection threshold of the IFNα ELISA (<i>i.e.</i>, 12.5 pg/ml). Results are representative of 4 independent experiments. Error bars represent the means ± SD. (<b>B</b>) Representative FACS analysis of pDC depletion and isolation from PBMCs using the pDC selective markers, CD123 and BDCA-2. (<b>C</b>) Quantification of IL6 in the supernatants of PBMCs and pDC-depleted PBMCs triggered by LPS (10 µg/mL for 20 hours). Results are expressed as percentage relative to LPS-treated PBMCs. 3 independent experiments in triplicate (error bars, means ± SD), paired Student's t test, ***p<0.0001, NS p>0.1, Δ; indicates separated group by ANOVA. (<b>D</b>) Total PBMC population containing a number of pDCs equivalent to the purified pDCs as determined by FACS analysis as described in (B) were co-cultured with DENV infected cells. IFNα productions were thus compared for equal numbers of pDCs. PBMCs and pDC-depleted PBMCs were compared using equal cell numbers. The results are expressed as IFNα levels relative to the co-culture with PBMCs, set at 100, 3 independent experiments in triplicate, paired Student's t test, ***p<0.0001, NS p>0.05, Δ; indicates separated group by ANOVA (error bars, means ± SD). (<b>E</b>) Quantification of IFNα secretion by pDCs isolated from the blood of a healthy donor cohort (n = 20) co-cultured with infected cells or treated with their supernatant. IFNα levels in the supernatants of pDCs co-cultured with uninfected (cont) cells or with DENV SN were all below the detection threshold (<i>i.e.</i>, 12.5 pg/ml).</p

IFITM3 is a <i>bona fide</i> virion-associated protein.

<p>Virion particles produced as described above were then analyzed by immuno-gold electron microscopy. Briefly, unfixed viral preparations purified by ultracentrifugation and produced in the presence or absence of IFITM3 were incubated with anti-Flag antibodies, followed by incubation with a gold-conjugated secondary antibody (arrows). Representative pictures are shown here. The graph displays the number of gold particles counted on a per virion basis.</p

CD45 depletion excludes a potentially confounding role of exosome-incorporated IFITMs on virion infectivity.

<p>SupT1 cells stably expressing the different IFITMs were infected with HIV-1 and VSV. At a late time after infection of the cell culture, supernatants containing newly-produced virions were harvested and divided in two fractions that were either incubated with CD45-conjugated microbeads or left untreated. After the microbeads removal, virion particles were purified by ultracentrifugation, normalized and then used for WB and infectivity analyses. The WB panels present typical results obtained, while the graph presents averages and SEM obtained in 3 independent experiments. No statistically significant differences were observed between depleted and non-depleted fractions, after a Student t test.</p

CCR5 usage relieves the negative effects of IFITM3 on HIV-1 replication and on its ability to decrease the virion particles infectivity.

<p>A) Human CCR5 was introduced in the IFITM3-stable SupT1 cells used before, by retroviral-mediated gene transduction and cells were challenged with the indicated viruses. HIV spreading was assessed by exo-RT activity over time (day 0 through 7). The panels and the histogram overlay present the patterns of expression obtained for IFITM3 and CCR5 following WB and flow cytometry analyses. The graph presents normalized data obtained in 2 to 3 independent experiments. B) Virions obtained at late times after infection were harvested, normalized and used to infect HeLaP5 cells that contain a β-galactosidase reporter gene under the control of the HIV-1 LTR.</p