Growth of codon shuffled RVFV variants.

<p>(A) Schematic presentation of the viruses with shuffled or codon-optimized genes. (B) Part of the shuffled M segment and codon-optimized N gene sequence. (C) Growth curve of the indicated viruses in Vero cells infected at MOI 0.01. Supernatants were harvested at different time points and titrated on Vero cells.</p

Colocalization coefficient of vRNAs in RVFV infected cells.

<p>Vero cells were infected at MOI 0.1 with RVFV and cells were fixed at 5 hpi. Cells were subsequently probed against (A) the S segment (N gene, green) using fluorescein labelled probes, the M segment (Gc gene, red) using quasar 670 labelled probes and the GAPDH mRNA using quasar 570 labelled (blue) probes or against (B) the S segment (N gene, green) using fluorescein labelled probes, the M segment (Gc gene, red) using quasar 670 labelled probes and the M-segment using quasar 570 labelled (Gn gene, blue) probes or against the (C) S segment (N gene, green) using fluorescein labelled probes, against the M segment (Gn gene, red) using quasar 670 labelled probes and against the L-segment (polymerase gene, blue) using quasar 570 labelled probes. Cell nuclei were visualized with dapi (cyan). Images were taken using a wide-field microscope. The level of colocalization is determined by calculation of the Pearson’s colocalization coefficient. Bars represent means and SDs of 4 independent measurements.</p

Single molecule vRNA FISH of NSR infected cells.

<p>(A) Schematic presentation of the experimental setup. (B) Maximal titers and SDs of wild-type RVFV and NSR stocks. (C) Spatio-temporal distribution of genome segments in NSR infected cells. Vero cells were infected at MOI 0.1 with NSR and cells were fixed at 2,4,6,8 and 10 hpi. Cells were subsequently probed against the S segment (N gene, red) using quasar 670 labelled probes and against the L-segment (polymerase gene, green) using quasar 570 labelled probes. Cell nuclei were visualized with dapi (blue). Images were taken using a wide-field microscope. Magnified images of the squared regions are shown at the right of each panel. The merge images show the spatial relationship between all the different channels.</p

Single molecule vRNA FISH of multiple RVFV infected cells.

<p>RVFV infected Vero cells (MOI 0.1) were fixed at 7 hpi. Cells were subsequently probed against the S segment (N gene) using fluorescein labelled probes (green), against the M segment (Gn and Gc gene) using quasar 670 labelled probes (red) and against the L-segment (polymerase gene) using quasar 570 labelled probes (blue). Cell nuclei were visualized with dapi (cyan). The picture shows that the molar ratios of different vRNAs vary among cells. Most likely the presented cells were infected with either a particle containing a single copy of each genome segment, a particle lacking the M-segment, and a particle with an additional M-segment, respectively. In <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005800#ppat.1005800.s002" target="_blank">S2 Fig</a>, additional images are presented.</p

Genome segment composition of immobilized virions.

<p>(A) Schematic presentation of the experimental setup. (B) Control experiment to validate the visualization of immobilized RVFV virions and their genome segments. Immobilized virions were incubated in the presence (left image) or absence (right image) of the 4-39-cc mAb targeting the Gn glycoprotein followed by incubation with a DyLight 350 labelled conjugate. (C) Validation of the ability to determine segment colocalization inside immobilized virions. Immobilized RVFV virions were hybridized with a quasar 570 labelled Gn gene-specific probe set (green) and a quasar 670 labelled Gc gene-specific probe set (red). Since the Gn and Gc coding regions are both present on the M-genome segment, Gn and Gc gene-specific spots should show a high level of colocalization (in yellow). Colocalization percentages were on average 80% (D) Immobilized RVFV virions were hybridized with S segment specific probe sets (N gene, fluorescein, green), M segment specific probes sets (Gn and Gc, quasar 670, red) and L segment specific probe sets (polymerase, quasar 570, yellow) and incubated with the 4-39-cc Gn specific mAb in combination with the DyLight 350 labelled conjugate (blue). In each channel, spots were subsequently detected with the ComDet plugin of ImageJ and merged images of the four different channels are presented. (E) Quantification of the different genome compositions inside virions. About 800 virions were analysed for their genome content using the ComDet plugin of ImageJ.</p

Visualization of GAPDH, CD80 and CD83 mRNAs in infected cells using FISH.

<p>DCs were stimulated for 24 h with LPS, infected with NSR or left untreated and then fixed and subjected to FISH. Shown are (A) representative cells of each treatment condition from three independently performed experiments with cells from three different donors and (B) average ±SD spot counts of cells probed for GAPDH, CD80 and CD83. Relevant statistical significance is indicated with an asterisk.</p

Infection of DCs by NSR.

<p>(A) DCs were infected with NSR for 24 h and evaluated for expression of GFP, using an EVOS fluorescence microscope. (B) Infection efficiency under optimal conditions as determined by flow cytometry. (C) Viability and percentage of infected cells at different time points after infection. Cells were infected with NSR or mock-infected with NSRmock, harvested at the indicated time points, stained with 7AAD and analysed by flow cytometry. The percentage of GFP expressing cells (bars) and the viability after NSR or NSRmock infections (lines) is depicted. Viability of the cells was calculated relative to the viability at 8 hpi, which was set at 100%. The data depict average values from two experiments with cells from two different donors ±SD. (D) Morphology of DCs stimulated with the indicated stimuli at 24 h post treatment.</p

Cytokine secretion by NSR-infected DCs.

<p>Supernatants of infected or control-treated DCs were harvested at 24 hpi and analysed with a luminex-based cytokine assay. Bars represent the mean cytokine concentrations ± SD of triplicates with cells from one donor. Statistical significance between infected (NSR) and mock-infected (NSRmock) conditions is indicated.</p

Expression of CD83 after inhibition of cellular protein degradation routes.

<p>(A) Flow cytometry analysis of CD83 surface expression after inhibition of the proteasome. DCs were stimulated with NSRmock, LPS+NSRmock or LPS+NSR for 8 h and then clasto Lactacystin β-lactone (CLBL) was added at two concentrations, as indicated. Control cells were left untreated or were treated with DMSO. Cells were analysed at 24 hpi for CD83 expression. Bars represent average MFI ±SD from two experiments with cells from one donor (B) Detection of total amounts of CD83 in cell lysates by Western blot. Cells were stimulated as described under point “A” and treatments are indicated above the image. (C) Inhibition of endocytosis. DCs were stimulated with LPS+NSRmock, LPS+NSR or left unstimulated. Cytochalasin D (Cyt D) or the solvent DMSO were subsequently added at different time points. The moments of adding Cyt D/DMSO and harvesting of cells are indicated above each graph. Bars represent average fold change of the MFI relative to unstimulated cells treated with DMSO ±SD. Average values of three experiments with cells from one donor are depicted. Relevant statistical significances are shown.</p

Effect of NSR infection on intracellular and extracellular CD83 levels.

<p>(A) The levels of soluble CD83 in supernatants from cells harvested 24 h after stimulation with LPS, infection with NSR, or from cells mock infected with NSRmock were determined by ELISA. Bars represent average CD83 concentrations ±SD. Results from one of two independently performed experiments with similar results are shown. (B) Detection of CD83 in cell lysates by Western blot at 24 hpi. The different treatments are shown above the top panel and the probed proteins are depicted at the right. The positions of molecular weight standard proteins are shown at the left. The top blot was stripped and re-probed with antibodies against GAPDH and GFP, which served as loading control and control to confirm NSR infection, respectively. Results from one of two independent experiments with cells from two donors are shown.</p