The gene P of TioV encodes for three proteins: V, W and P.

<p>Whereas conventional transcription and translation lead to the expression of TioV-V, co-transcriptional insertion of one G residue at the editing site by the viral RNA polymerase leads to the expression of a chimeric protein called W. Insertion of two G residues can also occur during transcription, thus leading to the expression of the phosphoprotein P.</p

Expression of IFN-inducible genes is impaired by MuV-V but not TioV-V expression.

<p>HEK-293T cells were transfected with pCI-neo-3xFLAG expression vectors encoding for 3xFLAG alone or fused to TioV-V, MuV-V, TioV-P or TioV-W (500 ng/well). 24 h after transfection, cells were left unstimulated or stimulated with 200 IU/ml of recombinant IFN-β. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. For each gene, data were normalized so that 100% corresponds to cells transfected with pCI-neo-3xFLAG empty vector and stimulated with recombinant IFN-β (dotted red line). Experiment was performed twice and data represent means ± SD. *indicates that differences observed with MuV-V relative to controls cells transfected with 3xFLAG alone and stimulated with IFN-β were statistically significant (p-value<0.05).</p

TioV-V fails to interact with _PrSTAT2.

<p>HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V or MuV-V (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged STAT2 from human (hSTAT2) or <i>Pteropus rodricensis</i> (_PrSTAT2). Total cell lysates from transfected cells were prepared 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting.</p

TioV-V fails to interact with human STAT2 and does not induce STAT1 degradation.

<p>(<b>A–B</b>) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to MuV-V, TioV-V (<b>A–B</b>) or NiV-V (<b>B</b>) (500 ng/well), and pCI-neo-3xFLAG expression vectors (300 ng/well) encoding for 3xFLAG-tagged human STAT2 (<b>A</b>) or STAT1 (<b>B</b>). Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panel). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. (<b>C</b>) HEK-293T cells were co-transfected with expression vectors encoding GST alone or fused to TioV-V, MuV-V or CHIKV-nsP4 (500 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG-tagged human STAT1 and STAT2 (150 ng/well of each vector). At 24 h post-transfection, cells were left untreated or stimulated with recombinant IFN-β at 200 IU/ml. Total cell lysates from transfected cells were prepared at 48 h post-transfection (cell lysate; middle and lower panels), and protein complexes were assayed by pull-down using glutathione-sepharose beads (GST pull-down; upper panels). 3xFLAG- and GST-tagged proteins were detected by immunoblotting. Upper and lower panels on top of figure C correspond to short and longer exposures of the same blot, respectively. (<b>D</b>) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V, TioV-P or TioV-W. Total cell lysates were prepared at 48 h post-transfection and endogenous STAT1 expression levels were determined by western-blot analysis. Actin expression was determined and used as a protein extraction and loading control. (<b>E</b>) HEK-293T cells were transfected with pCI-neo-3xFLAG expression vector (1 µg/well) either empty or encoding for 3xFLAG-tagged TioV-V, MuV-V or CHIKV-nsP4. Total cell lysates were prepared at 48 h post-transfection, and 3xFLAG-tagged viral proteins were purified using anti-FLAG antibodies conjugated to sepharose beads. Co-immunopurification of endogenous STAT2 with 3xFLAG-tagged viral proteins was determined by western-blot analysis (top and middle panel, respectively). Actin expression was determined prior to the immunoprecipitation on total cell lysates and used as a protein extraction control (lower panel).</p

TioV-V does not inhibit STAT1 nuclear translocation induced by IFN-β.

<p>Vero cells were transfected with 100 ng of each plasmid encoding Cherry alone or fused to TioV-V, MuV-V or NiV-V. After 48 h of culture, cells were stimulated with IFN-β for 30 min, and STAT1 was labeled by immunostaining to determine its subcellular localization pattern. Green color corresponds to STAT1 whereas red corresponds to Cherry alone or Cherry-tagged viral proteins. Data show representative fields for each culture condition, and white arrows indicate cells expressing Cherry or Cherry-tagged viral proteins. Scale bar  = 10 µm.</p

TioV-V inhibits IFN-β promoter activation by MDA5 and IL-6 signaling, but not IFN-α/β signaling.

<p>(<b>A</b>) HEK-293T cells were co-transfected with reporter plasmid pISRE-Luc (300 ng/well), pRL-CMV reference plasmid (30 ng/well), and pCI-neo-3xFLAG expression vectors encoding for 3xFLAG alone or fused to MuV-V or TioV-V (300 ng/well). After 24 h, recombinant IFN-β was added at 200 IU/ml. After an additional 24 h, relative luciferase activity was determined. (<b>B</b>) HEK-293T cells were co-transfected with reporter plasmid pSTAT3-Luc (300 ng/well), pRL-CMV (30 ng/well), and expression vectors encoding 3xFLAG-tagged MuV-V or TioV-V (300 ng/well). At 24 h post-transfection, recombinant IL-6 was added at 10 ng/ml. After an additional 24 h, relative luciferase activity was determined. (<b>C</b>) HEK-293T cells were co-transfected with IFN-β-pGL3 reporter plasmid (300 ng/well), pRL-CMV (30 ng/well), expression vectors encoding 3xFLAG-tagged MDA5 (300 ng/well) and MuV-V or TioV-V (300 ng/well). After 48 h, relative luciferase activity was determined. (<b>D</b>) Experiment was performed as in (A), but cells were co-transfected with expression vectors encoding 3xFLAG-tagged TioV-P or TioV-W. (<b>E</b>) Experiment was performed as in (A), but cells were co-transfected with pCI-neo expression vector, either empty or encoding untagged MuV-V, TioV-V, TioV-P or TioV-W. (<b>F</b>) Experiment was performed as in (A), but cells were stimulated with recombinant IFN-λ1 at 50 ng/ml. All experiments were performed in triplicates, and data represent means ± SD. *indicates that differences observed relative to controls (none) with IFN-β, IL-6, MDA5 or IFN-λ were statistically significant (p-value<0.01).</p

Activation of ISRE-dependent gene expression by MeV or TioV infection.

<p>HEK-293 cells stably transfected with an ISRE-luciferase reporter gene (STING-37 reporter cell line) were infected with MeV or TioV. (<b>A</b>) Bright field microscopy of cell cultures at 48 h post-infection (MOI  = 1). (<b>B</b>) Luciferase expression was determined at 24 h and 48 h post-infection. (<b>C</b>) Culture supernatants from (B) were collected at 48 h post-infection, clarified by centrifugation, UV-inactivated and added to culture wells containing STING-37 cells. Alternatively, culture medium was supplemented with increasing doses of recombinant IFN-β. After 24 h, luciferase expression was determined. Luciferase activity in culture supernatants from (B) was below 1,400 luciferase activity units (data not shown). Experiment was performed in triplicates, and data represent means ± SD. *indicates that differences observed between MeV and TioV-infected cells were statistically significant (p-value <0.01).</p

Inhibition of pyrimidine biosynthesis accounts for the amplified cellular response to ssRNA.

<p>(<b>A</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. Culture medium was supplemented or not with uridine. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. (<b>B</b>) Cells were transfected with 20 ng/well of ssRNA in 12-well plates, and incubated in the presence of DD264 (80 µM) or DMSO alone. Culture medium was supplemented or not with uridine. After 24 hours, expression levels of IFI6, IFI27, IFIT1 and IFI35 were determined by qRT-PCR. Data were normalized relative to control housekeeping genes (GAPDH, HPRT1, and 18S). Experiment was performed in duplicate, and data represent means ± SD. (<b>C</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected in 96-well plates with 50 ng of an expression vector encoding for DHODH (using two independent plasmid preparations of the same construct, #1 and #2). DHODH overexpression was assessed by western-blot analysis (upper right panel). Alternatively, cells were transfected with expression vectors encoding for control proteins (CT1 and CT2, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003678#s4" target="_blank">Materials and Methods</a> for details). After 48 hours, cells were transfected with 6 ng/well of ssRNA, and incubated in the presence of DD264 (80 µM) or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD. (<b>D</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of ssRNA, and incubated in the presence of brequinar (200 nM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD.</p

DD264 is a broad-spectrum antiviral molecule.

<p>(<b>A</b>) HEK-293T cells were infected with a recombinant strain of MV expressing EGFP (MOI = 0.1), and incubated for 48 hours in the presence of DD264 at 40 µM or DMSO alone. Scale bar = 200 µm. (<b>B</b>) HEK-293T cells were infected with a recombinant strain of MV expressing luciferase (MOI = 0.1), and incubated with increasing doses of DD264 or DMSO alone. After 24 hours, luciferase expression was determined. (<b>C</b>) HEK-293T cells were infected with CHIKV (MOI = 0.1), and incubated with DMSO alone or DD264 at 40 µM. After 24 hours, cells were fixed, and CHIKV E2 glycoprotein was detected by immunostaining. Cell nuclei were stained with DAPI. Scale bar = 200 µm. (<b>D</b>) HEK-293T cells were infected with a recombinant strain of CHIKV expressing <i>Renilla</i> luciferase (MOI = 0.2), and incubated with increasing doses of DD264 or DMSO alone. After 24 hours, <i>Renilla</i> luciferase expression was determined. Experiments in (B) and (D) were performed in triplicate, and data represent means ± SD. (<b>E</b>) HEK-293T cells were infected with WNV (MOI = 1), and incubated with DMSO alone or DD264 at 40 µM. After 24 hours, cells were fixed and WNV E glycoprotein was detected by immunostaining. Cell nuclei were stained with DAPI. Scale bar = 200 µm. (<b>F</b>) HEK-293T cells were infected with WNV (MOI = 10), washed, and incubated with increasing doses of DD264 or matching volumes of DMSO alone. After 24 hours, supernatants were recovered, clarified by centrifugation and titrated. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003678#s2" target="_blank">Results</a> are expressed as log₁₀ PFU (plaque-forming units) per ml. Experiment was performed twice, and data represent means ± SD. (<b>A–F</b>) In all experiments, DD264 (or control DMSO) was added to cell cultures at the time or few minutes after infection.</p

DD264 amplifies cellular response to transfection of PAMP-like ssRNA molecules or IFN-β stimulation, and this correlates with its antiviral activity.

<p>(<b>A</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of synthetic 5′-triphosphate RNA molecules (ssRNA), and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well cultures plates. After 24 hours, luciferase expression was determined. (<b>B</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with increasing doses of recombinant IFN-β, and incubated in the presence of DD264 (80 µM) or DMSO alone in 96-well culture plates. After 24 hours, luciferase expression was determined. Experiments (A) and (B) were performed in duplicate, and data represent means ± SD. (<b>C</b>) Eight molecules were randomly picked among analogs of DD264 (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003678#ppat.1003678.s012" target="_blank">Table S2</a>) to build a set of compounds with a range of antiviral activities from null to high as determined by their potency to inhibit MV-Luc replication. Then, the selected molecules were tested for the amplification ISRE-luciferase expression when stimulating cells with suboptimal doses of ssRNA (6 ng/well) as described in (A). Finally, for DD264 and selected analogs, the capacity to amplify cellular response to ssRNA was plotted as a function of the antiviral activity by means of the experimental IC₅₀ values (antiviral activity = 1/IC₅₀*100).</p