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 activates the ISRE-luciferase reporter gene.

<p>(<b>A</b>) Bar chart showing results for one 96-well screening plate. ISRE-luciferase reporter gene induction is expressed as a fold change compared to control wells treated with DMSO alone. Each bar represents one compound and the red bar corresponds to DD264 (induction factor = 6.9). Compounds from this representative plate were from Institut Curie library and were screened at 20 µg/ml, thus corresponding to a final concentration of 81.1 µM for DD264. (<b>B</b>) Chemical structure of DD264. (<b>C</b>) HEK-293 cells that express luciferase under control of five interferon-stimulated response elements (ISRE) were incubated with increasing doses of DD264 or DMSO alone. After 24 hours, luciferase expression was determined. (<b>D</b>) Same experiment as in (C), but cells were stimulated with increasing doses of recombinant IFN-β. Except for (A) that corresponds to one representative experiment, all experiments were performed in triplicate, and data represent means ± SD.</p

IRF1 is required for the expression of ISGs.

<p>(<b>A</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against <i>IRF1</i> and cultured for 48 hours in 96-well culture plates. Then, cells were transfected with increasing doses 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 triplicate, and data represent means ± SD. (<b>B</b>) HEK-293 cells with the ISRE-luciferase reporter gene (STING-37 cells) were transfected with control siRNA (CT) or siRNA directed against <i>IRF1</i> and cultured for 48 hours in 12-well culture plates. Cells were transfected with ssRNA (20 ng/well), and incubated in the presence of DD264 (40 µM) or DMSO alone. After 24 h of culture, total RNAs were extracted, and expression levels of indicated genes were quantified by qRT-PCR. <i>GAPDH</i>, <i>HPRT1</i> and <i>18S</i> correspond to control housekeeping genes, whereas others are well-known ISGs. Data were normalized using cells stimulated with ssRNA alone as a reference (red bars).</p

DD264 is an inhibitor of pyrimidine biosynthesis pathway.

<p>(<b>A</b>) Inhibition of uridine and cytidine in DD264-treated cells. HEK-293T cells were treated with DMSO alone or increasing concentrations of DD264. After 24 hours, cells were harvested and nucleoside concentrations were determined by HPLC. Concentration is expressed as a percentage relative to DMSO-treated cells. (<b>B–E</b>) HEK-293T cells were infected with recombinant MV strain expressing luciferase (MOI = 0.1), and incubated with DD264 (40 µM), mycophenolic acid (15 µM) or DMSO alone, and culture medium was supplemented with uridine (<b>B</b>), guanosine (<b>C</b>), orotate (<b>D</b>) or dihydroorotate (<b>E</b>). After 24 hours, luciferase expression was determined. Experiment was performed in duplicate, and data represent means ± SD.</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

Antiviral activity of DD264 and brequinar is abrogated when silencing IRF1 expression in HEK-293T cells.

<p>(<b>A–B</b>) HEK-293T cells were transfected with control siRNA (solid colors) or siRNA directed against <i>IRF1</i> (shaded colors) and cultured for 48 hours. Then, cells were infected with recombinant MV strain expressing luciferase (MOI = 0.1) or CHIKV strain expressing luciferase (MOI = 0.2), and incubated with increasing concentrations of DD264 or DMSO alone. After 24 hours, luciferase expression was determined. Experiment was performed in triplicate, and data represent means ± SD. (<b>C–D</b>) Same experiment was performed but cells were treated with increasing concentrations of brequinar instead of DD264. * corresponds to p-values<0.05.</p