Binding of IFNα2 and its mutants to the IFNAR1 and IFNAR2 receptor subunits.

<p>(A) Ribbon representation (based on Protein Data Bank #3SE3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.ref029" target="_blank">29</a>] of the ternary complex of IFNAR1 (R1), IFNAR2 (R2) and IFN, including the locations of the point mutations of the antagonists. On the right is a representation of the antagonist, which binds tighter to R2, but does not bind R1. (B) SPR sensograms of different IFNs (analyte) binding IFNAR2 (surface bound ligand) measured on a ProteOnXPR36. (C) IFNAR1-binding equilibrium analysis of the antagonists and IFNα2. The measurements were done using the ProteOn XPR36 Protein Interaction Array System (BioRad) with IFNAR1 and IFNAR2 immobilized on the sensor chip. For equilibrium binding analysis towards IFNAR1, six different concentrations of the interferon proteins were administrated, and the data were fitted using the mass action equation. (D) <i>In situ</i> measurements of the interferon antagonists towards the IFNAR receptor subunits. The 50% competition values for the four antagonists are ~1 nM, in line with their binding affinities to IFNAR2. 1000-fold lower than the EC₅₀ value determined for YNS. All extracted data from panels C-E are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.t001" target="_blank">Table 1</a>.</p

Gene expression in response to antagonist treatment.

<p>Gene expression in OVCAR3, WISH and T47D cells in response to antagonists, YNS and IFNα2. (A) Cells were treated for 8 hours with 200 nM antagonists or 1nM YNS, and analyzed by qPCR. The data presented are the relative expression levels compared to those of untreated cells, normalized against HPRT1. (B) As described in (A), but cells were treated for 24 hours. (C) Cells were treated with a combination of 200 nM antagonist and 200 pM IFMα2. Gene induction was analyzed as described in (A). Error bars represent standard deviation of the data.</p

STAT1 and STAT2 activation by the IFN antagonists.

<p>WISH cells were treated with either 200 nM antagonist or 1 nM YNS for 30 minutes and analyzed for levels of STAT1 and STAT2 phosphorylation by western blot assay using specific antibodies in comparison to total protein levels. Total and pSTAT2 levels upon 1 nM of YNS or IFNα2 are shown in the middle panel. pSTAT1 levels for these two proteins were previously shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.ref030" target="_blank">30</a>]. The blot was quantified using the Image Studio Lite software. The results were normalized using 1nM YNS as 100% phosphorylation and the non-treated as 0% (lower panel). The error bars are SE from three independent experiments.</p

Antiviral activity of IFN antagonists in WISH and OVCAR3 cells.

<p>(A) WISH cells were treated with the antagonists, YNS, IFNα2 or left untreated for four hours. VSV virus was added for 18 hours, which after cell survival was monitored by crystal violet staining. (B) OVCAR3 cells were treated with the antagonists, YNS, IFNα2 or left untreated for four hours. EMCV virus was added for 23 hours, which after cell survival was monitored by crystal violet staining. The experiments were performed 3 times and the fitting results (for the amplitude) are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.t001" target="_blank">Table 1</a>. (C) Reduction in antiviral activity upon combined treatment of WISH cells with 200 nM antagonists combined with 0.0001–100 pM IFNα2.</p

Antagonist activity in mice.

<p>Antagonist activity in mice was determined by their ability to induce or inhibit IFN-induced gene production 3 hours after injection. (A) WT C57bl/6 mice were injected with 1 μg of human (Hu) or mouse (m) IFNβ or 50 μg of each of the antagonists (1ant stands for IFN-1ant and the other antagonists are on top of the 1ant mutations). qPCR of the liver cDNA was performed to evaluate the gene induction levels of three robust (MX1, OAS2 and IFIT1) and two tunable (CXCL10 and CXCL11) genes. (B) WT C57bl/6 mice were injected I.P. with mIFNβ alone or in combination together with 50 μg of each of the antagonist. (C) HyBNAR mice were injected with 50 μg IFN-1ant, 1 μg HuIFNβ or a combination of the two. (D) Statistical analysis of the results in panel B (for genes that are differentially regulated by IFNβ versus IFN-1ant—marked in red) using Anova and Tukey post hoc test. Bars show mean difference between the treatments and error bars show 0.05 confidence intervals between the different treatments. The confidence interval for IFNβ vs combination is 0.005.</p

Binding constants and antiviral response of wild-type IFNα2 and its mutants.

<p><i>k</i>_a, <i>k</i>_d, and <i>K</i>_D values were determined using the XPR36 from measurements of six different protein concentrations as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.g001" target="_blank">Fig 1</a>, with the extracellular domain of IFNAR2 being the ligand. The fraction amplitude (Amp) of antiviral response (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.g002" target="_blank">Fig 2</a>) and the errors (in parenthesis) were calculated from the IFN AV dose-response curves. EC₅₀ values are calculated from <i>In situ</i> measurements of ¹²⁵I labelled IFNs binding to the cell surface. Standard Error for <i>k</i>_a values is 25%, for <i>k</i>_d is 15% and for <i>K</i>_D is 30%.</p><p>Binding constants and antiviral response of wild-type IFNα2 and its mutants.</p

Antiproliferative activities of the type 1 IFN antagonists.

<p>Cell survival was monitored 72 hours post IFN treatment by crystal violet staining. The fraction cell density is relative to untreated cells, with the line in panels A and B indicating cell survival upon 1nM YNS treatment. (A) OVCAR3 cells, (B) WISH cells. (C) Comparing cell viability as determined by crystal violet versus the colorimetric XTT assay, using 1nM YNS and 10nM IFNα2. (D) Reduction in cell count upon treatment of WISH cells with 2, 20 or 200 nM IFNα2 together with 200 or 500 nM antagonists for 72 hours. The grey line indicates cell survival upon 10 nM IFNα2 treatment. The antiproliferative activity of each IFN alone is presented at the right side of the chart (Control). The error bars are SE from 3 independent experiments.</p

–ΔC_T and ΔΔC_T of robust and tunable genes.

<p>Comparing –ΔC_T (A) to ΔΔC_T (B) values of IFN induced tunable (upper) and robust (lower) genes. The cells were treated with 200 nM antagonists, 1nM YNS or not treated (0). The data were analyzed with the NetWalker analysis tool [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130797#pone.0130797.ref031" target="_blank">31</a>].</p

B16F1-HyBNAR mouse melanoma cells are sensitive to human IFN-Is.

<p>(A & B) Mouse B16F1 melanoma cells were stably transfected with either the HyBNAR double transgenic construct, or a control GFP vector and tested for their anti-proliferative dose response to Hu-IFNα2, Hu-IFN-YNS and for comparison, mouse IFNβ. (A) HyBNAR transfected B16F1 cells were responsive to both human and mouse IFN-Is (EC50 values shown in parentheses) (B) In the GFP-transfected B16F1 control cells however, the human ligands only promoted a minor loss in proliferation and when administered at very high concentrations. (C) B16F1-HyBNAR and non-transfected control cells were incubated with an increasing dose of both human and mouse IFNβ. After 45 minutes incubation the cells were lysed and analyzed by Western Blot detection for phosphorylated STAT1. Total STAT1 measurements are shown as control. (D). B16F1-HyBNAR cells were treated for the indicated time-points with 100 pM human and mouse IFN-Is. Measurements of the IFN-response gene MX2 were performed by qPCR analysis. Relative fold-change was determined in comparison to untreated cells and normalized using the reference gene HPRT1. Error bars represent standard error of duplicate measurements.</p

Time and injection regiment affects response to IFN-Is.

<p>(A). Response of different injection regiments to 0.2 ug (weight adjusted per 20 g mouse weight) of human IFN<b>β</b>. Injections were by intraperitoneal (IP), intra venous (IV) or by sub-cutaneous (SC) route. (B). Mice were injected IP with the indicated increasing doses of YNSα8 and luminosity was measured in a time-course from live animal measurements. MX2-LUC mice doubly transgenic for HyBNAR (solid lines) or without presence of HyBNAR (dotted lines) were both measured. The relative luminosity values given are averaged from three animals (A) or two animals (B) per injection group and were measured from a defined region of maximal signal (found in the liver region) for each mouse as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084259#pone.0084259.s002" target="_blank">Figure S2</a>. In both experiments, baseline luminosity measurements were determined from the mice taken immediately prior to IFN induction.</p