FVB WT mice were obtained from Charles River Laboratories (France). GADD34 DC/DC FVB mice were obtained from L. Wrabetz (Milan). Mice were anesthetized and inoculated via the intradermal route with 10 6 PFU of CHIKV-21 isolate [72] . Viral titers in tissues and serum were determined as described before [59] , and expressed as tissue cytopathic infectious dose 50 (TCID50)/g or TCID50/ml, respectively. Organs including heart, liver, skeletal muscles and spleen were collected for histopathological procedures. organs were then fixed in 4% paraformalde-hyde solution, paraffin-embedded, sectioned coronally in 5-10 mm thickness and stained with hematoxylin-eosin. Figure S1 Poly I:C stimulation induces protein translation inhibition and IFN-b production in NIH3T3 cells. A) Protein synthesis was quantified in poly I:C-stimulated NIH3T3 using puromycin labeling followed by immunoblot with antipuromycin mAb 12D10. Protein synthesis was strongly reduced upon poly I:C stimulation. Immunoblot for phosphorylated (P-eIF2a) and total eIF2a were performed on the same NIH3T3 extract. Cycloheximide (chx) was added 5 min before puromycin incorporation. b-actin immunoblot is shown for equal loading control. B) Puromycin integration was analysed by immunofluorescence in NIH3T3 cells treated for 4 h with poly I:C and labeled with puromycin in the last 10 min. Figure S2 Protein translation in cells with non-phosphorylatable eIF2a. Protein synthesis was quantified in MEFs with non-phosphorylatable eIF2a, eIF2aA/A and the corresponding control cells, eIF2aS/S. After poly I:C or thapsigargin treatment, puromycin labeling followed by immunoblot, was performed. Puromycin labeling was quantified with ImageJ software and protein translation was depicted as percentage of steady state. Cycloheximide (chx) was added 5 min before puromycin incorporation. Tubulin immunoblot is shown for equal loading control. One of two independent experiments with similar results is shown. (TIF) Figure S3 GADD34 mRNA induction in ATF4-deficient MEFs stimulated with cytosolic poly I:C. The levels of GADD34 transcript were determined by qPCR in WT and ATF4 2/2 cells after 8 h of poly I:C stimulation. Treatment with tunicamycin and thapsigargin were used as positive controls for GADD34 induction. Results are displayed according to both WT internal reference (left) and ATF4 2/2 internal reference (right). (TIF) Figure S4 GADD34 mediates eIF2a dephosphorylation in MEFs stimulated with poly I:C. A) Wild-type and GADD34 DC/DC MEFs were treated for the indicated times with poly I:C (pI:C), tunicamycin (tun) or thapsigargin (th) and eIF2a phosphorylation was monitored by immunoblot. B) GADD34 expression was analyzed by immunoblot in samples treated for 1 or 6 hours with poly I:C alone or together with tunicamycin (tun) or thapsigargin (th). Data shown in (A) and (B) are representative of three independent experiments with similar results. (TIF) Figure S5 RNA integrity upon poly I:C exposure. WT MEFs were treated with poly I:C for the indicated times and RNA integrity evaluated by capillary electrophoresis (Agilent RNA 6000). RNA Integrity Numbers (RIN) between 8.2 and 9.2 were obtained, indicating a high level of RNA integrity. Data shown are representative of three independent experiments with similar results. (TIF) Figure S6 UPR-inducing drugs do not elicit IFN-b production. Cell culture supernatants of murine embryonic fibroblasts were tested for the presence of IFN-b, after treatment with poly I:C (8 h), tunicamycin and thapsigargin (6 h). The results shown are representative of 4 experiments. (TIF) Figure S7 Deletion of the constitutively-expressed PP1 co-factor, CReP, does not impact protein translation and IFN-b production in MEFs. A) WT and CReP 2/2 MEFs were treated with poly I:C (pI:C) for the indicated times and the levels of P-eIF2a and PKR were analyzed by immunoblot. Although basal levels of P-eIF2a were higher in CReP 2/2 MEFs, increase of phosphorylation upon poly I:C exposure was similar to the WT. PKR expression upon poly I:C treatment was equivalent in CReP 2/2 and WT MEFs. B) Protein synthesis was quantified using puromycin labeling followed by immunoblot with the antipuromycin mAb 12D10. Where indicated, cells were treated with cycloheximide (chx) 5 min before puromycin incorporation. No major differences were found between WT and CReP 2/2 cells at the level of translation inhibition following poly I:C exposure.  Targets and Intracellular Signaling Mechanisms for Deoxynivalenol-Induced Ribosomal RNA Cleavage The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3. The trichothecenes, a group of sesquiterpenoid mycotoxins produced by Fusarium that contaminate wheat, barley, and corn globally, are problematic because of their resistance to degradation during processing and their potential to adversely affect human and animal health (Pestka, 2010) . Among the over 200 trichothecenes discovered to date, deoxynivalenol (DON) is most frequently encountered in food, and human exposure to this toxin throughout the world has been well documented in a recent series of elegant biomarker studies (Hepworth et al., 2012; Turner et al., 2010a Turner et al., ,b, 2011 . A potential target for adverse health effects for DON is the innate immune system, with low doses of the toxin having immunostimulatory effects and high doses causing immunosuppression (Pestka, 2010) .