Similar FSE and BSE histopathological data in the TgOvPrP4 mice.

<p>A. Similar FSE and BSE lesion profiles and PrP^d mapping, observed in the brain of Tg(OvPrP4) mice (n = 5 to 6) infected with either cattle BSE or cheetah FSE at second passage. The nine gray matter sites of the lesion profiles were: 1. dorsal medulla nuclei, 2. cerebellar cortex, 3. superior colliculus, 4. hypothalamus, 5. central thalamus, 6. hippocampus, 7. lateral septal nuclei, 8. cerebral cortex at the level of thalamus, 9. cerebral cortex at the level of septal nuclei. The red color stands for the schematic representation of PrP^d within the 4 brain levels analyzed. The red stars symbolize the florid plaque type of PrP^d deposition. B.Typical florid plaques similarly detected in FSE and BSE transmission studies. Neuropathology of Tg(OvPrP4) mice inoculated with FSE agent from cheetah (case 1) (B1) or with BSE agent from cattle (B2 and B3) revealed the presence of typical florid plaques at first and second passage. Amyloid florid plaques sometimes formed clusters as in the cortex (B1 and B2: PrP^d immunohistochemistry, B3 and insert: Red Congo staining). In these structures, the diameter of the florid plaques ranged from 30 to 100 µm.</p

Transmission of FSE and BSE prions from natural host to Tg(OvPrP4) mice: survival data.

<p>Transmission of FSE and BSE prions from natural host to Tg(OvPrP4) mice: survival data.</p

Similar PrP^d distribution and type of deposition in nervous and lymphoid tissues.

<p>On the left, the purple and red areas on the schematic coronal brain sections designate regions where PrP^d was observed after immunohistochemistry using SAF84 and 3F4 mAb, in case 1 (A, C, E) compared to case 2 (B, D, F). On the right, illustrations of representative PrP^d type of deposition: PrP^d was seen in thalamic nuclei (H and I), in the hippocampus - although moderately (J and K), in the mesencephalon, e.g. in the periaqueductal gray matter or the substantia nigra (L and M). The deep cerebellar nuclei and the cerebellum within the molecular and granular layer also revealed strong PrP^d depositions (N and O). In both FSE cases, the most caudal brain regions were the most severely affected by PrP^d accumulation as illustrated by the level of the obex region with the example of the nucleus of the tractus solitarius (P and Q). PrP^d was apparently not associated with the vascular elements. In the lymphoid tissues, PrP^d was detected in tonsils of case 2 (G) similarly to what was seen in the lymph nodes of case 1. Scale bars, 50 µm (G, P, Q) and 20 µm (H-M, P, Q).</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

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

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

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 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