Journal: Scientific Reports

Article Title: Lyssavirus matrix protein cooperates with phosphoprotein to modulate the Jak-Stat pathway

doi: 10.1038/s41598-019-48507-4

Figure Lengend Snippet: Mutated P and/or M proteins attenuate RABV virulence correlating with differing innate immune-response activation. Three-week-old BALB/c mice (6 per experiment) were infected intramuscularly with 1000 FFUs of Tha, Th2P, Th4M or Th2P-4M RABVs and monitored for 21 days. At the end of the experiment, 3 mice survived when infected with Th2P-4M, named Th2P-4M survivors. ( A ) The mice were sacrificed day 9 post-infection (denoted D9) or when late infection symptoms appeared (endpoint). mRNA, extracted from their brains, was subjected to RT–qPCR to analyze expression of the indicated genes, normalized to the GAPDH -reporter gene level in Mock-infected mice. RABV mRNA ( B ), and ISG15 ( C ), MxA ( D ), IFNβ1 ( E ), Tyk2 ( F ), Jak1 ( G ) and Stat1 ( H ) gene expression was measured. Results show means ± standard deviation (T-bars) expressed in arbitrary units (AU). (NA): non applicable; *p < 0.05 compared to Mock-infected D9 samples and # p < 0.05 compared to Mock-infected End-point samples. Th2P-4Ms*: Th2P-4M survivors.

Article Snippet: ELISA assays were performed using “RayBio phospho-JAK1 and total JAK1 ELISA” kit, (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer recommendation.

Techniques: Activation Assay, Infection, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Scientific Reports

Article Title: Lyssavirus matrix protein cooperates with phosphoprotein to modulate the Jak-Stat pathway

doi: 10.1038/s41598-019-48507-4

Figure Lengend Snippet: RABV M protein inhibited Jak1 activation and P protein blocked Jak1-Stat1 interaction. Protein complementation assay: wild-type ( A ) or mutated M (Th4M) or P (Th2P) protein ( B ) was co-transfected with JAK1-protein–coding plasmids and stimulated for 1 or 24 h with IFNα or not. Two days post-transfection, the Gaussia luciferase activity, whose intensity corresponds to interaction, was determined. P and M proteins were also transfected as third partners in some experiments. The bold horizontal lines represent Gaussia significance threshold. Results are expressed as means ± standard deviation (T-bars) of at least four independent experiments. *p < 0.05 compared to unstimulated samples. ( C ) Phosphorylated JAK1 protein (pJAK1), JAK1, and P and M proteins were detected by immunoblotting of cell lysates with specific antibodies. HeLa cells were transfected with wild-type P or M-proteins and stimulated (+) with IFNα (1000 U/mL, 24 h) or not (−). ( D ) pJAK1 ELISA was run on HeLa cells infected with RABV wild-type (Tha) or P- and/or M-mutated proteins (Th2P, Th4M, Th2P-4M) and stimulated or without IFNα. *p < 0.05 compared to Tha-infected samples not stimulated by IFNα and # p < 0.05 compared to IFNα-stimulated Tha-infected samples. Protein complementation assay ( E ): STAT1 and JAK1 proteins were co-transfected and stimulated for 1 or 24 h with IFNα or not. P and M proteins were also co-transfected as third partners in some experiments. Two days post-transfection, Gaussia luciferase activity, whose intensity corresponds to interaction, was determined. The bold horizontal lines represent Gaussia significance threshold. Results are expressed as means ± standard deviation (T-bars) of at least four independent experiments. *p < 0.05 compared to the corresponding data for STAT1/JAK1 alone.

Article Snippet: ELISA assays were performed using “RayBio phospho-JAK1 and total JAK1 ELISA” kit, (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer recommendation.

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Standard Deviation, Western Blot, Enzyme-linked Immunosorbent Assay, Infection

Journal: Scientific Reports

Article Title: Lyssavirus matrix protein cooperates with phosphoprotein to modulate the Jak-Stat pathway

doi: 10.1038/s41598-019-48507-4

Figure Lengend Snippet: Dynamic interactions between RABV P and/or M proteins and pSTAT1 and Jak1 proteins. Protein complementation assay: P and M proteins were co-transfected with STAT1 or JAK1-coding plasmids. Two days post-transfection, cells were stimulated with IFNα for 15 or 30 min, or 1, 2, 4, 8 or 24 h. Gaussia luciferase activity, whose intensity corresponds to interaction, was determined. The bold horizontal lines represent the Gaussia significance threshold. Results are expressed as means ± standard deviation (T-bars) of five independent experiments.

Article Snippet: ELISA assays were performed using “RayBio phospho-JAK1 and total JAK1 ELISA” kit, (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer recommendation.

Techniques: Transfection, Luciferase, Activity Assay, Standard Deviation

Journal: Scientific Reports

Article Title: Lyssavirus matrix protein cooperates with phosphoprotein to modulate the Jak-Stat pathway

doi: 10.1038/s41598-019-48507-4

Figure Lengend Snippet: Model of the cooperation between M and P proteins to inhibit different Jak-Stat-pathway steps. ( A ) At infection onset, before the IFN response is triggered and IFNα binds to its receptor (IFNR), JAK1 and STAT1 are not phosphorylated. Then, both RABV M (red) and P (purple) proteins can interact with JAK1, although M-STAT is favoured. Only the mutant 2P (pink) but not 4M (orange) interact with JAK1 (phosphorylated or not). ( B ) In the presence of IFNα, JAK1 is phosphorylated which lead to the phosphorylation of STAT1 and translocation in the nucleus of STAT phosphorylated dimers and finally activation of ISRE promotors and increase of transcription of corresponding genes (ex: ISG15, MxA, IFNβ1 and Tyk2). This is inhibited in the context of a RABV infection by both M and P proteins. Neither the mutant 2P nor 4M are able to interact with STAT1 (phosphorylated or not). Less than 30 min after IFN stimulation (1), the M protein blocked JAK1 phosphorylation and then STAT1 phosphorylation. 30 min after the initial stimulation (2), P destabilises M-JAK1 and the M protein switches towards pSTAT1 interaction, which then enhances the capacity of P protein to also switch towards pSTAT1 interaction and inhibit the translocation of phosphorylated STAT dimers. ( C ) Several hours later, P and M proteins tend to interact preferentially with JAK1, although the initially high P/JAK1 levels were never again reached.

Article Snippet: ELISA assays were performed using “RayBio phospho-JAK1 and total JAK1 ELISA” kit, (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer recommendation.

Techniques: Infection, Mutagenesis, Translocation Assay, Activation Assay