Syncytia formation induces autophagy in MeV-infected cells.

<p>(A) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz wild-type (Sch-MeV) or deficient for MeV-C expression (Sch-MeVΔC). 24 h post infection, the number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy in infected cells detected by staining for the viral nucleoprotein N (MeV-N). (B) GFP-LC3 HeLa cells were infected or not with Ed-MeV (MOI 3) or treated with rapamycin (Rapa) (125 nM), and treated or not with the FIP peptide (10 µg/mL). 24 h post infection, the number of GFP+ vesicles per cell was assessed by confocal microscopy. (C) HeLa cells were co-transfected with a vector encoding for the H protein of Ed-MeV and one encoding for the F protein. 24 h post transfection H/F-co-transfected HeLa cells (H⁺F⁺ HeLa) were co-cultured with GFP-LC3-HeLa cells to induce cell-cell fusion. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy after 18 h of co-culture. (D) GFP-LC3 HeLa cells and HeLa cells were treated with the indicated siRNA for 24 h. HeLa were then co-transfected with MeV-H/F as in (C) and co-cultured with siRNA treated GFP-LC3 HeLa cells. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy. (E) GFP-LC3 HeLa cells were treated with Polyethylene Glycol (PEG) and treated or not with 250 nM rapamycin (Rapa) for 2 h. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy 6 h after PEG treatment. For each experiment, representative profiles are shown and are accompanied by a graph representing the number of GFP+ vesicles per cell ( = GFP+ vesicles per one nucleus). For syncytia, the number of dots was normalized to the number of nuclei. Error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

The MeV-C protein is involved in MeV-induced autophagy.

<p>(A–B) GFP-LC3-HeLa cells were infected or not with Ed-MeV (MOI 3) and treated or not with 0.5 µg/ml cycloheximide (CHX) during 1.5 h (A) or 24 h (B), in the presence of 125 nM rapamycin (Rapa) when indicated. (C) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz (Sch-MeV) at an MOI 1. Autophagy was monitored by the numeration of GFP+ autophagosomes at the indicated period of time post infection. (D) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz wild-type (Sch-MeV) or deficient for MeV-C expression (Sch-MeVΔC). 24 h post infection, the total number of GFP+ vesicles per cell was assessed by confocal microscopy in infected cells detected by staining for the viral nucleoprotein N (MeV-N). For each experiment, graph represents the number of GFP+ vesicles per cell ( = GFP+ vesicles per one nucleus). For syncytia, the number of dots was normalized to the number of nuclei. Error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

The late wave of autophagy induced by attenuated MeV is productive.

<p>(A) HeLa cells were infected with Sch-MeV (MOI 1). At the indicated time points post infection, cellular p62 expression was determined by western blot. (B) HeLa cells were infected with Ed-MeV (MOI 1). At the indicated time points post infection, cellular p62 expression was determined by western blot. The graph represents the intensity of p62 over actin expression normalized to the control condition (non infected). Error bars, mean ± MD of two independent experiments. (C) HeLa cells were infected with Ed-MeV (MOI 3). 24 h p.i., cellular p62 expression was determined by western blot. Representative results are shown and are accompanied by a graph representing the intensity of p62 over actin expression normalized to the control condition (non infected). (D) mRFP-GFP-LC3 HeLa cells were infected with Ed-MeV (MOI 3) or treated with 250 nM rapamycin (Rapa) or 75 µm Chloroquine (CQ) during 2 h. 24 h p.i., the total number of autophagic vesicles (mRPP+) and the number of autophagosomes (mRFP+/GFP+) were assessed by confocal microscopy. The number of autolysosomes was determined by subtracting the number of autophagosomes (mRFP+/GFP+) from the total number of vesicles (mRPP+). Representative profiles are shown and are accompanied by a graph representing the number of autophagic vesicles (white), autophagosomes (yellow) and autolysosomes (red) per nuclear profile for each condition. (E–F) HeLa cells were treated with the indicated si-RNA for 48 h and infected with Ed-MeV (MOI 3 (E) or MOI 0.1 (F)). 24 h (E) or 48 h (F) post-infection, cells were lysed and anti-N and anti-P western blot were performed to reveal MeV-N and MeV-P, respectively. Representative results are shown and are accompanied by a graph representing the intensity of MeV-N or MeV-P expression over cellular actin normalized to the control condition. (C–F) : error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

Ed-MeV exploits autophagy to replicate.

<p>(A) HeLa cells were treated with the indicated si-RNA for 48 h and infected with Ed-MeV (MOI 3). One, two or three days post infection infectious viral particles were titrated by plaque assay. (B–D) HeLa cells were treated or not with 125 nM rapamycin (Rapa) or 50 µM chloroquine (CQ) and infected with Ed-MeV (MOI 1 (B) or MOI 0.1 (C–D)). Three days post infection infectious viral particles were titrated by plaque assay (B) or 48 h post-infection, cells were lysed and MeV-N (C), MeV-P (D) expression were detected by western blot. (C–D) Representative results are shown and are accompanied by a graph representing the intensity of MeV-N (C) or MeV-P (D) expression over actin, normalized to the non-infected condition. For each experiment, error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

A schematic overview of the signaling pathway triggered by increased Sp1 level.

<p>Upon Sp1 overexpression, <i>Oas2</i> and <i>Rnasel</i> genes are upregulated and small self-RNAs are produced. Sensing of small self-RNAs leads to the activation of the sensor RIG-I, the IRF3/7 transcription factors and downstream effector targets such as ISGs.</p

Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

<div><p>Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells <i>in vitro</i> and <i>in vivo</i>. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.</p></div

Sp1 activates the OAS-RNAse L pathway and the production of small self-RNAs.

<p>(A) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for the indicated times, and relative mRNA levels of OAS2 and RNase L genes were quantified by RT-qPCR. (B, D, E) 3T3 (B) and MEFs (D, E) WT or deficient for the RNase L (RNase L KO) or MAVS (MAVS KO) were transduced with Sp1, Sp1 Zn^2,3 (Zn) or empty vector (EV). Transduced cells (CD2 positive) were purified 72 h post-transduction by magnetic selection and relative mRNA levels of indicated genes were quantified by RT-qPCR. (C) Small RNAs from BaF3-Sp1 cells following induction Sp1 or not were extracted at the indicated times, and transfected into 293HEK cells (5μg per condition). Luciferase reporter assay was performed to analyze the ISRE promoter activity. Data are mean values ± standard deviation (SD) from one experiment representative of two or three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01.</p

Sp1 overexpression activates genes of the RIG-I pathway.

<p>(A-B) BaF3 (A) and 3T3 (B) cells were transduced with retroviruses encoding wild-type Sp1, Sp1 Zn^2,3 mutant (Zn) that does not bind DNA, or empty vector (EV). Transduced cells (CD2 positive) were purified by magnetic selection 30 h (BaF3) or 72 h (3T3) later, and mRNA levels of indicated genes were measured by RT-qPCR (TLDA used in B) and normalized to housekeeping genes (HPRT in A and Rps-21 in B) mRNA levels. Data are representative of one out of three independent experiments in A. In B panel, data are representative of two independent experiments and statistical analysis was performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: **<i>P</i> <0.01. (C) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for indicated times, and relative mRNA levels of indicated genes were quantified by RT-qPCR (TLDA). Data are representative of one out of four independent experiments.</p

Functional analysis of Sp1 signature.

<p>The gene list enrichment analysis from the Gene Ontology of the SP1 specific signature was performed with g:Profiler. The moderate hierarchical filtering used here allows a compact representation of gene list enrichment results. Significantly enriched GO terms containing less than 5 genes were excluded.</p><p>Functional analysis of Sp1 signature.</p

Proteins of the antiviral RIG-I pathway are induced by increased Sp1 level.

<p>BaF3-Sp1 cells were grown in the presence or absence of dox. (A) Cell extracts were collected at indicated time and expression of the indicated proteins was analyzed by immunoblot. A549 cells treated 16 h with IFNa (1000U/ml) were used as positive control (IFN) for MDA5 and RIG-I induction. (B) BaF3-Sp1 cells were cultured for 20 h, fixed and stained for RIG-I (red) and analyzed by fluorescent microscopy. The percentage of cells presenting RIG-I punctuated staining is indicated, and corresponds to the mean of 8 independent acquisitions. Data are representative of one out of two independent experiments.</p