A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Robitaille, Alexa C.; Mariani, Mélissa K.; Fortin, Audray; Grandvaux, Nathalie

doi:10.3791/53723-v

Cited by 2 publications

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“…For native PAGE (adapted from (114)), 150,000 HEK293T cells were seeded and transfected the next day with 405 ng total DNA complexed with 1.6 µL FuGENE HD in 30µL Opti-MEM per well. Cells were lysed 20 h post transfection in 75 µL RIPA lysis buffer freshly supplemented with PI and incubated for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

Schwanke

Magalhães

Schmelz

et al. 2023

Preprint

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Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Using electrophoretic mobility shift assays (EMSA), we demonstrate that purified M35 protein specifically binds to the regulatory DNA element that governs transcription of the first type I IFN gene induced in non-immune cells, Ifnb1. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35's immunomodulatory activity. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signalling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signalling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labelled transcripts (SLAM-seq), and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically down-regulated basal expression of IRF3-dependent genes, and during MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonises gene induction by IRF3 and impairs the antiviral response more broadly than formerly recognised. Importance Replication of the ubiquitous human cytomegalovirus (CMV) in healthy individuals mostly goes unnoticed, but can impair foetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes. Unrelated proteins from other herpesviruses employ the same mechanism, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

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Section: Methodsmentioning

confidence: 99%

The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

Schwanke

Magalhães

Schmelz

et al. 2023

Preprint

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“…Quantification of proteins was performed using a Bradford assay (Biorad). Thirty micrograms of WCE were resolved by electrophoresis on SDS‐PAGE and immunoblotted on a nitrocellulose membrane according to the method detailed by Robitaille et al 26 The following primary antibodies were used: anti‐IRF‐3‐phospho‐Ser396 (described in Servant et al 27 ), anti‐IRF‐3 (Active Motif, cat.#39 033), anti‐P65‐phospho‐Ser536 (Cell Signaling, cat.#3033), anti‐p65 (Santa Cruz, cat.#SC‐372), anti‐phospho‐IκBα‐Ser32 (Cell Signaling, cat.#2859), anti‐IκBα (Cell Signaling, cat.#9242), anti‐phospho‐STAT1‐Tyr701 (Cell Signaling, cat.#7649), anti‐STAT1 (Cell Signaling, cat.#9176), anti‐SeV (MBL life Science, cat #PD029), and anti‐actin (clone AC‐15, Sigma, cat#A5441) diluted in either PBS containing 0.5% Tween (MP Biomedicals, PBS‐T) completed with 5% Bovine Serum Albumin (Millipore Sigma, cat# A7906) or 5% milk (Carnation).…”

Section: Methodsmentioning

confidence: 99%

DUOX2 regulates secreted factors in virus‐infected respiratory epithelial cells that contribute to neutrophil attraction and activation

et al. 2023

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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

Cited by 2 publications

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The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

The Cytomegalovirus M35 Protein Modulates Transcription ofIfnb1and Other IRF3-Driven Genes by Direct Promoter Binding

DUOX2 regulates secreted factors in virus‐infected respiratory epithelial cells that contribute to neutrophil attraction and activation

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