Biology subjects

Goncalves Magalhaes, V.

Publications and source records attributed to Goncalves Magalhaes, V..

3 recordsLinked to original sources

bioRxiv2023

RBM39 shapes innate immunity through transcriptional and splicing control of IRF3 and other key factors

RNA-binding motif protein 39 (RBM39) is an RNA-binding protein involved in tumorigenesis, cell metabolism, and development. Here, we performed a genome-wide CRISPR/Cas9 screen in two liver-derived cell lines and identified RBM39 as a regulator of cell intrinsic innate immune responses. The knockdown of RBM39 or the treatment with Indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections upon sensing by toll-like receptor 3 (TLR3) or cytosolic RIG-I-like receptors. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion. RBM39 knockdown further restrained type I and type III IFN pathways, by reducing expression of the type I IFN receptor subunit interferon alpha and beta receptor subunit 2 (IFNAR2), type III IFN receptor subunit interleukin 10 receptor subunit beta (IL-10RB) and transcription factor signal transducer and activator of transcription (STAT) 1 and 2. RBM39 overall orchestrates innate immunity by regulating basal expression of key factors of the interferon response via transcription and/or alternative splicing. SignificanceThe function of RBM39 in tumorigenesis has been investigated intensively in the last decade, but its immunological role is still largely unknown. In our study, we identified RBM39 as a regulatory factor of cell intrinsic signaling via a CRISPR/Cas9 screen. Depletion of RBM39 impairs TLR3, RIG-I/MDA5, and IFN pathways, and thus attenuates innate immune responses. Our omics analysis revealed that RBM39 governs the basal expression of several key factors within these pathways, such as RNA sensors RIG-I and MDA5, type I/III receptors, transcription factors IRF3, STAT1 and STAT2, via its transcriptional and splicing function. Therefore, RBM39 might be a therapeutic target to modulate innate immunity, e.g. in the context of autoimmune disorders.

immunology↗

bioRxiv2023

Enhanced Airway Epithelial Response to SARS-CoV-2 Infection in Children is Critically Tuned by the Cross-Talk Between Immune and Epithelial Cells

To cope with novel virus infections to which no prior adaptive immunity exists, the body strongly relies on the innate immune system. In such cases, including infections with SARS-CoV-2, children tend to fair better than adults. In the context of COVID-19, it became evident that a rapid interferon response at the site of primary infection is key for successful control of the virus and prevention of severe disease. The airway epithelium of children was shown to exhibit a primed state already at homeostasis and to respond particularly well to SARS-CoV-2 infection. However, the underlying mechanism for this priming remained elusive. Here we show that interactions between airway mucosal immune cells and epithelial cells are stronger in children, and via cytokine-mediated signaling lead to IRF-1-dependent upregulation of the viral sensors RIG-I and MDA5. Based on a cellular in vitro model we show that stimulated human peripheral blood mononuclear cells (PBMC) can induce a robust interferon-beta response towards SARS-CoV-2 in a lung epithelial cell line otherwise unresponsive to this virus. This is mediated by type I interferon, interferon-gamma and TNF, and requires induction of both, RIG-I and MDA5. In single cell-analysis of nasal swab samples the same cytokines are found to be elevated in mucosal immune cells of children, correlating with elevated epithelial expression of viral sensors. In vitro analysis of PBMC derived from healthy adolescents and adults confirm that immune cells of younger individuals show increased cytokine production and potential to prime epithelial cells. In co-culture with SARS-CoV-2-infected A549 cells, PBMC from adolescents significantly enhance the antiviral response. Taken together, our study suggests that higher numbers and a more vigorous activity of innate immune cells in the airway mucosa of children tune the set-point of the epithelial antiviral system. This likely is a major contributor to the robust immune response to SARS-CoV-2 in children. Our findings shed light on the molecular underpinnings of the stunning resilience of children towards severe COVID-19, and may propose a novel concept for immunoprophylactic treatments.

immunology↗

bioRxiv2023

The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

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 M35s 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 M35s crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35s 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 M35s 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. ImportanceReplication 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.

molecular biology↗