Respiratory Syncytial Virus Infection Induces Chromatin Remodeling to Activate Growth Factor and Extracellular Matrix Secretion Pathways

Abstract

Lower respiratory tract infection (LRTI) with respiratory syncytial virus (RSV) is associated with reduced lung function through unclear mechanisms. In this study, we test the hypothesis that RSV infection induces genomic reprogramming of extracellular matrix remodeling pathways. For this purpose, we sought to identify transcriptionally active open chromatin domains using assay for transposase-accessible-next generation sequencing (ATAC-Seq) in highly differentiated lower airway epithelial cells. High confidence nucleosome-free regions were those predicted independently using two peak-calling algorithms. In uninfected cells, ~12,650 high-confidence open chromatin regions were identified. These mapped to ~8700 gene bodies, whose genes functionally controlled organelle synthesis and Th2 pathways (IL6, TSLP). These latter cytokines are preferentially secreted by RSV-infected bronchiolar cells and linked to mucous production, obstruction, and atopy. By contrast, in RSV infection, we identify ~1700 high confidence open chromatin domains formed in 1120 genes, primarily in introns. These induced chromatin modifications are associated with complex gene expression profiles controlling tyrosine kinase growth factor signaling and extracellular matrix (ECM) secretory pathways. Of these, RSV induces formation of nucleosome-free regions on TGFB1/JUNB//FN1/MMP9 genes and the rate limiting enzyme in the hexosamine biosynthetic pathway (HBP), Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2). RSV-induced open chromatin domains are highly enriched in AP1 binding motifs and overlap experimentally determined JUN peaks in GEO ChIP-Seq data sets. Our results provide a topographical map of chromatin accessibility and suggest a growth factor and AP1-dependent mechanism for upregulation of the HBP and ECM remodeling in lower epithelial cells that may be linked to long-term airway remodeling.

Discovery of RSV-Induced BRD4 Protein Interactions Using Native Immunoprecipitation and Parallel Accumulation—Serial Fragmentation (PASEF) Mass Spectrometry

“…The human RSV long strain was grown in Hep-2 cells and prepared as described [ 7 , 24 ]. The viral titer of purified RSV pools was varied from 8 to 9 log PFU/ml, determined by a methylcellulose plaque assay [ 25 , 26 ]. Viral pools were aliquoted, quick-frozen on dry ice-ethanol, and stored at −70 °C until used.…”

Alternative mRNA Processing of Innate Response Pathways in Respiratory Syncytial Virus (RSV) Infection

“…The major focus of this study was to identify the effect of the RSV induced innate response on AS events. Previous work analyzing the global gene expression profile using high-density microarrays [ 2 , 16 ] and short-read RNA sequencing [ 17 ] has been published. These studies have been successful in identifying time-dependent, replication-sensitive [ 18 ], and NFκB-controlled gene expression programs [ 16 , 19 ] in response to RSV infection.…”

“…Here, RSV replication can affect the expression of >1000 epithelial genes in a time- and MOI-dependent manner, including up- and downregulation [ 2 ]. From these studies, it is well established that the inducible transcriptome encodes innate responses and growth-factor-associated remodeling pathways [ 17 ]. Although this gene-level analysis has been informative to elucidate key signaling mediators and characterize products of innate defense, we lack a deep understanding of how mRNA processing controls the diversity of the cell stress response.…”

“…The genomic response of RSV-infected airway epithelial cells has been studied systematically using high-density microarrays [ 2 , 16 ] and short-read RNA sequencing [ 17 ] to identify time-dependent, replication-sensitive [ 18 ], and NFκB-controlled gene expression programs [ 16 , 19 ]. Although these studies have provided information at the gene expression level, mRNA-processing events, including alternative splicing (AS) and alternative polyadenylation (APA), are recognized to generate transcriptome complexity and proteome diversity.…”

Nanoapproaches to Modifying Epigenetics of Epithelial Mesenchymal Transition for Treatment of Pulmonary Fibrosis

“…NFκB -dependent activation of the core EMT regulators results in expression of COL and FN, key ECM regulators of myofibroblast expansion, airway remodeling and expansion of the subepithelial basement membrane. Supporting this chromatin remodeling mechanism, recent analyses using transposase-accessible-next generation sequencing (ATAC-Seq) in highly differentiated lower airway epithelial cells infected with Respiratory Syncytial Virus demonstrate that the TGFβ-FN-JUN pathways controlling EMT are activated by chromatin decondensation at their proximal promoters ( Xu et al, 2020 ).…”