Abstract

Respiratory Syncytial Virus (RSV) causes severe inflammation and airway pathology in children and the elderly by infecting the epithelial cells of the upper and lower respiratory tract. RSV replication is sensed by intracellular pattern recognition receptors upstream of the IRF and NF-κB transcription factors. These proteins coordinate an innate inflammatory response via Bromodomain-containing protein 4 (BRD4), a protein that functions as a scaffold for unknown transcriptional regulators. To better understand the pleiotropic regulatory function of BRD4, we examine the BRD4 interactome and identify how RSV infection dynamically alters it. To accomplish these goals, we leverage native immunoprecipitation and Parallel Accumulation—Serial Fragmentation (PASEF) mass spectrometry to examine BRD4 complexes isolated from human alveolar epithelial cells in the absence or presence of RSV infection. In addition, we explore the role of BRD4’s acetyl-lysine binding bromodomains in mediating these interactions by using a highly selective competitive bromodomain inhibitor. We identify 101 proteins that are significantly enriched in the BRD4 complex and are responsive to both RSV-infection and BRD4 inhibition. These proteins are highly enriched in transcription factors and transcriptional coactivators. Among them, we identify members of the AP1 transcription factor complex, a complex important in innate signaling and cell stress responses. We independently confirm the BRD4/AP1 interaction in primary human small airway epithelial cells. We conclude that BRD4 recruits multiple transcription factors during RSV infection in a manner dependent on acetyl-lysine binding domain interactions. This data suggests that BRD4 recruits transcription factors to target its RNA processing complex to regulate gene expression in innate immunity and inflammation.

Highlights

  • Respiratory Syncytial Virus (RSV) is an enveloped, single-stranded, negative-senseRNA virus that infects ciliated epithelial cells in the respiratory tract [1]

  • To investigate the effects of RSV infection on the interactome of Bromodomaincontaining protein 4 (BRD4), we utilized native immunoprecipitation and online Parallel Accumulation—Serial Fragmentation (PASEF)-MS to quantify members of the BRD4 complex isolated from A549 human alveolar epithelial cells

  • A549 cells were infected with sucrose-cushion purified RSV at a multiplicity of infection (MOI) of 1 for a total duration of 24 h before harvest

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Summary

Introduction

Respiratory Syncytial Virus (RSV) is an enveloped, single-stranded, negative-senseRNA virus that infects ciliated epithelial cells in the respiratory tract [1]. The innate immune response induced by RSV-infection is mediated by pattern recognition receptors (PRRs) such as the Toll-like receptors (e.g., TLR3) and Retinoic acid inducible gene (RIG-I) that monitor the airway lumen and cellular cytoplasm [3,7]. These PRRs recognize double-stranded RNA (dsRNA) and 50 -phosphorylated RNA as products of effective RSV replication and stimulate the nuclear translocation of the interferon regulatory factor IRF3 and the various nuclear factor kappa beta (NF-κB) transcription factors into the nucleus, where they cooperate to induce rapid expression of pro-inflammatory cytokines and anti-viral interferons [8,9,10].

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