Identification of NF-κB-Dependent Gene Networks in Respiratory Syncytial Virus-Infected Cells

Abstract

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. In epithelial cells, RSV replication activates nuclear translocation of the inducible transcription factor nuclear factor κB (NF-κB) through proteolysis of its cytoplasmic inhibitor, IκB. In spite of a putative role in mediating virus-inducible gene expression, the spectrum of NF-κB-dependent genes induced by RSV infection has not yet been determined. To address this, we developed a tightly regulated cell system expressing a nondegradable, epitope-tagged IκBα isoform (Flag-IκBα Mut) whose expression could be controlled by exogenous addition of nontoxic concentrations of doxycycline. Flag-IκBα Mut expression potently inhibited IκBα proteolysis, NF-κB binding, and NF-κB-dependent gene transcription in cells stimulated with the prototypical NF-κB-activating cytokine tumor necrosis factor alpha (TNF-α) and in response to RSV infection. High-density oligonucleotide microarrays were then used to profile constitutive and RSV-induced gene expression in the absence or presence of Flag-IκBα Mut. Comparison of these profiles revealed 380 genes whose expression was significantly changed by the dominant-negative NF-κB. Of these, 236 genes were constitutive (not RSV regulated), and surprisingly, only 144 genes were RSV regulated, representing numerically ∼10% of the total population of RSV-inducible genes at this time point. Hierarchical clustering of the 144 RSV- and Flag-IκBα Mut-regulated genes identified two discrete gene clusters. The first group had high constitutive expression, and its expression levels fell in response to RSV infection. In this group, constitutive mRNA expression was increased by Flag-IκBα Mut expression, and the RSV-induced decrease in expression was partly inhibited. In the second group, constitutive expression was very low (or undetectable) and, after RSV infection, expression levels strongly increased. In this group, NF-κB was required for RSV-inducible expression because Flag-IκBα Mut expression blocked their induction by RSV. This latter cluster includes chemokines, transcriptional regulators, intracellular proteins regulating translation and proteolysis, and secreted proteins (complement components and growth factor regulators). These data suggest that NF-κB action induces global cellular responses after viral infection.