Redox-sensitive pathways Mediating RSV Pathogenesis

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The respiratory epithelium represents the principal cellular barrier between the environment and the intemal milieu of the airways. Following inhalation of infectious agents, the epithelium is able to secrete a variety of molecules involved in inflammatory and immune responses, like cytokines and chemokines. Respiratory syncytial virus (RSV) is a potent stimulus of cytokines and chemokines gene expression in airway epithelial cells. We have shown that during the process of viral uncoating, transcription and viral replication, RSV induces several intracellular signals leading to activation of a subset of transcription factors, including NFkappaB, AP-1, STAT and IRF, which are necessary for RSV-induced chemokine gene expression. Activation of NF-kappaB, STAT and IRF, following RSV infection, occurs through redox-sensitive pathways since it is inhibited by antioxidant treatment of airway epithelial cells. RSV infection is a strong inducer of reactive oxygen species (ROS), ubiquitous, highly diffusable and reactive molecules which function as important regulators of intracellular signaling. In this project, we will investigate the role of ROS in modulating intracellular protein phosphatase (PTP) activity, as well as in activation of lKKepsilon, a recently cloned IKK-like kinase, as novel signaling pathways leading to STAT, IRF and NF-kappaB induction in airway epithelial cell, following RSV infection. We will test the relationship between ROS production and modulation of IKKepsilon, PTPs and downstream transcription factor activation in the following specific aims: Aim 1. To investigate the role of ROS in protein tyrosine phosphatase activity and its relationship to RSV-induced STAT activation. We will investigate PTP1B and SHP-2 enzymatic activity in cells infected with RSV in the presence or absence of antioxidants, and we will then determine their role in STAT activation by over-expressing catalytically-inactive proteins or down-regulating their expression by small interfering RNA (siRNA). Aim 2. To determine the role of ROS in RSV-induced IKKepsilon activation. We will investigate IKKepsilon gene expression, protein synthesis and kinase activity in airway epithelial cells infected with RSV in the absence or presence of antioxidants. We will clone the IKKepsilon promoter to determine if IKKepsilon induction is transcriptionally mediated and if it is dependent on viral-induced ROS generation. Aim 3. To identify ROS-dependent mechanisms regulating NF-kappaB driven transcription. We will analyze viral-induced levels of NF-kappaB phosphorylation in airway epithelial cells treated with or without antioxidant. Similar experiments will be performed in cells where IKKepsilon signaling has been disrupted. We will determine whether specific serine phosphoacceptor sites are required for IKKepsilon-mediated NF-kappaB transcriptional activation. Finally, we will perform IKKepsilon kinase assays, in the absence or presence of antioxidants, using wild type or serine mutated NF-kappaB as substrate, to determine the ability of IKKepsilon to directly phosphorylate NF-kappaB and if the kinase activity is redox-dependent. Insights gained from these studies will be instrumental in formulating novel therapeutic approaches for the treatment of viral-induced lung inflammation and post-infectious asthma.