Respiratory syncytial virus (RSV) causes epidemic upper respiratory tract disease worldwide. Mucosal infection by RSV produces expression of interleukin-8 (IL-8), a cytokine responsible for bronchiolar inflammation. Our studies on the mechanism for IL-8 expression in airway epithelial cells indicate that multiple nuclear proteins are induced to bind a viral-inducible enhancer within the IL-8 promoter. Although mutation of the nuclear factor-kbeta (NF-KB) site completely blocks inducible IL-8 transcription, intact IRF- 1, AP-1 and NF-IL6 sites are all required for maximal promoter activity. We hypothesize that RSV induces both NF-KB nuclear translocation and an ROS-stimulated signaling pathway (both required for inducible IL-8 transcription) that initiate the formation of a multiprotein complex """"""""enhanceosome"""""""" composed of DNA-remodeling proteins, transcription factors, and coactivators on the IL-8 viral enhancer. In this project, we will pursue five aims relating to this hypothesis: 1. Examine the mechanism for RSV-inducible IKB kinase (IKK) activity. Components of the multisubunit IKB Kinase (IKK) activated by RSV infection and their requirement for upstream kinase activity in IKK activity will be determined. 2. Determine the mechanism for enhanced IKK activity by the alternatively spliced IKKy subunit and its role in RSV infection. deltaIKKgamma is expressed in airway cells and is associated with enhanced IKK activity. We will determine if its increased activity occurs through differential association with upstream kinases, enhanced oligomerization, or tendency for membrane partitioning. 3. Analyze the formation and composition of the IL-8 enhanceosome following RSV infection. The requirement for the DNA-remodeling protein HMG I(Y), and IRF family members in the RSVRE-binding complex will be identified by affinity assays. 4. Identify inducible NF-KB coactivators recruited to the IL-8 promoter following RSV infection and chemokine stimulation. The effects of RSV and TNF on inducible coactivator BCL-3, p300 and CBP recruitment, will be determined as well as the kinetics of histone acetylase recruited to the IL-8 gene. 5. Examine the effects of dominant-negative NF-KB and coactivator proteins on promoter recruitment. Expression of dominant-negative NF-KB and CBP/p300 coactivators will be used to determine their effect on coactivator recruitment. These studies will identify signaling mechanisms and protein interactions in viral-induced inflammation.
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