Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory disease in U.S. children, resulting in the hospitalization of 100,000 children per year. A hallmark of RSV infection is the pronounced inflammatory response in infected airways; this is due, in part, to soluble mediators synthesized by airway epithelial cells. We have demonstrated that RSV-infected alveolar epithelial cells inducibly transcribe the potent cytokine interleukin-8 (IL-8). IL-8 plays a role in initiating the inflammatory response to this mucosal virus. In this project, we will investigate the hypothesis that RSV-induced transcriptional regulation of the IL-8 gene is the result of a combinatorial interaction between two cis-regulatory elements (each is necessary for RSV induction) These are 1. the tumor necrosis factor response element (TNFRE), a TNF-inducible element that binds the transcription factors nuclear factor-16 (NF-16) and nuclear factor-kappaB (NF-kappaB) subunit Rel A, and 2. the RSV-inducible response element (RSVIRE). RSV infection activates (through distinct mechanisms) the transcription factors that.bind and regulate both the TNFRE and RSVIRE. Our project has four Specific Aims: 1. NF-IL6 is synthesized in RSV- infected A549 cells through enhanced translation of preformed mRNA.
In Aim 1, we will identify RNA control regions and RNA binding proteins mediating RSV-inducible translational regulation of the NF-IL6 mRNA. 2. Rel A, the activator subunit of NF-kappaB, is translocated into the nucleus upon RSV infection.
In Aim 2, we will determine mechanism(s) for RSV-induced Rel A nuclear translocation by analysis of the inhibitory proteins (IkappaB) that retain Rel A in the cytoplasm of uninfected cells. 3. We have identified a previously unrecognized element in the 5'flanking region of the IL-8 gene necessary for RSV induction (the RSVIRE).
In Aim 3, we will characterize and identify the transcription factor binding to the RSVIRE. 4. Nuclear factor activation is unique to RSV infection and not seen by parainfluenza, influenza, or polio virus infection.
In Aim 4, we will express unique RSV genes to identify those involved in NF-IL6, Rel A and RSVIRE transcription factor activation. This project will define RSV-inducible mechanisms for transcription factor activation, and so should identify new cellular targets that can be therapeutically blocked to inhibit pro-inflammatory cytokine production.
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