title: Respiratory syncytial virus inhibits apoptosis and induces NF-kappa B activity through a phosphatidylinositol 3-kinase-dependent pathway.
abstract: Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-kappaB activity and inflammatory cytokine synthesis. Both NF-kappaB activity and apoptosis regulation have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes, AKT and GSK-3. This study evaluates the role of PI 3-K and its downstream mediators in apoptosis and inflammatory gene induction during RSV infection of airway epithelial cells. Whereas RSV infection alone did not produce significant cytotoxicity until 24-48 h following infection, simultaneous RSV infection and exposure to LY294002, a blocker of PI 3-K activity, resulted in cytotoxicity within 12 h. Furthermore, we found that RSV infection during PI 3-K blockade resulted in apoptosis by examining DNA fragmentation, DNA labeling by terminal dUTP nick-end labeling assay, and poly(ADP-ribose) polymerase cleavage by Western blotting. RSV infection produced an increase in the phosphorylation state of AKT, GSK-3, and the p85 regulatory subunit of PI 3-K. The activation of PI 3-K by RSV and its inhibition by LY294002 was confirmed in direct PI 3-K activity assays. Further evidence for the central role of a pathway involving PI 3-K and AKT in preserving cell viability during RSV infection was established by the observation that constitutively active AKT transfected into A549 cells prevented the cytotoxicity and apoptosis of combined RSV and LY294002 treatment. Finally, both PI 3-K inhibition by LY294002 and AKT inhibition by transfection of a dominant negative enzyme blocked RSV-induced NF-kappaB transcriptional activity. These data demonstrate that anti-apoptotic signaling and NF-kappaB activation by RSV are mediated through activation of PI 3-K-dependent pathways. Blockade of PI 3-K activation resulted in rapid, premature apoptosis and inhibition of RSV-stimulated NF-kappaB-dependent gene transcription.
authors.affiliation: Department of Internal Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242, USA. karl.thomas@uiowa.edu
authors.forename: Karl W
authors.initials: KW
authors.lastname: Thomas
authors.forename: Martha M
authors.initials: MM
authors.lastname: Monick
authors.forename: Janice M
authors.initials: JM
authors.lastname: Staber
authors.forename: Timor
authors.initials: T
authors.lastname: Yarovinsky
authors.forename: A Brent
authors.initials: AB
authors.lastname: Carter
authors.forename: Gary W
authors.initials: GW
authors.lastname: Hunninghake
mesh_terms: D017209:Apoptosis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002867:Chromones / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3; D006801:Humans; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt; D012136:Respiratory Syncytial Viruses / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002867:Chromones; D038362:Glycogen Synthase Kinase 3; D009025:Morpholines; D016328:NF-kappa B; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human