The major component of IκBα proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells

M. Jamaluddin, Antonella Casola, Roberto Garofalo, Y. Han, T. Elliott, P. L. Ogra, A. R. Brasier

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Abstract

Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IκB inhibitor and can be released only following IκB degradation. In this study, we examined the spectrum of IκB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-α), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IκB inhibitory subunits IκBα, IκBβ, and p105; however, following either stimulus, only the IκBα and IκBβ steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IκBα in control cells was shortened to 5 min in TNF-α-stimulated cells and to 90 min in pRSV-infected cells. Although IκBα was resynthesized within 30 min following recombinant human TNFα treatment due to a robust 25-fold increase of IκBα mRNA expression (the RelA:IκBα positive feedback loop), following pRSV infection, there was no reaccumulation of IκBα protein, as infected cells produced only a 3-fold increase in IκBα mRNA at 24 h, indicating the RelA:IκBα positive feedback loop was insufficient to restore control IκBα levels. IκBα proteolysis induced by TNF-α occurred through the 26S proteasome, as both 26S proteasome activity and IκBα proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLFCHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90% inhibited by the proteasome inhibitors; surprisingly, however, IκBα proteolysis was not. We conclude that RSV infection produces IκBα proteolysis through a mechanism primarily independent of the proteasome pathway.

Original languageEnglish (US)
Pages (from-to)4849-4857
Number of pages9
JournalJournal of Virology
Volume72
Issue number6
StatePublished - 1998

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Respiratory Syncytial Viruses
proteasome endopeptidase complex
Proteasome Endopeptidase Complex
proteolysis
Proteolysis
epithelial cells
Epithelial Cells
lungs
Lung
viruses
Respiratory Syncytial Virus Infections
Tumor Necrosis Factor-alpha
tumor necrosis factor-alpha
cells
infection
Transcription Factor RelA
Protein Isoforms
Infection
Messenger RNA
Proteasome Inhibitors

ASJC Scopus subject areas

  • Immunology

Cite this

The major component of IκBα proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells. / Jamaluddin, M.; Casola, Antonella; Garofalo, Roberto; Han, Y.; Elliott, T.; Ogra, P. L.; Brasier, A. R.

In: Journal of Virology, Vol. 72, No. 6, 1998, p. 4849-4857.

Research output: Contribution to journalArticle

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abstract = "Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IκB inhibitor and can be released only following IκB degradation. In this study, we examined the spectrum of IκB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-α), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IκB inhibitory subunits IκBα, IκBβ, and p105; however, following either stimulus, only the IκBα and IκBβ steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IκBα in control cells was shortened to 5 min in TNF-α-stimulated cells and to 90 min in pRSV-infected cells. Although IκBα was resynthesized within 30 min following recombinant human TNFα treatment due to a robust 25-fold increase of IκBα mRNA expression (the RelA:IκBα positive feedback loop), following pRSV infection, there was no reaccumulation of IκBα protein, as infected cells produced only a 3-fold increase in IκBα mRNA at 24 h, indicating the RelA:IκBα positive feedback loop was insufficient to restore control IκBα levels. IκBα proteolysis induced by TNF-α occurred through the 26S proteasome, as both 26S proteasome activity and IκBα proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLFCHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90{\%} inhibited by the proteasome inhibitors; surprisingly, however, IκBα proteolysis was not. We conclude that RSV infection produces IκBα proteolysis through a mechanism primarily independent of the proteasome pathway.",
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AU - Jamaluddin, M.

AU - Casola, Antonella

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AU - Elliott, T.

AU - Ogra, P. L.

AU - Brasier, A. R.

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