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J Virol. 2011 Nov;85(22):11752-69. doi: 10.1128/JVI.05360-11. Epub 2011 Sep 7.

RelA Ser276 phosphorylation-coupled Lys310 acetylation controls transcriptional elongation of inflammatory cytokines in respiratory syncytial virus infection.

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1
MRB 8.126, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060, USA. arbrasie@utmb.edu

Abstract

Respiratory syncytial virus (RSV) is a negative-sense single-stranded RNA virus responsible for lower respiratory tract infections (LRTIs) in humans. In experimental models of RSV LRTI, the actions of the nuclear factor κB (NF-κB) transcription factor mediate inflammation and pathology. We have shown that RSV replication induces a mitogen-and-stress-related kinase 1 (MSK-1) pathway that activates NF-κB RelA transcriptional activity by a process involving serine phosphorylation at serine (Ser) residue 276. In this study, we examined the mechanism by which phospho-Ser276 RelA mediates expression of the NF-κB-dependent gene network. RelA-deficient mouse embryonic fibroblasts (MEFs) complemented with the RelA Ser276Ala mutant are deficient in CXCL2/Groβ, KC, and interleukin-6 (IL-6) expression, but NFKBIA/IκBα is preserved. We show that RSV-induced RelA Ser276 phosphorylation is required for acetylation at Lys310, an event required for transcriptional activity and stable association of RelA with the activated positive transcriptional elongation factor (PTEF-b) complex proteins, bromodomain 4 (Brd4), and cyclin-dependent kinase 9 (CDK9). In contrast to gene loading pattern of PTEF-b proteins produced by tumor necrosis factor (TNF) stimulation, RSV induces their initial clearance followed by partial reaccumulation coincident with RelA recruitment. The RSV-induced binding patterns of the CDK9 substrate, phospho-Ser2 RNA polymerase (Pol) II, follows a similar pattern of clearance and downstream gene reaccumulation. The functional role of CDK9 was examined using CDK9 small interfering RNA (siRNA) and CDK inhibitors, where RSV-induced NF-κB-dependent gene expression was significantly inhibited. Finally, although RSV induces a transition from short transcripts to fully spliced mRNA in wild-type RelA (RelA WT)-expressing cells, this transition is not seen in cells expressing RelA Ser276Ala. We conclude that RelA Ser276 phosphorylation mediates RelA acetylation, Brd4/CDK9 association, and activation of downstream inflammatory genes by transcriptional elongation in RSV infection.

PMID:
21900162
PMCID:
PMC3209292
DOI:
10.1128/JVI.05360-11
[Indexed for MEDLINE]
Free PMC Article
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