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. 2006 Nov;80(21):10763-71.
doi: 10.1128/JVI.01195-06.

Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression

R Travis Taylor  1 , Wade A Bresnahan
Affiliations

Human cytomegalovirus IE86 attenuates virus- and tumor necrosis factor alpha-induced NFkappaB-dependent gene expression

R Travis Taylor et al. J Virol. 2006 Nov.

Abstract

Human cytomegalovirus (HCMV) infection regulates a number of genes involved in the host antiviral response. We have previously reported that HCMV attenuates the expression of beta interferon (IFN-beta) and a number of proinflammatory chemokines, and this attenuation is mediated by the HCMV immediate-early protein IE86. The present study seeks to identify the mechanism by which IE86 blocks IFN-beta expression. We demonstrate that the induction of IFN-beta during HCMV infection requires the activation of both the IRF-3 and the NFkappaB pathways. Therefore, IE86 may target either pathway to block IFN-beta expression. Our results show that IE86 does not block IRF-3 phosphorylation, dimerization, nuclear translocation, or target gene expression. However, using gel shift analysis, we demonstrate that IE86 efficiently inhibits virus-induced binding of NFkappaB to the IFN-beta promoter, resulting in attenuation of IFN-beta and NFkappaB-dependent gene expression. Furthermore, IE86 expression inhibits tumor necrosis factor alpha-induced NFkappaB DNA binding and target gene expression. Together, these results identify IE86 as a NFkappaB antagonist, which results in the suppression of NFkappaB-dependent cytokine and chemokine gene expression.

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Figures

FIG. 1.
FIG. 1.
IRF-3 and NFκB are both required for IFN-β expression. HFF cells were transduced with adenoviruses expressing GFP, IE86 (A), and IκBαSR (B) or retroviruses expressing IRF-3ΔN and empty vector (C). Transduced cells were then mock infected or infected with WT-HCMV or UV-inactivated HCMV. RNA was isolated 8 h postinfection and analyzed by Northern blot analysis for IFN-β and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) transcripts.
FIG. 2.
FIG. 2.
IE86 does not block IRF-3 activation or target gene expression. (A) HFF cells were mock infected or infected with WT-HCMV, UV-HCMV, or Sendai virus. Cell lysates were prepared 6 h postinfection and assayed for IRF-3 and tubulin expression by Western blot analysis. (B) HFF cells were transduced with adenoviruses expressing IE86 or GFP. Twenty-four hours posttransduction, cells were mock infected or infected with WT-HCMV or UV-HCMV at a multiplicity of 5 PFU/cell. Cell extracts were prepared 6 h postinfection and assayed for IRF-3 dimerization by native gel electrophoresis and Western blot analysis using an IRF-3 antibody. HFF cells (C) or HFF cells transduced with adenovirus expressing IE86 (D) were seeded onto coverslips and either mock infected, infected with WT-HCMV, or infected with UV-HCMV. Cells were fixed 3 h postinfection and assayed for IRF-3 and IE86 localization by immunofluorescence assay. Nuclei were stained with Hoechst. (E) HFF cells were transduced with adenoviruses expressing IE86 or GFP for 24 h. Cells were then infected with WT-HCMV or UV-HCMV at a multiplicity of 5 PFU/cell. RNA was isolated 8 h postinfection and assayed for ISG15 and GAPDH expression by Northern blot analysis.
FIG. 3.
FIG. 3.
NFκB is activated during HCMV infection. (A) HFF cells were mock transduced or transduced with adenoviruses expressing IE86 or GFP and treated with 50 ng/ml TNF-α. Cell lysates were prepared 30 min posttreatment and assayed for phosphorylated IκBα, total IκBα, and tubulin by Western blot analysis. HFF cells (B) or HFF cells transduced with adenovirus expressing IE86 (C) were seeded onto coverslips and either mock infected or infected with WT-HCMV or UV-HCMV. Cells were fixed 3 h postinfection and assayed for NFκB (p50) and IE86 localization by an immunofluorescence assay. Nuclei were stained with Hoechst.
FIG. 4.
FIG. 4.
IE86 attenuates NFκB DNA binding activity. (A) HFF cells were mock infected or infected with WT-HCMV or UV-HCMV at a multiplicity of 5 PFU/cell. Nuclear extracts were prepared 6 h postinfection and assayed by EMSA for binding of NFκB to the PRDII region of the IFN-β promoter. NS indicates a nonspecific shift. (B) HFF cells were infected with WT-HCMV or UV-HCMV. Six hours postinfection, nuclear lysates were isolated and assayed for NFκB binding. A competition analysis was performed on UV-HCMV extracts by adding unlabeled specific-competitor oligonucleotide probe (PRDII) or a mutated probe sequence (mPRDII) in increasing concentrations to the binding mixture to confirm the specificity of the NFκB DNA binding. (C) HFF cells were infected with HCMV, UV-HCMV, UL83Stop virus, or IE2ΔSX virus at a multiplicity of 5 PFU/cell. Nuclear extracts were prepared 6 h postinfection and assayed for NFκB binding by EMSA. A Western blot is also included to confirm the expression of the various viral proteins. (D) HFF cells were transduced with adenoviruses expressing IE86, pp65, GFP, or IκBαSR and then infected at a multiplicity of 5 PFU/cell with UV-HCMV. Nuclear lysates were prepared 6 h postinfection and assayed for NFκB binding by EMSA. A Western blot is included to confirm the expression of IE86, pp65, IκBα, GFP, adenovirus hexon, and tubulin.
FIG. 5.
FIG. 5.
IE86 attenuates NFκB target gene expression. Cells were transduced with adenovirus expressing either IE86 or GFP and then infected at a multiplicity of 5 PFU/cell with UV-HCMV. RNA was isolated 8 h postinfection and assayed for TRAIL, IL-6, and GAPDH expression by Northern blot analysis.
FIG. 6.
FIG. 6.
IE86 attenuates NFκB DNA binding following TNF-α treatment. HFF cells were transduced with adenovirus expressing IE86, GFP, or IκBαSR and then infected with Sendai virus at 100 HAU/ml (A) or treated with 50 ng/ml TNF-α (B). Nuclear extracts were prepared 6 h postinfection or -treatment and assayed for NFκB binding by EMSA. NS indicates a nonspecific shift. (C) Cells were transduced with adenovirus expressing IE86, GFP, or IκBαSR for 24 h. Transduced cells were then treated with TNF-α. RNA was isolated 6 h posttreatment and assayed for IL-8 and RANTES expression by Northern blot analysis.
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