Skip to main content
. 2019 Feb 12;10(1):e02510-18. doi: 10.1128/mBio.02510-18

FIG 7.

FIG 7

HCMV IE86 impairs transcription of endogenous but not constitutively expressed ectopic pro-IL-1β. (A) Pro-IL-1β mRNA levels in THP-1-IE72 or THP-1-IE86 cells left untreated (mock) or infected with UV-inactivated HCMV-TB40/E (MOI of 3) for 18 h in the presence (gray) or absence (black) of doxycycline. (B) Immunoblot assay for NF-κB subunits P65 and P50, as well as GAPDH, from parental THP-1 cells or those from which the indicated proteins were deleted using CRISPR-Cas9. (C) Pro-IL-1β mRNA levels in control (ΔHUMCYC) THP-1 cells or those from which P65/P50 have been deleted, as indicated, following treatment with PMA or UV-HCMV (MOI of 3). Values presented are mean mRNA fold changes ± SEM calculated relative to levels in control cells not treated with PMA (PMA−). (D) THP-1 NF-κB Quanti-Blue reporter cells were transduced with recombinant adenovirus expressing IE72 or IE86 (AdIE72 and AdIE86) overnight and then mock treated or treated with UV-HCMV (MOI of 3) for an additional 18 h. Values are averages ± SEM from three replicates. One-way ANOVA with Bonferroni correction was performed. n.s., not significant; **, P < 0.01. (E) Levels of Myc-specific mRNA in THP-1-IE86 cells ectopically expressing Myc-tagged pro-IL-1β that were either left untreated (mock) or infected with UV-inactivated HCMV-TB40/E (MOI of 3) for 18 h in the presence (gray) or absence (black) of doxycycline. Data are expressed as average levels of transcript ± SEM relative to levels in untreated cells (mock) from three biological replicates. (F) Constitutive ectopic pro-IL-1β mRNA degradation was examined by quantifying transcript levels at indicated times after the addition of actinomycin D using qPCR in the presence (gray) or absence (black) of IE86. Transcript levels are expressed relative to levels in DMSO-treated cells. (G) IL-1β secretion in THP-1-IE86 cells ectopically expressing Myc-tagged pro-IL-1β that were either left untreated (mock) or infected with UV-inactivated HCMV (MOI of 3) for 18 h in the presence (gray) or absence (black) of doxycycline. (H) Immunoblot assay for pro-IL-1β (ectopic and endogenous, as indicated), Myc (ectopic pro-IL-1β), IE86, and GAPDH in THP-1-IE86 cells ectopically expressing Myc-tagged pro-IL-1β under a constitutive promoter. (I) Immunoblot assay for pro-IL-18, pro-IL-1β, IE86, and GAPDH in THP-1-IE86 cells ectopically expressing pro-IL-18 under a constitutive promoter.