Fig 1.
EBV deleted for IE locus allows independent assessment of the role of Rta and Zta in lytic gene regulation.
(A) Western blots probed for the indicated EBV, or cell proteins of lysates prepared from 293 cells infected with WT Akata (239-EBV-WT) or AkataΔRZ virus (293-EBVΔRZ) transfected with empty vector, or expression plasmid for Zta, Rta, or expression plasmids for both Rta and Zta (Rta+Zta). Data are representative of two independent experiments. Lanes between 3 & 4 and 7 & 8 are deliberately empty. (B) Longer exposure of the Rta western blot shown in (A) reveals that transfected Zta had induced Rta expression in 293-EBV-WT (but not 293-EBVΔRZ) cells that was not apparent on the shorter exposure.
Fig 2.
Rta and Zta dependence of EBV lytic gene expression in 293 cells.
(A) Western blots showing the specified EBV or cell proteins from 293-EBVΔRZ cells transfected with vector only, Zta, Rta, or both (Rta+Zta). Data are two independent experiments from the same 293-EBVΔRZ clone. (B) UCSC genome browser images displaying RNA-seq data mapped to the EBV Akata_BAC_GFP genome. Each track corresponds to complementation with Rta and/or Zta as indicated on the left as described for (A). Black stars indicate reads arising from transfected Rta and/or Zta (i.e., not expressed from the EBV genome).
Fig 3.
Rta and Zta dependence of EBV lytic gene expression in NOKs.
(A) Western blots showing the specified EBV or cell proteins from NOKs-EBVΔRZ cells transfected with either Zta, Rta, Rta+Zta, or vector-only. The data are representative of two independent experiments. The data are from two different NOKs-EBVΔRZ clones and representative of two independent biologic replicates. (B) UCSC genome browser images displaying RNA-seq data mapped to the EBV Akata_BAC_GFP genome. Trans-complemented proteins are indicated to the left of each track and correspond to those used in (A). Black stars indicate reads arising from transfected Rta and/or Zta.
Fig 4.
Classification of EBV lytic genes based on their Rta and Zta dependencies.
Real time-qPCR data displaying the relative expression of various EBV lytic transcripts in 293-EBVΔRZ, AGS-EBVΔRZ and NOKs-EBVΔRZ. (A) Rtaresponsive transcripts (BSLF1 and BARF1) depend only on Rta for expression. (B) Rtasynergy transcripts (BMRF1 and BALF2) are activated by Rta, but expressed at higher levels with Zta co-expression. (C) Rta+Zta transcripts (BcLF1 and BILF2) require both Rta and Zta transfection to be expressed. Results are expressed as mean values ± the standard error of the mean across two (AGS-EBVΔRZ, NOKs-EBVΔRZ) or one (293-EBVΔRZ) biological replicate. Data shown are representative of two independent experiments. Significant differences are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P>0.05 (ns).
Fig 5.
Responsiveness of EBV lytic transcriptome to Rta and Zta in NOKs.
(A) Heatmap displaying expression levels of individual EBV genes derived from RNA-seq of two biological replicates of NOKs-EBVΔRZ (cl1, cl2) transfected either with vector, Zta, Rta, or Rta+Zta as indicated. Transcript names are indicated to the right and kinetic class to the left. For each gene, responsiveness to Rta and Zta is indicated as follows: Rtaresponsive transcripts (*), Rtasynergy transcripts (**), Rta+Zta transcripts (***). Transcripts were quantified by log2 of the RPKM and color coded as indicated at bottom right. Transcripts that were subject to further analysis (Figs 4, 9 and 10) are shown in bold font.
Fig 6.
Characterization of Rta co-activation by Zta.
(A) Western blots showing expression levels of the indicated EBV proteins in 293-EBVΔRZ trans-complemented with either Rta alone or Rta plus Zta-wt (Zta) or a methyl-DNA binding defective Zta mutant (Zta-S186A). Western blot images are representative of two independent experiments. (B) Western blot data from 293 cells infected with an EBV Bacmid (B95-8 strain) deleted for oriLyt transfected with vector control, Zta, Rta, or Rta and Zta and probed for the indicated proteins. (C) Western blots for the indicated proteins from AGS-EBVΔRZ transfected with Rta alone, Rta and Zta, or Rta and KHSV K8alpha.
Fig 7.
Rta increases Zta binding at Rtaresponsive and Rtasynergy gene promoters.
Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) data for (A) 293-EBVΔRZ or (B) NOKs-EBVΔRZ showing Rta association with control (Qp), Rtaresponsive (SMp and BARF1p) and Rtasynergy (BMRF1p and BALF2p) promoters. Cross-linked chromatin prepared from cells were trans-complemented with vector, Zta, or Zta and Rta as indicated. Cross-linked chromatin was immunoprecipitated with anti-Zta antibody (Zta Ab) or left untreated (No Ab) and quantified relative to input DNA. For each chromatin prep, western blots were performed (far right panels) to ensure early gene induction (BMRF1 and SM) and that equivalent amounts of Zta were expressed in the Zta and Zta+Rta conditions. Data shown in (A) are representative of two independent experiments and the NOKs ChIP (B) was performed once. Error bars indicate standard error of the mean and significant differences are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P>0.05 (ns).
Fig 8.
Zta increases Rta binding at Rtaresponsive and Rtasynergy gene promoters.
Chromatin Immunoprecipitation quantitative PCR (ChIP-qPCR) data from (A) 293-EBVΔRZ or (B) AGS-EBVΔRZ showing Zta association with control (Qp), Rtaresponsive (SMp and BARF1p) and Rtasynergy (BMRF1p and BALF2p) promoters. Crosslinked-chromatin was prepared from cells trans-complemented with vector, Rta-flag (Rta) or Rta-flag and Zta (Rta+Zta) and immunoprecipitated using anti-Flag antibody (Flag Ab) or left untreated (No Ab) and quantified relative to input DNA. For each chromatin prep, western blots were performed (far right panels) to ensure early gene induction (BMRF1 and SM) and that equivalent amounts of Rta were expressed in the Rta and Zta+Rta conditions. Significant differences are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P>0.05 (ns).
Fig 9.
Zta does not affect RNA stability of the early lytic Rtasynergy transcripts.
(A) Western blots of AGS-EBVΔRZ transfected with empty vector, Rta with non-targeting siRNA vector (Rta+siNT), Rta with UPF1 siRNA vector (Rta+siUPF1), or Rta with Zta (Rta+Zta) and probed with the indicated antibodies. (B) Real-time qPCR quantification from the same cells described in (A) showing expression levels of the indicated early Rtasynergy transcripts. (C) Western blot images of NOKs-EBVΔRZ cells harboring either wild-type YTHDF2 (lane 1–3) or knockout protein (lane 4–5) deleted by CRISPR/Cas9 using two different YTHDF2 sgRNA targets 1 and 2 (YTHDF2 KO T1 and YTHDF2 KO T2). Cells were transfected with Rta alone or Rta plus Zta (Rta+Z) where indicated, then probed for the indicated EBV proteins, YTHDF2, and beta actin control. (D) Real-time qPCR data of the trans-complemented NOKs-EBVΔRZ shown in (C) quantifying the expression levels of representative Rtasynergy transcripts as indicated. (E) Western blot of AGS-EBVΔRZ cells transfected with Rta alone, Rta and Zta, or Rta and constitutively active MKK6 mutant (MKK6(glu)) and probed for the indicated proteins. Significant differences are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P>0.05 (ns).
Fig 10.
Zta increases nascent transcription of the early lytic Rtasynergy but not Rtaresponsive gene transcripts.
(A) BrU-labelling assay for nascent transcription of the SM, BSLF1, and BARF1 Rtaresponsive transcripts. Forty-eight hours following transfection with vector, Rta, or Rta+Zta, AGS-EBVΔRZ cells were pulse-labelled with BrU for 1 hour, subjected to anti-BrU immunoprecipitation, and labelled transcripts assayed by quantitative RT-PCR. (B) Relative levels of nascent transcription of the BHRF1, BMRF1, and BALF2 Rtasynergy transcripts in AGS-EBVΔRZ cells examined by BrU-labelling assay. (C) Control western blot of AGS-EBVΔRZ cells used in panels A-B and probed for the indicated proteins. (D) Control BrU-assays performed in AGS-EBVΔRZ cells to demonstrate lack of BrU labelling of the Actinomycin-D (ActD) sensitive cellular transcripts DUSP1 and SGK1 in the presence of ActD. For all bar graphs, values are expressed relative to cellular labelled GAPDH mRNA and error bars indicate standard error of the mean. Data are representative of two independent experiments. Significant differences are indicated as follows: P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P>0.05 (ns).