Fig 1.
EBV lytic reactivation in both B- and epithelial cells downregulates E2F1 expression.
(A) EBV lytic cycle replication was induced by with 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3 mM sodium butyrate (NaBu) treatment in EBV transformed B-lymphocytes LCL#89 and subjected to Immunoblot analysis. (B) qRT-PCR analysis was performed on cDNA isolated from TPA-NaBu treated LCL#89 cells. (C) Immunoblot analysis of whole cell lysates (WCLs) from EBV+ EB3 cells treated with combination of TPA and NaBu for lytic reactivation induction for the indicated time points (0–72 h). (D) qRT-PCR analysis was performed on cDNA isolated from TPA-NaBu treated EBV+ EB3 cells for the indicated time points (0–72 h). (E) EBV lytic cycle replication was induced by TPA-NaBu treatment in EBV+ BL line P3HR1 for the indicated time points (0–72 h) and subjected to immunoblot analysis. (F) Immunoblot analysis of WCLs from P3HR1 cells treated with TPA-NaBu for lytic reactivation induction for 72 h in the presence and absence of 25 µg/ml ganciclovir (GCV). (G) EBV lytic cycle replication was induced in P3HR1 with 10 µg/ml anti-human IgG for the indicated time points (0–72 h) and subjected for immunoblot analysis. (H) Reanalysis of scRNA-Seq data (GSE272763) of P3HR1-ZHT cells (n = 8965) undergoing EBV lytic reactivation. Cell clusters were identified using unsupervised clustering and visualised using UMAP expression profiles (top panel) and bar plots (bottom panel) showing differential gene expression of BZLF1 and E2F1 across different clusters. (I) P3HR1 cells stably expressing BZLF1 under doxycycline responsive promoter were subjected to immunoblot analysis without or with doxycycline (-/ + DOX) treatment for 48 h. (J) Representative pictures of bright field and GFP fluorescence of TPA-NaBu treated HEK293T-BAC-GFP-EBV cells. Scale bars, 100 μm. (K) EBV lytic cycle replication was induced by TPA-NaBu treatment in HEK293T-BAC-GFP-EBV cells for the indicated time points (0–72 h) and subjected to immunoblot analysis. (L) qRT-PCR analysis was performed on cDNA from TPA-NaBu treated HEK293T-BAC-GFP-EBV cells. (M) Heat map representation of differential gene expression of BZLF1 and E2F1 from two bulk RNA-Seq datasets (GSE231687 and GSE155811) for EBV lytic reactivation in nasopharyngeal and gastric carcinoma cell lines, HK1-EBV and AGSiZ, respectively. (N) EBV lytic cycle replication was induced by 1 μM MG132 treatment in HEK293T-BAC-GFP-EBV cells for the indicated time points (0–48 h) and subjected to immunoblot analysis. (O) qRT-PCR analysis was performed on cDNA from 1 μM MG132 treated HEK293T-BAC-GFP-EBV cells for the indicated time points (0–48 h). (P) qRT-PCR analysis was performed on cDNA from 1 μM MG132 treated LCL#89. Immunoblots are representative of n = 3 replicates. For qRT-PCR analysis, the relative changes in transcripts of the selected genes were quantified using the 2−ΔΔCT method and normalized with B2M as internal control. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig 2.
BZLF1 transcriptionally represses E2F1 expression.
(A) Reanalysis of ChIP-Seq data (E-MTAB-7788) showing enrichment of BZLF1 on E2F1 promoter. Bottom panel indicates the MACS2 identified peaks (Sites 1–3) for BZLF1/AP-1 binding on E2F1 promoter. (B) BZLF1 homologue AP-1 binding motif identified on the MACS2 peaks of E2F1 promoter region. (C) ChIP-qPCR data showing recruitment of BZLF1 on E2F1 promoter upon EBV lytic cycle reactivation by TPA-NaBu treatment for 72 h in P3HR1 cells. (D) Immunoblot analysis of whole cell lysates (WCLs) from EBV+ P3HR1 cells reactivated to lytic cycle replication as similar to (C). (E) HEK293 cells transiently transfected with empty vector or increasing concentrations of flag-tagged BZLF1 expression plasmid for 36 h were harvested and subjected to immunoblot analysis. (F) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type E2F1 promoter in the presence of increasing concentrations of BZLF1 expression plasmid in transiently transfected HEK293 cells. (G) Schema showing three wild-type BZLF1/AP-1 binding sites (Sites 1–3) and their corresponding mutations (Muts 1–3) on E2F1 promoter for cloning into pGL3 luciferase reporter vector. (H) Luciferase reporter activity of the wild-type and the mutant E2F1 promoters in the presence of either vector control or BZLF1 expression plasmid in HEK293 cells. A fraction of the total protein was evaluated by immunoblot analysis. (I) Schema showing different structural domains of BZLF1 for cloning in flag-tagged expression vector. (J) Luciferase reporter activity and the corresponding immunoblot analysis of the E2F1 promoter in the presence of empty vector, wild-type (WT) or transactivation domain deleted (ΔTAD) BZLF1 expression plasmids in HEK293 cells. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig 3.
E2F1 directly targets EBV genome to differentially control both latent and lytic transcriptional programs.
(A) ChIP-Seq analysis of E2F1 binding in two EBV transformed lymphoblastoid cell lines – LCL#1 and LCL#89. Tracks are aligned with the annotated EBV genome shown at the bottom. (B) ChIP-qPCR analysis of E2F1 occupancy at different EBV promoters and genomic regions. Anti-E2F1 ChIP was performed on chromatin extracted from LCL#1 and LCL#89, followed by qPCR using primers specific for Wp, Cp, Qp, LMP1p, LMP2p, OriLytL, OriLytR and Zp regions. Data are presented as % input. (C) E2F1 ChIP-Seq tracks and the corresponding MACS2 identified peaks on BZLF1 promoter region (Zp) in LCL#1 and LCL#89. Bottom panel indicates different Zp elements and three putative E2F1 binding motifs obtained from JASPAR database in Zp. (D) Schema showing three wild-type E2F1 binding sites (Sites 1–3) and their corresponding mutations (Muts 1–3) on Zp for cloning into pGL3 luciferase reporter vector. (E) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type (WT) and mutant (Mut) Zp in the absence and presence of flag-tagged E2F1 expression vector in HEK293 cells. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig 4.
E2F1 suppresses EBV lytic cycle reactivation.
(A) Immunoblot analysis of whole cell extracts of four EBV+ BL lines – P3HR1, Jiyoye, EB3 and Namalwa with the indicated antibodies against viral and cellular proteins. (B) P3HR1 and Jiyoye cells were reactivated to lytic cycle replication by TPA-NaBu treatment for 72 h were subjected to immunoblot analysis. (C) EBV intracellular or DNase-treated extracellular genome copy number was quantified by qRT-PCR from P3HR1 and Jiyoye cells treated with TPA-NaBu for 72h. (D) P3HR1 cells stably expressing sh-RNA specific for E2F1 under doxycycline responsive promoter were subjected to immunoblot analysis without or with doxycycline (-/ + DOX) treatment. (E) qRT-PCR of EBV intracellular or extracellular genome copy number from P3HR1-sh-E2F1 cells without or with doxycycline (-/ + DOX) treatment. (F) Immunoblot analysis of P3HR1 cells expressing either control (sgCon) or two E2F1 specific sgRNAs. (G) qRT-PCR of EBV intracellular or extracellular genome copy number quantified from P3HR1 cells in the presence of control or two E2F1-specific sgRNAs. (H) Jiyoye cells transiently transfected either control vector or flag-tagged E2F1 expression plasmid, followed by TPA-NaBu treatment for 72 h were subjected to immunoblot analysis. (I) EBV intracellular or DNase-treated extracellular genome copy number analysis was performed in Jiyoye cells transiently transfected either control vector or flag-tagged E2F1 expression plasmid, followed by TPA-NaBu treatment for 72 h. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig 5.
E2F1, but not E2F2, transcriptionally deactivates BZLF1 expression through its transactivation domain.
(A) Schema showing known structural domains of all eight E2F genes (E2F1-8). Right panel indicates the % sequence similarities of E2F1 and other E2F family members. (B) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged E2F1 expression plasmid. (C) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged E2F2 expression plasmid. (D) Luciferase reporter activity and the corresponding immunoblot analysis of the BZLF1 promoter (Zp) in the presence of control vector, or flag-tagged E2F1 or E2F2 expression plasmids in HEK293 cells. (E) Pairwise 3D structure alignment of E2F1 and E2F2. Bottom panel indicates sequence similarly (Blue) and dissimilarity (Grey) between E2F1 and E2F2 structure alignment. (F) Luciferase reporter activity and the corresponding immunoblot analysis of the Zp in the presence of empty vector, wild-type (WT) or transactivation domain deleted (ΔTAD) E2F1 expression plasmids. (G) Luciferase reporter activity and the corresponding immunoblot analysis of the Zp in the presence of empty vector, WT E2F1, E2F1 fused with E2F2-TAD domain (TAD2), WT E2F2 or E2F2 fused with E2F1-TAD domain (TAD1) expression plasmids in HEK293 cells. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. ND, not detected.
Fig 6.
E2F1 positively regulates c-Myc expression.
(A) Whole transcriptome analysis of P3HR1 stably expressing E2F1 sh-RNA (P3HR1-sh-E2F1) in the absence and presence of doxycycline (-/ + DOX) were either left untreated or treated with TPA/sodium butyrate (NaBu) for 72 h. Left panel indicates heatmap analysis of top 100 downregulated genes. Right panel bar diagram indicates most significantly affected pathways (p < 0.05, FDR < 0.05) based on top 100 downregulated genes in the RNA-Seq data of E2F1 knockdown and EBV lytic cycle reactivation. (B) Heat map visualization of c-Myc transcripts in the P3HR1 RNA-Seq data. (C) Two-tailed unpaired Student’s t-test and two-sided Pearson’s correlation were employed to analyse the association between E2F1 and c-Myc transcripts in 19 EBV+ BL lines from DepMap portal. Right panel indicates heatmap representation of E2F1 and c-Myc expressions in P3HR1 and Jiyoye cells. (D) qRT-PCR analysis of cDNA isolated from P3HR1-sh-E2F1 cells without or with doxycycline (-/ + DOX) treatment. (E) Immunoblot analysis of P3HR1-sh-E2F1 cells without or with doxycycline (-/ + DOX) treatment. (F) qRT-PCR analysis of cDNA isolated from Jiyoye cells transiently transfected with either control vector or flag-tagged E2F1 expression plasmid. (G) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged E2F1 expression plasmid. (H) EBV+ LCL#89 and Raji (GSE76191) ChIP-Seq tracks of E2F1 occupancy at c-Myc gene locus. (I) ChIP-qPCR analysis of E2F1 occupancy at c-Myc promoter region in LCL#89 and P3HR1 cells. (J) Schema showing two wild-type E2F1 binding sites (Red, Sites 1–2) and their corresponding mutations (Blue, Muts 1–2) on c-Myc promoter region for cloning into pGL3 luciferase reporter vector. (K) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type (WT) and mutant (Mut) c-Myc promoter in the absence and presence of flag-tagged E2F1 expression vector in HEK293 cells. (L) Immunoblot analysis of Jiyoye cells transiently transfected with either control vector or flag-tagged c-Myc expression plasmid. (M) Immunoblot analysis of P3HR1 cells expressing either control (sgCon) or two c-Myc specific sgRNAs. (N) Reanalysis of RNA-Seq data (GSE140653) of E2F1 transcripts in Akata cells expressing either control (sgCon) or c-Myc specific sgRNA. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Fig 7.
Together with c-Myc, E2F1 transcriptionally repress BZLF1 expression and EBV lytic cycle reactivation.
(A) E2F1 (red, sites 1–3) and c-Myc (blue, site 4) binding motifs obtained from JASPAR database in BZLF1 promoter (Zp) region. Schema showing single wild-type c-Myc binding site (Site 4) and the corresponding mutation (Mut 4) on Zp for cloning into pGL3 luciferase reporter vector. (B) Luciferase reporter activity and the corresponding immunoblot analysis of the wild-type Zp in the absence and presence of E2F1 and c-Myc expression plasmids either in increasing concentrations or in combination in HEK293 cells. (C) Luciferase reporter activity and the respective immunoblot analysis of the mutant Zp in the absence and presence of E2F1 and c-Myc expression plasmids either in increasing concentrations or in combination in HEK293 cells. (D) Schematic model depicting E2F1’s role in repressing EBV lytic cycle reactivation. During EBV latency, E2F1 promotes c-Myc expression, together which bind to the BZLF1 promoter and repress its leaky expression that impedes subsequent expression of other lytic genes. Upon EBV lytic reactivation, BZLF1, on the contrary, transcriptionally repress E2F1 and thereby c-Myc expression, highlighting a unidirectional regulatory hierarchy. BZLF1 mediated E2F1 depletion deploys an effective lytic cycle environment that facilitates a cascade of EBV lytic gene expression. The results are presented as the mean ± SD, n = 3 biological replicates. Statistical significance was determined by a two-sided Student’s t-test, *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Table 1.
Key resources table.