Fig 1.
Whole transcriptome analysis of EBV infected resting B-cells during initial phase of infection.
(A) Schematic representation of whole transcriptome analysis of PBMCs infected with GFP-EBV for 0, 2 and 4 days post-infection (DPI) in Illumina platform as described in the “Materials and Methods” section. (B) Pie chart and scattered plot analyses of differentially expressed gene sets in 2 and 4 DPI. (C) Differentially expressed gene sets were uploaded on DAVID v6.8 webserver for functional analysis. Gene Ontology (GO) was selected from the hits table for DAVID clustering. The bar diagrams (upregulated: red; downregulated: blue) represent top 20 most significantly affected pathways.
Fig 2.
EBV infection of naïve B-lymphocytes specifically enhances expressions of two membrane associated carbonic anhydrase isoforms—CA9 and CA12.
(A) Heat map analysis of differentially expressed genes under “Cellular response to Hypoxia pathway” GO: 0001666 from RNA-Seq analyses of PBMCs infected with GFP-EBV. Differential gene expressions were performed based on p-value as < 0.05 and log2 Fold Change as 2 and above (upregulated, red) and -2 and below (downregulated, blue). (B) Heat map analysis (log2 Fold Change) of all carbonic anhydrase (CA) isoforms in RNA-Seq analyses of PBMCs infected with GFP-EBV for 0, 2 and 4 days post-infection (DPI). (C-E) PBMCs from two individual donors were infected with GFP-EBV for the indicated time points and subjected to (C) quantitative real-time PCR (qPCR), (D) western blot and (E) immunofluorescence analyses. (C) The relative changes in transcripts (log10) of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to uninfected PBMC-control using B2M as housekeeping gene. Two independent experiments were carried out in similar settings and results represent as an average value for each transcript. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (D) 0, 2 and 4 days post-infected cells were harvested, washed with 1 x PBS, lysed in RIPA buffer and subjected for western blot analyses with the indicated antibodies. GAPDH blot was used as loading control. Representative gel pictures are shown of at least two independent experiments. (E) For immunofluorescence studies, ~5 x 104 post-infected cells at the indicated time points were fixed with 4% paraformaldehyde and incubated with the indicated primary antibodies followed by Alexa Fluor conjugated secondary antibodies for visualization in a Zeiss fluorescence microscope with Apotome attachment. Nuclei were counterstained using DAPI (4’,6’-diamidino-2-phenylindole) before mounting the cells. All panels are representative pictures of two independent experiments. Scale bars, 5 μm.
Fig 3.
Elevated levels of CA9 and CA12 expressions are detected in EBV transformed B-lymphocytes.
(A-B) Total RNA or (C) total proteins isolated from two LCL clones–LCL#1 and LCL#89 along with PBMCs from two different donors were subjected for either (A) qPCR or (B) western blot analyses. (A-B) The relative changes in transcripts (log10) of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to uninfected PBMC-control using B2M as housekeeping gene. Two independent experiments were carried out in similar settings and results represent as an average value for each transcript. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (C) For Western blot analyses, ~ 5 million cells were harvested, washed with 1 x PBS, lysed in RIPA and fractionated using appropriate SDS-PAGE followed by incubation with the indicated antibodies. GAPDH blot was performed as loading control. Representative gel pictures are shown of at least two independent experiments. (D) Heat map and dot plot analysis of the transcripts profile of the indicated genes in whole blood cells and LCLs using ‘Genotype-Tissue Expression (GTEx)’ project (https://gtexportal.org/home/). (E) ~ 20 million LCLs were fractionated into nuclear, membrane, mitochondrial and cytoplasmic cell fractions using centrifugation methods, followed by carbonic anhydrase assay according to the manufacturer’s instructions. Bar diagrams are the average of two independent experiments. A small portions were subjected to western blot analyses with the indicated antibodies. GAPDH and histone blots were used as reference proteins for cytoplasmic and nuclear fractions, respectively.
Fig 4.
Carbonic anhydrase inhibitors induce cell death in EBV transformed B-lymphocytes.
(A-B) LCLs (LCL#1 and LCL#89) were either left untreated (DMSO control) or treated with increasing concentrations (10–1000 μM) of CA9/CA12 specific inhibitors—(A) SLC-0111/U-104 or (B) S4 for 24 h and measured carbonic anhydrase activity, intracellular pH, cell viability and colony formation ability as described in the “Materials and Methods section”. Carbonic anhydrase activity and intracellular pH determination assays were performed using kits as per manufacturer’s protocols. For cell viability assays, viable cells from each well of 6-well plates were measured by Trypan blue exclusion method using an automated cell counter. For the colony formation assay, 14 days post-treatment colonies on the soft agar were photographed (bright-field) using a Fluorescent Cell Imager. Scale bars, 100 μm. The number of colonies were measured by ImageJ2 software and plotted as bar diagrams. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively.
Fig 5.
Carbonic anhydrase inhibitor retards EBV induced B-cell transformation.
(A-B) PBMCs isolated from two individual donors (donor #1 and donor #2) were infected with GFP-EBV (MOI ~10) in the absence (DMSO control) or in the presence of 0.1 mM carbonic anhydrase inhibitor S4 for 28 days. At the indicated time points post-infected cells were photographed using a Fluorescent Cell Imager. Scale bars, 100 μm. (C-D) In a similar experimental set up, cells at the indicated time points were harvested and subjected to either (C) carbonic anhydrase activity assay, (D) intracellular pH detection and (E) qPCR analyses. (C) Carbonic anhydrase activity and (D) intracellular pH determination assays were performed using kits as per manufacturer’s protocols. (E) The relative changes in transcripts (log10) of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to uninfected PBMC-control using B2M as housekeeping gene. (C-E) Average values +/- SEM are plotted of two independent experiments performed in similar settings. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (F) Reanalyses of RNA-Seq data (GSE125974 and DRA011328) of B-cells infected with EBV for the indicated time points showing temporal expression prolife of CA9 transcript.
Fig 6.
Carbonic anhydrase inhibitor causes cell apoptosis in EBV transformed cells.
(A-F) ~ 10 million LCLs (LCL#1 and LCL#89) were either left untreated (DMSO control) or treated with 0.1 mM carbonic anhydrase inhibitor S4 for 24 h and subjected to (A-B) apoptosis assay using annexin V/propidium iodide (PI) staining, (C) western blot analyses using the indicated antibodies, (D-E) real-time qPCR analyses for the indicated genes and (F) semi-quantitative PCR analyses as described in the “Materials and Methods” section. (A-B) Representative images of (A) fluorescence microscopy and (B) flow cytometry analyses showing cell apoptosis in LCLs after 0.1 mM S4 treatment. (A) DAPI stained cell nucleus, FITC-labelled Annexin V and PI were visualized by blue (Ex = 359 nm; Em = 457 nm), green (Ex = 488 nm; Em = 350 nm) and red (Ex = 586 nm; Em = 603 nm) signals, respectively using a Ziess fluorescence microscope. Scale bars, 10 μm. (B) FITC labelled annexin V binding and PI staining were detected using a BD cell analyzer. Results were analysed using Floreada.io, an online based tool for flow cytometry data analyses. (C) Representative western blot images of two independent experiments. GAPDH blot was used as loading control. Protein bands were quantified by Odyssey imager software and indicated as bar diagrams at the bottom of corresponding lanes. (D) The relative changes in transcripts of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to DMSO treated samples using B2M as housekeeping gene. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. (E) Representative images of melt curve analyses of real-time PCR amplifications of the indicated genes in (D). (F) Representative ethidium bromide stained 2.5% agarose gel images of PCR amplification of the indicated genes using the similar primer sets used in qPCR amplifications in (D).
Fig 7.
EBNA2 and several B-cell specific transcriptional factors colocalize onto CA9 promoter region.
(A) ChIP-seq data for the indicated viral antigens and B-cell specific transcription factors were reanalysed and displayed using IGV (Integrative Genomics Viewer) software for CA9 gene locus. (B-C) RBP-Jκ binding motif obtained from JASPAR database in the EBNA2 binding region of CA9 promoter. (C) Cartoon representation of genomic location for three RBP-Jκ binding and non-binding sites on the CA9 promoter region for ChIP-qPCR primer designing. Transcription start site (TSS) is indicated by red arrow. (D-E) ChIP-qPCR showing recruitment of (D) EBNA2 and (E) RBP-Jκ on CA9 promoter region. Site 1, 2 and 3 are the binding region and site 4 is non-binding region. ChIP-qPCR primers were designed by NCBI primer BLAST application. Two independent experiments were carried out in similar settings and results represent as an average value for each genomic segment. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively.
Fig 8.
EBNA2 mediated CA9 transcriptional activation is essential for B-cell survival.
(A-D) PBMCs infected (MOI: ~10) with EBNA2 deleted and wild-type EBV generated from P3HR1 and Jiyoye cells, respectively for the indicated time points were subjected to (B) qPCR, (C) carbonic anhydrase activity and (D) intracellular pH analyses. (E, G and H) Comparative analyses of P3HR1 and Jiyoye cells by (E) western blot, (G) qPCR and (H) carbonic anhydrase activity analyses with or without the treatment of 0.1 mM compound S4, respectively. (F) ~20 million P3HR1 cells were transiently transfected with empty vector or pSG5-EBAN2 construct by electroporation. 48 h post-transfection cells were harvested and subjected to western blot analyses with the indicated antibodies. (I) ~20 million Jiyoye were fractionated into nuclear, membrane, mitochondrial and cytoplasmic cell fractions using centrifugation methods, followed by carbonic anhydrase activity assay. Bar diagrams are the average of two independent experiments. (J) ~5 x 104 P3HR1 or Jiyoye cells either left untreated (DMSO control) or treated with increasing concentrations (10–1000 μM) of CA9/CA12 specific inhibitors S4 for 24 h and cell viability was measured by Trypan blue exclusion method using an automated cell counter. (K-N) ~10 x 106 Jiyoye cells were treated with DMSO or 0.1 mM S4 for 24 h and subjected to (K-L) cell apoptosis, (M) western blot and (N) qPCR analyses. (K-L) Representative images of (K) flow cytometry and (L) fluorescence microscopy analyses showing cell apoptosis in Jiyoye after 0.1 mM S4 treatment for 24 h. (K) FITC labelled annexin V binding and PI staining were detected using a BD cell analyzer. Results were analysed using Floreada.io, an online based tool for flow cytometry data analyses. (L) DAPI stained cell nucleus, FITC-labelled Annexin V and PI were visualized by blue (Ex = 359 nm; Em = 457 nm), green (Ex = 488 nm; Em = 350 nm) and red (Ex = 586 nm; Em = 603 nm) signals, respectively using a Ziess fluorescence microscope. Scale bars, 10 μm. (O-T) Jiyoye cells stably transduced with lentiviruses expressing either sh-control or sh-RNA (2) against CA9 gene were (O) photographed using a using a Fluorescent Cell Imager and subjected to (P) western blot, (Q) qPCR, (R) carbonic anhydrase activity, (S) intracellular pH and (T) cell proliferation analyses. (T) For cell proliferation, ~1 x 105 Jiyoye cells stably expressing either sh-control or sh-CA9-2 were grown in 6-well plates and counted the viable cells every 24 h for the indicated time points using Trypan blue exclusion method in an automated cell counter. (C, H-I and R) Carbonic anhydrase activity and (D and S) intracellular pH determination assays were performed using kits according to the manufacturer’s instructions. (E-F, M and P) Western blot analyses were performed with the indicated antibodies, where GAPDH represents as loading control. (B, G, N and Q) For qPCR analyses, the relative changes in transcripts of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to control samples using B2M as housekeeping gene. Error bars represent standard deviations of duplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. ns: non-significant; N/A: not amplified.
Fig 9.
RNA-Seq analyses of EBV lytic cycle reactivation from LCLs reveal CA9 downregulation.
(A-C) Whole transcriptome analysis of LCLs reactivated to lytic cycle replication through chemical induction with 3 mM sodium butyrate (NaBu) and 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for the indicated time points using Ion S5 XL platform as described in the “Materials and Methods” section. (B-C) Volcano plot and heat map analyses of differentially expressed gene sets in 48 and 72 h post-induction. Differential gene expressions were performed based on p-value as < = 0.05 and log2 Fold Change as 3 and above (upregulated, red) and -3 and below (downregulated, blue). (D-F) LCLs reactivated to lytic cycle replication in a similar experimental set up were subjected to (D) western blot, (E) qPCR and (F) carbonic anhydrase activity analyses. (D) Western blot analyses were performed with indicated antibodies, where GAPDH was used as loading control. (E) For qPCR analyses, the relative changes in transcripts of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to control samples using B2M as housekeeping gene. (F) Carbonic anhydrase activity was performed using kit according to the manufacturer’s instructions. Error bars represent standard deviations of triplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. ns: non-significant.
Fig 10.
EBV lytic cycle master regulator BZLF1 transcriptionally represses CA9 expression.
(A-C) ~10 x 106 HEK293 cells were transiently transfected with either empty vector (pA3F) or increasing concentrations of pA3F-BZLF1 expressing flag-tagged BZLF1. 36 h post-transfection cells were harvested and subjected to (A) qPCR, (B) western blot and (C) carbonic anhydrase activity analyses. (D) HEK293 cells transiently transfected with empty vector or BZLF1 expression plasmid, were either left untreated (DMSO control) or treated with 20 μM MG132 for additional 4 h and subjected to western blot analyses. (E) Reanalyses of ChIP-Seq data (E-MTAB-7788) showing enrichment of BZLF1 on CA9 promoter region. Bottom panel indicates the MACS identified peaks (site 1 and site 2) for BZLF1 binding on CA9 promoter. Schema showing BZLF1 homologue AP-1 binding motifs on the MACS identified peaks of CA9 promoter region. (F) ChIP-qPCR data showing recruitment of BZLF1 on the CA9 promoter upon EBV lytic cycle reactivation using with 3 mM sodium butyrate (NaBu) and 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in Jiyoye cells. (G-I) Jiyoye cells reactivated to lytic cycle replication as similar to (F) were subjected to (G) western blot, (H) qPCR and (I) carbonic anhydrase activity analyses. (J) Schema illustrating known structural domains of BZLF1 and CA9 promoter region used for cloning in pA3F expression vector and pGL3 luciferase based reporter vector, respectively. (K) Luciferase reporter activity of the wild-type (blue) and mutant (grey) CA9 promoters in transiently transfected HEK293 cells. (L) A fraction of the total protein from (K) were evaluated by western blot analyses. (A and H) For qPCR analyses, the relative changes in transcripts of the selected genes using the 2-ΔΔCt method are represented as bar diagrams in comparison to control samples using B2M as housekeeping gene. Error bars represent standard deviations of triplicate assays of two independent experiments. (B, D, G and L) For western blot analyses, cells were harvested, washed with 1 x PBS, lysed in RIPA and fractionated using appropriate SDS-PAGE. For transient transfection studies, cells were additionally transfected with GFP expression vector to monitor the transfection efficiency. Western blots were performed with the indicated antibodies. GAPDH blot was performed as loading control. The relative intensities (RI) of protein bands shown as bar diagrams were quantified using the software provided by Odyssey CLx Imaging System. Representative gel pictures are shown of at least two independent experiments. (C and I) Carbonic anhydrase activity was performed using kit according to the manufacturer’s instructions. Error bars represent standard deviations of triplicate assays of two independent experiments. *, **, *** = p-value < 0.01, 0.005 and 0.001 respectively. ns: non-significant.
Fig 11.
Schematic representation of EBV mediated dynamic CA9 expression and pH regulation during B-cell transformation and lytic cycle reactivation.
The current model depicts while during EBV latent infection of naïve B-lymphocytes EBNA2 transcriptionally activates CA9 expression, lytic cycle reactivation by BZLF1 transcriptionally represses CA9 expression. Dynamic CA9 expression alters intracellular pH that affects viral pathogenesis and subsequent EBV-induced B-cell lymphomagenesis.
Table 1.
Key resources table.