EBNA3C knockdown in LCLs leads to an increase in apoptotic cell death.

<p>A–C) Two EBV negative Burkitt's lymphoma lines - Ramos and DG75 and two EBV transformed cell lines - LCL1 and LCL2; D–F) Short hairpin (Sh) RNA mediated knockdown EBV transformed LCLs (Sh-Con or Sh-E3C) were subjected for genotoxic stress with serum starvation and 5 µM etoposide treatment for 12 h. A and D) Propidium iodide stained cells were analyzed by flow cytometry for a quantitative measurement of apoptosis (subG1 value). A and D) Bar diagrams below histograms represent the mean value of two independent experiments with standard deviation. B and E) Panels indicate representative western blots (WB) of 10% of the total cell lysates with indicated antibodies. C and F) Approximately 0.1 million of indicated cells were grown in 6 well plate for 6 days in RPMI medium containing 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. Viable cells from each well were counted by trypan blue exclusion method every 2nd day using an automated cell counter.</p

Co-expression of EBNA3C blocks E2F1 mediated transcriptional activity.

<p>A) The schematic represents three wild-type (WT) and three mutant (Mut) copies of the E2F1 responsive promoter element fused with the luciferase gene in pGL2Basic. B) HEK 293 (pRb^+/+) cells were co-transfected with either 10 µg of WT (blue) or Mut (red) E2F1 reporter plasmids in combinations of expression plasmids for myc-EBNA3C and flag-E2F1 as indicated. C–E) Saos-2 (pRb^−/−) cells were transfected with WT E2F1 reporter plasmid in the presence of different expression constructs as indicated. Cells were additionally transfected with 5 µg of pCMV-βgal and pEGFP-C1 expression vectors to normalize transfection efficiency. At 36 h post-transfection, cells were harvested and lysed in reporter lysis buffer. Total amount of proteins were normalized by Bradford assay and both luciferase and β-galactosidase activities were measured as described in ‘<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002573#s2" target="_blank">Materials and Methods</a>’ section. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. GAPDH blot was done as an internal loading control. E2F1 protein bands were quantified using Odyssey imager software as indicated as arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on both GFP and GAPDH loading controls. F) HEK293 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids were subjected to ChIP assay as described in ‘<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002573#s2" target="_blank">Materials and Methods</a>’ section. The eluted DNA samples were subjected to qPCR analysis using primers directed for either E2F1-responsive promoter fused with luciferase gene (top) or SV-40 promoter region (bottom). Panels show representative pictures from two independent experiments.</p

EBNA3C expression leads to E2F1 destabilization via an ubiquitin-proteasome dependent pathway.

<p>A) HEK 293 cells were co-transfected with flag-E2F1 and either vector control (lanes 1 and 3) or myc-EBNA3C (lanes 2 and 4) with GFP expressing vector. At 36 h posttransfection, samples were treated with either 20 µM MG132 (+lanes) or DMSO (−lanes) for 6 h and resolved by 10% SDS-PAGE and probed with the indicated antibodies. B) HEK 293 cells were similarly transfected with expression plasmids for flag-E2F1, myc-tagged EBNA3C and GFP plasmids as indicated. At 36 h post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated lengths of time. 5% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. C) 15 million HEK 293 cells transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with flag antibody and samples were resolved by 9% SDS-PAGE. D) Approximately 10 million of stably generated LCLs with either Sh-control (Sh-Con) or Sh-EBNA3C (Sh-E3C) were incubated with 100 µg/ml CHX for indicated lengths of time in RPMI medium containing either 10% FBS (+serum/DMSO) or 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. 10% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. E) Approximately 50 million LCLs with either control Sh-RNA (Sh-Con) or EBNA3C directed Sh-RNA (Sh-E3C) were harvested after 10 h incubation with proteasome inhibitor MG132 (40 µM). Cells were lysed and E2F1 was immunoprecipitated (IP). Samples were resolved by 9% SDS-PAGE and western blotting (WB) was done by stripping and reprobing the same membrane. F) Saos-2 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids followed by incubated with either DMSO or MG132 (20 µM) were subjected for reporter assay as essentially described in ‘<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002573#s2" target="_blank">Materials and Methods</a>’. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. E2F1 protein bands were quantified using Odyssey imager software as indicated as either bar diagrams (A, B and D) or arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on GFP or GAPDH loading controls, where applicable. G–H) Approximately 10 million of indicated cells incubated with either DMSO or MG132 (40 µM) for 10 h, harvested and 10% of total lysates were subjected for western blots with indicated antibodies. For all western blots, where appropriate, GAPDH serves as an internal loading control and GFP as a transfection efficiency control. Western blotting was done by stripping and reprobing the same membrane. Protein bands were quantified using Odyssey imager software as indicated either as arbitrary numerical values at the bottom of gel or as bar diagrams based on either GAPDH or GFP loading control. I) In response to DNA damage signals E2F1 gets stabilized and transcriptionally activates pro-apoptotic genes p73 and Apaf-1, which eventually induces apoptosis. In EBV transformed cells, by forming a stable complex with E2F1, EBNA3C inhibits its DNA binding activity and inhibits the transcriptional activation of p73 and Apaf-1. Moreover, EBNA3C specifically targets E2F1 for an ubiquitin-proteasome mediated degradation, which altogether blocks apoptotic induction. Moreover, sh-RNA designed against E2F1 showed reverse consequences and augments the apoptotic resistance of the cells.</p

EBNA3C binding region is located at the N-terminal DNA binding domain of E2F1.

<p>A) 10 million HEK293 (pRb^+/+) cells were co-transfected with myc-EBNA3C expressing construct with plasmids expressing either vector control or different truncated versions of E2F1 as indicated. After 36 h of transfection, cells were harvested, lysed and immunoprecipitated (IP) with anti-flag antibody (M2). Samples were resolved by a 9% SDS-PAGE and detected by western blot (WB) for the indicated proteins by stripping and reprobing the same membrane. B–C) GST-fused wild-type or different truncated recombinant proteins of E2F1 were incubated with either S³⁵-labeled (top) or ectopically expressed myc-EBNA3C in HEK293 lysate (bottom). After preclearing of all S³⁵-radiolabeled translated proteins with GST-beads for 1 h at 4°C, samples were subjected to GST-pull-down by incubating with either recombinant GST alone or wild-type GST-E2F1 protein as indicated. Samples were electrophoretically separated on 8% SDS-PAGE and were subjected to either autoradiography or western blot using anti-myc antibody. A parallel coomassie stained 12% SDS-PAGE resolving purified GST-proteins is shown at the bottom panel of B. C) The schematic illustrates different structural and interaction domains of E2F1 and summarizes the binding studies among EBNA3C and E2F1. +, binding; −, no binding. NLS, nuclear localization signal; DBD, DNA binding domain; LZ, leucine zipper motif; MB, Marked box. The asterisk indicates the immunoglobulin bands.</p

EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs.

<p>A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC GFP-EBV or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting <i>E2F1</i> and <i>EBNA3C</i> transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2^{− Δ(ΔCt)}, where ΔCt = Ct_target−Ct_GAPDH and Δ(ΔCt) = ΔCt_{test sample}−ΔCt_{control sample}. C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were transfected with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting <i>E2F1, p73 and Apaf-1</i> transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53^−/− pRb^−/−) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002573#s2" target="_blank">Materials and Methods</a>’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.</p

LCLs with E2F1 knockdown are less responsive to apoptosis.

<p>A) Approximately 10 million of Sh-RNA directed control or E2F1 knock-down LCLs (Sh-Con and Sh-E2F1 #1, respectively) were harvested and total proteins were subjected to western blot (WB) analysis using indicated antibodies. B–C) Approximately 0.1 million of indicated cells were grown in 6 well plate for 6 days in RPMI medium containing either B) 10% FBS (+serum/DMSO) or C) 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. Viable cells from each well were counted by trypan blue exclusion method every 2nd day using an automated cell counter. D) Cells were treated with serum starvation and an increasing dose of etoposide (0, 5, 10 and 20 µM) for 12 h, harvested, stained with propidium iodide and analyzed by flow cytometry. E) The bar diagram represents the change in G0 phase due to serum starvation and etoposide treatment in D). F) In a parallel experiment similar as D) approximately 2 million of indicated cells were subjected for western blot to detect the endogenous expression levels of EBNA3C, E2F1, PARP and GAPDH.</p

Both N- and C-terminal domains of EBNA3C bind to E2F1.

<p>A and C) 10 million HEK293 (pRb^+/+) cells were co-transfected with plasmids expressing pRb binding deficient flag-E2F1 (residues 1–400) in presence of different myc-EBNA3C constructs as indicated. Cells were harvested, lysed and immunoprecipitated (IP) with anti-flag antibody (M2). Samples were resolved by a 9% SDS-PAGE and detected by western blot (WB) for the indicated proteins by stripping and reprobing the same membrane. B and D) Wild-type or different truncated mutant expression plasmids of EBNA3C were <i>in vitro</i> translated in presence of S³⁵-Met as per manufacturer's instructions. After preclearing of all S³⁵-radiolabeled translated proteins with GST-beads for 1 h at 4°C, samples were subjected to GST-pull-down by incubating with either recombinant GST alone or wild-type GST-E2F1 protein as indicated. Reactions were resolved by a 9% SDS-PAGE, exposed to phosphoimager plate for overnight and scanned using Typhoon 9410 imaging system. Coomassie staining of a parallel SDS-PAGE resolving purified GST-proteins is shown at the bottom panel of B. E) The schematic illustrates different structural and interaction domains of EBNA3C and summarizes the binding studies between different domains of EBNA3C with E2F1. +, binding; −, no binding. ND, not determined. NLS, nuclear localization signal. The asterisks indicate the immunoglobulin bands.</p

KSHV infection leads to Par3 nuclear localization.

<p>(A) KSHV negative BJAB, Ramos, (B) KSHV positive BCBL1, BC-3, JSC-1 cell lines and (D) PBMCs uninfected and infected with KSHV at day 6 were used to determine the localization of Par3. LANA staining was used as positive control. In KSHV positive cells graphs were plotted with the percent of LANA foci colocalized with Par3. (C) Co-localization of LANA with Par3 was performed in KSHV negative Ramos cell line. The graphs represent the percent of LANA foci co-localized with Par3.</p

KSHV infection leads to Par3 up-regulation.

<p>(A) Expression of Par3 was examined at the transcript and protein levels by real-time PCR and Western blot in KSHV infected PBMCs at day 2 and 6. Here RQ and RD terms are using for relative quantification and relative density, respectively. (B) Par3 were measured at the transcript and protein levels in BJAB (KSHV negative) and BCBL1 and BC-3 (KSHV positive) cell lines. (C) Par3 levels were measured in HEK-293 and HEK-293-BACKSHV at the transcript and protein levels. (D) KSHV positive cells (BC-3 and JSC-1) were knockdown with LANA and compared with controls to assessed transcript of Par3.</p

KSHV can induce epithelial to mesenchymal transition markers in infected B-cells.

<p>(A, B) HEK-293 and BAC-KHSV cells were probed with the antibodies against the EMT markers E-cadherin, and SNAIL. DAPI was used to stain the nucleus. (C) Ramos cells were transfected with an increasing dose of LANA to evaluate the transcript levels of E-cadherin and SNAIL. (D) KSHV infection was carried out in primary PBMCs and monitored up to 7 days post-infection. Control cells without KSHV infection was measured for days 1, 2, 4 and 7 for the measurement of SNAIL and E-cadherin transcripts. (E) Stable LANA knockdown cells were compared to controls for the measurement of Par3, E-cadherin, MMP9 and SNAIL. LANA blots were used as a positive control to monitor its expression in cells, and GAPDH was used as a protein loading control. (F) HEK-293-BAC-KSHV compared to HEK293 cells were evaluated at the protein levels for the expression of Par3, E-cadherin, MMP9 and SNAIL. LANA blots were used as a positive control for the expression of cells and GAPDH was used as a protein loading control. (G) SNAIL knockdown in BC-3 cells were evaluated with control vector for the measurement of the expression of LANA, Par3, and E-cadherin. GAPDH was used as a protein loading control.</p