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EBV nuclear antigen EBNA3C stabilizes Cyclin D1 protein level. A) 10 million human peripheral blood mononuclear cells (PBMC) were infected by BAC GFP-EBV for 4 h. At 3-days post-infection cells were lysed in RIPA buffer and western blots of endogenous proteins were probed with the indicated antibodies. B-E) 20 million cells of (B) Burkitt's lymphoma (BL) cell line BL41 and BL41 cells infected with wild-type EBV strain B95.8 (BL41/B95.8); (C) type I and III latency BL cell lines-MutuI cells (latency I gene expression program) and MutuIII cells (latency III gene expression program); (D) BJAB cells and BJAB cells stably expressing EBNA3C (BJAB_E3C#7); (E) EBNA3C and cyclin D1 knock-down LCL1 cells (LCL1_Sh-E3C and LCL1_Sh-CyD1 respectively) were harvested and total cell proteins were subjected to Western blot (WB) analysis using indicated antibodies. F-I) Total RNA was isolated from cells F) BJAB, Ramos, LCL1 and LCL2; G) BL41 and BL41/B95.8; H) MutuI and MutuIII; I) BJAB and BJAB E3C3 7 and were individually subjected to quantitative real-time PCR analysis for detecting cyclin D1, D2 and D3 transcript levels. Each sample was tested in triplicate and data obtained from three independent experiments were 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 cyclin D mRNA relative to GAPDH was calculated based on the threshold cycle (Ct) as 2-D(DCt) , where DCt = Ct target-Ct GAPDH and D(DCt) = DCt test sample-DCt control sample. J) HEK 293 cells were transfected with an increasing amount (0, 2, 5, 20 mg) of EBNA3C expressing construct and western blot analysis was performed to detect EBNA3C, Cyclin D1 and GAPDH. K) HEK 293 cells were co-transfected with flag-Cyclin D1 and either vector control (lanes 1 and 3) or myc-EBNA3C (lanes 2 and 4). At 36 h posttransfection, samples were treated with either 40 mM MG132 (+ lanes) or DMSO (-lanes) for 6 h and resolved by 10% SDS-PAGE and probed with the indicated antibodies. L) HEK 293 cells were similarly transfected with expression plasmids for flag-tagged both Cyclin D1 and CDK6 and myctagged EBNA3C as indicated. At 36 h post-transfection, cells were treated with 40 mg/ml cyclohexamide (CHX) for indicated lengths of time. 10% of the lysate from each sample were resolved by 10% SDS-PAGE. GAPDH blot was done for loading 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 (A-E) or as bar diagrams (J-L) based on GAPDH loading control. doi:10.1371/journal.ppat.1001275.g001
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EBNA3C, one of the Epstein-Barr virus (EBV)-encoded latent antigens, is essential for primary B-cell transformation. Cyclin D1, a key regulator of G1 to S phase progression, is tightly associated and aberrantly expressed in numerous human cancers. Previously, EBNA3C was shown to bind to Cyclin D1 in vitro along with Cyclin A and Cyclin E. In the pr...
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... cells (PBMC) were infected by BAC GFP- EBV as previously described [40] for 4 h and western blot analysis was performed on samples collected 3 days after infection. The results showed that EBV infection leads to a significant induction of all three Cyclin D protein levels 3 days post-infection, with no preference for any particular D-type cyclins (Fig. 1A). Similarly, western blot results of Burkitt's lymphoma (BL) cell line BL41 and BL41 infected with wild-type EBV strain B95.8 (BL41/B95.8) also showed elevated levels of Cyclin Ds with Cyclin D1 expression more dramatically changed compared to other Cyclin Ds (Fig. 1B). Since Cyclin D1 expression was induced significantly after EBV ...
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... 3 days post-infection, with no preference for any particular D-type cyclins (Fig. 1A). Similarly, western blot results of Burkitt's lymphoma (BL) cell line BL41 and BL41 infected with wild-type EBV strain B95.8 (BL41/B95.8) also showed elevated levels of Cyclin Ds with Cyclin D1 expression more dramatically changed compared to other Cyclin Ds (Fig. 1B). Since Cyclin D1 expression was induced significantly after EBV infection in both PBMC and BL cell line, we next wanted to determine if the induction was related to a specific EBV latent protein expressed during type III latency. The results showed that the levels of both Cyclin D1 and Cyclin D2 proteins were induced in type III ...
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... infection in both PBMC and BL cell line, we next wanted to determine if the induction was related to a specific EBV latent protein expressed during type III latency. The results showed that the levels of both Cyclin D1 and Cyclin D2 proteins were induced in type III latency BL cell line MutuIII compared to latency I expressing MutuI BL cell line (Fig. 1C). These results differ with previously published observations which suggested that B-cells infected with EBV do not express Cyclin D1 [43,44,45]. However, in agreement with previously published results [32], our real-time PCR data showed that EBV infection led to a significant increase of cyclin D2 mRNA level in LCLs (LCL1 and LCL2) ...
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... not express Cyclin D1 [43,44,45]. However, in agreement with previously published results [32], our real-time PCR data showed that EBV infection led to a significant increase of cyclin D2 mRNA level in LCLs (LCL1 and LCL2) when compared to EBV negative BL cells (BJAB and Ramos) whereas, there was little or no detectable change for cyclin D1 mRNA (Fig. 1F). Real-time PCR data obtained from two other matched sets of cell lines BL41 -BL41/B95.8 and MutuI -MutuIII also showed similar results as above ( Fig. 1G and 1H, respectively). These results suggest that D-type cyclins are regulated through distinctly different mechanisms in EBV infected B-cells. EBV effects on Cyclin D2 are at the ...
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... increase of cyclin D2 mRNA level in LCLs (LCL1 and LCL2) when compared to EBV negative BL cells (BJAB and Ramos) whereas, there was little or no detectable change for cyclin D1 mRNA (Fig. 1F). Real-time PCR data obtained from two other matched sets of cell lines BL41 -BL41/B95.8 and MutuI -MutuIII also showed similar results as above ( Fig. 1G and 1H, respectively). These results suggest that D-type cyclins are regulated through distinctly different mechanisms in EBV infected B-cells. EBV effects on Cyclin D2 are at the level of its transcript stability whereas the effects on Cyclin D1 or D3 seem to be post- ...
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... effects of the EBV encoded essential nuclear antigen, EBNA3C on Cyclin D1, BL lines BJAB and E3C #7, a BJAB stably expressing EBNA3C were analyzed. The western blot results showed a significant increase in Cyclin D1 protein expression among D-type Cyclins in E3C #7 cells compared to the BJAB control cells and smaller changes in Cyclin D2 and D3 (Fig. 1D). The effect of EBNA3C on Cyclin D1 steady-state levels was not due to changes in the transcription as EBNA3C expression did not alter the level of cyclin D1 mRNAs in these cells as seen above (Fig. 1I). To further verify the role of EBNA3C on Cyclin D1 protein accumulation, we determined the levels of Cyclin Ds in a lymphoblastoid ...
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... in Cyclin D1 protein expression among D-type Cyclins in E3C #7 cells compared to the BJAB control cells and smaller changes in Cyclin D2 and D3 (Fig. 1D). The effect of EBNA3C on Cyclin D1 steady-state levels was not due to changes in the transcription as EBNA3C expression did not alter the level of cyclin D1 mRNAs in these cells as seen above (Fig. 1I). To further verify the role of EBNA3C on Cyclin D1 protein accumulation, we determined the levels of Cyclin Ds in a lymphoblastoid cell line with the EBNA3C mRNA specifically targeted by short-hairpin RNA (Sh-E3C). The western blot data showed that the expression level of Cyclin D1 in the LCLs stably knocked-down for EBNA3C (Sh-E3C) ...
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... we determined the levels of Cyclin Ds in a lymphoblastoid cell line with the EBNA3C mRNA specifically targeted by short-hairpin RNA (Sh-E3C). The western blot data showed that the expression level of Cyclin D1 in the LCLs stably knocked-down for EBNA3C (Sh-E3C) was significantly dimin- ished as compared to the control cell line (Sh-Control) (Fig. 1E), ; (E) EBNA3C and cyclin D1 knock-down LCL1 cells (LCL1_Sh-E3C and LCL1_Sh-CyD1 respectively) were harvested and total cell proteins were subjected to Western blot (WB) analysis using indicated antibodies. F-I) Total RNA was isolated from cells F) BJAB, Ramos, LCL1 and LCL2; G) BL41 and BL41/B95.8; H) MutuI and MutuIII; I) BJAB and ...
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... the expression levels of other Cyclin Ds was not altered (Fig. 1E). These results indicate that EBNA3C can contribute to Cyclin D1 accumulation in latently infected EBV positive ...
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... demonstrate that EBNA3C can stabilize Cyclin D1 protein levels, HEK 293 cells were transfected with an increasing amount of an expression construct expressing EBNA3C and tested for endogenous Cyclin D1 protein level. The results showed that EBNA3C stabilizes Cyclin D1 protein expression in a dose dependent manner (Fig. ...
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... because of the inhibition of Ub-proteasome mediated destabilization by EBNA3C, transiently co-transfected cells were treated with the proteasome inhibitor, MG132. The results showed that both the treatment with MG132, and presence of EBNA3C led to a significant accumulation (six fold) of Cyclin D1 when compared to mock treatment or vector control (Fig. 1K). Therefore the increased levels of Cyclin D1 observed in the presence of EBNA3C and MG132 is a result of stabilization of Cyclin D1 likely by EBNA3C inhibition of the Ub-proteasome degradation system. Importantly, both CDK6 and EBNA3C levels were not altered by MG132 (Fig. ...
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... fold) of Cyclin D1 when compared to mock treatment or vector control (Fig. 1K). Therefore the increased levels of Cyclin D1 observed in the presence of EBNA3C and MG132 is a result of stabilization of Cyclin D1 likely by EBNA3C inhibition of the Ub-proteasome degradation system. Importantly, both CDK6 and EBNA3C levels were not altered by MG132 (Fig. ...
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... treated with protein synthesis inhibitor cycloheximide, and samples were collected at 0, 1, and 2 hours. Western blots probed with flag antibody showed that the stability of Cyclin D1 protein was significantly enhanced by EBNA3C co-expression, whereas in the absence of EBNA3C, Cyclin D1 was degraded to near completion by 2-h after addition of CHX (Fig. 1L, grey bar). Cyclin D1 half life was determined to be 2 h in EBNA3C expressing cells; however, it shortened noticeably to less than 1 h when Cyclin D1 was expressed alone (Fig. 1L, bar diagram). The results also indicated that both EBNA3C and CDK6 were notably stable throughout the experimental period of time and had no sign of protein degradation ...
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... D1 protein was significantly enhanced by EBNA3C co-expression, whereas in the absence of EBNA3C, Cyclin D1 was degraded to near completion by 2-h after addition of CHX (Fig. 1L, grey bar). Cyclin D1 half life was determined to be 2 h in EBNA3C expressing cells; however, it shortened noticeably to less than 1 h when Cyclin D1 was expressed alone (Fig. 1L, bar diagram). The results also indicated that both EBNA3C and CDK6 were notably stable throughout the experimental period of time and had no sign of protein degradation (Fig. 1L, CDK6 indicated as black bar). Overall, the results of these experiments suggest EBNA3C can stabilize Cyclin D1 by regulating its targeted degradation likely through the ...
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... grey bar). Cyclin D1 half life was determined to be 2 h in EBNA3C expressing cells; however, it shortened noticeably to less than 1 h when Cyclin D1 was expressed alone (Fig. 1L, bar diagram). The results also indicated that both EBNA3C and CDK6 were notably stable throughout the experimental period of time and had no sign of protein degradation (Fig. 1L, CDK6 indicated as black bar). Overall, the results of these experiments suggest EBNA3C can stabilize Cyclin D1 by regulating its targeted degradation likely through the Ub-proteasome degradation ...
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... EBNA3C expression constructs were fused in frame with a myc epitope tag at the C-terminus of the protein. As expected, the results showed that Cyclin D1 co-immunoprecipitated with full-length EBNA3C as well as with the N-terminal domain of EBNA3C (residues 1-365) (Fig 4A, left- middle panel, lanes 2 and 3, respectively) whereas no co-IP was detected with vector control or other truncated versions of EBNA3C (Fig 4A, left-middle panel, lanes 1, 4 and 5). To further corroborate the binding data, an in vitro GST-pulldown experi- ment was performed using in vitro translated 35 S-radiolabeled fragments of EBNA3C (residues 1-100, 1-129, 1-159 and 1-200) within the N-terminal domain. ...
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... 35 S-radiolabeled fragments of EBNA3C (residues 1-100, 1-129, 1-159 and 1-200) within the N-terminal domain. In vitro precipitation experiments with bacterially expressed GST-Cyclin D1 showed strong association with residues 1-159 and 1-200 of EBNA3C (Fig. 4B, bottom panel, lanes 3 and 4, respectively), but not with EBNA3C residues 1-100 or 1-129 (Fig. 4B, bottom panel, lanes 1 and 2, respectively). All fragments of EBNA3C failed to interact with the GST control, indicating that the observed binding was specific for Cyclin D1 (Fig. 4B, middle panel, lanes 1 to ...
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... showed strong association with residues 1-159 and 1-200 of EBNA3C (Fig. 4B, bottom panel, lanes 3 and 4, respectively), but not with EBNA3C residues 1-100 or 1-129 (Fig. 4B, bottom panel, lanes 1 and 2, respectively). All fragments of EBNA3C failed to interact with the GST control, indicating that the observed binding was specific for Cyclin D1 (Fig. 4B, middle panel, lanes 1 to ...
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... D1 was predominantly nuclear. To further support these data, transiently transfected HEK 293 cells were subjected to sub-cellular fractionation and fractionated proteins were analyzed by immunoblot analysis. The result showed that flag-tagged Cyclin D1 alone was detected approximately 50% in both cytoplasmic and nuclear fractions, respectively (Fig. 6A, compare lanes 1 and 4). However, when co- transfected with EBNA3C, flag-Cyclin D1 was detected predom- inantly within the nuclear fraction (Fig. 6A, compare lanes 3 and 6), with an approximately 50% increase compared to flag-Cyclin D1 alone (Fig. 6A, compare lanes 1 and 3). EBNA3C was detected completely within nuclear fraction (Fig. 6A, lanes 2 and 3). The ...
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... D1 alone was detected approximately 50% in both cytoplasmic and nuclear fractions, respectively (Fig. 6A, compare lanes 1 and 4). However, when co- transfected with EBNA3C, flag-Cyclin D1 was detected predom- inantly within the nuclear fraction (Fig. 6A, compare lanes 3 and 6), with an approximately 50% increase compared to flag-Cyclin D1 alone (Fig. 6A, compare lanes 1 and 3). EBNA3C was detected completely within nuclear fraction (Fig. 6A, lanes 2 and 3). The efficiency of cytoplasmic and nuclear fractionation was confirmed Figure 3. EBNA3C forms a complex with Cyclin D1 in human cells. A-B) 15 million HEK 293T cells were co-transfected with myc-tagged EBNA3C and flag-tagged Cyclin D1 vectors. In each case ...
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... 293 cells were transfected with expression vectors encoding flag-tagged Cyclin D1, with or without GSK-3b and myc-tagged EBNA3C. Fractionated cell lysates were analyzed by western blot to clarify flag-tagged Cyclin D1 localization. As expected, Cyclin D1 was primarily present in the cytoplasmic fraction both in the absence and presence of GSK- 3b (Fig. 6B, lanes 1 and 4). In contrast, Cyclin D1 was largely detected within the nuclear fraction when co-expressed with EBNA3C (Fig. 6B, lane 3). Interestingly, even in the presence of GSK-3b nuclear fractionation of Cyclin D1 was greatly increased when co-expressed with EBNA3C compared with the vector control (Fig. 6B, compare lanes 1 and 3). GSK-3b has been ...
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... the absence and presence of GSK- 3b (Fig. 6B, lanes 1 and 4). In contrast, Cyclin D1 was largely detected within the nuclear fraction when co-expressed with EBNA3C (Fig. 6B, lane 3). Interestingly, even in the presence of GSK-3b nuclear fractionation of Cyclin D1 was greatly increased when co-expressed with EBNA3C compared with the vector control (Fig. 6B, compare lanes 1 and 3). GSK-3b has been shown to phosphorylate Cyclin D1 on Thr- 286 in vitro [31], and is postulated to be a major regulator of protein levels and intracellular distribution of Cyclin D1 [31]. To establish a plausible explanation for the inhibitory effects of Figure 4. A small N-terminal region of EBNA3C binds to two different sites of ...
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... where GSK-3b was immuno-precipitated in the absence and presence of EBNA3C, and tested for its ability to phosphorylate recombinant GST-Cyclin D1 proteins (wild-type and T286A mutant Cyclin D1). The results showed that the phosphorylation level of wild-type GST-Cyclin D1 by GSK-3b was reduced by more than 4 fold in the presence of EBNA3C (Fig. 6D, compare lanes 1 and 2). As expected, no phosphorylation bands were observed in case of mutant GST-Cyclin D1 (T286A) indicating the specificity of this experiment (Fig. 6D, lanes 3 and 4). Parallel blots showed the protein expression levels in whole cell- lysate (Fig. 6D), and the amount of purified GST-Cyclin D1 used in this experiment (Fig. 6D). These ...
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... flag-tagged Cyclin D1, flag-tagged CDK6, and EBNA3C (Fig. 7C). The results clearly showed that independent expression of either Cyclin D1/CDK6 complex or EBNA3C reduced pRb expression levels (Fig. 7C [upper panel], compare lanes 1-9). Surprisingly, when both EBNA3C and Cyclin D1/ CDK6 complex were expressed together, little or no pRb was detected (Fig. 7C [lower panel], lanes 1-3), indicating that EBNA3C can also facilitate pRb degradation in cooperation with Cyclin D1/CDK6 either through stabilization of Cyclin D1 (Fig. 7C [lower panel], compare lanes 4-9) or increasing kinase activity of Cyclin D1/CDK6 ...
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... has been shown earlier that both EBV positive cells and cells stably expressing EBNA3C can bypass G1/S phase checkpoint caused by serum starvation [20,35]. Cell-cycle profiles of cells cultured in medium with 0.1% FBS were analyzed by flow cytometry (Fig. 10). Analyses of serum-starved, EBV negative cell lines BJAB and DG75 and LCLs sh-E3C and sh-CyD1 revealed an increased percentage of cells at the G0/G1 phase of the cell cycle (Fig. 10A, B) and decreased percentage of cells in the G2/M phases (Fig.10A, C). Fig. 10B and 10C represents the difference in both G0/G1 and G2/M phases due to ...
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... can bypass G1/S phase checkpoint caused by serum starvation [20,35]. Cell-cycle profiles of cells cultured in medium with 0.1% FBS were analyzed by flow cytometry (Fig. 10). Analyses of serum-starved, EBV negative cell lines BJAB and DG75 and LCLs sh-E3C and sh-CyD1 revealed an increased percentage of cells at the G0/G1 phase of the cell cycle (Fig. 10A, B) and decreased percentage of cells in the G2/M phases (Fig.10A, C). Fig. 10B and 10C represents the difference in both G0/G1 and G2/M phases due to serum starvation, respectively. However, under the same culture conditions, the EBV-positive LCLs -LCL1, LCL2, LCL1-with Sh-control and BJAB-cells stably expressing EBNA3C (E3C# 7 and E3C# ...
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... Cell-cycle profiles of cells cultured in medium with 0.1% FBS were analyzed by flow cytometry (Fig. 10). Analyses of serum-starved, EBV negative cell lines BJAB and DG75 and LCLs sh-E3C and sh-CyD1 revealed an increased percentage of cells at the G0/G1 phase of the cell cycle (Fig. 10A, B) and decreased percentage of cells in the G2/M phases (Fig.10A, C). Fig. 10B and 10C represents the difference in both G0/G1 and G2/M phases due to serum starvation, respectively. However, under the same culture conditions, the EBV-positive LCLs -LCL1, LCL2, LCL1-with Sh-control and BJAB-cells stably expressing EBNA3C (E3C# 7 and E3C# 10) continued through the cell-cycle without being arrested at any ...
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... profiles of cells cultured in medium with 0.1% FBS were analyzed by flow cytometry (Fig. 10). Analyses of serum-starved, EBV negative cell lines BJAB and DG75 and LCLs sh-E3C and sh-CyD1 revealed an increased percentage of cells at the G0/G1 phase of the cell cycle (Fig. 10A, B) and decreased percentage of cells in the G2/M phases (Fig.10A, C). Fig. 10B and 10C represents the difference in both G0/G1 and G2/M phases due to serum starvation, respectively. However, under the same culture conditions, the EBV-positive LCLs -LCL1, LCL2, LCL1-with Sh-control and BJAB-cells stably expressing EBNA3C (E3C# 7 and E3C# 10) continued through the cell-cycle without being arrested at any particular ...
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... 10B and 10C represents the difference in both G0/G1 and G2/M phases due to serum starvation, respectively. However, under the same culture conditions, the EBV-positive LCLs -LCL1, LCL2, LCL1-with Sh-control and BJAB-cells stably expressing EBNA3C (E3C# 7 and E3C# 10) continued through the cell-cycle without being arrested at any particular phase (Fig. 10A histograms, B and C). Furthermore, the results also indicated that upon knockdown of both EBNA3C and Cyclin D1, LCLs underwent a substantial degree of apoptosis (Sub G0) in response to serum starvation, similar to EBV negative cell lines, BJAB and DG75 (Fig. 10A). However, there was no sign of apoptosis observed either in BJAB cells ...
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... 10) continued through the cell-cycle without being arrested at any particular phase (Fig. 10A histograms, B and C). Furthermore, the results also indicated that upon knockdown of both EBNA3C and Cyclin D1, LCLs underwent a substantial degree of apoptosis (Sub G0) in response to serum starvation, similar to EBV negative cell lines, BJAB and DG75 (Fig. 10A). However, there was no sign of apoptosis observed either in BJAB cells stably expressing EBNA3C or wild-type LCLs (Fig. 10A). Altogether, this experiment demonstrated that EBNA3C and Cyclin D1 positively contribute to cell growth in EBV transformed cells and are critical for overriding the G1 block as a result of serum ...
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... the results also indicated that upon knockdown of both EBNA3C and Cyclin D1, LCLs underwent a substantial degree of apoptosis (Sub G0) in response to serum starvation, similar to EBV negative cell lines, BJAB and DG75 (Fig. 10A). However, there was no sign of apoptosis observed either in BJAB cells stably expressing EBNA3C or wild-type LCLs (Fig. 10A). Altogether, this experiment demonstrated that EBNA3C and Cyclin D1 positively contribute to cell growth in EBV transformed cells and are critical for overriding the G1 block as a result of serum ...
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... promote G1-S phase transition, nuclear localization of Cyclin D1 is critical and it occurs either via decreased proteolysis in cytoplasm which facilitates nuclear import or through inhibition Figure 11. A schematic illustration of how EBNA3C regulates Cyclin D1 stability and functions to facilitate G1 to S phase transition in EBV positive cells. ...
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... this report that the essential EBV latent antigen, EBNA3C physically interacts with and stabilizes Cyclin D1 by blocking nuclear export or inhibiting the poly- ubiquitination. In addition, EBNA3C alters pRb phosphorylation as well as stability by enhancing Cyclin D1/CDK6 kinase activity, thereby nullifying pRb mediated growth suppressive activity (Fig. 11). Furthermore, knockdown of both EBNA3C and Cyclin D1 expression by lentivirus-delivered sh-RNA demonstrated that both EBNA3C and Cyclin D1 play a critical role in cell proliferation in EBV transformed cells. Thus, the present study provides an insight into the mechanisms linked to the development of EBV-associated B-cell lymphomas ...
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... EBNA3C promotes cell proliferation by stabilizing cyclin D1 and cyclin D2. It stabilizes cyclin D1 by inhibiting its polyubiquitination, inhibiting GSK-3β, which enhances cyclin D1 nuclear localization, and promoting the proteasomal degradation of RASSF1A, a tumor suppressor protein involved in cyclin D1 regulation [60][61][62]. EBNA3C also increases cyclin D2 stability by affecting the proteasomal degradation of Bcl6 [63]. Additionally, EBNA3C promotes cell proliferation by stabilizing Pim-1, inhibiting its proteasomal degradation, and ultimately leading to the downregulation of the cell cycle inhibitor p21/WAF1 [64]. ...
... Direct Interactions: USP46/USP12: EBNA3C interacts with the USP46/USP12 DUB complex, leading to EBV-associated oncogenesis [66]. Cyclin D1: EBNA3C inhibits the polyubiquitination of cyclin D1 and stabilizes it [60][61][62]. Bcl6: EBNA3C modifies the proteasomal degradation Bcl6, causing a secondary increase in cyclin D2 stability [63]. ...
The Herpesviridae include the Epstein–Barr Virus (EBV) and the Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are oncogenic gamma-herpesviruses. These viruses manipulate host cellular mechanisms, including through ubiquitin-mediated pathways, to promote viral replication and oncogenesis. Ubiquitin, a regulatory protein which tags substrates for degradation or alters their function, is manipulated by both EBV and KSHV to facilitate viral persistence and cancer development. EBV infects approximately 90% of the global population and is implicated in malignancies including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma. EBV latency proteins, notably LMP1 and EBNA3C, use ubiquitin-mediated mechanisms to inhibit apoptosis, promote cell proliferation, and interfere with DNA repair, contributing to tumorigenesis. EBV’s lytic proteins, including BZLF1 and BPLF1, further disrupt cellular processes to favor oncogenesis. Similarly, KSHV, a causative agent of Kaposi’s Sarcoma and lymphoproliferative disorders, has a latency-associated nuclear antigen (LANA) and other latency proteins that manipulate ubiquitin pathways to degrade tumor suppressors, stabilize oncogenic proteins, and evade immune responses. KSHV’s lytic cycle proteins, such as RTA and Orf64, also use ubiquitin-mediated strategies to impair immune functions and promote oncogenesis. This review explores the ubiquitin-mediated interactions of EBV and KSHV proteins, elucidating their roles in viral oncogenesis. Understanding these mechanisms offers insights into the similarities between the viruses, as well as provoking thought about potential therapeutic targets for herpesvirus-associated cancers.
... EBNA3C stabilizes CCND2 to regulate cell cycle progression [126]. EBNA3C also stabilizes CCND1 through inhibition of its polyubiquitination and EBNA3C enhances the kinase activity of Cyclin D1/CDK6 [127] ( Figure 1). EBNA3C amino acids 130-160 bind to two different sites on CCND1. ...
Epstein–Barr virus (EBV) is the first human DNA tumor virus identified from African Burkitt’s lymphoma cells. EBV causes ~200,000 various cancers world-wide each year. EBV-associated cancers express latent EBV proteins, EBV nuclear antigens (EBNAs), and latent membrane proteins (LMPs). EBNA1 tethers EBV episomes to the chromosome during mitosis to ensure episomes are divided evenly between daughter cells. EBNA2 is the major EBV latency transcription activator. It activates the expression of other EBNAs and LMPs. It also activates MYC through enhancers 400–500 kb upstream to provide proliferation signals. EBNALP co-activates with EBNA2. EBNA3A/C represses CDKN2A to prevent senescence. LMP1 activates NF-κB to prevent apoptosis. The coordinated activity of EBV proteins in the nucleus allows efficient transformation of primary resting B lymphocytes into immortalized lymphoblastoid cell lines in vitro.
... These intracellular pathogens have evolved impressive strategies to modulate the kinase activity of the Cyclin/CDK complex by interacting with the individual subunits of the heterodimer or altering the activity of CKIs. For instance, the EBV nuclear antigen-3C (E3C) interacts with the D-type cyclins D1 and D2 (Saha et al., 2011;Pei et al., 2018). E3C can form stable complexes with G 1 phase cyclins-D1 and D2/CDK-6 heterodimer [ Fig. 2a] (Saha et al., 2011;Pei et al., 2018). ...
... For instance, the EBV nuclear antigen-3C (E3C) interacts with the D-type cyclins D1 and D2 (Saha et al., 2011;Pei et al., 2018). E3C can form stable complexes with G 1 phase cyclins-D1 and D2/CDK-6 heterodimer [ Fig. 2a] (Saha et al., 2011;Pei et al., 2018). By upregulating the expression of D-type cyclin, EBV can deregulate the activities of the G 1 phase. ...
... Furthermore, an in-vitro study revealed that the N-terminal amino acids (130-159) of E3C are responsible for binding with Cyclin A and restricting the p27 KIP1 -mediated inhibition of Cyclin A/CDK-2 kinase activity (Tursiella et al., 2014). At the same time, C-terminal domain amino acids (957-990) might be playing a role in stabilizing the elements of this complex [ Fig. 2a] Saha et al., 2011). E3C might serve as a bridge between SCF Skp2 and p27 KIP1 , resulting in the degradation of p27 KIP1 , thereby relieving its inhibitory effect on the Cyclin A/CDK-2 complex (Jason S. Knight Jason et al., 2005). ...
The Epstein-Barr virus is a well-known cell cycle modulator. To establish successful infection in the host, EBV alters the cell cycle at multiple steps via antigens such as EBNAs, LMPs, and certain other EBV-encoded transcripts. Interestingly, several recent studies have indicated the possibility of EBV's neurotrophic potential. However, the effects and outcomes of EBV infection in the CNS are under-explored. Additionally, more and more epidemiological evidence implicates the cell-cycle dysregulation in neurodegeneration. Numerous hypotheses describe the triggers that force post-mitotic neurons to re-enter the cell cycle are prevalent. Apart from the known genetic and epigenetic factors responsible, several reports have shown the association of microbial infections with neurodegenerative pathology. Although, studies implicating the herpesvirus family members in neurodegeneration exist, the involvement of Epstein-Barr virus (EBV), in particular, is under-evaluated. Interestingly, a few clinical studies have reported patients of AD or PD to be seropositive for EBV. Based on the findings mentioned above, in this review, we propose that EBV infection in neurons could drive it towards neurodegeneration through dysregulation of cell-cycle events and induction of apoptosis.
... Frequent dysregulation of Bcl6 expression is involved in various B ce Furthermore, previous studies showed that EBNA3C can induce the degradation of Bcl6 protein through the ubiquitin-proteasome-dependent signaling pathway, further promoting cell proliferation, and the cell cycle by targeting Bcl2 and cyclin D1 [21]. EBNA3C directly interacts with cyclin D1 and inhibits its ubiquitination [22]. EBNA3C also stabilizes cyclin D2 by suppressing its ubiquitin-dependent degradation to facilitate cell proliferation [23]. ...
Simple Summary
Epstein–Barr virus (EBV) is the first discovered human tumor virus, which contributes to the oncogenesis of many human cancers. The ubiquitin–proteasome system is a key player during EBV-mediated oncogenesis and has been developed as a crucial therapeutic target for treatment. In this review, we briefly describe how EBV antigens can modulate the ubiquitin–proteasome system for targeted protein degradation and how they are regulated in the EBV life cycle to mediate oncogenesis. Additionally, the developed proteasome inhibitors are discussed for the treatment of EBV-associated cancers.
Abstract
Deregulation of the ubiquitin–proteasome system (UPS) plays a critical role in the development of numerous human cancers. Epstein–Barr virus (EBV), the first known human tumor virus, has evolved distinct molecular mechanisms to manipulate the ubiquitin–proteasome system, facilitate its successful infection, and drive opportunistic cancers. The interactions of EBV antigens with the ubiquitin–proteasome system can lead to oncogenesis through the targeting of cellular factors involved in proliferation. Recent studies highlight the central role of the ubiquitin–proteasome system in EBV infection. This review will summarize the versatile strategies in EBV-mediated oncogenesis that contribute to the development of specific therapeutic approaches to treat EBV-associated malignancies.
... Knockdown of Gli2 reversed the cancer-promoting effect of circcd151. cyclin d1 is a key protein regulating G 1 /S phase transformation, which can cause dna replication and division (50,51). Studies have shown that the expression of cyclind1 and Bax decreases and the expression of Bcl-2 increases following Gli2 gene silencing (52,53). ...
To investigate the changes of circular (circ)RNA circCD151 expression in lung cancer tissues and cells and its effects on proliferation, migration and invasion of lung cancer cells. The relative expression levels of circCD151 in lung cancer tissues and lung cancer cells (A549 and NCI‑H292) were determined by reverse transcription‑quantitative PCR. The effects of silencing or upregulation of circCD151 on the activity and clonal forming ability of A549 and NCI‑H292 cells were detected by CCK‑8 and cloning formation experiments. Transwell invasion assay detected the effects of silencing or upregulation of circCD151 on the migration and invasion ability of A549 and NCI‑H292 cells. The regulatory effect of circCD151 on miR‑30d‑5p was detected by dual luciferase reporter gene. The relative expression level of circCD151 in lung cancer tissues was significantly higher compared with that in adjacent tissues. The relative expression level of circCD151 in A549 and NCI‑H292 cells was significantly higher compared with that in human lung epithelial cells. In A549 and NCI‑H292 cells, silencing circCD151 decreased cell activity and clonal formation ability and invasion ability was also significantly decreased. circCD151 was upregulated in A549 and NCI‑H292 cells and the activity and clonal formation ability of A549 and NCI‑H292 cells were significantly increased and the invasion ability was also significantly increased. Double luciferase reporter assay confirmed the ceRNA regulatory mechanism of circCD151/miR‑30d‑5p/GLI2. In the present study, in vivo and in vitro functional studies demonstrated that circCD151 may promote the proliferation, invasion and cell stemness of lung cancer cells. Further molecular mechanism studies demonstrated that circCD151 could promote the malignant proliferation of lung adenocarcinoma by targeting miR‑30d‑5p and upregulating GLI2 expression. From the perspective of circRNA, the present study will provide new clues to the pathogenesis and prognostic judgment of lung adenocarcinoma and provide a new target for clinical treatment.
... Flow cytometry suggested that apatinib was involved in the inhibition of G1/S cell cycle transition in GBM cells (Fig. 5B). Cyclin D1 is an important marker of G1 phase arrest in the cell cycle 31 . Western blotting revealed that the expression (see figure on previous page) Fig. 3 NDUFA4L2 knockdown induces apoptosis, which is further enhanced by autophagy inhibitor treatment in vitro and vivo. ...
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.
... In addition, a prominent role of EBNA3C is pointed in this context as it can interact and transcriptionally regulate a wide array of cellular and viral transcriptional factors (153). For instance, EBNA3C N-terminal region was shown to complex with the N-terminal pRb binding domain and C-terminal domain of cyclin D1, which not only stabilizes cyclin D1 through the inhibition of its ubiquitin-mediated proteasomal degradation, but also enhances the functional kinase activity of cyclin D1/CDK6, thus facilitating the G1-S transition (154). Repression of p53 transcriptional activity and sparing p53-induced apoptosis in osteosarcoma cells was mediated by EBNA3C direct interaction of the latter N-terminal domain with p53 C-terminal DNA-binding and tetramerization domain (155). ...
Tumors are renowned as intricate systems that harbor heterogeneous cancer cells with distinctly diverse molecular signatures, sizes and genomic contents. Among those various genomic clonal populations within the complex tumoral architecture are the polyploid giant cancer cells (PGCC). Although described for over a century, PGCC are increasingly being recognized for their prominent role in tumorigenesis, metastasis, therapy resistance and tumor repopulation after therapy. A shared characteristic among all tumors triggered by oncoviruses is the presence of polyploidy. Those include Human Papillomaviruses (HPV), Epstein Barr Virus (EBV), Hepatitis B and C viruses (HBV and HCV, respectively), Human T-cell lymphotropic virus-1 (HTLV-1), Kaposi's sarcoma herpesvirus (KSHV) and Merkel polyomavirus (MCPyV). Distinct viral proteins, for instance Tax for HTLV-1 or HBx for HBV have demonstrated their etiologic role in favoring the appearance of PGCC. Different intriguing biological mechanisms employed by oncogenic viruses, in addition to viruses with high oncogenic potential such as human cytomegalovirus, could support the generation of PGCC, including induction of endoreplication, inactivation of tumor suppressors, development of hypoxia, activation of cellular senescence and others. Interestingly, chemoresistance and radioresistance have been reported in the context of oncovirus-induced cancers, for example KSHV and EBV-associated lymphomas and high-risk HPV-related cervical cancer. This points toward a potential linkage between the previously mentioned players and highlights PGCC as keystone cancer cells in virally-induced tumors. Subsequently, although new therapeutic approaches are actively needed to fight PGCC, attention should also be drawn to reveal the relationship between PGCC and oncoviruses, with the ultimate goal of establishing effective therapeutic platforms for treatment of virus-associated cancers. This review discusses the presence of PGCCs in tumors induced by oncoviruses, biological mechanisms potentially favoring their appearance, as well as their consequent implication at the clinical and therapeutic level.
... Cyclin D and cyclindependent kinase (CDK) have been identified as potential therapeutic targets in cancers [19]. The EBV essential antigen EBNA3C facilitates G1/S transition and cell proliferation by stabilizing Cyclin D1 and Cyclin D2 proteins in EBV-transformed cells [20,21]. LMP1 can also induce Cyclin D2 ...
... Cyclin D and cyclin-dependent kinase (CDK) have been identified as potential therapeutic targets in cancers [19]. The EBV essential antigen EBNA3C facilitates G1/S transition and cell proliferation by stabilizing Cyclin D1 and Cyclin D2 proteins in EBV-transformed cells [20,21]. LMP1 can also induce Cyclin D2 expression to mediate uncontrolled cell proliferation [22]. ...
Simple Summary
Epstein-Barr virus (EBV) is the first-discovered and important human tumor virus. It infects more than 90% of human population and induces various lymphomas. Development of specific targeted therapies is very critical for treatment of EBV-induced lymphomas, but it remains a great challenge. In this review, we introduced the current progress of EBV-specific therapies and the promising approaches that can be developed as novel targeted therapies, which involve protective or therapeutic strategies to target these lymphomas on different levels. This work will provide new insights into the development of new targeted therapies against EBV-associated lymphomas.
Abstract
The Epstein-Barr virus (EBV) is the first human tumor virus identified that can transform quiescent B lymphocytes into lymphoblastoid cell lines (LCLs) in vitro. EBV can establish asymptomatic life-long persistence and is associated with multiple human malignancies, including non-Hodgkin lymphoma and Hodgkin lymphoma, as well as infectious mononucleosis. Although EBV-associated lymphomagenesis has been investigated for over 50 years, viral-mediated transformation is not completely understood, and the development of EBV-specific therapeutic strategies to treat the associated cancers is still a major challenge. However, the rapid development of several novel therapies offers exciting possibilities to target EBV-induced lymphomas. This review highlights targeted therapies with potential for treating EBV-associated lymphomas, including small molecule inhibitors, immunotherapy, cell therapy, preventative and therapeutic vaccines, and other potent approaches, which are novel strategies for controlling, preventing, and treating these viral-induced malignances.
... Mdm2, Cyclin D1, c-Myc, Pim1) or enhance degradation of tumor suppressor proteins (viz. p53, p27 Kip1 , pRb, p21 CIP1 ) [27,28,29,30,31,32]. In addition, EBNA3C is poly-ubiquitinated at the N-terminal region and interacts with the C8/α7 subunit of the 20S proteasome [28,33]. ...
... Earlier, we have demonstrated that EBNA3C expression increases basal level of K63-linked polyubiquitination, which further increases in absence of growth promoting signals [36]. Additionally, EBNA3C was shown to recruit UPS to regulate turn-over of many important cell oncoproteins and tumor suppressors [27,28,29,30,31,74,75,76]. Initially Knight et al. demonstrated the poly-ubiquitination on the N-terminal domain of EBNA3C [28]. ...
... Myc-, flag-, GFP-and RFP-tagged EBNA3C constructs in pA3M, pA3F, pEGFP-C1 and pDsRED-Monomer-N1 vectors, respectively were previously described [27,29,36]. Myc-tagged EBNA1 expressing construct in pA3M was described earlier [90]. ...
Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.
... In Vitro cell transformation, up-regulate chemokines, (White et al., 2012) EBNA3C Co-activates ENBA2, Interact with cell cycle regulation, apoptosis and tumor suppressor proteins (Lin et al., 2002;Piovan et al., 2005;Saha et al, 2011aSaha et al, , 2011bSaha and Robertson, 2011) EBNA5 Helps in B cell transformation, Act as a transcriptional activator (Harada and Kieff, 1997;Mannick et al., 1991) LMP1 Mimic CD40 signaling, Act as an oncogene. (F Hu et al., 1993;Izumi et al., 1997;Mancao et al., 2005;Mosialos et al., 1995;Wang et al., 1985) LMP2A ...
p53, p63, and p73, the members of the p53 family of proteins, are structurally similar proteins that play central roles regulating cell cycle and apoptotic cell death. Alternative splicing at the carboxyl terminus and the utilization of different promoters further categorizes these proteins as having different isoforms for each. Among such isoforms, TA and ΔN versions of each protein serve as the pro and the anti-apoptotic proteins, respectively. Changes in the expression patterns of these isoforms are noted in many human cancers. Proteins of certain human herpesviruses, like Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), interact with p53 family members and alter their expressions in many malignancies. Upon infections in the B cells and epithelial cells, EBV expresses different lytic or latent proteins during viral replication and latency respectively to preserve viral copy number, chromosomal integrity and viral persistence inside the host. In this review, we have surveyed and summarised the interactions of EBV gene products, known so far, with the p53 family proteins. The interactions between P53 and EBV oncoproteins are observed in stomach cancer, non-Hodgkin's lymphoma (NHL) of the head and neck, Nasopharyngeal Cancer (NPC), Gastric carcinoma (GC) and Burkitt's lymphoma (BL). EBV latent protein EBNA1, EBNA3C, LMP-1, and lytic proteins BZLF-1 can alter p53 expressions in many cancer cell lines. Interactions of p63 with EBNA-1, 2, 5, LMP-2A and BARF-1 have also been investigated in several cancers. Similarly, associations of p73 isoform with EBV latent proteins EBNA3C and LMP-1 have been reported. Methylation and single nucleotide polymorphisms in p53 have also been found to be correlated with EBV infection. Therefore, interactions and altered expression strategies of the isoforms of p53 family proteins in EBV associated cancers propose an important field for further molecular research.
