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. 2015 Nov 18;90(3):1359-68.
doi: 10.1128/JVI.02794-15. Print 2016 Feb 1.

Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis

Hyoji Kim  1 , Hoyun Choi  1 , Suk Kyeong Lee  2
Affiliations

Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by Inhibiting BAD-Mediated caspase-3-Dependent Apoptosis

Hyoji Kim et al. J Virol. .

Abstract

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and the in vivo triggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genes BRLF1 and BZLF1 as well as Bcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels of BRLF1 and BZLF1 were suppressed in cells following BAD knockdown and increased after BAD overexpression. Progeny virus production was also downregulated by specific knockdown of BAD. Our results demonstrated that caspase-3-dependent apoptosis is a prerequisite for BAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibiting BAD-mediated caspase-3-dependent apoptosis, which would trigger immediate early gene expression.

Importance: EBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressing BAD-induced caspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

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Figures

FIG 1
FIG 1
Effect of LNA-miR-BART20-5p(i) and siBAD on BRLF1, BZLF1, and BAD expression. AGS-EBV cells were transfected with 50 nM control LNA, LNA-miR-BART20-5p(i), or LNA-miR-BART20-5p(i) plus siBAD. After 24 h, the cells were treated with 5 nM TPA for 48 h and then harvested for RNA and protein preparation. (A) Real-time RT-PCR analysis of BRLF1, BZLF1, and BAD was carried out by using a SYBR green qPCR kit. The RT-PCR results from three independent experiments were normalized to GAPDH levels and are expressed as ratios relative to the values obtained from control LNA-transfected cells. (B) BRLF1, BZLF1, and BAD protein levels were analyzed by Western blot analysis. (C) Western blot results similar to those shown in panel B were obtained by using two additional sets of independently transfected AGS-EBV cells. Western blot results were normalized to α- tubulin values and are expressed as ratios relative to the values obtained from control LNA-transfected cells. Mean values from all three experiments are plotted. Error bars indicate SD (n = 3). *, P < 0.05; †, P < 0.01.
FIG 2
FIG 2
Effect of dually specific siBRLF1/BZLF1 and siBAD on BRLF1, BZLF1, and BAD expression. (A) Schematic drawing showing the binding sites of siBRLF1/BZLF1 on BRLF1 and BZLF1. (B to D, left) AGS-EBV cells were treated with the vehicle (dimethyl sulfoxide [DMSO]) or 5 nM TPA for 48 h and harvested for RNA (B) and protein (C and D) preparation. AGS-EBV cells were transfected with 30 nM siBAD, siBRLF1/BZLF1, siBAD plus siBRLF1/BZLF1, or the scrambled control. (Right) After 24 h, transfected cells were stimulated with TPA for 48 h. (B) BRLF1, BZLF1, and BAD mRNA levels were detected by real-time RT-PCR. The RT-PCR results from three independent experiments were normalized to GAPDH values and are expressed as ratios relative to the values obtained from scrambled control-transfected cells. (C) BRLF1, BZLF1, and BAD protein levels were analyzed by Western blot analysis. (D) Western blot results similar to those shown in panel C were obtained by using two additional sets of independently transfected AGS-EBV cells. Mean values from all three experiments are plotted. Error bars indicate SD (n = 3). *, P < 0.05; †, P < 0.01.
FIG 3
FIG 3
Overexpression of BAD upregulates BRLF1 and BZLF1 expression. AGS-EBV cells were transfected with pCEP4-BAD-CDS, pCEP4-BAD-CDS+3′-UTR, or the empty vector (pCEP4). (A) Real-time RT-PCR and Western blot results confirm that BAD is overexpressed in AGS-EBV cells transfected with pCEP4-BAD-CDS or pCEP4-BAD-CDS+3′-UTR. RT-PCR results have been normalized to GAPDH values and are expressed as ratios relative to the values obtained from cells transfected with pCEP4. (B) At the indicated times after transfection, 10 μl of CCK-8 solution was added to each well to assess cell proliferation. O.D, optical density. (C and D) Appropriately transfected cells were treated with 5 nM TPA for 48 h and harvested for real-time RT-PCR (C) or Western blotting (D). (E) Western blot results similar to those shown in panel D were obtained by using two additional sets of independently transfected AGS-EBV cells. Mean values from all three experiments are plotted. Error bars indicate SD (n = 3). *, P < 0.05; †, P < 0.01.
FIG 4
FIG 4
Effect of BAD, BRLF1, and BZLF1 on apoptosis. (A to C) AGS-EBV cells were transfected with 30 nM siBAD, siBRLF1/BZLF1, siBAD plus siBRLF1/BZLF1, or the scrambled control. (D to F) AGS-EBV cells were transfected with 50 nM control LNA, LNA-miR-BART20-5p(i), or LNA-miR-BART20-5p(i) plus siBAD. Twenty-four hours after transfection, transfected cells were treated with 5 nM TPA for 48 h. (A and D) Expression levels of the cleaved forms of the PARP and caspase-3 proteins were detected by Western blot analysis. (B and E) The ratio of cleaved PARP to the uncleaved form observed in the three independent Western blot experiments shown in panels A and D. (C and F) The expression levels of cleaved caspase-3 in transfected cells were normalized to α-tubulin levels and are expressed as ratios relative to the values obtained from the control. Error bars indicate SD (n = 3). *, P < 0.05; †, P < 0.01.
FIG 5
FIG 5
Regulation of BRLF1 and BZLF1 by caspase-3-dependent apoptosis. (A and C to E) AGS-EBV cells were transfected with 30 nM siCASP3 or the scrambled control. (B and F to H) AGS-EBV cells were transfected with 50 nM control LNA, LNA-miR-BART20-5p(i), or LNA-miR-BART20-5p(i) plus siCASP3. After 24 h, transfected cells were treated with 5 nM TPA for 48 h. (A and B) BRLF1, BZLF1, and caspase-3 mRNA levels were quantified by real-time RT-PCR. (C and F) Protein levels of BRLF1 and BZLF1, as well as cleaved forms of PARP and caspase-3, were measured by Western blotting. (D and G) Expression levels of cleaved caspase-3, BRLF1, and BZLF1 in transfected cells were normalized to α-tubulin levels and are expressed as ratios relative to the values obtained from the control. (E and H) Ratios of cleaved PARP to the uncleaved form observed in three independent Western blot experiments shown in panels C and F. Error bars indicate SD (n = 3). *, P < 0.05; †, P < 0.01.
FIG 6
FIG 6
Suppression of viral production by siRNAs for BAD and caspase-3. (A and C) AGS-EBV cells were transfected with 30 nM siRNAs or the scrambled control. (B and D) AGS-EBV cells were transfected with 50 nM control LNA, LNA-miR-BART20-5p(i), or LNA-miR-BART20-5p(i) plus siRNA. Twenty-four hours after transfection, cells were treated with 5 nM TPA for 72 h. Cell supernatants were assayed in order to detect the EBV genome by real-time PCR. The ratios of the levels of viral production to the amount obtained from the control are shown. Error bars indicate SD (n = 3). †, P < 0.01.
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