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

Cell culture and reagents.

AGS-EBV is a gastric carcinoma cell line derived from AGS cells infected with a recombinant Akata virus (40–42). Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml of G418 (Gibco, Carlsbad, CA, USA). BAD expression plasmid-transfected cells were cultured in RPMI 1640 medium supplemented with 200 μg/ml hygromycin B (Invitrogen, San Diego, CA, USA) in order to select transfectants for 2 weeks before analysis. All cells were maintained at 37°C in a 5% CO₂ incubator.

Plasmid construction.

The BAD coding sequences (BAD-CDSs) with or without the 3′ UTR (NCBI GenBank accession number NM_004322) were amplified by using cDNA prepared from AGS-EBV cells to obtain BAD-CDS+3′-UTR and BAD-CDS, respectively. The amplicons were cloned into the HindIII/BamHI sites of the pCEP4 vector (Invitrogen) by using an EZ-Fusion cloning kit (Enzynomics, Daejeon, South Korea). The constructed BAD expression vectors (pCEP4-BAD-CDS and pCEP4-BAD-CDS+3′-UTR) contained a hygromycin selection marker for enrichment of transfected cells. The sequences of the primers used for each plasmid construct were as follows: 5′-CCAGCTGCTAGCAAGCTTATGTTCCAGATCCCAGAGTT-3′ and 5′-CTTATCATGTCTGGATCCTCACTGGGAGGGGGCGGAGC-3′ for pCEP4-BAD-CDS and 5′-CCAGCTGCTAGCAAGCTTATGTTCCAGATCCCAGAGTT-3′ and 5′-CTTATCATGTCTGGATCCCGGCGGCACAGACGCGGGCTT-3′ for pCEP4-BAD-CDS+3′-UTR.

Transfection and TPA treatment.

The locked nucleic acid (LNA)–miR-BART20-5p inhibitor [LNA-miR-BART20-5p(i)] (5′-GAATGAAGACATGCCTGCT-3′) (catalog number 426096-00) and the control LNA-miRNA inhibitor (control LNA) (5′-GTGTAACACGTCTATACGCCCA-3′) (catalog number 199004-00) were purchased from Exiqon (Vedbaek, Denmark). The scrambled control (5′-ACGUGACACGUUCGGAGAAUU-3′) was purchased from Genolution Pharmaceuticals (Seoul, South Korea). The scrambled control and control LNAs were used as negative controls for miRNA mimics and LNA inhibitors, respectively. For experiments, 1 × 10⁶ cells were seeded into 100-mm-diameter dishes containing 10 ml culture medium 24 h prior to transfection. All transfection experiments were performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols. After 24 h, cells were treated with 5 nM TPA for 48 or 72 h to induce the EBV lytic cycle.

Quantitative reverse transcription-PCR (RT-PCR).

AGS-EBV cells were harvested and total RNA was extracted by using RNAzol B reagent (Tel-Test, Friendswood, TX, USA) according to the manufacturer's instructions. cDNA was synthesized by using 1 μg total RNA, oligo(dT) primers (Ahram Biosystems, Seoul, South Korea), and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Real-time PCR was carried out by using a SYBR green quantitative PCR (qPCR) kit (TaKaRa, Tokyo, Japan) with an Mx3000p real-time PCR system (Stratagene, La Jolla, CA, USA). The sequences of the primers used for each gene were as follows: 5′-GCTCCGGCAAGCATCATC-3′ and 5′-GGTAGGAGCTGTGGCGACT-3′ for BAD, 5′-GGCTAACCAAGGACAACAGC-3′ and 5′-GAAGCCACCCGATTCTTGTA-3′ for BZLF1, 5′-GTGTTCCACAGCCTGCAC-3′ and 5′-GAAGCCACCCGATTCTTGTA-3′ for BRLF1, 5′-TCGGTCTGGTACAGATGTCG-3′ and 5′-GGCTCAGAAGCACACAAACA-3′for caspase-3, and 5′-ATGGGGAAGGTGAAGGTCG-3′ and 5′-GGGGTCATTGATGGCAACAATA-3′ for the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). PCR conditions were 95°C for 5 min followed by 40 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. To confirm the purity of the PCR products, dissociation curves were checked routinely. To accomplish this, reaction mixtures were incubated at 95°C for 60 s and ramped from 60°C to 95°C at a rate of 0.1°C/s with continuous measurement of fluorescence. Relative gene expression was calculated by the comparative threshold cycle (C_T) method using GAPDH as an internal loading control.

Cell proliferation assay.

Cell proliferation was analyzed by using Cell Counting kit 8 (CCK-8; Dojindo Molecular Technologies, Tokyo, Japan). BAD-overexpressing cells (1 × 10³ cells/well) were seeded into a 96-well plate. After the indicated periods, 10 μl of CCK-8 solution was added to each well. The absorbance at a wavelength of 450 nm was measured after 2 h by using a SoftMax apparatus (Molecular Devices, Sunnyvale, CA, USA).

Small interfering RNA sequences.

Small interfering RNAs (siRNAs) and a negative-control siRNA lacking any known target gene product were synthesized by Genolution Pharmaceuticals. The siBAD siRNA was specific for BAD, while siBRLF1/BZLF1 had dual specificity for BRLF1 and BZLF1. The siRNA specific for caspase-3 (siCASP3) was purchased from Bioneer Corporation (Daejeon, South Korea). The sequence of the negative-control siRNA was 5′-ACGUGACACGUUCGGAGAAUU-3′. The sequences of the siRNAs were as follows: 5′-CGACAUAACCCAGAAUCAACA-3′ for siBRLF1/BZLF1, 5′-GUACUUCCCUCAGGCCUAU-3′ for siBAD, and 5′-AGUAUGCCGACAAGCUUGA-3′ for siCASP3.

Western blot analysis.

Cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 10 μg/ml aprotinin). The cell lysate (50 μg protein) was mixed with 5× loading buffer (Fermentas, Waltham, MA, USA) and heated at 95°C for 5 min. Samples were separated by electrophoresis on 12.5% sodium dodecyl sulfate-polyacrylamide gels, and the separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). To detect the expression of EBV lytic proteins and apoptosis-related genes, anti-BZLF1 (1:500; Dako, Glostrup, Denmark), anti-BRLF1 (1:500; Argene, Verniolle, France), anti-BAD (1:1,000; Cell Signaling Technology, Beverly, MA, USA), anti-poly(ADP-ribose) polymerase (PARP) (1:500; Becton Dickinson, San Diego, CA, USA), and anti-cleaved caspase-3 (1:500; Cell Signaling Technology) antibodies were used. After washing, the blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) at a dilution of 1:5,000 for 1 h at room temperature. Anti-α-tubulin antibody (Cell Signaling Technology) was used to confirm comparable loading between gel lanes. Protein bands were visualized by using an enhanced chemiluminescence detection system (Amersham Bioscience), and the membrane was exposed to X-ray film (Agfa, Mortsel, Belgium). The density of each protein band was quantified by using Fujifilm Multi Gauge software (version 3.0).

Quantitative PCR to assess EBV genome copy numbers.

AGS-EBV cells (1 × 10⁶) were seeded into a 100-mm-diameter dish. After 24 h, the cells were transfected with siRNAs (30 nM) or the LNA-miR-BART20-5p inhibitor (50 nM). After 24 h, the cultures were refreshed with new RPMI 1640 medium containing 5 nM TPA to induce EBV lytic replication. The cells were incubated at 37°C in a 5% CO₂ incubator for 3 days, allowing the production of progeny viruses. Cells were removed by centrifugation for 5 min at 800 × g at room temperature, and the supernatant was then passed through a 0.45-μm-pore-size filter (Nalgene, Rochester, NY, USA). The filtrate was ultracentrifuged by using an SW41 rotor (Beckman Instruments, Fullerton, CA, USA) at 75,000 × g for 2 h at 4°C. The pellet was then resuspended in 200 μl 0.2× phosphate-buffered saline and heated to 95°C for 15 min. Next, 20 μl of 20 mg/ml proteinase K was added to the suspension, and the mixture was incubated at 56°C for 1 h, followed by heat inactivation at 95°C for 30 min. Real-time PCR amplification of EBNA1 was carried out by using a SYBR green qPCR kit (TaKaRa, Tokyo, Japan) with an Mx3000P real-time PCR system (Stratagene). The sequences of the EBNA-1 primers were 5′-AGTCGTCTCCCCTTTGGAAT-3′ (sense) and 5′-TCCTCACCCTCATCTCCATC-3′ (antisense). The relative viral copy number was calculated as described previously (27).

Statistical analyses.

Data were analyzed by using the Student t test. Cell proliferation assay results were analyzed by two-way repeated-measure analysis of variance. Curve fitting and analyses were performed by using GraphPad Prism (GraphPad Software, San Diego, CA, USA). P values of <0.05 were considered to reflect statistically significant differences. All results were expressed as means ± standard deviations (SD).

siBAD abrogates the enhancing effect of the miR-BART20-5p inhibitor on expression of EBV immediate early genes.

To confirm that miR-BART20-5p targeted the EBV immediate early genes and BAD, AGS-EBV cells were transfected with LNA-miR-BART20-5p(i). After induction of the EBV lytic cycle by TPA treatment, expression levels of the EBV immediate early genes BRLF1 and BZLF1, as well as the BH3-only protein gene BAD, were analyzed by real-time RT-PCR and Western blotting. Expressions of the BRLF1 and BZLF1 proteins in AGS-EBV cells were readily detected by Western blotting after 48 h when tested at different time points after treatment with 5 nM TPA. In addition, when cells were treated with TPA for 48 h, the most clear effect of miR-BART20-5p was observed (data not shown). Both mRNA and protein levels of BRLF1, BZLF1, and BAD were increased following transfection with LNA-miR-BART20-5p(i) compared with the control LNA (Fig. 1A to C).

When AGS-EBV cells were cotransfected with siBAD and LNA-miR-BART20-5p(i), siBAD abrogated the enhancing effect of LNA-miR-BART20-5p(i) on the mRNA expression of BRLF1, BZLF1, and BAD (Fig. 1A). Western blot results also showed that the effect of LNA-miR-BART20-5p(i) on the expression of these three genes was completely abolished by siBAD (Fig. 1B and C).

BAD affects the expression of BRLF1 and BZLF1.

To investigate whether BAD affected the expression of BRLF1 and BZLF1 in AGS-EBV cells, siBAD and siBRLF1/BZLF1 were used. It should be noted that siBRLF1/BZLF1 is capable of targeting the two genes simultaneously, as BZLF1 is encoded within the 3′ UTR of BRLF1 (Fig. 2A). Effective induction of the EBV lytic cycle in AGS-EBV cells was confirmed by the expression of BRLF1 and BZLF1 following TPA treatment (Fig. 2B to D, left). TPA treatment showed a tendency to reduce BAD mRNA and protein levels slightly. However, no statistical significance was found on the protein level.

The expression levels of BAD, BRLF1, and BZLF1 were determined by real-time RT-PCR and Western blotting of TPA-treated AGS-EBV cells following transfection with the scrambled control, siBAD, siBRLF1/BZLF1, or siBAD plus siBRLF1/BZLF1. The expression levels of BRLF1 and BZLF1 were reduced to similar levels by siBAD and siBRLF1/BZLF1 (Fig. 2B to D, right). An additive effect on the expression of the BRLF1 and BZLF1 mRNAs was observed when AGS-EBV cells were cotransfected with siBAD and siBRLF1/BZLF1 compared to cells transfected with either of these siRNAs (Fig. 2B, right). In contrast, siBRLF1/BZLF1 did not affect the expression of BAD (Fig. 2B, right). Similar results were observed in Western blot experiments (Fig. 2C and D, right). However, the additive effect of siBAD and siBRLF1/BZLF1 shown for the mRNA levels of the two EBV immediate early genes was not obvious when the protein levels of BRLF1 and BZLF1 were assessed by Western blotting (Fig. 2C and D, right).

Overexpressed BAD upregulates the expression of BRLF1 and BZLF1.

The BAD expression vectors pCEP4-BAD-CDS and pCEP4-BAD-CDS+3′-UTR were used to examine the effect of BAD overexpression on the expression of BRLF1 and BZLF1. AGS-EBV cells were transfected with each of the BAD expression vectors or an empty vector (pCEP4) as a control. After selection of transfected cells following 2 weeks of treatment with 200 μg/ml hygromycin, the cells were harvested to assess BAD expression levels. Real-time RT-PCR and Western blotting revealed that transfection with pCEP4-BAD-CDS and pCEP4-BAD-CDS+3′UTR significantly upregulated BAD expression in AGS-EBV cells (Fig. 3A). The BAD expression level was slightly higher following transfection with pCEP4-BAD-CDS than with pCEP4-BAD-CDS+3′-UTR (Fig. 3A). We then investigated the effect of BAD overexpression on cell proliferation. A CCK-8 assay showed that cell proliferation decreased following BAD overexpression and that cell proliferation seemed to inversely correlate with the level of BAD expression (Fig. 3B).

Next, we performed real-time RT-PCR and Western blot analysis to assess the expression levels of BRLF1 and BZLF1 in 5 nM TPA-treated BAD-overexpressing cells. BAD expression levels in BAD-overexpressing cells were reduced by TPA treatment (Fig. 3A versus C). This is similar to the results shown in Fig. 2B (left). Real-time RT-PCR and Western blotting revealed that the expression levels of BRLF1, BZLF1, and BAD in BAD-overexpressing cells were significantly higher than those in control cells. The expression levels of BRLF1, BZLF1, and BAD were noticeably higher in cells transfected with pCEP4-BAD-CDS than in those transfected with pCEP4-BAD-CDS+3′-UTR (Fig. 3C to E).

Induction of BRLF1 and BZLF1 by BAD is mediated by caspase-3.

To investigate the effect of EBV immediate early genes and BAD on apoptosis, the expression levels of PARP and caspase-3 were analyzed by Western blotting following TPA treatment in cells transfected with the siRNAs for BAD and/or BRLF1/BZLF1. When AGS-EBV cells were transfected with siBAD, the cleaved forms of PARP and caspase-3 were reduced by 60%. In contrast, siBRLF1/BZLF1 did not affect the ratios of cleaved PARP and caspase-3 (Fig. 4A to C). In addition, expression levels of both cleaved PARP and caspase-3 were increased following transfection with LNA-miR-BART20-5p(i), and this LNA inhibitor effect was completely counteracted by siBAD cotransfection (Fig. 4D to F).

We also conducted experiments to test whether the expression of the EBV immediate early genes was dependent on caspase-3. To accomplish this, AGS-EBV cells were transfected with a siRNA specific for caspase-3 (siCASP3) or a scrambled control and then treated with TPA before the expression levels of caspase-3, BRLF1, and BZLF1 were compared. Real-time RT-PCR results showed that the expression levels of BRLF1, BZLF1, and caspase-3 were reduced by siCASP3 (Fig. 5A). Similarly, AGS-EBV cells treated with TPA showed reduced BRLF1 and BZLF1 protein levels when transfected with siCASP3 compared with those transfected with the scrambled control (Fig. 5C and D). Similar reductions were also observed for cleaved PARP and caspase-3 following siCASP3 transfection (Fig. 5C). The ratio of cleaved PARP to the uncleaved form was decreased by up to 60% by siCASP3 (Fig. 5E). In contrast, transfection with LNA-miR-BART20-5p(i) increased the expression levels of cleaved caspase-3 and PARP as well as BRLF1 and BZLF1; however, these effects were eliminated by cotransfection with siCASP3 (Fig. 5B and F to H).

miR-BART20-5p inhibits virus production by targeting viral and cellular genes.

In order to determine whether apoptosis induced not only the expression of EBV lytic genes but also viral particle production, EBV genome copy numbers were analyzed. The level of viral particle production was significantly decreased following TPA treatment when cells were transfected with siBAD and/or siBRLF1/BZLF1 (Fig. 6A). Conversely, EBV genome copy numbers were increased >2.5-fold by TPA treatment in cells transfected with LNA-miR-BART20-5p(i) compared with cells transfected with the control LNA (Fig. 6B). When AGS-EBV cells were cotransfected with LNA-miR-BART20-5p(i) and siBAD, the level of viral particle production was significantly decreased compared with that in cells transfected with LNA-miR-BART20-5p(i) only (Fig. 6B). Results similar to those shown in Fig. 6A and B were also obtained when digital droplet PCR was used to measure viral DNA titers (data not shown).

The effect of siCASP3 on progeny virus production was also assessed following TPA treatment. The level of viral particle production was reduced by 50% when cells were transfected with siCASP3 compared to that in cells transfected with the scrambled control (Fig. 6C). In addition, cotransfected siCASP3 completely abolished the enhancing effect of LNA-miR-BART20-5p(i) on viral production (Fig. 6D).