N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay (2021)
Figure 5

Figure 5. Knockdown of YTHDF1 prolongs the half-life of EBV transcripts

A, B. The relative YTHDF1-RIP enrichment ratio of the indicated genes in C666 cells (A) and CNE2EBV cells with induced EBV reactivation (24 h post TPA/NaB treatment) (B). The EEF1A1 mRNA known to contain m6A sites and RPL30 mRNA containing no m6A modification were used as positive and negative controls, respectively. BALF1 and BCRF1 containing no m6A modification, identified in this study, were used as viral negative controls. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample. The mean of the fold enrichment by IgG was normalized to 1. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. ns, **P<0.01 and ***P<0.001 compared to IgG according to unpaired Student's t-test.

C. The relative Myc-RIP enrichment ratios of wild-type and mutant BZLF11-262nt mRNA in CNE2EBV cells. CNE2EBV cells were co-transfected with wild-type or mutant BZLF11-262nt and myc-YTHDF1 or vector control for 24 h. The fold Myc-RIP enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample. The mean of the fold Myc-RIP enrichment level on WT-BZLF1 mRNA was normalized to 1. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. **P<0.01 according to unpaired Student's t-test.

D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. ns, not significant according to unpaired Student's t-test.

E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates. *P<0.05, **P<0.01 and ***P<0.001 according to unpaired Student's t-test.

G. Residual mRNA levels of BZLF1 and BRLF1 after termination of transcription via ActD treatment in C666 cells transfected with YTHDF1-specific siRNAs or the siNC control. After 24 h of siRNA transfection, the C666 cells were treated with ActD and the mRNA levels analyzed by qRT-PCR. The relative mRNA level at 0 h after the ActD treatment was normalized to 1. Experiments were independently repeated four times, and the results are represented as the means ± SD of n=4 biological replicates.

H. Residual mRNA levels of BZLF1 and BRLF1 after termination of transcription via ActD treatment in CNE2EBV cells transfected with YTHDF1-specific siRNAs or the siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were induced with NaB (2 mM) for 24 h, treated with ActD and analyzed by qRT-PCR. The relative mRNA level at 0 h after the ActD treatment was normalized to 1. Experiments were independently repeated three times, and the results are represented as the means ± SD of n=3 biological replicates.