N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay (2021)
Figure 2

Figure 2. The BZLF1 mRNA is m6A modified

A. m6A peak distribution of BZLF1 in different EBV infection stages was analyzed based on MeRIP-seq data. The data presented from the top down are the EBV acute infected (24 hpi) NPEC1-Bmi1 sphere-like cells (NPEC-sp), and HK1 cells; latently infected CNE2EBV cells; and CNE2EBV cells with reactivated EBV induced using NaB alone or TPA/NaB together. For acute EBV infection, RNA was harvested at 24 hpi. TPA (30 ng/ml) and NaB (2 mM) were used alone or together to treat the cells for 24 h to reactivate EBV. The PA-m6A-seq peak is indicated by the green box, and the potential m6A sites on the indicated sequence are shown in red. The region indicated by the dotted box was PCR-amplified to construct the wild-type BZLF11-262nt plasmid.

B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells 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 and ***P<0.001 compared to mock-treated cells according to unpaired Student's t-test.

C. The relative product abundance of SELECT at the predicted m6A sites in the latently infected or lytic reactivated CNE2EBV cells. TPA (30 ng/ml) and NaB (2 mM) were used to treat the CNE2EBV cells for 24 h to reactivate EBV. The mean value of results in mock-treated cells 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<0.001 compared to the mock-treated group according to unpaired Student's t-test.

D. The validation of the BZLF1 m6A peaks by MeRIP-qPCR in PDXs, NPC1 and NPN. One nontumor control biopsy (NPN), one NPC sample (NPC1), and two PDX samples (PDX-NPC04L and PDX-NPC03W) were used to perform the MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The data represent the means ± SD of n=3 technical replicates.

E. Construction of the WT-BZLF1 and mut-BZLF1 plasmids. The WT-BZLF1 plasmid was constructed by inserting the BZLF11-262nt fragment (1-262 nt of the BZLF1 mRNA) into pcDNA3.1+ plasmid. The mut-BZLF1 plasmid was constructed by introducing A>T mutations at the putative m6A sites of BZLF11-262nt.

F. The IgG or anti-m6A antibody enrichment of the WT-BZLF1 and mut-BZLF1 mRNAs was measured by RIP-qPCR in the CNE2EBV cell lines. Blank indicates CNE2EBV cells without plasmids transfection. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample. The mean value of the m6A enrichment level on WT-BZLF1 mRNA was normalized to 1. Experiments were independently repeated four times, and the results are represented as the means ± SD of n=4 biological replicates. ***P<0.001 according to unpaired Student's t-test.