N(6)-methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay (2021)
Figure 3

Figure 3. Knockdown of YTHDF1 promotes EBV infection and replication

A. The knockdown efficiency of METTL3, METTL14, FTO, YTHDF1, YTHDF2 and YTHDF3 in HK1 and CNE2EBV cells at 24 h after siRNA transfection was determined by western blot analysis. ACTB was used as loading control.

B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 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. ns, **P<0.01 and ***P<0.001 compared to siNC according to unpaired Student's t-test.

C. The ratio of GFP-positive HK1 cells transfected with ALKBH5-specific siRNAs (siA5-1 and siA5-2) or the siNC control, was quantified using FACS. The experiments were performed similarly as described for (B) except for the different siRNA transfection. At 24 h post siRNA transfection, the protein level of ALKBH5 was determined by western blot analysis. ACTB was used as loading control. 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 siNC according to unpaired Student's t-test.

D. The GFP-positive cells of the EBV reactivated CNE2EBV cells transfected with indicated siRNAs. After 24 h of siRNA transfection, the cells were induced with NaB (2 mM) for 24 h. The GFP-positive cells were quantified using FACS. The mean value of the GFP-positive rates in the siNC transfected 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. ns, *P<0.05, **P<0.01 and ***P<0.001 compared to siNC according to unpaired Student's t-test.

E. The GFP-expressed cells were visualized by fluorescence microscopy after YTHDF1 knockdown in HK1 cells following EBV infection and CNE2EBV cells with induced EBV reactivation. The experiments were performed similarly as described for (B) in HK1 cells and (D) in CNE2EBV cells. Representative data from three independent experiments are shown. Scale bars: 50 μm.

F. The knockdown efficiency of YTHDF1 in HK1, CNE2EBV and C666 cells, transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control for 24 h, was determined by qRT-PCR. The mRNA levels of YTHDF1 were normalized to the housekeeping gene ACTB. 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 compared to siNC according to unpaired Student's t-test.

G, H. The HK1 cells were transfected with YTHDF1-specific siRNA (siY1-1 and siY1-2) or the siNC control for 24 h, and then infected with EBV-GFP virus. At 24 hpi, the ratio of GFP-positive cells was quantified using FACS (G), and the number of EBV copies was quantified using qPCR (H). The EBV copies were determined using a specific primers targeting BamHI-W fragment region of EBV, and GAPDH was used as a reference genome copy. 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 siNC according to unpaired Student's t-test.

I, J. CNE2EBV cells were transfected with YTHDF1-specific siRNA (siY1-1 and siY1-2) or the siNC control for 24 h, and then induced with NaB (2 mM). At 24 h post NaB-induced EBV reactivation, the GFP-positive cells (I) and the EBV copies (J) were quantified as described for (G and H). 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 siNC according to unpaired Student's t-test.

K. The relative number of EBV copies in C666 cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was quantified using qPCR. At 24 h after siRNA transfection, the EBV copies were quantified as described for (H). The mean value of EBV copies in the siNC cells 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<0.05 and **P<0.01 compared to siNC according to unpaired Student's t-test.

L. The relative production of EBV virions in CNE2EBV cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control following EBV reactivation, was measured by qPCR. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) for 24 h, and the DNA from the cell-free culture supernatant was harvested for qPCR. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC 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<0.05 and **P<0.01 compared to siNC according to unpaired Student's t-test.

M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells 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<0.01 and ***P<0.001 compared to siNC according to unpaired Student's t-test.

N. FACS analysis of gp350 expression in CNE2EBV cells transfected with YTHDF1-specific siRNA (siY1-1 and siY1-2) or the siNC control following EBV reactivation. To measure gp350 expression in the reactivated CNE2EBV cells, cells were stained with anti-gp350 mAb (72A1) followed by an Alexa Fluor 594-conjugated secondary anti-mouse antibody and analyzed by FACS. The quantification of the ratio of GFP-positive and gp350-positive (GFP+/gp350+) cells from three independent experiments was shown. 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 siNC according to unpaired Student's t-test.