Mutated m⁶A sites in VPm function in the ORF of VP1.

<p>(A) The SV40 VP1 gene was cloned from SV40 WT or VPm using PCR and then inserted into an expression plasmid with the addition of a FLAG epitope tag. Transfected 293T cells were probed for VP1 expression by Western blot, with endogenous GAPDH used as a loading control and GST used as a transfection control. (B) The expression level of VP1 from three independent transfection experiments was quantified and normalized to the GST control. The level of VP1 protein detected in cells transfected with the wild type VP1 cDNA were then set at 1.0. (C) VP1 mRNA levels were quantified in parallel using qRT-PCR. No reverse transcriptase (RT-) values shown in red as a negative control. (D) Subcellular fractionation of vector (-), VP1 (WT), or VPm-transfected cells, with Lamin A/C as the nuclear fraction marker and GAPDH as the cytosolic marker. (E) VP1 mRNA levels in the nuclear and cytosolic fractions were quantified in parallel using qRT-PCR. Error bars = SD. **p<0.005 by two-tailed T test.