FIG 5.

Viral m⁵C modifications are installed in the nucleus. (A) Western blot of cellular fractionation. Nuclear and cytoplasmic RNAs and proteins were isolated in parallel from 3T3 cells infected with MLV. Antibodies against tubulin and lamin B1 were used to assess the purity of cytoplasmic and nuclear isolations, respectively. (B) Sanger sequencing analysis of bisulfite-treated nuclear and cytoplasmic fractions isolated from infected 3T3 cells. Blue arrows indicate m⁵C site location. Results for two independent replicates are shown for each fraction. (Top) Schematic of the MLV genome with a red asterisk indicating the location of the examined m⁵C sites. The m⁵C sites are located at 817, 818, and 819 of the MLV genome (pNCA).