Fig 3.

ORF2 interacts with DNA following extraction from transfected Neuro-2A cells. Neuro-2A cells (5 × 10⁶) were transfected with plasmids expressing wt ORF2 (A) or ORF2 mutants (B and C). DNA chromatography was performed as described in Materials and Methods, with ssDNA-cellulose, dsDNA-cellulose, or blank cellulose beads. As a negative control, Neuro-2A cells were transfected with just transfection reagent. One milligram of cell extract was used for cells that expressed wt and mutant ORF2, except for the ORF2-P and ORF2-AP mutants, for which 300 μg of cell extract was incubated with DNA-cellulose. A lower concentration of cell extract was used after transfection with ORF2-AP or ORF2-P because the respective plasmids expressed higher levels of ORF2. Input (I) equivalent to 10% cell extract used in the experiments was loaded in the respective panels to verify that ORF2 was expressed. Expression of wt or mutant ORF2 in Neuro-2A cells was analyzed by Western blotting using an anti-Flag antibody. Asterisks denote ORF2-specific bands. Molecular size standards denote the positions of ORF2-specific bands.