Figure 5.

Direct evidence for the role of E2 in chromosomal attachment. E2 protein colocalized with mitotic chromosomes, whereas the A4 mutant protein did not. Cells were cotransfected with the neomycin-resistance gene and BPV genomes that do not express the repressor forms of E2 and fixed after drug selection at day 10 (at which point about half of the cells were neo^r and contained BPV DNA because of the time course of neomycin killing). E2 protein was detected by B201 monoclonal antibody followed by Cy3-conjugated secondary antibody, and DNA was detected by DAPI staining. The B201 antibody recognizes the wild-type and mutant forms of E2 equally, as demonstrated by the equivalent interphase signals obtained (panels labeled Inter and cells labeled with ∗). Arrows indicate the positions of condensed chromosomes in cells at the metaphase or anaphase stages of mitosis. Two metaphase and two anaphase examples are shown for the A3 and A4 samples. Quantitatively, 14 of 26 mitotic cells transfected with the A3 virus contained chromosomal staining, whereas 0 of 17 mitotic cells transfected with the A4 virus showed this colocalization. Interphase nuclei for both A3 and A4 samples stain for E2. Panels labeled Neo show that the E2 reagents do not stain interphase or anaphase nucleoprotein when BPV genomes were not cotransfected.