FIG 6.

Prediction and mapping of an NLS in p012. (A) Schematic representation of the C-terminal 50 amino acids (aa) of p012. The position of two putative overlapping nuclear localization signals is indicated (boxed and grey/underlined). (B) Mutational disruption of the p012 NLS in MDV v20 inhibits nuclear accumulation. CEFs were infected with v20_012Flag, a C-terminal deletion mutant (v20_012ΔNLSFlag), or an alanine substitution mutant corresponding to the predicted short NLS (v20_012mutshortNLS) (bottom). At 5 days p.i., the cells were fixed, permeabilized, and stained with polyclonal rabbit anti-Flag antibody. Arrows indicate cells with representative localization of p012. (C) Mutational mapping of essential NLS regions in p012. Alanine substitutions in the NLS are indicated in gray. DF-1 cells were transfected with pc012Flag, pc012FlagmutRS, or pc012FlagmutshortNLS. Images of representative results, as well as the quantification of three independent experiments, are shown.