Figure 3

Binding domain identification of PCV3 Cap with NPM1. A Schematic representation of the truncation mutants of PCV3 Cap used in this study. B, C The N-terminal nuclear localization signal of PCV3 Cap interacted with NPM1. HEK293T cells were cotransfected with plasmids encoding full-length PCV3 Cap or truncation mutants fused with a GFP-, or Flag-gst tag, along with Flag-NPM1; cell lysates were subjected to immunoprecipitation or GST pull-down and immunoblotting using the indicated antibodies. D, E The nuclear localization signals (NLSs) within capsid protein of porcine circovirus type 1, 2, 3, 4 and circoviruses from other species were responsible for the binding to NPM1. HEK293T cells were cotransfected with plasmids encoding NLSs of PCV1, 2, 3, 4 (D) and circoviruses from terrestrial, aquatic and avian species, including pigs, canaries, canines, minks, dragonflies, pigeons, ducks, bats, geese, and parrots (E), along with Flag-NPM1; cell lysates were subjected to immunoprecipitation and immunoblotting using the indicated antibodies. F Identification of the nucleolar localization signal in PCV3 Cap. HEK293T cells were cotransfected with GFP-NCL and mCherry-PCV3-Cap-WT, -M1, or -M2 for 24 h. The resultant cells were fixed, stained with DAPI and subjected to confocal microscopy analysis. Scale bar, 10 μm. G Amino acid sequence alignment of the nucleolar localization signal from different PCV3 genotypes.