Next, to confirm that VP1 not only has DNA binding activity but also has nuclear localization activity, we constructed a transit expression plasmid, pEGFP-VP1, which is a pcDNA3.1 vector carrying the VP1 gene fused to a GFP gene for cell transfection (Fig. 3a). When pEGFP-VP1 was respectively transfected into chicken lymphocytes, MDCC-MSB1 cells and Chinese Hamster Ovary (CHO) K1, the localization of GFP fluorescence was observed using confocal microscopy (Fig. 3b and c). As illustrated in Fig. 3b and c, GFP-VP1 and DAPI staining coincided significantly in the nuclei of MDCC-MSB1 cells (Fig. 3c). Additionally, GFP-VP1 was partially distributed and displayed in the cytoplasm of MDCC-MSB1 cells (Fig. 3c). A similar pattern of the distribution of GFP-VP1 was also presented in the CHO-K1 cells (Fig. 3b). These results clearly demonstrated VP1 is also a nuclear protein and distributed within the nucleocytoplasmic compartment. Taken together, these results indicated that CAV VP1 is a DNA-binding protein with nuclear localization activity, and its DNA binding is not specific to a particular sequence.Fig. 3The nucleocytoplasmic distribution characterization of VP1 protein in CHO-K1 and MDCC-MSB1 cells. To realize the subcellular distribution of VP1, the full-length VP1 gene included the fused whole EGFP gene at the 5′-terminus to generate the EGFP-VP1 expressing plasmid pEGFP-VP1 as illustrated in a schematic diagram (a). After 48 h post-transfection with the above plasmid in CHO-K1 cells (b) and MDCC-MSB1 cells (c), both cell types were fixed and stained with DAPI to reveal the nuclei. The subcellular localization of VP1 was determined by green fluorescence detection through confocal fluorescence microscopy