Characterization of novel cell-penetrating peptides derived from the capsid protein of beak and feather disease virus

Beak and feather disease virus (BFDV) is a 17-20 nm icosahedral virus belonging to the Circoviridae family. Psittacine beak and feather disease (PBFD) is caused by BFDV and its common symptoms include abnormal feather, beak, and claw development, as well as immunosuppression in various bird species. In this study, novel cell-penetrating peptides (CPPs) in the capsid protein (Cap) of BFDV were identified through bioinformatic analyses, after which they were experimentally characterized. The cell-penetrating activities of both CPP1 and CPP2 of BFDV were analyzed through flow cytometry and image analysis. The internalization of CPP1 and CPP2 was both dose- and time-dependent but their uptake efficiencies varied depending on the cell type. The cellpenetrating activities of BFDV CPP1 and CPP2 were both superior to that of a typical CPP-TAT originating from the viral protein of human immunodeficiency virus. The cellular uptake of 5 mu M CPP1 was close to that of 25 mu M TAT, albeit with less cytotoxicity. Using the identified CPPs, the pc-mCheery, pc-Rep, and pc-Cap plasmids were successfully delivered into the target cells for expression. Moreover, both the replication-associated protein with the tag and the Cap protein with the tag could also be successfully delivered into the cells by CPP1 and CPP2. Multiple endocytosis pathways and direct translocation were involved in the cell internalization of CPP1 and CPP2. Furthermore, the delivery of the apoptin gene using CPP1 and CPP2 effectively triggered apoptosis, thus confirming the potential of these CPPs as delivery vehicles. Similarly, green fluorescent protein (GFP) fused with CPP1 or CPP2 at their N-terminus successfully entered the cells. However, the cell internalization efficiency of CPP2-GFP was higher than that of CPP1-GFP. Taken together, our findings demonstrated that both CPP1 and CPP2 of BFDV have promising potential as novel CPPs.