Identification and characterization of complex dual nuclear localization signals in human bocavirus NP1

Human bocavirus (HBoV), closely related to canine minute virus (MVC) and bovine parvovirus (BPV), is a new member of the Bocavirus genus within the Parvoviridae family. The non-structural protein NP1 of HBoV is a nuclear localized protein and plays an important role in DNA replication as well as in the evasion of host innate immunity. In the current study, we provide the first evidence that NP1 possesses a non-classical nuclear localization signal (ncNLS) (amino acids 7-50). Embedded within this ncNLS is a classical bipartite nuclear localization signal (cNLS) (amino acids 14-28), capable of transporting a heterologous cytoplasmic protein,beta-galactosidase fusion protein (beta-gal-EGFP) to the nucleus via the classical importin alpha/beta 1 -mediated pathway. Amino acids 7-50 containing the cNLS and the ncNLS of NP1 or full-length NP1 interact with importin alpha 1, importin beta 1 and importin beta 1 Delta, which lacks the importin a binding domain, indicating that the nuclear import of NP1 is through both conventional importin alpha/beta 1 heterodimer- and non-classical importin beta 1-mediated pathways. Given that the arrangement of a cNLS embedded within an ncNLS is unusual in viral proteins, our data together reveal a novel molecular mechanism underlying the nuclear import of HBoV NP1, providing a basis for further understanding its biological function.