(Background): Herpes simplex virus 1(HSV-1)ICP22 is a multifunctional protein and important for HSV-1replication.Pseudorabies virus(PRV)ICP22(P-ICP22)is a homologue of HSV-1ICP22 and is reported to be able to selectively modify the transcription of different kinetic classes of PRV genes,however,the subcellular localization,localization signal and molecular determinants for its transportto execute this function is less well understood.Findings:In this study,by utilizing live cells fluorescent microscopy,P-ICP22 fused to enhanced yellow fluorescent protein(EYFP)gene was transient expressed in live cells and shown to exhibit a predominantly nucleus localization in the absence of other viral proteins.By transfection of a series of P-ICP22 deletion mutants fused to EYFP,a bona fide nuclear localization signal(NLS)and its key amino acids(aa)of P-ICP22 was,for the first time,determined and mapped to aa 41 to 60(PASTPTPPKRGRYVVEHPEY)and aa 49 to 50(KR),respectively.Besides,the P-ICP22 was demonstrated to be targeted to thenucleusvia Ran-,importin α1-,and α7-mediated pathway.Conclusions:Our findings reported herein disclose the NLS and molecular mechanism for nuclear transport of P-ICP22,these results will uncover new avenues for depicting the biological roles of P-ICP22 during PRV infection.