Characterization of the nuclear import and export signals, and subcellular transport mechanism of varicella-zoster virus ORF9

Varicella-zoster virus (VZV) open reading frame 9 (ORF9) mRNA is one of the most abundantly expressed messages during VZV infection. However, little is known concerning the function of ORF9 protein. Here, we found that transient expression of ORF9 fused to enhanced yellow fluorescent protein (EYFP) in COS-7 cells showed a predominantly cytoplasmic localization in the absence of other viral proteins. By constructing a series of ORF9 variants fused to EYFP, a bona fide bipartite nuclear localization signal of ORF9 was, for the first time, determined and mapped to aa 16-32 (RRKTTPSYSGQYRTARR). Additionally, the nuclear export signal (NES) was identified and found to be in a leucine-rich region at aa 103-117 (LRHELVEDAVYENPL). Finally, ORF9 was demonstrated to be targeted to the cytoplasm through the functional NES by Ran and the chromosomal region maintenance 1-dependent pathway, and to the nucleus via an importin β-dependent pathway that does not require importin α5.