The varicella zoster virus (VZV) ORF4 protein, one of immediate-early genes protein, is associated with the tegument in purified virions. ORF4 protein functions at both transcriptional and post-transcriptional levels, present during different phase of whole VZV life cycle. ORF4 protein acts as a nucleocytoplasm shuttle protein, the precise nuclear location signals (NLS) and molecular mechanisms of nucleocytoplasm transport are not elucidated. At this study, we constructed a series of mutants, used fluorescence microscopy and Co-IP analysis to identify an unconventional bipartite NLS ((130)RKHRDRSLSNRRRRP(144)) in VZV ORF4. This study also demonstrates that nuclear import of VZV ORF4 occurs via a Ran-dependent pathway with importin-α5 and importin-β1. Additionally, NLS function of ORF4 is independent from VZV ORF62 protein. ORF62 protein cannot influence the intracellular distribution of ORF4 protein without NLS. So interaction between ORF4 and ORF62 protein is speculated to occur in nucleus. Thus, NLS is indispensable for the post-transcriptional function of ORF4.
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