Nuclear Localization Signal Its 6 U69 Gene Product and Identification of Characterization of the Human Herpesvirus

To elucidate the function of the U69 protein kinase of human herpesvirus 6 (HHV-6) in vivo, we firstanalyzed its subcellular localization in HHV-6-infected Molt 3 cells by using polyclonal antibodies against theU69 protein. Immunofluorescence studies showed that the U69 signal localized to the nucleus in a mesh-likepattern in both HHV-6-infected and HHV6-transfected cells. A computer program predicted two overlappingclassic nuclear localization signals (NLSs) in the N-terminal region of the protein; this NLS motif is highlyconserved in the N-terminal region of most of the herpesvirus protein kinases examined to date. An N-terminaldeletion mutant form of the protein failed to enter the nucleus, whereas a fusion protein of green fluorescentprotein (GFP) and/or glutathione S-transferase (GST) and the U69 N-terminal region was transported into thenucleus, demonstrating that the predicted N-terminal NLSs of the protein actually function as NLSs. ThenucleartransportoftheGST-GFPfusionproteincontainingtheN-terminalNLSofU69wasinhibitedbywheatgerm agglutinin and by the Q69L Ran-GTP mutant, indicating that the U69 protein is transported into thenucleus from the cytoplasm via classic nuclear transport machinery. A cell-free import assay showed that thenuclear transport of the U69 protein was mediated by importin