Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.
During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.
Habran
Lionel
L
Laboratory of Virology and Immunology, Center for Biomedical Genoproteomics, Institute of Pathology B23, University of Liège, B-4000, Liège, Belgium.
Bontems
Sébastien
S
Di Valentin
Emmanuel
E
Sadzot-Delvaux
Catherine
C
Piette
Jacques
J
D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D051357:Cyclin-Dependent Kinase 2; D051360:Cyclin-Dependent Kinase 5; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D014645:Herpesvirus 3, Human / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D047468:Immunoprecipitation; D009154:Mutation; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D014162:Transfection; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D047428:Protein Kinase Inhibitors; D011687:Purines; D014759:Viral Envelope Proteins; C074551:immediate early protein 63, Human herpesvirus 3; D000077546:Roscovitine; D013912:Threonine; D012694:Serine; D004247:DNA; D005982:Glutathione Transferase; D051360:Cyclin-Dependent Kinase 5; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495905:CDK5 protein, human; C467969:CDK9 protein, human; D051357:Cyclin-Dependent Kinase 2; D042863:Cyclin-Dependent Kinase 9; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase