MAPPING OF INTRACELLULAR-LOCALIZATION DOMAINS AND EVIDENCE FOR COLOCALIZATION INTERACTIONS BETWEEN THE IE110 AND IE175 NUCLEAR TRANSACTIVATOR PROTEINS OF HERPES-SIMPLEX VIRUS

Transcriptional regulation by the IE175 (ICP4) and IE110 (ICPO) phosph orylated nuclear proteins encoded by herpes simplex virus (HSV) appear s to be a key determinant for the establishment of successful lytic cy cle infection. By indirect immunofluorescence in transient DNA transfe ction assays, we have examined the intracellular distribution of delet ion and truncation mutants of both IE175 and IE110 from HSV-1. Inserti on of short oligonucleotides encoding the basic amino acid motifs 726- GRKRKSP-732 from IE175 and 500-VRPRKRR-506 from IE110 into deleted cyt oplasmic forms of the two proteins restored the karyophilic phenotype and confirmed that these motifs are both necessary and sufficient for proper nuclear localization. Analysis of IE110 deletion mutants and a panel of IE110/IE175 hybrid proteins was also used to evaluate the cha racteristic IE110 distribution within nuclear punctate granules as see n by immunofluorescence and phase-contrast microscopy. The phase-dense punctate pattern persisted with both large C-terminal truncations and deletions of the Cys rich zinc finger region and even with a form of IE110 that localized in the cytoplasm, implying that the punctate char acteristic is an intrinsic property of the N-terminal segment of the I E110 protein, Transfer of the full IE110-like punctate phenotype to th e normally uniform diffuse nuclear pattern of the IE175 protein by exc hange of the N-terminal domains of the two proteins demonstrated that the first 105 to 244 amino acids of IE110 represent the most important region for conferring punctate characteristics. Surprisingly, cotrans fection of a wild-type nuclear IE175 gene together with the IE110 gene revealed that much of the IE175 protein produced was redistributed in to a punctate pattern that colocalized with the IE110-associated punct ate granules seen in the same cells. This colocalization did not occur after cotransfection of IE110 with the IE72 (IE1) nuclear protein of human cytomegalovirus and therefore cannot represent simple nonspecifi c trapping. Evidently, the punctate phenotype of IE110 represents a do minant characteristic that reveals the potential of IE110 and IE175 to physically interact with each other either directly or indirectly wit hin the intracellular environment.