IDENTIFICATION OF A DIMERIZATION DOMAIN IN THE C-TERMINAL SEGMENT OF THE IE110 TRANSACTIVATOR PROTEIN FROM HERPES-SIMPLEX VIRUS

The 775-amino-acid IE110 (or ICPO) phosphoprotein of herpes simplex vi rus (HSV) functions as an accessory transcription factor during the ly tic cycle and plays a critical role in reactivation from latent infect ion. By immunofluorescence analysis, IE110 localizes in a novel patter n consisting of several dozen spherical punctate granules in the nucle i of DNA-transfected cells. We constructed a hybrid version of IE110 t hat contained an epitope-tagged domain from the N terminus of the HSV IE175 protein and lacked the IE110 N-terminal domain that confers punc tate characteristics. This hybrid IE175(N)/IE110(C) protein gave an ir regular nuclear diffuse pattern on its own but was redistributed very efficiently into spherical punctate granules after cotransfection with the wild-type HSV-1 IE110 protein. Similar colocalization interaction s occurred with internally deleted forms of IE110 that lacked the zinc finger region or large segments from the center of the protein, inclu ding both cytoplasmic and elongated punctate forms, but C-terminal tru ncated versions of IE110 did not interact. In all such interactions, t he punctate phenotype was dominant. Evidence that C-terminal segments of IE110 could also form stable mixed-subunit oligomers in vitro was o btained by coimmunoprecipitation of in vitro-translated IE110 polypept ides,vith different-size hemagglutinin epitope-tagged forms of the pro tein. This occurred only when the two forms were cotranslated, not whe n they were simply mixed together. An in vitro-synthesized IE110 C-ter minal polypeptide also gave immunoprecipitable homodimers and heterodi mers when two different-size forms were cross-linked with glutaraldehy de and reacted specifically with a bacterial glutathione S-transferase /lE110 C-terminal protein in far-Western blotting experiments. The use of various N-terminal and C-terminal truncated forms of IE110 in the in vivo assays revealed that the outer boundaries of the interaction d omain mapped between codons 617 and 711, although inclusion of adjacen t codons on either side increased the efficiency severalfold in some a ssays. We conclude that the C-terminal region of IE110 contains a high -affinity self-interaction domain that leads to stable dimer and highe r-order complex formation both in DNA-transfected cells and in in vitr o assays. This segment of IE110 is highly conserved between HSV-1 and HSV-2 and appears to have the potential to play an important role in t he interaction with the IE175 protein, as well as in correct intracell ular localization, but it is not present in the equivalent proteins fr om varicella-zoster virus, pseudorabies virus, or equine abortion viru s.