Differences in determinants required for complex formation and transactivation in related VP16 proteins.
VP16-H is an essential structural protein of herpes simplex virus type 1 (HSV-1) and is also a potent activator of virus immediate-early (IE) gene expression. Current models of functional determinants within VP16-H indicate that it consists of two domains, an N-terminal domain involved in recruiting VP16-H to a multicomponent DNA binding complex with two host proteins, Oct-1 and host cell factor (HCF), and an acidic C-terminal domain exclusively involved in transactivation. VP16-E, from equine herpesvirus 1 (EHV-1), exhibits strong conservation with the N-terminal domain of VP16-H but, with the exception of a short segment at the extreme C terminus, lacks almost the entire acidic C-terminal domain. Studies of key activation determinants within the C terminus of VP16-H would predict that VP16-E may activate poorly, if at all. However, VP16-E is a potent activator of both EHV-1 and HSV-1 IE gene transcription. We show that VP16-E does not follow the simple two-domain model of VP16-H. Thus, despite the conservation in the N-terminal domains, this region in VP16-E is not sufficient for assembly into the DNA binding complex with Oct-1 and HCF. The short conserved determinant close to the C terminus is completely dispensable in VP16-H but is absolutely required in VP16-E. In activation studies, the potency of intact VP16-E was not recapitulated in chimeric proteins in which it was fused with a GAL4 DNA binding domain. Furthermore, a chimeric protein consisting of the C-terminal region of VP16-E fused to the N-terminal domain of VP16-H, while able to promote complex formation, nevertheless exhibited very weak activation. These results indicate that the mode of recruitment of the activation domain, i.e., through complex formation with Oct-1 and HCF, may be crucial for activation and that key determinants required for activation in VP16-E, and possibly VP16-H, may involve interactions between regions of the C terminus and the N terminus rather than discrete domains with independent functions.
Grapes
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Marie Curie Research Institute, Oxted, Surrey RH8 OTL, United Kingdom.
O'Hare
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D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004861:Herpesvirus 1, Equid / Q000235:genetics / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011817:Rabbits; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D017405:Herpes Simplex Virus Protein Vmw65; C050678:IE1 protein, Human herpesvirus 1; D017874:Immediate-Early Proteins; D015534:Trans-Activators; C415845:VP16-E protein, Equine herpesvirus 1; D014764:Viral Proteins