Mutational analysis of the sequence encoding ICP0 from herpes simplex virus type 1.
In-frame codon insertion and deletion mutants were constructed in a plasmid containing the sequence that encodes ICP0, a transcriptional activator of herpes simplex virus type 1 (HSV-1). The effect of these mutations was analyzed in a transient expression assay using the promoters for, the IE-0 gene (an immediate early (alpha) gene), the thymidine kinase gene (an early (beta) gene), and the glycoprotein C gene (a late (gamma) gene) fused to reporter cassettes that encoded either beta-galactosidase or chloramphenicol acetyl transferase. Assays were performed in the presence or absence of a plasmid encoding ICP4, the major regulatory protein of HSV-1. Our results demonstrate that ICP0-mediated transactivation varied depending on the position of the insertion in the gene. One region of this protein was consistently shown to be required for full activation of each promoter examined either in the presence or in the absence of ICP4. This region overlaps with a cysteine-rich region and coincides with a transactivator domain identified in another extensive mutational analysis of this sequence. Analysis of the deletion mutants generated in this study demonstrated that the carboxy-terminal regions were required for activation in certain circumstances and that this varied depending on the promoter being assayed and the cell type in which the analysis was performed.
Chen
J X
JX
Department of Microbiology, Columbia University, New York, New York 10032.
Zhu
X X
XX
Silverstein
S
S
D000595:Amino Acid Sequence; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection / Q000235:genetics; D044767:Ubiquitin-Protein Ligases; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics
D017874:Immediate-Early Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1; D013937:Thymidine Kinase; D001616:beta-Galactosidase