The regions important for the activator and repressor functions of herpes simplex virus type 1 alpha protein ICP27 map to the C-terminal half of the molecule.
The herpes simplex virus type 1 (HSV-1) alpha or immediate-early proteins ICP4 (IE175), ICP0 (IE110), and ICP27 (IE63) are trans-acting proteins which affect HSV-1 gene expression. We previously showed that ICP27 in combination with ICP4 and ICP0 could act as a repressor or an activator in transfection assays, depending on the target gene (R. E. Sekulovich, K. Leary, and R. M. Sandri-Goldin, J. Virol. 62:4510-4522, 1988). To investigate the regions of the ICP27 protein which specify these functions, we constructed a series of in-frame insertion and deletion mutants in the ICP27 gene. These mutants were analyzed in transient expression assays for the ability to repress or to activate two different target genes. The target plasmids used consisted of the promoter regions from the HSV-1 beta or early gene which encodes thymidine kinase and from the beta-gamma or leaky late gene. VP5, which encodes the major capsid protein, each fused to the chloramphenicol acetyltransferase gene. Our previous studies showed that induction of pTK-CAT expression by ICP4 and ICP0 was repressed by ICP27, whereas the stimulation of pVP5-CAT expression seen with ICP4 and ICP0 was significantly increased when ICP27 was also added. In this study, a series of transfection assays was performed with each of the ICP27 mutant plasmids in combination with plasmids containing the ICP4 and ICP0 genes with each target. The results of these experiments showed that mutants containing insertions or deletions in the region from amino acids 262 to 406 in the carboxy-terminal half of the protein were unable to stimulate expression of pVP5-CAT but were able to repress induction of pTK-CAT activity by ICP4 and ICP0. Mutants in the carboxy-terminal 78 amino acids lost both activities; that is, these mutants did not show repression of pTK-CAT activity or stimulation of pVP5-CAT activity, whereas mutants in the hydrophilic amino-terminal half of ICP27 were able to perform both functions. These results show that the carboxy-terminal half of ICP27 is important for the activation and repression functions. Furthermore, the carboxy-terminal 62 amino acids are required for the repressor activity, because mutants with this region intact were able to repress. Analysis of the DNA sequence showed that there are a number of cysteine and histidine residues encoded by this region which have some similarity to zinc finger metal-binding regions found in other eucaryotic regulatory proteins. These results suggest that the structural integrity of this region is important for the function of ICP27.
Hardwicke
M A
MA
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
Vaughan
P J
PJ
Sekulovich
R E
RE
O'Conner
R
R
Sandri-Goldin
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002872:Chromosome Deletion; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits; D012097:Repressor Proteins / Q000235:genetics*; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics*; D012867:Skin / Q000166:cytology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins / Q000096:biosynthesis; D015678:Viral Structural Proteins / Q000235:genetics*
D004251:DNA Transposable Elements; C057120:ICP27 protein, human herpesvirus 1; D017874:Immediate-Early Proteins; D009841:Oligonucleotides; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D015678:Viral Structural Proteins; C048925:herpes simplex virus, type 1 protein ICP4