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An isoform variant of the cytomegalovirus immediate-early auto repressor functions as a transcriptional activator
E Baracchini1, E Glezer, K Fish
1Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
Abstract:
The major immediate-early promoter (MIEP) of human cytomegalovirus directs the expression of several differentially spliced and polyadenylated mRNAs. These mRNAs encode nuclear phosphorproteins (IE55, IE72, and IE86), which consist of common and unique amino acid sequences. To date, very little is known of the functional role of the 55-kDa (IE55) protein. Here we present evidence that the IE55 protein is a positive activator of the MIEP. In human fibroblast cells IE55 protein activated the MIEP between 10- and 30-fold. Fusion of IE55 to the GAL4 DNA binding domain resulted in a chimeric protein capable of trans-activating a reporter with GAL4 recognition sequences. These results strongly suggest that IE55 is a bona fide transcriptional activator protein. In addition, the IE55 protein was found not to act synergistically with the IE72 activator protein. The IE55 protein shares the same amino acid sequence as IE86 except for a 154-amino-acid deletion at the C-terminal end of the protein. These proteins were functionally antagonistic; IE55 relieved repression by IE86 and, conversely, IE86 negated IE55 activation. Mutagenesis of the MIEP revealed that the target sequence for activation by IE55 is different from the IE86 autorepressive response element. These experiments suggest that the mechanism of action of the IE55 and IE86 isoforms is distinct. Moreover, from these results it is apparent that the interplay of these factors might be critical in determining the level of HCMV replication in the host.
Insights
The human cytomegalovirus IE55 protein activates its major immediate-early promoter (MIEP). IE55 and IE86 isoforms have distinct mechanisms and antagonistic functions, impacting viral replication.
Area of Science:
- Molecular Virology
- Gene Regulation
- Human Cytomegalovirus Biology
Background:
- Human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) controls viral gene expression.
- HCMV encodes nuclear phosphoproteins including IE55, IE72, and IE86 from MIEP transcripts.
- The function of the IE55 protein remains largely uncharacterized.
Purpose of the Study:
- To investigate the functional role of the IE55 protein in HCMV MIEP regulation.
- To determine the relationship between IE55, IE72, and IE86 proteins.
- To elucidate the mechanism of action for IE55 and IE86 isoforms.
Main Methods:
- Transfection of human fibroblast cells with IE55 expression constructs.
- Reporter gene assays to quantify MIEP activity.
- GAL4-IE55 fusion protein experiments.
- Site-directed mutagenesis of the MIEP.
- Analysis of protein-protein interactions and functional antagonism.
Main Results:
- IE55 protein demonstrated a 10- to 30-fold activation of the HCMV MIEP in human fibroblasts.
- A GAL4-IE55 fusion protein confirmed IE55's role as a transcriptional activator.
- IE55 did not act synergistically with IE72.
- IE55 and IE86 exhibited functional antagonism, with distinct MIEP target sequences.
- IE55 relieved IE86-mediated repression, while IE86 negated IE55 activation.
Conclusions:
- IE55 is a bona fide transcriptional activator of the HCMV MIEP.
- IE55 and IE86 isoforms possess distinct mechanisms of action and antagonistic functions.
- The interplay between IE55 and IE86 is crucial for regulating HCMV replication levels.