The 86-kilodalton IE-2 protein of human cytomegalovirus is a sequence-specific DNA-binding protein that interacts directly with the negative autoregulatory response element located near the cap site of the IE-1/2 enhancer-promoter.
The 86-kDa IE-2 protein of human cytomegalovirus is able to autoregulate its own expression via a short nucleotide sequence, termed the cis repression signal (CRS), that is located between the TATA box and the cap site of the IE-1/2 enhancer-promoter. Here we report that the 86-kDa IE-2 protein can interact directly with the CRS, thus demonstrating that IE-2 is a DNA-binding protein. This could be shown by both DNase I protection and gel retardation experiments using a procaryotically expressed IE-2 protein that was purified to near homogeneity. The interaction was sequence specific since a mutated form of the CRS that had previously been reported to be defective in mediating negative regulation could no longer compete for binding in DNase I protection experiments. In addition, an IE-2-reactive monoclonal antibody was able to elicit a supershift in gel retardation experiments, thus proving the presence of IE-2 within the protein-DNA complex. These results suggest that formation of a specific complex between an IE protein and a target sequence located near the cap site of its own gene promoter may be a common mechanism used by both alphaherpesviruses and betaherpesviruses to autoregulate IE gene transcription, although the sequence requirements differ between the two herpesviral subgroups.
Lang
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Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Germany.
Stamminger
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics*; D001667:Binding, Competitive; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012315:RNA Caps / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D013379:Substrate Specificity; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D012315:RNA Caps; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C044582:immediate-early proteins, cytomegalovirus; D003850:Deoxyribonuclease I