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.
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Germany.
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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