title: Binding and repression of the latency-associated promoter of herpes simplex virus by the immediate early 175K protein.
abstract: We demonstrate that the immediate early 175K protein (IE175K) of herpes simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP cap site was significantly larger than that formed on the IE175K cap site and the requirements for binding were qualitatively distinct with respect to both the primary sequence determinants at the site, and the regions of IE175K protein required for binding compared to those for the IE175K cap site. Although purified IE175K was sufficient for this large complex formed on the LAP cap site, the DNA-binding domain was unable to bind efficiently. This contrasted strikingly with the IE175K cap site where, using precisely analogous probes, the DNA-binding domain exhibited a strong interaction. Surprisingly, from dissociation kinetics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap site (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expression than IE175K promoter-driven expression by IE175K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region differed from previously identified IE175K recognition sequences in that in addition to a partially conserved consensus sequence, neighbouring bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to this region. Together these results indicate that IE175K forms an unusual complex at the LAP cap site, broadening the range of previously defined sequences recognized by IE175K.
authors.affiliation: Marie Curie Research Institute, Oxted, Surrey U.K.
authors.forename: A H
authors.initials: AH
authors.lastname: Batchelor
authors.forename: K W
authors.initials: KW
authors.lastname: Wilcox
authors.forename: P
authors.initials: P
authors.lastname: O'Hare
mesh_terms: D000911:Antibodies, Monoclonal; D001483:Base Sequence; D001667:Binding, Competitive; D016384:Consensus Sequence; D015342:DNA Probes / Q000138:chemical synthesis; D004279:DNA, Viral / Q000032:analysis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007700:Kinetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014162:Transfection; D017735:Virus Latency / Q000235:genetics*
chemical_list: D000911:Antibodies, Monoclonal; D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; C048925:herpes simplex virus, type 1 protein ICP4