title: Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function.
abstract: Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.
authors.affiliation: Laboratory of Tumor Virus Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
authors.forename: S W
authors.initials: SW
authors.lastname: Wong
authors.forename: P A
authors.initials: PA
authors.lastname: Schaffer
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells
chemical_list: D004279:DNA, Viral; D015534:Trans-Activators