CHARACTERIZATION OF A BINDING-SITE FOR THE HERPES-SIMPLEX VIRUS TYPE-1 UL9 ORIGIN-BINDING PROTEIN WITHIN THE UL9 GENE

Gene UL9 of herpes simplex virus type 1 (HSV-1) encodes a sequence-spe cific origin-binding protein (OBP) that plays a direct and essential r ole in viral DNA synthesis. A search of the complete HSV-1 genomic seq uence for possible OBP binding sites lying outside the known origins o f replication revealed the presence of a very close match to the OBP r ecognition sequence within the UL9 coding region. The ability of OBP t o bind to this site (referred to as the 'UL9 box') was confirmed by DN ase I footprinting and gel retardation assays, and filter binding expe riments demonstrated that the affinity of OBP for the UL9 box was of t he same order as for its high affinity sites within the three replicat ion origins. To investigate whether binding of OBP to the UL9 box play ed a role during viral replication we constructed a mutant virus in wh ich the sequence was altered in such a way as to preserve the encoded amino acid sequence whilst abolishing the ability of OBP to bind. Grow th of the virus was indistinguishable from wild-type and no alteration s were observed in the accumulation of transcripts from the UL9 region of the genome. In addition, a DNA fragment containing the UL9 box seq uence did not exhibit origin activity in a transient assay for viral D NA synthesis. We therefore conclude that binding of OBP to the UL9 box is not essential for virus growth and that expression of the UL9 gene is unlikely to be autoregulated through this site.