Herpes simplex virus type 1 origin-specific binding protein: oriS-binding properties and effects of cellular proteins

The herpes simplex virus type 1 (HSV-1) origin of replication, oriS, contains three highly homologous sequences, sites I, II, and III. The HSV-1 origin-binding protein (OBP), the product of the UL9 gene, has been shown to bind specifically to sites I and II. In this study, gel shift analysis was used to characterize interactions between site I DNA and proteins in infected and uninfected cell extracts. The formation of two protein-DNA complexes, bands A and B, was demonstrated with infected cell extracts, and one predominant protein-DNA complex, band M, was identified with mock-infected extracts. Protein interactions with the highly homologous site II and III DNAs were also characterized. Incubation of infected cell extracts with the lower-affinity site II DNA as a probe resulted in the appearance of two protein-DNA complexes with mobilities identical to those of the A and B complexes, while incubation with site III DNA resulted in the formation of a single complex with the mobility of band B; no A-like band was observed. Incubation of high concentrations of partially purified OBP with site I DNA resulted in the formation of two novel complexes, bands 9-1 and 9-2. Addition of uninfected or HSV-1-infected cell extracts to the purified OBP-site I DNA mix significantly enhanced the formation of complex 9-1. The enhanced formation of complex 9-1 by uninfected cell extracts implicates a cellular factor or factors in the formation or stabilization of the OBP-site I DNA complex. The linear double-stranded DNA genome of herpes sim-plex virus type 1 (HSV-1) is 152 kb in length and encodes at least 72 proteins which are expressed in a sequential and highly regulated fashion (24, 32). Among these proteins are seven essential DNA replication proteins (Fig. 1A) as well as a variety of enzymes involved in DNA precursor metabolis