Biochemical, functional, and genetic analysis of the HSV-1 origin binding protein

The origin binding protein (OBP) is one of seven essential virus-encoded proteins required for HSV DNA replication. Based on biochemical observations, OBP is thought to function as an initiator of HSV DNA replication by binding to HSV origins, inducing localized unwinding of origin DNA, and recruiting other essential replication proteins to the site of initiation. One major obstacle in studying OBP is the extremely low level of OBP expressed in HSV-infected cells. Using cell-culture based assays optimized to overcome this problem, the work presented here addresses the function of OBP and its regulation during HSV infection.;Using gel shift assays, three OBP-containing complexes that bind to oriS site I were identified. To isolate and characterize the protein components within these complexes, a streptavidin-based protein pull-down method was developed. Additionally, binding of OBPC, a truncated form of OBP, to oriS site I was shown to be insufficient to support oriS-dependent DNA replication. Based on these and other observations, a model in which OBPC facilitates a switch in the mode of HSV DNA replication from theta to rolling circle is proposed. Using a metabolic labeling and immunoprecipitation approach, OBP was shown to be phosphorylated during HSV infection, and phosphorylation was demonstrated to be mediated, in part, by a virus encoded early protein. Phosphorylation was shown not to influence the origin binding activity of OBP although it may regulate an alternative activity of OBP. Preliminary phosphopeptide analysis designed to identify the phosphorylation sites within OBP is presented. Lastly, a region of OBP located between helicase motifs II and III that is essential for the DNA replication function of OBP was identified. Mutagenesis of this region revealed that the motif II-III region is required for the ability of OBP to support origin-dependent DNA replication, but is dispensable for origin binding activity. Based on these data and the observation that this region is highly conserved among OBP homologs in other alphaherpesviruses, we propose that the motif II-III region of OBP serves as an "optional" domain and specifies an essential DNA replication function other than origin binding.