Stepwise evolution of the herpes simplex virus origin binding protein and origin of replication.

The herpes simplex virus replicon consists of cis-acting sequences, oriS and oriL, and the origin binding protein (OBP) encoded by the UL9 gene. Here we identify essential structural features in the initiator protein OBP and the replicator sequence oriS, and we relate the appearance of these motifs to the evolutionary history of the alphaherpesvirus replicon. Our results reveal two conserved sequence elements in herpes simplex virus type 1, OBP; the RVKNL motif, common to and specific for all alphaherpesviruses, is required for DNA binding, and the WP XXXGAXXFXX L motif, found in a subset of alphaherpesviruses, is required for specific binding to the single strand DNA-binding protein ICP8. A 121-amino acid minimal DNA binding domain containing conserved residues is not soluble and does not bind DNA. Additional sequences present 220 amino acids upstream from the RVKNL motif are needed for solubility and function. We also examine the binding sites for OBP in origins of DNA replication and how they are arranged. NMR and DNA melting experiments demonstrate that origin sequences derived from many, but not all, alphaherpesviruses can adopt stable boxI/boxIII hairpin conformations. Our results reveal a stepwise evolutionary history of the herpes simplex virus replicon and suggest that replicon divergence contributed to the formation of major branches of the herpesvirus family.