Stepwise Evolution of the Herpes Simplex Virus Origin Binding Protein and Origin of Replication

Abstract

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 WPXXXGAXXFXXL motif, found in a subset of alphaherpesviruses, is required for specific binding to the single strand DNAbinding 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.

Replication and Recombination of Herpes Simplex Virus DNA

“…A dimer of OBP can simultaneously bind two double-stranded DNA ligands but only one copy of a box I/box III hairpin with a 10-nucleotide single-stranded tail (26). It has been argued that this finding reflects a conformational change in OBP, from an origin-binding conformation to a helicase conformation, which can be facilitated by the interaction between ICP8 and the WPXXXGAXXFXXL motif found in the extreme C terminus of OBP (20,26). When ICP8 is in contact with ssDNA, this interaction can no longer be sustained (27).…”

“…sites are always separated by an AT-rich spacer sequence, and at least for HSV-1, the efficiency of initiation of DNA replication varies periodically with the length of spacer sequence (21,22). The recognition sequence for OBP, TTCGCAC, is highly conserved among alphaherpesviruses but differs from its counterpart in roseoloviruses (20). OBP binds cooperatively to oriS, and the assembly of a stable complex containing two OBP dimers on oriS as well as ICP8 has been visualized by electron microscopy (23).…”

“…oriS from HSV-1 has two binding sites, boxes I and II, for the initiator protein OBP and an additional weak site, box III. Boxes I and III together form a stable hairpin (20). The binding FIGURE 1.…”

“…OBP is a superfamily II DNA helicase with a C-terminal domain for sequence-specific binding to origins of replication. The replicon has evolved from an ancestor common to the roseoloviruses, human herpesviruses 6 and 7, and the alphaherpesviruses (20). oriS from HSV-1 has two binding sites, boxes I and II, for the initiator protein OBP and an additional weak site, box III.…”

Identification of Conserved Amino Acids in the Herpes Simplex Virus Type 1 UL8 Protein Required for DNA Synthesis and UL52 Primase Interaction in the Virus Replisome

“…UL8 binds to the UL5-UL52 dimer and facilitates unwinding of long stretches of DNA in the presence of ICP8 (8). A conserved C-terminal motif of OBP binds specifically to ICP8 (9).…”