The herpes simplex virus type 1 (HSV-1) origin binding protein (UL9 protein) interacts specifically with the HSV-1 encoded single strand DNA-binding protein ICP8 (Boehmer, P.E. and Lehman, I.R. (1994) Proc. Natl. Acad. Sci. U.S.A. 90, 8444-8448). A UL9 mutant protein (UL9DM27) that lacks the C-terminal 27 amino acids shows normal origin-specific DNA binding and retains its DNA-dependent ATPase and helicase activities, but has a greatly reduced affinity for ICP8. The extreme C-terminal portion of the UL9 protein is therefore required for ICP8 binding. The helicase activity of the UL9DM27 protein is approximately 8-fold greater than that of the wild type UL9 protein and is not stimulated by ICP8. The UL9DM27 protein has a reduced ability to replicate origin-containing plasmids in vivo. Consequently, the interaction between the UL9 protein and ICP8 is likely to be important for origin-dependent DNA replication in vivo, presumably to promote efficient unwinding of the DNA at an HSV-1 origin of DNA replication.