IDENTIFICATION OF RESIDUES WITHIN THE HERPES-SIMPLEX VIRUS TYPE-1 ORIGIN-BINDING PROTEIN THAT CONTRIBUTE TO SEQUENCE-SPECIFIC DNA-BINDING

Gene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid prot ein which is essential for viral DNA synthesis and functions as a sequ ence-specific origin-binding protein and DNA helicase. We generated mo noclonal antibodies against purified UL9 protein and identified one su ch antibody (MAb 13924) that can block the interaction of the UL9 C-te rminal DNA-binding domain (amino acids 534-851) with its recognition s equence. MAb 13924 interacted with immobilized peptides containing res idues 780-786 of UL9. Although the corresponding region of the homolog ous protein encoded by varicella-zoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesi s experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-s pecific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-bin ding properties of the mutated proteins were examined. The results sho wed that lysine residues that are located close to the peptide recogni zed by MAb 13924 or lie within the region of the DNA-binding domain mo st highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysin e residue 18 amino acids from the recognized peptide prevented the int eraction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Thre e helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924. (C) 1998 Academic Press.