Structure-function analysis of the herpes simplex virus type 1 origin binding protein, UL9

UL9 is the origin binding protein of Herpes Simplex Virus type-1 (HSV-1). It binds cooperatively to two high affinity sites within the HSV-1 origin, exists as a dimer in solution and exhibit ATPase and helicase activities. The goal of this thesis is to define and map the functional domains of UL9. The identification and analysis of transdominant mutants of UL9 provided a genetic approach to identify domains and functional sites. UL9 contains six sequence motifs, conserved in a superfamily of RNA and DNA helicases, mutations within these abolish UL9 function in vivo. Overexpression of wild-type UL9 is inhibitory to plaque formation in a transfection assay which measures viral plaque formation by infectious HSV-1 DNA. Constructs containing mutations in motif I, II or VI are strong transdominant inhibitors of plaque formation by the wild-type virus. The transdominant phenotype can be relieved by introducing a second mutation in the DNA-binding domain or by deleting the N-terminal 35 amino acids of the protein. The inhibitory effects of wild type UL9 can also be partially relieved by deletion of amino acids 292-404. We propose that the N-terminal 35 amino acids of UL9 and residues 292-404 may define new functional domains of the UL9 protein.^ A UL9-specific monoclonal antibody (17B) whose epitope maps to the N-terminal 33 amino acids was used to study the intracellular localization of UL9. We demonstrate the colocalization of UL9 and the HSV-1 single-strand DNA binding protein in replication compartments, sites of viral DNA synthesis. Cells transfected with various deletion or pyruvate kinase fusion constructs were analyzed by indirect immunofluorescence assay to define the nuclear localization signal (NLS) of UL9. A fusion construct containing the carboxy-terminal 107 residues (amino acids 745-851) localized efficiently to the nucleus. Mutations designed to alter a potential NLS sequence (793-KREFAGARFKLR-804) within the C-terminal 107 residues result in a mutant UL9 protein which fails to localize efficiently to the nucleus. These results suggest that the major NLS of UL9 maps within the C-terminal 107 amino acids.