INTRACELLULAR-LOCALIZATION 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). A UL9-specific monoclonal antibody (17B) whose epitope maps to the N-terminal 33 amino acids was used to study the localization of UL9 i n infected and transfected cells. We demonstrate the colocalization of UL9 and the HSV-1 single-strand DNA binding protein (ICP8 or UL29) in replication compartments, sites of viral DNA synthesis. On the other hand, UL9 does not completely colocalize with ICP8 in prereplicative s ites, structures observed under conditions that inhibit viral DNA poly merase. Cells transfected with various deletion or pyruvate kinase fus ion constructs were analyzed by indirect immunofluorescence assay to d efine the nuclear localization signal (NLS) of UL9, Deletion analysis showed that the region required for nuclear localization lies within t he C-terminal DNA binding domian (amino acids 535-851). Various region s of UL9 were tested in fusion constructs for their ability to direct the normally cytoplasmic Chicken pyruvate kinase protein to the nucleu s. A fusion construct containing the carboxy-terminal 107 residues (am ino acids 745-851) localized efficiently to the nucleus, whereas a fus ion construct containing the N-terminal 660 amino acids of UL9 was una ble to do so. Mutations designed to alter a potential NLS sequence (79 3-KREFAARFKLR-804) within the C-terminal 107 residues result in a muta nt UL9 protein which fails to localize efficiently to the nucleus, The se results suggest that the major NLS of UL9 maps within the C-termina l 107 amino acids. (C) 1996 Academic Press, Inc.