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 in 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 sites, structures observed under conditions that inhibit viral DNA polymerase. 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. Deletion analysis showed that the region required for nuclear localization lies within the C-terminal DNA binding domian (amino acids 535-851). Various regions of UL9 were tested in fusion constructs for their ability to direct the normally cytoplasmic chicken pyruvate kinase protein to the nucleus. A fusion construct containing the carboxy-terminal 107 residues (amino acids 745-851) localized efficiently to the nucleus, whereas a fusion construct containing the N-terminal 660 amino acids of UL9 was unable to do so. Mutations designed to alter a potential NLS sequence (793-KREFAGARFKLR-804) within the C-terminal 107 residues result in a mutant UL9 protein which falls to localize efficiently to the nucleus. These results suggest that the major NLS of UL9 maps within the C-terminal 107 amino acids.
Malik
A K
AK
Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA.
Shao
L
L
Shanley
J D
JD
Weller
S K
SK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D002522:Chlorocebus aethiops; D004265:DNA Helicases / Q000378:metabolism; D019915:DNA Primase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C064565:UL9 protein, Human herpesvirus 1; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases