CHARACTERIZATION OF NUCLEAR-STRUCTURES IN CELLS INFECTED WITH HERPES-SIMPLEX VIRUS TYPE-1 IN THE ABSENCE OF VIRAL-DNA REPLICATION

Herpes simplex virus type 1 DNA replication occurs in nuclear domains termed replication compartments, which are areas of viral single-stran ded DNA binding protein (UL29) localization (M. P. Quinlan, L. B. Chen , and D. M. Knipe, Cell 36:857-868, 1984). In the presence of herpesvi rus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-prim ase complex proteins containing short peptide epitopes which can be de tected in an immunofluorescence assay, we have found that the helicase -primase complex localizes to prereplicative sites and replication com partments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have stu died UL29 localization in cells infected with replication-defective vi ruses. Cells infected with viruses that fail to express one of the thr ee helicase-primase subunits or the origin-binding protein show a diff use nuclear staining for UL29. However, in the presence of polymerase inhibitors, mutant-infected cells contain UL29 in prereplicative sites . Replication-defective viruses containing subtle mutations in the hel icase or origin-binding proteins behaved identically to their null mut ant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites i n the absence and presence of polymerase inhibitors. We propose that a ctive viral polymerase prevents the formation of prereplicative sites. Models of the requirement of essential viral replication proteins in the assembly of prereplicative sites are presented.