Herpes simplex virus type 1 prereplicative sites are a heterogeneous population: only a subset are likely to be precursors to replication compartments.

When herpes simplex virus type 1 (HSV-1) DNA replication is blocked by viral polymerase inhibitors, such as phosphonoacetic acid (PAA) or acyclovir (ACV), UL29 (ICP8) localizes to numerous punctate nuclear foci which are called prereplicative sites. Since this pattern can form in cells infected with mutants which are defective in UL5, UL8, UL9, or UL52 in the presence of polymerase inhibitors (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996; L. M. Liptak, S. L. Uprichard, and D. M. Knipe, J. Virol. 70:1759-1767, 1996), we previously proposed that it is unlikely that these numerous UL29 foci actually represent a functional subassembly of viral replication proteins that could lead to the formation of replication compartments (C. J. Lukonis and S. K. Weller, J. Virol. 70:1751-1758, 1996). In this paper, we have investigated the requirement for formation of the prereplicative site pattern by using double mutants of HSV. From the analysis of mutants lacking both UL5 and UL9, we conclude that neither viral helicase is required for the prereplicative site pattern to form as long as a polymerase inhibitor is present. From the analysis of mutants defective in both UL30 and UL5, we suggest that the prereplicative site pattern can form under conditions in which viral and/or cellular polymerases are inhibited. Furthermore, reexamination of the UL29 staining pattern in cells infected with wild-type virus in the presence of PAA reveals that at least two different UL29 staining patterns can be detected in these cells. One population of cells contains numerous (greater than 20) punctate UL29 foci which are sites of cellular DNA synthesis. In another population of cells, fewer punctate foci (less than 15) are detected, and these structures do not colocalize with sites of cellular DNA synthesis. Instead, they colocalize with PML, a component of nuclear matrix structures known as ND10. We propose that ND10-associated UL29 sites represent domains at which replication compartments form.

Formation of herpes simplex virus type 1 replication compartments by transfection: requirements and localization to nuclear domain 10.

During infection, the seven essential herpes simplex virus type 1 (HSV-1) replication proteins are found in globular nuclear structures called replication compartments. Replication compartments form adjacent to ND10, nuclear matrix-bound domains which are present in most cell types but whose function is unknown (G. G. Maul, I. M. Ishov, and R. D. Everett, Virology 217:67-75, 1996). We now demonstrate that replication compartments can be formed by cotransfecting Vero cells with constructs expressing the seven essential viral replication proteins and a plasmid containing an HSV-1 origin of DNA replication. Like replication compartments in infected cells, replication compartments formed by cotransfection contain all of the essential viral replication proteins, are sites of DNA synthesis, and are found adjacent to ND10. However, neither the viral origin-binding protein nor a plasmid containing an HSV-1 origin of DNA replication is individually required for the formation of transfection replication compartments, although the presence of each increases the efficiency of replication compartment formation. Further, we provide evidence that UL29 independently localizes adjacent to ND10 and so may play a role in directing replication compartments to these preexisting nuclear structures.

The herpes simplex virus type 1 transactivator ICPO mediates aberrant intracellular localization of the viral helicase/primase complex subunits.

The infected cell polypeptide 0 (ICP0) protein of herpes simplex virus type 1 (HSV-1) is a promiscuous transactivator. When expressed by transfection, ICP0 forms spherical structures in the nucleus. Using a double-label immunofluorescence assay, we have found that the HSV-1 helicase/primase complex subunits accumulate within ICP0 structures in cotransfected cells. This phenomenon was also observed in cells coexpressing ICP0 and UL6, a protein thought to be involved in the cleavage and/or packaging of viral genomes. ICP0 structures were found to be proteinaceous by immunoelectron microscopy. These results suggest that ICP0 may interact nonspecifically with a variety of viral proteins.

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-stranded DNA-binding protein (UL29) localization (M.P. Quinlan, L. B. Chen, and D. M. Knipe, Cell 36:857-868). In the presence of herpesvirus-specific polymerase inhibitors, UL29 localizes to punctate nuclear foci called prereplicative sites. Using versions of the helicase-primase complex proteins containing short peptide epitopes which can be detected in an immunofluorescence assay, we have found that the helicase-primase complex localizes to prereplicative sites and replication compartments. To determine if prereplicative site formation is dependent upon these and other essential viral replication proteins, we have studied UL29 localization in cells infected with replication-defective viruses. Cells infected with viruses that fail to express one of the three helicase-primase subunits or the origin-binding protein show a diffuse 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 helicase or origin-binding proteins behaved identically to their null mutant counterparts. In contrast, cells infected with viral mutants which fail to express the polymerase protein contain prereplicative sites in the absence and presence of polymerase inhibitors. We propose that active 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.