During replication of herpes simplex virus type 1 (HSV-1), viral DNA is synthesized in the infected cell nucleus, where DNA-free capsids are also assembled. Genome-length DNA molecules are then cut out of a larger, multigenome concatemer and packaged into capsids. Here we report the results of experiments carried out to test the idea that the HSV-1 UL6 gene product (pUL6) forms the portal through which viral DNA passes as it enters the capsid. Since DNA must enter at a unique site, immunoelectron microscopy experiments were undertaken to determine the location of pUL6. After specific immunogold staining of HSV-1 B capsids, pUL6 was found, by its attached gold label, at one of the 12 capsid vertices. Label was not observed at multiple vertices, at nonvertex sites, or in capsids lacking pUL6. In immunoblot experiments, the pUL6 copy number in purified B capsids was found to be 14.8 ± 2.6. Biochemical experiments to isolate pUL6 were carried out, beginning with insect cells infected with a recombinant baculovirus expressing the UL6 gene. After purification, pUL6 was found in the form of rings, which were observed in electron micrographs to have outside and inside diameters of 16.4 ± 1.1 and 5.0 ± 0.7 nm, respectively, and a height of 19.5 ± 1.9 nm. The particle weights of individual rings as determined by scanning transmission electron microscopy showed a majority population with a mass corresponding to an oligomeric state of 12. The results are interpreted to support the view that pUL6 forms the DNA entry portal, since it exists at a unique site in the capsid and forms a channel through which DNA can pass. The HSV-1 portal is the first identified in a virus infecting a eukaryote. In its dimensions and oligomeric state, the pUL6 portal resembles the connector or portal complexes employed for DNA encapsidation in double-stranded DNA bacteriophages such as φ29, T4, and P22. This similarity supports the proposed evolutionary relationship between herpesviruses and double-stranded DNA phages and suggests the basic mechanism of DNA packaging is conserved.

Herpes simplex virus (HSV) encodes seven proteins necessary for viral DNA synthesis—UL9 (origin-binding protein), ICP8 (single-strand DNA [ssDNA]-binding protein), UL30/UL42 (polymerase), and UL5/UL8/UL52 (helicase/primase). It is our intention to provide an up-to-date analysis of our understanding of the structures of these replication proteins and how they function during HSV replication. The potential roles of host repair and recombination proteins will also be discussed.

In an earlier report, we described a DNA helicase that is specifically induced upon infection of Vero cells with herpes simplex virus 1. We have purified this enzyme to near homogeneity and found it to consist of three polypeptides with molecular weights of 120,000, 97,000, and 70,000. Immunochemical analysis has shown these polypeptides to be the products of three of the genes UL52, UL5, and UL8 that are required for replication of a plasmid containing a herpes simplex 1 origin (oriS). In addition to helicase activity, the enzyme contains a tightly associated DNA primase. Thus, the three-subunit enzyme is a helicase-primase complex that may prime lagging-strand synthesis as it unwinds DNA at the viral replication fork.

Herpes simplex virus type 1 (HSV-1) DNA replication is associated with nuclear domains called ND10, which contain host recombination proteins such as RPA, RAD51, and NBS1 and participate in the cell's response to DNA damage. The stages of HSV-1 infection have been described previously. Infected cells at stage IIIa are observed after the initial disruption of ND10 and display nuclear foci, or prereplicative sites, containing the viral single-stranded-DNA-binding protein (UL29), the origin-binding protein (UL9), and the heterotrimeric helicase-primase. At stage IIIb, the viral polymerase, its processivity factor, and the ND10, protein PML, are also recruited to these sites. In this work, RPA, RAD51, and NBS1 were observed predominantly in stage IIIb but not stage IIIa prereplicative sites, suggesting that the efficient recruitment of these recombination proteins is dependent on the presence of the viral polymerase and other replication proteins within these sites. On the other hand, Ku86 was not found in any of the precursors to replication compartments, suggesting that it is excluded from the early stages of HSV-1 replication. Western blot analysis showed that RPA and NBS1 were (hyper)phosphorylated during infection, indicating that infection induces the host response to DNA damage. Finally, RPA, RAD51, and NBS1 were found to be associated with UL29 foci observed in transfected cells expressing UL29 and the helicase-primase heterotrimer and containing intact ND10. The ability to recruit recombination and repair proteins to various subassemblies of viral replication proteins thus appears to depend on several factors, including the presence of the viral polymerase and/or UL9 within prereplicative sites and the integrity of ND10.