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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 phi29, 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.

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The structures of bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 1 (HPV-1) were determined at 2.5 nm resolution by cryoelectron microscopy and three dimensional image reconstruction techniques. As expected, the reconstructions showed that both viruses consist of a T = 7 icosahedral capsid (approximately 60 nm in diameter) which surrounds a nucleohistone core. The capsid morphologies of the two viruses are nearly indistinguishable. Each capsid consists of a shell layer (approximately 2 nm thick) of nearly continuous density from which capsomers project radially to a maximum height of approximately 5.8 nm. The five-coordinate (pentavalent) and six-coordinate (hexavalent) capsomers both exhibit distinct five-fold axial symmetry as was observed for SV40 and polyoma viruses. Thus, both genera (papilloma and polyoma) of the papovavirus family have now been shown to have the characteristic "all-pentamer" capsid construction. BPV-1 and HPV-1 capsomers consist of a thick (8.6 nm diameter) trunk that broadens distally to form a regular five-pointed, star-shaped head, and proximally to create the shell layer where capsomers associate. A cylindrical channel (approximately 2.8 nm diameter) extends along the axis of each capsomer from the interior of the virus to a point approximately half way to the capsomer surface. Computationally sectioned views of individual capsomers displayed at decreasing radii show that each of the five capsomer subunits (in both pentavalent and hexavalent capsomers) makes a pronounced (30 degrees) left-handed twist just above the outer surface of the capsid shell. Similar views of the reconstructions also clarify the morphology of intercapsomer contacts. For example, they show how hexavalent capsomers coordinate six neighboring capsomers despite the fact that they contain only five subunits. The system of intercapsomer contacts is indistinguishable in BPV-1 and HPV-1, but quite different from that reported for polyoma virus capsids assembled in vitro from the major capsid protein, VP1 (D. M. Salunke, D. L. D. Caspar, and R. L. Garcea. 1989. Biophys. J. 56:887-900). Thus, because both polyoma and papilloma viruses have all-pentamer capsids, it appears that intracapsomer subunit-subunit interactions which stabilize pentameric capsomers are better preserved evolutionarily than those involved in capsomer-capsomer contacts.

UL25 and UL17 are two essential minor capsid proteins of HSV-1, implicated in DNA packaging and capsid maturation. We used cryo-electron microscopy to examine their binding to capsids, whose architecture observes T = 16 icosahedral geometry. C-capsids (mature DNA-filled capsids) have an elongated two-domain molecule present at a unique, vertex-adjacent site that is not seen at other quasiequivalent sites or on unfilled capsids. Using SDS-PAGE and mass spectrometry to analyze wild-type capsids, UL25 null capsids, and denaturant-extracted capsids, we conclude that (1) the C-capsid-specific component is a heterodimer of UL25 and UL17, and (2) capsids have additional populations of UL25 and UL17 that are invisible in reconstructions because of sparsity and/or disorder. We infer that binding of the ordered population reflects structural changes induced on the outer surface as pressure builds up inside the capsid during DNA packaging. Its binding may signal that the C-capsid is ready to exit the nucleus.