The UL25 and UL17 proteins are minor components of the HSV-1 capsid but play essential roles in packaging and retaining the viral genome. The UL25-null mutant appears to package DNA only transiently, yielding empty capsids and near-unit-length genomes, whereas the UL17-null mutant completely abolishes DNA packaging (3
). These two proteins, being essential and highly specific to herpesviruses, are therefore of great interest as structural drug targets for new classes of antivirals. We have pursued biochemical and structural characterization of these proteins, aiming to discover their location and conformation on the capsid as well as their binding partners, interfaces, and accessible surfaces. We have previously identified regions of the UL25 N terminus that are essential for binding to capsids (2
) and confirmed by cryo-EM that the UL25 protein occupies a portion of the CVSC density that is arrayed around the capsid vertices (4
). Its location within the CVSC molecule was constrained to the penton-distal portion by visualization of a GFP tag within the N-terminal portion of the UL25 sequence (). The UL17 protein is proposed to partner with UL25 in comprising the CVSC molecule, presumably occupying the remaining portion of CVSC that is closest to the penton (36
). We have now confirmed this by several approaches involving TAP- and GFP-tagged mutants that remain viable on noncomplementing cells, although at yields lower than that of wild-type KOS virus. In particular, we have demonstrated that the tagged UL17 and UL25 proteins are present on A, B, and C capsids and that they copurify in TAP pulldown assays. Further, cryo-EM-based reconstructions of the UL17-GFP mutant virus definitively localize UL17 in the penton-proximal density of the CVSC molecule, in an approximate 1:1 ratio with the UL25 protein and with its C terminus abutting the UL25-containing portion.
The UL17-tagged mutants produce an abundance of empty capsids, as does the UL25-null mutant, and presumably are impaired in the packaging reaction. The evident roles for the UL17 and UL25 proteins in stably packaging DNA would seem at odds with their localization on the capsid exterior around penton vertices, from where packaged viral DNA is presumably inaccessible. Given the imposition of icosahedral symmetry in the density maps calculated for this study, the disposition of CVSC molecules around the unique portal vertex remains unknown, but it is reasonable to assume their presence at this site where the DNA enters the capsid and likely their activity in packaging and maintaining the DNA. However, as is common for virus structural subunits, these proteins also have additional functions. UL25 is implicated in signaling nuclear exit for the DNA-filled mature capsid (15
) and in docking at nuclear pores during infection (24
). UL17 has been proposed to stabilize the procapsid and prevent premature angularization that would be detrimental to DNA packaging (30
). Together, these functions appear consistent with the global coverage of the capsid by the CVSC molecule observed in cryo-EM studies, including its presence on A and B capsids, as well as the mature, DNA-filled C capsid. Detection of the CVSC on procapsids would further support the structural roles in capsid stabilization proposed.
The cryo-EM density maps allow assignment of the UL17 and UL25 proteins to distinct regions of the CVSC molecule as well as their approximate orientation within this molecule and the probable capsid proteins with which they interact. Localization of the UL17 C-terminal GFP tag and UL25 N-terminal GFP tag supports the UL17 and UL25 proteins binding primarily in a C-terminal-to-C-terminal fashion and with these domains of both proteins contacting the underlying penton-proximal triplex (). UL25 extends away from the penton, bridging the gap to the neighboring triplex and contacting one of the adjacent hexons. The density ascribed to UL17 weakens as it extends toward the vertex, suggesting that it is not stabilized by contact with the penton but is instead flexible—although contact at the portal vertex cannot be determined. Nonetheless, occupancy of CVSC density by UL17 is fairly constant at ~70% for wild-type and UL17-GFP capsids, while the UL25 protein ranges from 80% to 94%. Given the more extensive contacts with the capsid by the UL25 protein, we suspect that the single-triplex and UL25 binding site employed by the UL17 protein may result in greater loss during capsid purification, and indeed the CVSC molecule as a whole appears to be easily dislodged since it is absent from the early structural cryo-EM studies on herpesvirus capsids. While UL17 has been proposed to aid in recruiting UL25 to the capsid, and indeed UL25 does not bind efficiently to capsids in the absence of UL17 (30
), UL17 would subsequently appear to be lost from the capsid independently of UL25. However, despite immunodetection of UL25 on capsid preparations of the UL17-null mutant, no CVSC density has been detected in cryo-EM reconstructions, suggesting that the occupancy of the CVSC site is low or that some UL25 may bind to non-CVSC sites on the capsid surface.
The cryo-EM density map indicates that while UL17 is present at relatively high levels in the CVSC, the putative N-terminal penton-proximal domain is smeared out as if it were flexible. This may have functional significance around the portal where the DNA-terminase complex binds and together function as an ATP-driven motor to pump the viral DNA into the capsid and where UL17 may function to support postpackaging cleavage of the DNA. The mechanism of action for such a motor has been modeled for bacteriophages in several different ways, generally involving relative rotation between a capsid-supported stator element and a rotor, and which may exploit the symmetry mismatch between the icosahedral 5-fold vertex and dodecahedral portal complex (13
), although alternatives have been proposed (29
). Recognition of a terminal DNA sequence or stalling of the motor may separately or together signal cleavage, and the flexibility of the UL17 N-terminal domain may be essential to enable cleavage no matter what part of its cycle the motor and DNA substrate are in. Low-symmetry data analyses will need to target the portal vertex specifically to distinguish it on capsids from the similarly sized penton in order to resolve the conformation of the vertex-proximal CVSC density. In the vicinity of penton vertices, the flexibility of UL17 may have a role in binding the first layers of the irregular tegument and, in particular, the UL37 and UL36 (VP1/2) proteins (11
). Nuclear-purified capsids do not address this situation, for which capsids from partially stripped virions (19
), or extranuclear capsids, will be needed.
While localization of the UL17 protein on the herpesvirus capsid is an important step in understanding the capsid assembly and DNA packaging processes, specific investigation of its conformation at the portal vertex is a crucial sequel that we are currently pursuing. Nonetheless, identification of the UL17 C termini as accessible and the likely C-terminal-to-C-terminal domain organization with the UL25 protein offer several bases for exploitation in drug targeting. Abolishment of capsid association of either subunit, such as by tightly binding a highly specific molecule or poisoning the CVSC molecule with a defective subunit, would be expected to arrest the formation of C capsids and potentially neutralize viral propagation. In addition to further characterization of CVSC structure and function, we will further identify essential regions for UL17-UL25 dimer formation and for binding to the herpesvirus capsid. This will allow better understanding of interfaces that may potentially be disrupted as well as defining exposed regions that may be targeted for additional labeling experiments that will more comprehensively detail subunit organization.