Structure of the Human Cytomegalovirus B Capsid by Electron

JOURNAL OF STRUCTURAL BIOLOGY ARTICLE NO. SB984055

124, 70–76 (1998)

Structure of the Human Cytomegalovirus B Capsid by Electron Cryomicroscopy and Image Reconstruction S. J. Butcher,*,1 J. Aitken,† J. Mitchell,* B. Gowen,‡ and D. J. Dargan* *MRC Virology Unit and †Institute of Biomedical and Life Sciences, Division of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, United Kingdom; and ‡Department of Biochemistry, Imperial College of Science, Technology, and Medicine, London SW7 2AZ, United Kingdom Received June 22, 1998, and in revised form October 23, 1998

kinetics, and ability to spread in culture) and genetic relatedness. Herpesviruses genomes vary considerably in size (125–245 kb), G 1 C content (32–75%), and sequence organization, but these characteristics are independent of subfamily classification (Roizman et al., 1992). DNA sequence and genetic analysis of viruses from each subfamily have revealed a set of common ‘‘core’’ genes that are conserved in the viral DNA sequence. Core genes provide common functions and encode proteins required for viral DNA metabolism, DNA replication, and assembly of the viral capsid structure and some virus envelope glycoproteins with a role in virus entry (Davison, 1993). Other genes appear to be specifically conserved only within subfamilies and must have a role in conferring subfamily biological characteristics. Herpesviruses share a common virion architecture. The double-stranded DNA genome is contained within an icosahedrally symmetric capsid, though it is not yet clear how this large DNA molecule folds itself into the liquid crystalline packaged form (Booy et al., 1991). However, there is no convincing evidence that the DNA is spooled around a protein core in the shape of a torus as earlier reported (Furlong et al., 1972). The nucleocapsid is surrounded by a complex protein layer known as the tegument and enveloped by a lipid-rich membrane decorated with virus glycoprotein spikes. The capsid shell architecture of the a-herpesviruses herpes simplex virus type 1 (HSV-1) (Booy et al., 1988; Schrag et al., 1989; Zhou et al., 1994), equine herpesvirus type 1 (EHV-1) (Baker et al., 1989), and the more distantly related channel catfish virus (CCV) (Booy et al., 1996) has been studied by cryomicroscopy and image reconstruction. The capsid is organized on a T 5 16 lattice with 150 hexamers (hexons) and 12 pentamers (pentons). Intercapsomeric linkage is facilitated by triplex complexes. The HSV-1 capsid shell proteins have been localized by image reconstruction combined with

The three-dimensional structure of B capsids of the b-herpesvirus human cytomegalovirus (HCMV) was investigated at a resolution of 3.5 nm from electron cryomicrographs by image processing and compared with the structure obtained for the a-herpesvirus herpes simplex virus type 1 (HSV-1). The main architectural features of the HSV-1 and HCMV capsids are similar: the T 5 16 icosahedral lattice consists of 162 capsomers, composed of two distinct morphological units, 12 pentamers and 150 hexamers, with triplex structures linking adjacent capsomers at positions of local threefold symmetry. The main differences in the HSV-1 and HCMV capsids are found in the diameter of the capsids (125 and 130 nm, respectively); the hexamer spacing and relative tilt (center-to-center hexon spacing at outer edge, 17.9 and 15.8 nm, respectively); the morphology of the tips of the hexons (similar in length but 33% thinner in HCMV); and the average diameter of the scaffold (44 and 76 nm, respectively). By analogy with HSV-1, the mass on the HCMV hexon tip is attributed to the smallest capsid protein (HCMV gene UL48/49). The differences in capsid structure are discussed in relation to the ability of the HCMV structure to package a genome some 60% larger than that of HSV-1. r 1998 Academic Press Key Words: human cytomegalovirus; three-dimensional structure; electron cryomicroscopy. INTRODUCTION

Herpesviridae is a family of large and complex viruses ubiquitous in nature and infecting diverse animal species. The family is classified into a-, b-, and g-herpesvirus subfamilies on the basis of shared biological properties (i.e., host range, replication

1 Current address: Institute of Biotechnology and Department of Biosciences, Division of Genetics, Biocenter 2, FIN-00014 University of Helsinki, Finland.

1047-8477/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.

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TABLE I Location of the Four Major Components of the HSV Capsid Shell and Comparison to the HCMV Homologues (Gibson, 1996) HSV-1

HCMV

Location in capsid

Copy number

Gene

Gene product

Gene

Gene product

Hexons and pentons Triplexes Triplexes Hexon tips

960 640 320 900

UL19 UL18 UL38 UL35

VP5 (149 kDa) VP23 (34 kDa) VP19c (50 kDa) VP26 (12 kDa)

UL86 UL85 UL46 UL48/49

Major capsid protein (154 kDa) Minor capsid protein (35 kDa) Minor capsid protein-binding protein (33 kDa) Smallest capsid protein (8.5 kDa)

difference imaging (Trus et al., 1995; Zhou et al., 1988b, 1995) and antibody labeling (Trus et al., 1992) and are shown in Table I. Herpesvirus capsid assembly and viral DNA packaging takes place in the nucleus of infected cells and is dependent on the virus scaffolding proteins [e.g., the product of HSV-1 genes UL26 and UL26.5: and HCMV genes UL80 and UL80.5 (Desai et al., 1994; Preston et al., 1992; Tatman et al., 1994; Welch et al., 1991a,b)]. Three types of capsid structure are generated: A, which are devoid of any core structure; B, which contain a scaffold structure but lack viral DNA; and C, which are mature DNA-containing structures (Gibson and Roizman, 1972). The B-capsid structures are the most abundant in the infected cell nucleus and most easily isolated. This paper extends the investigation of capsid shell architecture to the b-herpesvirus subfamily. Using electron cryomicroscopy and image reconstruction, we have examined and compared the B-capsid shell structures of two important human pathogens: human cytomegalovirus (HCMV), the prototype virus of the b-herpesvirus subfamily, and HSV-1, the prototype virus of the a-herpesvirus subfamily. The HCMV capsid shell proteins are compared with those of HSV-1 in Table I. The HSV-1 and HCMV major capsid proteins (MCPs) are of similar molecular size and their respective genes share 25% nucleotide and 26% amino acid homology (Chee et al., 1989). The HSV-1 triplex structures are composed of two proteins, VP23 (gene UL18) and VP19c (gene UL38), in the ratio of 2:1. Similarly, the HCMV triplex structures are composed of the minor capsid protein-binding protein (mC-BP, gene UL46) and the minor capsid protein (mCP, gene UL85), also in the ratio of 2:1. However, while the HCMV mCP shares 22.5% amino acid homology with HSV-1 UL18, the HCMV mC-BP protein is only two-thirds of the size of its HSV-1 functional homologue and shares no appreciable amino acid homology (Gibson et al., 1996a). The small HSV-1 protein, VP26, located at the tip of the hexons is not required for capsid shell assembly (Tatman et al., 1994). HSV-1 VP26 and its HCMV counterpart, the ‘‘smallest capsid protein’’ (SCP, reported as gene UL48.5, or UL48/49), have

only limited amino acid homology, but both are small proteins with a basic pI (Baldick and Shenk, 1996; Gibson et al., 1996b). We report differences between the HCMV and HSV-1 capsid shell structures that are relevant to the observation that the HCMV capsid structure accommodates a DNA molecule up to 60% larger (at 245 kb) (Cha et al., 1996; Chee et al., 1990; Dargan et al., 1997) than that of the HSV-1 genome (152 kb) (McGeoch et al., 1988). MATERIALS AND METHODS Cells Human fetal foreskin fibroblast cells (HFFF-2; European Collection of Animal Cell Cultures, No. 86031405) were propagated in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (DMEM 10% FB). Virus Propagation The AD169 (Glasgow) stock of HCMV (Dargan et al., 1997) was used throughout. Virus-infected cell preparations were produced by infecting HFFF-2 cell monolayers (4 3 106 cells) with HCMV at a multiplicity of infection of 0.01 PFU/cell and, when extensive cytopathic effect was evident, seeding virus-infected cells (,105 cells) onto subconfluent monolayers of HFFF-2 cells in roller bottles (,2 3 107 cells/bottle) and incubating at 37°C. At 24 h postinfection (PI) the culture medium was replaced with fresh DMEM 10% FB and the infected cultures were incubated again at 37°C. Cytopathic effect was usually apparent by the fifth day PI and was extensive by the ninth day. The infected cells were detached from the surface of the roller bottle by gentle shaking and then pelleted by centrifugation at 1000g. Capsid Preparation Nuclei were isolated from the infected cell pellets by washing the cells with NTE buffer (0.5 M NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA) containing 1% Nonidet P-40. (NP40). The resulting nuclei were pooled and stored at 270°C until required. The nuclei were resuspended in NTE containing 1% NP40 and subjected to three cycles of freeze–thawing. This suspension was then probe sonicated to break residual nuclei open and the cellular debris pelleted at 1000g. The virus particles present in the supernatant were pelleted through a 40% (w/v in NTE) sucrose cushion (Sorvall TST41, 25 000 rpm, 1 h, 4°C), resuspended in 1% NP40 in NTE, and banded on a 10–40% sucrose gradient in NTE (Sorvall TST41, 40 000 rpm, 20 min, 4°C). The B-capsid band was harvested, pelleted (Sorvall TST41, 25 000 rpm, 1 h, 4°C), and resuspended in 0.15 M NaCl, 20 mM Tris, pH 7.4, 1 mM EDTA. HSV capsids were purified from baby hamster kidney cells

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infected for 16 h at 37°C with 5 PFU HSV-1 (strain 17) per cell, as described previously (Zhou et al., 1994). Electron Cryomicroscopy Electron cryomicroscopy allows us to examine samples under native, hydrated conditions free from some of the artifacts that are commonly associated with negatively stained specimens, as well as reveal internal details. Freshly prepared capsids were vitrified on holey carbon coated grids by plunge freezing into liquid ethane (Adrian et al., 1984; Fukami and Adachi, 1965). Samples were held at less than 2170°C in an Oxford CT3500 cryostage and observed using low-dose techniques in a Jeol JEM-1200 EX II electron microscope (120 kV, tungsten filament) fitted with an Oxford TAC100 twin-blade anticontaminator. Focal pairs were recorded on Kodak SO163 film using a dose of 10–15 e/Å2 at a nominal magnification of 25 000 or 30 000 at defocus values between 3 and 4 µm. Image Processing Selected micrographs were scanned on a Zeiss-SCAI scanner using a step size of 14 µm per pixel, corresponding to 0.58 nm per pixel (14/24 100) and 0.48 nm per pixel (14/29 000) on the specimen. Image quality was assessed and the defocus estimated by evaluating the position of the first zero in the sum of power spectra from the particles of each micrograph. In total six micrographs were scanned from which 144 particles were selected, boxed, floated, and normalized using SPIDER (Frank et al., 1981). Orientations and centers were determined and refined using the common lines procedure (Crowther, 1971; Fuller et al., 1996). The polar Fourier transform method was used to check the accuracy of the model, further refinement, and orientation searches for new data (Baker and Cheng, 1996). Three-dimensional reconstructions were calculated using cylindrical expansion methods (Crowther, 1971). Only particles with icosahedral phase residuals below 75° at 3.5-nm resolution were included in the reconstructions. The map of HCMV was calculated to 3.5-nm resolution from a subset of 59 particles from four micrographs (taken at 3 µm under focus). All the eigenvalues were greater than 10 and 97% were greater than 100, showing that reciprocal space was adequately sampled. Independent reconstructions calculated from subsets of the data were consistent with all the features described below. For comparison, a micrograph of HSV-1 B capsids (taken at 3 µm under focus) was also processed in a similar fashion. Here, 38 of 43 particles were included in the reconstruction that was also calculated to 3.5-nm resolution. For this reconstruction, all of the eigenvalues were greater than 1 and 87% were greater than 100, reflecting the lower number of particles included in the reconstruction. Visualization and rendering of the reconstructions were done in SPIDER. All of the programs were run on a Digital Alpha 255/233 AXP running Digital Unix OSF1 v3.2, an SGI Indigo Elan 4000 running IRIX 5.2, and a Digital Alpha 2100 running Open VMS 7.0. To address the scaling of the micrographs, additional data were collected on a CM200 FEG microscope at 200 kV, using preparations mixed with tomato bushy stunt virus [TBSV, 38.5 nm in diameter in the presence of 1 mM EDTA, pH 7.4 (Robinson and Harrison, 1982)] to act as an internal standard. These data (taken at defocus values of 5605 and 6120 nm for HSV-1 and 5200, 5605, and 5610 nm for HCMV, at a magnification of 319 400, and scanned with a 7-µm step size) could be corrected for the microscope contrast transfer function due to the presence of several minima in the power spectra, unlike the original data collected on the Jeol 1200EX with a tungsten filament. For each micrograph, at least 90 TBSV particles were aligned and a radial profile made of the average to check the magnification. The HSV1 data (134 particles) and the HCMV data (70 particles) were averaged separately and gave rise to radial profiles similar to those shown in Fig. 2, where the shell peaks for HCMV are at a larger radius

FIG. 1. Electron cryomicrograph of B capsids from HCMV.

than those for HSV1 (see Results). The data from the Jeol micrographs were then scaled accordingly. In addition the scaling of the HSV-1 reconstruction was checked against that of a 1.9-nm HSV-1 reconstruction kindly provided by Dr. Hong Zhou (Zhou et al., 1995). RESULTS

Electron Cryomicroscopy and Image Reconstruction We have studied the structure of the HCMV capsid by electron cryomicroscopy and image processing. Figure 1 shows part of an electron micrograph of vitrified HCMV B capsids. The capsids have a diameter of approximately 130 nm. This can also be seen from the radial density profile (Fig. 2). The capsomeres covering the surface of each capsid are clearly evident as projections at the edge of the particle and as regularly spaced dots across the surface (,15 nm in diameter). In addition the scaffold can be seen as a dense area, roughly in the center of each particle. The HCMV capsids were difficult to fully dissociate from the nuclear matrix without breakage, and could not be stored at 270, 220, or 4°C without further breakage occurring. Consequently, only relatively few particles from each field could be used (typically 30 particles were processed per micro-

FIG. 2. Graph showing the radial density distribution of B capsids from HCMV (solid line) and HSV-1 (broken line) calculated from the reconstructions shown in Fig. 3.


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FIG. 3. Comparison of HCMV and HSV-1 reconstructions. Surface representations of HCMV (A) and HSV-1 (B) viewed down an icosahedral twofold axis of symmetry contoured at one standard deviation above the mean density value. The inset shows the positions of hexamers (white dots) and pentamers (green dots) within 1 facet of a T 5 16 lattice, in an orientation similar to that of the reconstructions. Central 0.58-nm-thick sections through the HCMV (C) and the HSV-1 (D) reconstructions. Bar 5 50 nm.

graph), so data from several micrographs were combined to yield the final reconstruction. There was also a high rejection rate during the common lines refinement as up to 60% of the particles per micrograph gave icosahedral phase residuals greater than 75°, even at 3.5-nm resolution, and were therefore excluded from the reconstruction. Three-Dimensional Structure The major structural components of the HCMV B capsid are 150 hexons, 12 pentons, and 320 triplexes

organized on a T 5 16 icosahedral lattice (Fig. 3A). The relative positions of the capsomers (hexamers and pentamers) within a facet are shown in the top inset of Fig. 3. Capsomers The HCMV hexons and pentons form cylindrical protrusions (Figs. 3A, 3C, 4) penetrated by an axial channel constricted 7.4 nm from the base of the capsomer. The hexons appear slightly skewed, so that the axial channel is oval rather than round. The

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Comparison with HSV-1 B Capsids

FIG. 4. (A) Enlarged view of a HCMV1 hexamer from an icosahedral 2f axis. (B) Enlarged view of a HCMV1 pentamer from an icosahedral 5f axis. Adjacent triplexes can also be seen at the base of each capsomer.

tip of each hexon subunit is topped by a rod-shaped protrusion (Figs. 3A, 3C, 4A), so the hexon looks like an open flower with six petals (Figs. 3A, 4A). In contrast, the penton subunit tips are more pointed, giving a triangular cross section (Figs. 3A, 3C, 4B). Triplexes The triplexes are the structures found at the local threefold axes of symmetry, interacting with the hexon and penton walls (Figs. 3A, 3C, 4). The triplexes do not appear to be threefold symmetric despite their position. Instead they vary depending on the location within the asymmetric unit (Figs. 3A, 4). The most striking triplexes are those interacting with the pentons, where the triplex forms a buttress against the penton wall (Figs. 3A, 3C, 4B). Floor of the Capsid The floor of the capsid is seen as a peak of density in the radial profiles (Fig. 2) at a radius of 50 nm in HCMV. This floor is penetrated by holes at the base of each capsomer and underneath the triplexes (Fig. 3C). The thinnest part of the shell occurs between the triplexes. Internal Details The scaffold core is a very strong feature in the HCMV micrographs (Fig. 1). However, it is not evident in the reconstructions (Fig. 3C). Inside the capsid, there is only a low signal from icosahedrally ordered mass above the background noise level, which varies between different HCMV reconstructions, suggesting that it is not significant (data not shown). Although the density from the scaffold proteins does not reveal an icosahedrally ordered structure (Fig. 3C), we can still estimate the extent of the majority of the scaffold from the radial density plot (Fig. 2). This gives an average scaffold core radius of 38 nm The dips seen at low radii (less than 5 nm) are probably artifacts of the Fourier Bessel procedures, as analysis of the aligned raw data did not show similar fluctuations.

The basic elements of the HSV-1 reconstruction described here are similar to those reported by others (Booy et al., 1994; Newcomb et al., 1993; Trus et al., 1992, 1995; Zhou et al., 1998a, 1995) although the structure was calculated independently. The HCMV B capsid has a diameter larger than that of HSV-1 B capsids. This is illustrated from the radial density plots shown in Fig. 2. HCMV has an average outer radius of 65 nm compared with 62.5 nm for HSV-1. The internal radius of the capsid was estimated using the peak from the radial density plot (Fig. 2) corresponding to the floor of the shell in Figs. 3C and 3D (HCMV, 50 nm; HSV-1, 47.5 nm). The average height of the capsomers is 15 nm in both cases. In addition the maximum hexon diameter is similar in both structures (13.7 nm, Figs. 3C, 3D). However, a distinct difference is the relative tilt between adjacent hexons. For instance, the angle between the hexon on the 2f axis (9 o’clock, Figs. 3C, 3D) and the adjacent hexon is 9.5° in HCMV but only 5° in HSV-1. Consequently, the separation between the hexons at the base, measured from the center of one hexon to the center of the next, is 14.7 nm in both structures. However, at the tip of the hexons, it has increased to 17.9 nm in HCMV and only 15.8 nm in HSV-1, directly accounting for a change in capsid diameter. The tips of the HSV-1 hexons that have been identified as a single protein species, VP26 (Booy et al., 1994; Trus et al., 1995; Zhou et al., 1995), are not as prominent as those of HCMV at this resolution (Figs. 3, 4A). Those on the HCMV hexons are similar in length, but are 33% thinner (Figs. 3C, 3D); the choice of contour level did not appreciably affect the appearance or dimensions of these features. The HSV-1 pentons lack VP26, giving the pentons a distinctive top compared with the hexons. In HCMV, the penton tips are also distinct from those of the hexons (Fig. 4). This may reflect a difference in composition between the hexons and the pentons due to the SCP, or a conformational difference between the hexameric form and the pentameric form of the MCP. The final difference between the two capsids is the distribution of the scaffold core, which can be seen from the radially averaged profiles (Fig. 2). The profile of the HSV-1 B capsid agrees well with that of Zhou et al. (1998b). However, the average density falls off at radii of 22 nm in HSV-1 and 38 nm in HCMV. DISCUSSION

The overall architectural design of the herpesvirus capsid has been conserved among different subfamilies. However, our studies have demonstrated that limited changes can be made to the capsid structure by virus-specific differences in the component proteins.

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The HCMV capsid is made up of hexamers and pentamers arranged on a T 5 16 lattice. By analogy with HSV-1, we expect that the major capsid protein (MCP) forms the major part of these structures and that the smallest capsid protein (SCP) forms the tips of the hexons. The minor capsid protein (mCP) and the mCP-binding protein (mC-BP), which associate together in a ratio of 2:1, are analogous to the triplex proteins of HSV-1 and probably form the equivalent structures seen in the HCMV reconstruction at the positions of local threefold symmetry (Fig. 4). One of the motivating questions for performing this study was: How does the capsid of HCMV package a genome that is 60% larger than that of HSV-1? There is, as predicted from a comparative study between HSV-1 and channel catfish virus (Booy et al., 1996), no change in the T number of the capsid to accommodate the larger genome; however, there is an increase in the capsid volume. The internal radius of the capsid was estimated using the peak from the radial density plot which corresponds to the floor of the shell (HCMV, 50 nm; HSV-1, 47.5 nm) because the edge of the shell density is harder to estimate. Assuming a sphere, this gives a volume ratio of 1.17 for HCMV compared with HSV-1. This is much smaller than the ratio of the genome lengths (1.51) of the two virus strains used in this study [HCMV AD169, 230 kb (Dargan et al., 1997); HSV-1 strain 17, 152 kb (McGeoch et al., 1988)]. Interestingly, HCMV assembles this larger shell with proteins that have molecular weights similar to those of HSV-1 (Table I). In fact one of the HCMV triplex proteins is appreciably smaller (mC-BP). It has been shown for HSV-1 that the triplex proteins are pivotal in in vitro capsid assembly, linking the capsomers together (Booy et al., 1996; Newcomb et al., 1996). Thus it is possible that the HCMV triplex proteins are important in determining the greater center-tocenter spacing of the HCMV capsomers and their relative tilt. The size of the scaffold core may also be important for the assembly of a larger capsid. The observed difference in the HCMV and HSV-1 B capsid volumes is insufficient by itself to account for packaging of the larger HCMV genome. There are at least three additional factors that have been shown to be important for DNA packaging in other viruses: DNA density, variable capacity of the capsid, and expansion associated with DNA packaging. The average interduplex spacing for most viruses, including HSV-1 (Booy et al., 1991), has been reported to be ,2.6 nm, with L-A virus (at 4–4.5 nm) as an exception (Caston et al., 1997). HCMV could have more densely packed DNA than HSV-1. In support of this, studies of bacteriophage T7 have shown that interduplex spacing increased as genome length fell (Cerritelli et al., 1997), but on the contrary, HSV-1 and the

smaller channel catfish virus were reported to have similar genome packing densities (Booy et al., 1996). Alternatively HSV-1 may underutilize its packaging capacity like bacteriophage l, which can package DNA 9% larger than its genome without affecting viability (Weil et al., 1972). A third possibility is that an expansion of the HCMV capsid is associated with DNA packaging. B capsids from simian CMV have been estimated to have a diameter 7% smaller than that of A capsids when examined in thin sections of infected cell nuclei (Lee et al., 1988). Studies of the DNA-containing capsid are required to address these issues in more detail. We have endeavored to isolate such capsids from both virions and infected cells, but invariably ended up with damaged, empty capsids. This implies that HCMV is more fragile than HSV-1. In conclusion, this preliminary study of HCMV emphasizes the similarities between evolutionary distinct herpesviruses. It also supports the idea that although the three-dimensional structures of the capsid proteins are similar at the gross level, we can expect changes in the interactions between monomers at the atomic level, giving rise to differences in spacing between capsomers. Dr. Hong Zhou is thanked for providing a reconstruction of HSV-1 B capsids. Dr. Frazer Rixon is thanked for helpful discussions. Dr. Stephen Fuller kindly provided his programs, as did Felix deHaas and Ralph Heinkel. Ilaria Ferlenghi and Erica Mancini are thanked for help with scanning of the micrographs. This work was funded by the U.K. Medical Research Council. REFERENCES Adrian, M., Dubochet, J., Lepault, J., and McDowall, A. W. (1984) Cryo-electron microscopy of viruses, Nature (London) 308, 32–36. Baker, T. S., and Cheng, R. H. (1996) A model-based approach for determining orientations of biological macromolecules imaged by cryo-electron microscopy, J. Struct. Biol. 116, 120–130. Baker, T. S., Newcomb, W. W., Booy, F. P., Brown, J. C., and Steven, A. C. (1989) Three-dimensional reconstructions of ‘light’ and ‘intermediate’ capsids of equine herpes virus, Electron Microsc. Soc. Am. Proc. 47, 822–823. Baldick, C. J., and Shenk, T. (1996) Proteins associated with purified human cytomegalovirus particles, J. Virol. 70, 6097–6105. Booy, F. B., Newcomb, W. W., Brown, J. C., and Steven, A. C.(1988) Herpesvirus nucleocapsids imaged in the frozen-hydrated state, Electron Microsc. Soc. Am. Proc. 46, 164–165. Booy, F. P., Newcomb, W. W., Trus, B. L., Brown, J. C., Baker, T. S., and Steven, A. C. (1991) Liquid-crystalline, phage-like packing of encapsidated DNA in herpes simplex virus, Cell 64, 1007–1015. Booy, F. P., Trus, B. L., Davison, A. J., and Steven, A. C. (1996) The capsid architecture of channel catfish virus, an evolutionary distant herpesvirus, is largely conserved in the absence of discernible sequence homology with herpes simplex virus, Virology 215, 134–141. Booy, F. P., Trus, B. L., Newcomb, W. W., Brown, J. C., Conway, J. F., and Steven, A. C. (1994) Finding a needle in a haystack: Detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus), Proc. Natl. Acad. Sci. USA 91, 5652–5656. Caston, J. R., Trus, B. L., Booy, F. P., Wickner, R. B., Wall, J. S.,

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