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Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 3Comparative virion structures of human herpesviruses

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Introduction

The herpesvirus family consists of a group of viruses distinguished by the large size of their linear double-stranded DNA genomes (∼130–250 kbp) and a common architecture of infectious particles (Fig. 3.1) (Chiu and Rixon, 2002; Gibson, 1996; Steven and Spear, 1997). Indeed, before the birth of molecular biology and the availability of genomic sequencing, the common hallmark structural features shared by these viruses were the most important criteria for the classification of a herpesvirus (Roizman and Pellett, 2001). All herpesviruses identified to date, which include eight different types that are known to infect human, and more than 170 other viruses that are found in animals as well as in fish and amphibians (Roizman and Pellett, 2001), exhibit identical structural design as illustrated using human cytomegalovirus shown in Fig. 3.1. These viruses have a highly ordered icosahedral-shape nucleocapsid of about 125–130 nm in diameter, which encases the viral DNA genome. The nucleocapsid is surrounded by a partially ordered proteinaceous layer called the tegument, which in turn is enclosed within the envelope, a polymorphic lipid bilayer containing multiple copies of more than 10 different kinds of viral glycoproteins that are responsible for viral attachment and entry to host cells.

Fig. 3.1. Herpesvirus architecture.

Fig. 3.1

Herpesvirus architecture. (a) Electron cryomicrograph of a human cytomegalovirus virion showing the different compartments of a herpes virion. (b) Schematic diagram illustrating the multilayer organization of human herpesviruses. Also shown are the electron (more...)

Based on their biological properties such as growth characteristics and tissue tropism, herpesviruses can be further divided into three subfamilies. Among the eight human herpesviruses, the alpha subfamily includes neurotropic viruses and contains the herpes simplex virus (HSV) 1 and 2, and Varicella zoster virus (VZV). The members of the gamma subfamily are lymphotropic viruses and include Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV). The viruses of the beta subfamily appear to be able to establish infections in many different types of cells and tissues, and include human cytomegalovirus (HCMV), and human herpesvirus 6 and 7. This subfamily classification system is largely consistent with the extensive genomic information that is now available (McGeoch et al., 2000). While studies have been attempted to investigate the structure and architecture of each of the eight human herpesviruses, virion and virus-related particles of herpes simplex virus 1 (HSV-1), the prototype of all herpesviruses, have been subjected to the most extensive structural studies (Booy et al., 1991; Newcomb et al., 1993, 2000; Schrag et al., 1989; Trus et al., 1996; Zhou et al., 1999, 2000). During the last several years, significant progress has also been made in understanding the structure of cytomegaloviruses (Bhella et al., 2000; Chen et al., 1999; Trus et al., 1999), the prototype of the beta herpesvirus family, and KSHV, a representative of the gamma herpesvirus family (Lo et al., 2003; Nealon et al., 2001; Trus et al., 2001; Wu et al., 2000). Using HSV-1, HCMV, and KSHV as examples for each of the subfamilies, this chapter focuses primarily on the structures of these three viruses, and discusses the recent progress on understanding the structures of human herpesviruses.

Different virus-related particles found in infected cells

Summary of virion assembly pathway

Each of the herpesviruses encodes a specific set of proteins that form the different compartments of the virion (e.g. capsid, Table 3.1). Although many of the primary amino acid sequences of these proteins are not highly conserved among different viruses, the assembly pathway of the virus particles is highly similar (Fig. 3.2) (Gibson, 1996; Roizman and Knipe, 2001; Yu et al., 2003). The nucleocapsid is formed in the nucleus and follows a pathway that bears a marked resemblance to those of DNA bacteriophages (Casjens and Hendrix, 1988). First, a procapsid is assembled with the formation of the capsid shell and the internal scaffolding structure. Second, the procapsid is converted into mature nucleocapsid, during which time, the morphogenic internal scaffolding protein is released and replaced by the viral DNA genome, concomitant with a major conformation change of the capsid shell (Newcomb et al., 1999; Yu et al., 2005). Subsequent events, however, differ from the phage assembly pathway (Fig. 3.2). The mature nucleocapsid exits the nucleus and acquires its tegument and envelope, through repeated fusion with and detachment from nuclear membranes and other cellular membranous structures. Eventually, the mature infectious virion particles are released into the extracellular space via cellular secretory pathways. During this assembly process, different virus-related particles and structures, including the mature nucleocapsids and virions as well as the intermediate and aberrant products, can be found in the infected cells and the extracellular media (Figs. 3.1(c)–(e) and 3.2).

Table 3.1. Major virion proteins present in HSV-1, HCMV and KSHV.

Table 3.1

Major virion proteins present in HSV-1, HCMV and KSHV.

Fig. 3.2. Different virus-like particles and structures during lytic cycle of herpesvirus replication.

Fig. 3.2

Different virus-like particles and structures during lytic cycle of herpesvirus replication. The infectious virion initializes infection by either endocytosis or fusion with the cell membrane, which releases the nucleocapsid and some tegument proteins (more...)

Different virus-like particles secreted from infected cells

Since the discovery of the herpesviruses, it has been long recognized that, in addition to producing infectious virus particles, the infected host cells also generate non-infectious particles such as noninfectious enveloped particles (NIEP, Fig. 3.1(c)) and dense bodies (DB) (Figs. 3.1(d) and 3.2) (Gibson, 1996; Steven and Spear, 1997). Both NIEP and DB are commonly found in the culture media of cells that are lytically infected with HSV-1 and HCMV. The ratio of these particles to mature infectious virion particles can sometimes reach 20:1, suggesting that they are produced in great excess (Gibson, 1996; Steven and Spear, 1997). The exact function of these non-infectious particles in viral infection and replication is currently unknown, although they have been proposed to act as decoys that saturate and overwhelm the immune surveillance thereby facilitating the survival of the infectious virions in the hosts (Gibson, 1996; Steven and Spear, 1997)

Structurally, both the NIEP and DB are significantly different from the infectious virion (cf. Fig. 3.1(a), (c), (d)). They can be easily distinguished using electron cryomicroscopy (cryoEM) and separated from the mature infectious virions using ultracentrifugation approaches. As described above, all infectious herpesvirus virions share four common structural features (Fig. 3.1(b)). First, all herpesviruses contain a large double-stranded DNA (dsDNA) genome. The genomic DNA represents a dense core of ∼90 nm in diameter, which can be stained with uranyl acetate and visualized using electron microscopy (Gibson, 1996; Steven and Spear, 1997) and appears as “fingerprint” patterns when examined by electron cryomicroscopy (Fig. 3.1(a)) (Booy et al., 1991; Zhou et al., 1999). Second, a capsid of icosahedral shape, which primarily consists of many copies of four different viral proteins, encases the genomic DNA. Third, a protein layer structure, named as the tegument first by Roizman and Furlong (Roizman and Furlong, 1974), surrounds the capsid and occupies the space between the capsid and the envelope. The tegument structure contains many virus-encoded factors that are important for initiating viral gene transcription and expression as well as modulating host metabolism and shutting down host antiviral defense mechanism (for a brief review, see Roizman and Sears, 1996). Finally, a lipid-bilayer envelope constitutes the outermost perimeters of the particles, and contains all the surface virion glycoproteins that are responsible for viral infectivity and entry (Fig. 3.1).

Unlike infectious virion particles, a NIEP does not contain a genomic DNA core and its capsid core appears to be B-capsid-like under electron microscopy (Figs. 3.1(c) and 3.2). In contrast, a dense body does not contain a capsid and appears as a cluster of tegument proteins encased by the lipid-bilayer membranous envelope (Fig. 3.1(d)). The presence of NIEP and DB indicates that neither packaging of viral genome nor capsid formation is required for viral envelopment.

Different capsid-like structures inside the infected cells

The capsid assembly is a continuous sequential process, leading to the synthesis of the highly ordered capsid structures. In cells that are lytically infected with herpesviruses, several kinds of virus capsid-like structures have been identified as representing stable endpoints or long-lived states (Figs. 3.1 and 3.2). Gibson and Roizman first introduced the terms A-, B-, and C-capsids to describe these intracellular capsid-like structures in HSV-1 infected cells (Gibson and Roizman, 1972). Similar capsid structures have been observed in cells infected with HCMV (Gibson, 1996; Irmiere and Gibson, 1985). Recent work has revealed A-, B- and C-capsids of comparable chemical composition and structural features in the nuclei of gammaherpesvirus infected cells and this suggests that the gammaherpesvirus capsid assembly probably also proceeds in a similar manner (O’Connor et al., 2003; Yu et al., 2003). These capsids all have a distinctive polyhedral shape when examined under electron microscope. Another capsid type, termed procapsid, can be obtained from in vitro assembly experiments using recombinant capsid proteins or from cells infected by a HSV-1 mutant containing a temperature sensitive mutation at the gene encoding the viral protease (Rixon and McNab, 1999; Trus et al., 1996). The procapsid has a distinctive spherical shape and is only transiently stable. They undergo spontaneous structural rearrangement to become the stable angular or polyhedral form similar to the other types of capsids (Heymann et al., 2003; Yu et al., 2005; Zhou et al., 1998b). A-capsids represent empty capsid shells that contain neither viral DNA nor any other discernible internal structure. They are thought to arise from abortive, dead-end products derived from either the inappropriate loss of viral DNA from a C-capsid or the premature release of scaffolding protein from a B-capsid without concurrent DNA packaging (Gibson, 1996). B-capsids are capsid shells containing an inner array of scaffolding protein. C-capsids are mature capsid shells that are packaged with viral DNA and do not contain the scaffolding proteins. B-capsids are believed to be derived from the procapsids upon proteolytic cleavage of the scaffolding protein, and their fate in viral maturation is controversial. Early pulse-chase experiments have suggested that B-capsids can mature to C-capsids, which in turn serve as the infectious virus precursors (Perdue et al., 1976), and recent studies suggest that they might also be a dead-end product in capsid assembly similar to A-capsids (Trus et al., 1996; Yu et al., 2004). It remains unclear whether the spherical procapsids first adapt to the stable angular form before or after the cleavage of its scaffolding protein. The C-capsid buds through the nuclear membrane using an envelopment and de-envelopment process and acquires an additional layer of proteins that forms the tegument in the cytoplasm (for review, see Mettenleiter, 2002). Enveloped virions are then released by exocytosis (Fig. 3.2).

Assembly of viral capsid

A-, B- and C-capsids represent the stable intermediates or the end products of the herpesvirus capsid assembly process (Figs. 3.1(e) and 3.2). In HSV-1, capsid assembly begins with the formation of the spherical procapsid through the association of the carboxyl terminus of the scaffolding protein with the amino terminus of the viral major capsid protein (MCP), similar to bacteriophage proheads (Conway et al., 1995; Jiang et al., 2003). Previous experiments have shown that the procapsid can be assembled in vitro from the capsid and scaffolding proteins, in the absence of the viral capsid maturation protease (Newcomb et al., 1999) or from cells infected with viruses containing a temperature-sensitive protease mutant (Heymann et al., 2003). These procapsids can spontaneously rearrange into a large-cored, angular particle resembling the B-capsid, but these large-cored particles do not encapsidate DNA or become mature virions. Past studies have also shown that cells infected with a HSV-1 mutant containing a temperature-sensitive mutation in the protease gene produced capsids that assemble at the non-permissive temperature, similar to the in vitro-assembled procapsids (Rixon and McNab, 1999). The capsids matured when protease activity was restored (Rixon and McNab, 1999), demonstrating that the procapsid is the precursor to the angular capsid (Fig. 3.2). The proteolytic cleavage of the intra-capsid scaffolding proteins at their C-termini by the viral protease (Hong et al., 1996; Liu and Roizman, 1991, 1992; Preston et al., 1992; Welch et al., 1991) interrupts the interactions between the scaffolding proteins and the major capsid proteins (Zhou et al., 1998b). The interactions between the scaffolding protein, the major capsid protein, and viral protease are important targets for antiviral drug design in treating and controlling herpesvirus infections (Flynn et al., 1997; Qiu et al., 1996; Shieh et al., 1996; Tong et al., 1996, 1998). Proteolytic cleavage of the scaffolding protein is followed by the recruitment of the smallest capsid protein, VP26, through an ATP-dependent process (Chi and Wilson, 2000), leading to the formation of the intermediate or B-capsids. The mature procapsids are believed to arise spontaneously by packaging the viral genome DNA, a process that is currently not completely understood (Yu et al., 2005).

Compositions and three-dimensional structural comparisons of alpha, beta and gammaherpesvirus capsids

A-, B-, and C-capsids (Yu et al., 2005) can be isolated from the nucleus of the host cells lytically infected by herpesviruses and they have been subjected to three-dimensional structure studies for HSV-1 (Zhou et al., 1998a, 1994), HCMV (Butcher et al., 1998; Chen et al., 1999; Trus et al., 1999), and KSHV (Nealon et al., 2001; Trus et al., 2001; Wu et al., 2000; Yu et al., 2003). While these three types of capsids have different composition (e.g., viral DNA and internal scaffolding protein), they all have a common shell structure that consists of 150 hexameric (hexon) and 12 pentametric (penton) capsomers, which are connected in groups of three by the triplexes, asymmetric structures that lie on the capsid floor (Fig. 3.3). During the last few years, considerable progress of the three-dimensional structure of the capsids and the assembly of the capsomers and triplexes has been made on the studies.

Fig. 3.3. HSV-1 capsid at 8 Å resolution (Zhou et al.

Fig. 3.3

HSV-1 capsid at 8 Å resolution (Zhou et al., 2000) and atomic model of upper domain of the major capsid protein (MCP), VP5 (Bowman et al., 2003). (a) Radially color-coded surface representation of the HSV-1 B capsid structure at 8.5 Å. (more...)

The capsid, approximately 1250–1300 Å in diameter, is a T = 16 icosahedron with 12 pentons forming the vertices, 150 hexons forming the faces and edges, and 320 triplexes interconnecting the pentons and hexons (Rixon, 1993; Steven and Spear, 1997). One of the 20 triangular faces of the icosahedral capsid is indicated by the dotted triangle in Fig. 3.3(a) with three fivefold (‘5’), a twofold (‘2’) and threefold (through triplex Tf) symmetry axes labeled. The six fivefold axes pass through the vertices, the ten threefold (3f) axes pass through the centers of the faces, and the 15 twofold (2f) axes pass through the middle of the edges. The structural components in one asymmetric unit are labeled, including 1/5 of a penton (‘5’), one P (peri-pentonal) hexon, one C (center) hexons a half E (edge) hexon (Steven et al., 1986), and one each of Ta, Tb, Tc, Td and Te triplex and 1/3 of Tf triplex (Fig. 3.3(a)) (Zhou et al., 1994).

HSV-1 is the easiest to grow among all human herpesviruses and has been subjected to the most thorough structural analyses, and its capsid has been reconstructed to 8.5 Å resolution (Fig. 3.3(a)) (adapted from Zhou et al., 2000 with permission from the publisher). The capsid shell has a total mass of about 200 MDa. The structural features of the capsid are built from four of the six capsid proteins: 960 copies of the major capsid protein (MCP), VP5; 320 copies of triplex monomer protein (TRI-1), VP19c; 640 copies of triplex dimer protein (TRI-2), VP23; and 900 copies of the smallest capsid protein (SCP), VP26. At this high resolution, details of secondary structure can be resolved that are not visible at lower resolution. Alpha-helices, for example, appear as extended, cylindrical rods of 5–7 Å diameter. The VP5 major capsid protein of HSV-1 was found to contain 24 helices. These assignments of helices to densities were corroborated by docking the cryoEM structure with X-ray crystallographic data which were subsequently obtained for the upper domain of VP5 (Fig. 3.3(c)) (Baker et al., 2003; Bowman et al., 2003). A group of seven helices is clustered near the area of the protein that forms the narrowest part of the axial channel of the pentons and hexons (indicated by the red arrowhead in Fig. 3.3(b)). Shifts in these helices might be responsible for the constriction that closes off the channel to prevent release of packaged DNA. The floor domain of VP5 also contains several helices, including an unusually long one that interacts with the scaffolding core and may also interact with adjacent subunits to stabilize the capsid (arrow in Fig. 3.3(b)). Structural studies of in vitro assembled capsids that are representatives of capsid maturation stages suggest that substantial structural rearrangement at this region is directly related to the reinforcement of penton and hexons during morphogenesis (Heymann et al., 2003).

The higher resolution of this reconstruction also revealed the quaternary structure of the triplexes, which are composed of two molecules of VP23 and one molecule of VP19c (Fig. 3.3(d), (e)). The lower portion of the triplex, which interacts with the floor of the pentons and hexons, are threefold symmetric with all three subunits roughly equivalent. This arrangement alters through the middle of the triplex such that the upper portion is composed mostly of VP23 in a dimeric configuration. It appears that all three subunits of the triplex are required for the correct tertiary structure to form because VP23 in isolation exists only as a molten globule with no distinct tertiary structure (Kirkitadze et al., 1998).

The capsids of other human herpesviruses have also been studied by electron cryomicroscopy, including HCMV and simian cytomegalovirus (SCMV), and KSHV, members of the beta and gammaherpesviruses, respectively (Fig. 3.4) (Bhella et al., 2000; Chen et al., 1999; Trus et al., 1999, 2001; Wu et al., 2000). The HCMV capsid structure is very similar to HSV-1 in overall organization, with four homologous structural proteins at the same stoichiometries (Fig. 3.4(a) and (b)). The main difference is that the HCMV capsid had a larger diameter (650 Å) than HSV-1 (620 Å), resulting in a volume ratio of 1.17 (Bhella et al., 2000; Chen et al., 1999; Trus et al., 1999). The increased size of the HCMV capsid despite the similar molecular mass of its component proteins results in a greater center-to-center spacing of the capsomers compared to HSV-1 (Fig. 3.4(b)).

Fig. 3.4. Comparison of the three-dimensional structures of alpha, beta and gammaherpesvirus capsids.

Fig. 3.4

Comparison of the three-dimensional structures of alpha, beta and gammaherpesvirus capsids. The capsid maps of HSV-1 (a), HCMV (b) and KSHV (c) are shown as shaded surfaces colored according to particle radius and viewed along an icosahedral three-fold (more...)

The structure of KSHV capsids was also determined by cryoEM to 24 Å resolution and exhibit structural features very similar to those of HSV-1 and HCMV capsids (Fig. 3.4(c)) (Trus et al., 2001; Wu et al., 2000). The KSHV and HSV-1 capsids are identical in size and capsomer organization. However, some notable differences are seen upon closer inspection. The KSHV capsid appears slightly more spherical than the HSV-1 capsid, which exhibits a somewhat angular, polyhedral shape. When viewed from the top, the hexons in the KSHV capsid appear flower-shaped, whereas those of HSV-1 have slightly tilted subunits and as a result appear more gear-shaped (see below). Also, the KSHV triplexes are slightly smaller and deviate less from threefold symmetry than the much-elongated triplexes in the HSV-1 capsid. The differences in the upper domains of HSV-1 and KSHV triplexes indicate that the HSV-1 triplexes are slightly taller. The radial density profiles show that the KSHV and HSV-1 capsids have identical inner radii of 460 Å (Wu et al., 2000). Because both viruses also have similar genome sizes, their identical inner radii suggest that their DNA packing densities inside the capsids are similar. In contrast, betaherpesvirus capsids, such as those of HCMV, have a somewhat larger internal volume than HSV-1 or KSHV capsids (Bhella et al., 2000; Chen et al., 1999; Trus et al., 1999). However, the increase in volume is disproportionate to the large increase in the size of the HCMV genome over the HSV-1 and KSHV genomes. This implies that the viral DNA is more densely packed into HCMV virions than into HSV-1 or KSHV virions.

In herpesvirus capsids, both the penton and hexon have a cylindrical shape (about 140-Å diameter, 160-Å height) with a central, axial channel approximately 25 Å in diameter (Fig. 3.4). The penton and hexon subunits both have an elongated shape with multiple domains, including upper, middle, lower, and floor domains. The middle domains of the subunits interact with the triplexes. The lower domains connect the subunits to each other and form the axial channels. While the upper domains of adjacent hexon subunits interact with one another, adjacent penton subunits are disconnected at their upper domains, resulting in the V-shaped side view of the pentons (Fig. 3.4). Another major difference between the penton and hexon concerns their floor domains. These domains play an essential role in maintaining capsid stability, as suggested by the higher-resolution structural studies of the HSV-1 capsid (Zhou et al., 2000), where a long α-helix inserts into the floor domain of the adjacent subunit (Fig. 3.3(b)). The relative angle between the floor and lower domains is about 110° in the penton subunit and becomes less than 90° in the hexon subunit, making the penton to appear longer in its side view.

The HSV-1 penton and hexon subunits have the same basic shape as the HCMV and KSHV subunits (Fig. 3.4). Each consists of upper, middle, lower, and floor domains. However, the upper domains of the HSV-1 penton subunits point inward toward the channel, whereas those of the HCMV and KSHV penton subunits point outward. The upper domain of the KSHV subunit has a rectangular shape, while that of the HSV-1 penton subunit appears as a triangle. The most striking difference is that the HSV-1 hexon subunits contain an extra horn-shaped density which is not found in the HSV-1 penton (Fig. 3.4(a), arrow in right panel). This extra density binds to the top of each HSV-1 hexon subunit and has been shown to be the SCP, VP26, by difference imaging (Trus et al., 1995; Zhou et al., 1995), which associate with one another to form a hexameric ring around the hexon at a radius of approximately 600 Å. This accounts for the tilted or gear-like appearance of the HSV-1 hexon top view. The KSHV homolog of HSV-1 VP26 is ORF65. Difference map of anti-ORF65 antibody labeled and unlabeled KSHV capsids also showed that ORF65 binds only to the upper domain of the major capsid proteins in hexons but not to those in pentons (Lo et al., 2003). The lack of horn-shaped densities on the hexons indicates that KSHV SCP exhibits substantially different structural features from HSV-1 SCP. The location of SCP at the outermost regions of the capsid suggests a possible role in mediating capsid interactions with the tegument and cytoskeleton proteins during infection.

Structure and packaging of viral genomic DNA

The sizes of the dsDNA genomes of different human herpesviruses vary substantially, e.g., the HCMV genome is 51% longer than HSV-1 (Davison et al., 2003; McGeoch et al., 2000). The major point of interest concerns the packing of their genomes within the capsids. The HCMV capsid is 117% larger than HSV-1. Besides the volume, factors such as DNA density, capsid capacity, and capsid expansion can also influence DNA packaging in viruses. The genome of HCMV might be more densely packed than that of HSV-1, or might induce expansion of the capsid upon packaging. Alternatively, the two viruses might have a similar capacity but differ in the amount of unoccupied space at the center of the capsids.

In HSV-1, the genomic DNA within the nucleocapsid is closely packed into multiple shells of regularly spaced densities, with 26 Å between adjacent DNA duplexes (Zhou et al., 1999). The central slice and radial density plot in Fig. 3.5 indicate that the C-capsid of Rhesus rhadinovirus (RRV), a gammaherpesvirus, has an almost identical pattern of DNA organization to those observed in HSV-1, though slightly more compact, with a 25-Å inter-duplex distance (Yu et al., 2003). Although the RRV capsid, like the KSHV capsid, has nearly the same diameter as the HSV-1 capsid (1250 Å), RRV has a slightly larger genome size than HSV-1, ∼165 vs. 153 kb, respectively (Alexander et al., 2000; Lagunoff and Ganem, 1997; Renne et al., 1996; Searles et al., 1999). Therefore, the smaller inter-duplex distance may merely reflect the need to compact this greater amount of DNA into the same volume within the capsid. HCMV has the largest genome (∼230 kb) of all human herpesviruses but has a capsid that is only slightly larger (1300 Å diameter), and its DNA was shown to pack with an interduplex distance of only 23 Å (Bhella et al., 2000). Based on the interduplex spacing and the genome sizes, we estimate that the closely packed DNA genomes of HSV-1, RRV, and HCMV would occupy a total volume of 3.52 × 108 Å3, 3.51 × 108Å3, and 4.05 × 108Å3, respectively (Yu et al., 2003). These volumes would measure approximately 92%, 92%, and 90% of the total available spaces inside the HSV-1, RRV, and HCMV capsids, as estimated on the basis of their inner diameters of 900 Å, 900 Å and 950 Å, respectively. The 23–26 Å packing of strands of herpesvirus dsDNA is very close to the 20-Å diameter of B-type dsDNA, suggesting that herpesvirus genomes are packed as “naked” DNA without any bound histone-like basic proteins. In this regard, SDS-PAGE analyses demonstrated that the A-capsids and C-capsids have the same protein composition (Booy et al., 1991; O’Connor et al., 2003). In the absence of histone-like proteins, close packing of naked DNA would lead to a potentially strong electrostatic repulsion between the juxtaposed negatively charged DNA duplexes. This would make the packaging of DNA into procapsid energetically unfavorable, supporting the need for an energy-dependent DNA packaging machinery such as the bacteriophage-like connector recently reported in HSV-1 capsids (Newcomb et al., 2001). Even so, it is conceivable that the negative charge of DNA may at least be partially neutralized by binding polyamines (Gibson and Roizman, 1971) or some other undiscovered small basic molecules to reduce the strong electrostatic repulsion.

Fig. 3.5. Packing of dsDNA inside herpesvirus capsid (Yu et al.

Fig. 3.5

Packing of dsDNA inside herpesvirus capsid (Yu et al., 2003). (a) The upper half of a 100-Å thick central slice extracted from the 21 Å resolution reconstruction of the C-capsid of the rhesus rhadinovirus (RRV), a gammaherpesvirus and (more...)

Structure and assembly of tegument

Composition of viral tegument

The tegument occupies the space between the capsid and the envelope. Since the capsid and virion are ∼125 nm and ∼220 nm in diameter, respectively, the tegument represents a significant part of the virion space and indeed, contains approximately 40% of the herpesvirus virion protein mass (Gibson, 1996;). Since they are components of virions, tegument proteins are delivered to cells at the very initial stage of infection and they have the potential to function even before the viral genome is activated. Extensive studies, including amino acid sequencing and mass spectrometric analyses, have been carried out to determine the protein content of the tegument. These results have revealed the compositions of the teguments of HSV and HCMV, and provided insight into its function.

The tegument of HSV-1 contains more than 20 virus-encoded proteins (Roizman and Knipe 2001). The most notable proteins include the α-trans-inducing factor (αTIF, VP16), the virion host shutoff (vhs) protein (UL41), and a very large protein (VP1–2). VP16 functions as a transcription activator to induce the transcription of viral immediate–early genes, and in addition, plays an essential function as a structural component in the tegument (McKnight et al., 1987; Preston et al., 1988; Weinheimer et al., 1992). The protein vhs is a non-sequence specific RNase that degrades most of the host mRNAs during the initial stage of viral infection, and facilitates the translation of viral mRNAs and viral gene expression (Everly et al., 2002; Read and Frenkel, 1983). VP1–2 is found to be associated with a complex that binds to the terminal a sequence of the viral genome, which contains the signal for packaging the genome into the capsid (Chou and Roizman, 1989).

At least 30 virus-encoded proteins have been found in the HCMV tegument (Gibson, 1996; Mocarski and Courcelle, 2001). Significant progress has been made to delineate the function of these HCMV-encoded tegument proteins. For example, the UL69 protein acts to block cell cycle progression, while the UL99-encoded pp28 protein is required for cytoplasmic envelopment of the nucleocapsids (Hayashi et al., 2000; Sanchez et al., 2000; Silva et al., 2003).

There are five predominant protein species found in the HCMV tegument: the high molecular weight protein (HMWP) encoded by UL48, the HMWP-binding protein encoded by UL47, the basic phosphoprotein (BPP or pp150) encoded by UL32, the upper matrix protein (UM or pp71) encoded by UL82, and the lower matrix protein (LM or pp83) encoded by UL83 (Gibson, 1996; Mocarski and Courcelle, 2001). Although their organization within the virion is not completely understood, these abundant proteins are believed to form the structural backbone of the tegument. UL48 and UL32 products, both of which are essential for viral replication (Dunn et al., 2003; Meyer et al., 1997), have been proposed to interact intimately with nucleocapsids (see below). Blocking UL32 expression resulted in accumulation of the nucleocapsid, suggesting that this protein is essential for tegument formation (Meyer et al., 1997).

UL82 is also believed to be involved in direct interaction with the newly synthesized nucleocapsid, and is important for initiation of tegument assembly (Trus et al., 1999). Moreover, the UL82-encoded pp71 protein is a transcriptional activator that helps to induce the transcription of the immediate-early genes within the infected cells (Liu and Stinski, 1992). UL83, the most abundant tegument protein, accounts for more than 15% of the virion protein mass (Gibson, 1996). The encoded pp65 protein has been reported to block major histocompatibility complex class Ⅰ presentation of a viral immediate–early protein, and more recently, has been implicated to inhibit the induction of host interferon response (Browne and Shenk, 2003; Gilbert et al., 1996). Remarkably, pp65 is not essential for viral replication and infectious virion production (Schmolke et al., 1995). However, UL83 constitutes 90% of the protein mass in the noninfectious dense bodies, which have similar envelope structure, but lack a capsid core (Gibson, 1996). Non-infectious envelope particles, which contain B-capsid like core without the viral DNA genome, have a reduced amount (30–60% lower) amount of pp65, as do low passage clinical isolates, compared to the laboratory-adapted AD169 and Towne strains (Gibson, 1996; Klages et al., 1989). These observations suggest that UL83 serves as a nonstringent, volume-filling function in facilitating the assembly of virions, non-infectious enveloped particles, and dense bodies. Furthermore, the abundance of this protein in the viral particles and its function in blocking host immune response is believe to allow the virus to escape immune surveillance and significantly contributes to CMV survival (Browne and Shenk, 2003; Gilbert et al., 1996).

Comparative structure of viral tegument

Overview of tegument structure

While significant progress has been made during the last few years to identify tegument proteins and study their functions, little is currently known about the structure of the tegument and the organization of the proteins within the tegument. Equally elusive is the pathway of the assembly and formation of the tegument, which involves the packaging of all the tegument proteins and is certainly a highly regulated, ordered process.

Recent electron cryomicroscopy studies on the virus-related particles of HSV-1, HCMV, and simian CMV (SCMV) provide significant insight into the structure and organization of the herpesvirus tegument (Chen et al., 1999; Trus et al., 1999; Zhou et al., 1999). In these studies, the three-dimensional structures for the infectious virions or cytoplasmic tegumented capsids were reconstructed, and compared to the structures of the intranuclear capsids. The tegument can be seen in the virion as a region of relative low density covering an area in the 60–100 nm radius (Figs. 3.1(a), (c) and 3.6(a), (c)) (Chen et al., 1999; Zhou et al., 1999). Although the diameters of the nucleocapsids in different particles appear uniform, the sizes and shapes of the virus particles and the relative locations of nucleocapsids inside the particles vary. These observed variations suggest that most of the tegument proteins do not maintain rigid interactions with the enclosed nucleocapsids, and thus the bulk of the tegument layer does not possess icosahedral symmetry (Chen et al., 1999; Zhou et al., 1999).

Fig. 3.6. Difference of the anchored tegument proteins between HSV-1 ((a) and (b)) and HCMV ((c)–(e)).

Fig. 3.6

Difference of the anchored tegument proteins between HSV-1 ((a) and (b)) and HCMV ((c)–(e)). ((a) and (c)) Radially color-coded shaded surface views of the three-dimensional reconstruction of HSV-1 (a) and HCMV (c) virions as viewed along an icosahedral (more...)

The protein densities are also unevenly distributed across the tegument space. Studies on the localization of the tegument proteins have been reported using immunoelectron microscopy with antibodies specifically against tegument proteins and chemical treatment approaches for step-wise removal of layers of virion particles (Gibson, 1996; Steven and Spear, 1997). Several proteins have been found to be located at the tegument space distant to the nucleocapsids. For example, UL23 and UL24 are localized in the HCMV tegument space close to the inner side of the envelope membrane (Adair et al., 2002).

Detailed comparison of the electron cryomicroscopic images of the intact virion particles, the cytoplasmic tegumented capsids, and the nucleocapsids, revealed the unique tegument densities that are present in virion and tegumented capsids but not in capsid preparations. Some of these tegument densities, which are closely associated with nucleocapsid, also exhibit a certain degree of symmetry, and their structures were reconstructed to a resolution of 18–30 Å (Fig. 3.6) (Chen et al., 1999; Trus et al., 1999; Zhou et al., 1999). Since the surface of the nucleocapsid represents the starting site for tegument acquisition and envelopment, these tegument densities are believed to involve specific and direct interactions with capsid proteins and serve as anchors to recruit other tegument proteins for initiation of tegument formation. The tegument densities of HSV1 that are closely associated with the capsids exhibit a dramatic difference from those of HCMV and SCMV (Chen et al., 1999; Trus et al., 1999; Zhou et al., 1999) (Fig. 3.6). This may not be unexpected since there is little evolutionary conservation in the sequence of tegument proteins between HSV-1 and CMV, and many CMV tegument proteins do not have sequence homologues in HSV-1 (Davison et al., 2003; McGeoch et al., 2000).

Tegument structure of HSV-1

Comparison of the maps of HSV-1 intact virion particles and B capsids revealed the marked differences between the two maps in the region of the pentons, which are highlighted in color in the superposition of the difference map on the B-capsid map (Fig. 3.6(b)). The most obvious difference is the presence of additional material extending from the surface of the pentons. The extra material has a molecular mass of 170–200 kDa, extends from the interface between the upper domains of two adjacent VP5 subunits in the penton and connects to the nearby triplexes that are made up of VP19C and VP23 proteins (Fig. 3.6(b)). The restriction of the tegument contacts to the pentons is consistent with previous observations of tightly attached tegument material at the vertices of capsids in negative stain and freeze-etching images of detergent-treated equine herpevirus virions (Vernon et al., 1982). An identical pattern of tegument protein interaction was observed in a VP26-minus virion mutant (Chen et al., 2001). This result indicates that the lack of tegument association of the HSV-1 hexons is not due to the presence of VP26 on the hexon upper domain, but rather likely due to the inherent structural difference on the upper domains of penton and hexon VP5.

Based on its close association with the capsid and relative abundance in the tegument, the essential tegument protein VP1–3 has been proposed to constitute a major part of the protein complexes representing the tegument material (Zhou et al., 1999). VP1–3 is an interesting yet poorly characterized protein. It has been shown that VP1–3 is associated with a complex that binds to the terminal a sequence of the viral genome, which contains the signal for genome packaging into the capsid (Chou and Roizman, 1989). A temperature-sensitive mutant (ts B7) with a mutation in VP1–3 fails to release viral DNA from the infecting capsids into the nucleus during viral decoating process (Batterson and Roizman, 1983). Since the penton has been suggested to be the route by which viral DNA leaves the capsid (Newcomb and Brown, 1994), an interaction between VP1–3 and the penton proteins would place it in an appropriate position to influence the passage of the viral genome. Further studies are needed to test these hypotheses and completely reveal the identity of the proteins coding for the tegument material.

Tegument structure of CMV

The tegument densities of HCMV that are closely associated with nucleocapsid are dramatically different from those of HSV-1 (cf. Fig. 3.6 (a) and (c)) (Chen et al., 1999). A difference map between the HCMV particles and B-capsids revealed a thin shell of loosely connected filamentous densities, representing the icosahedrally ordered, capsid-proximal portion of the tegument in HCMV (Fig. 3.6(c)–(e)). Unlike HSV-1, the tegument densities interact with all of the structural components of the nucleocapsid: penton (made up of major capsid protein UL86), hexon (consisted of UL86 and smallest capsid protein UL48.5), and triplex (composed of minor capsid protein UL85 and its binding protein UL46). Figure 3.6(d) shows the close-up views of a region from the intact virus reconstruction that includes one penton (red), one P hexon (blue), and two representative adjacent triplexes Ta and Tc (green). Superimposed on the penton and hexon are their associated tegument densities (yellow). Clusters of five and six tegument densities attach to the pentons and hexons, respectively. Moreover, neighboring clusters associate with each by bridging over the intercapsomer space, apparently using triplexes as piers. Each of the filamentous tegument densities, which is about 12 nm in length and 2–3 nm in diameter, acts as the bridge arch. Thus, the capsid appears to act as the scaffold of the ordered tegument protein layer. These results imply that the ordered tegument layer cannot form without the underlying capsid and are consistent with the observations that no such tegument layer was found in dense bodies (Chen et al., 1999).

Detailed examination of the interactions between triplexes and tegument densities further revealed minor differences between the structures of SCMV cytoplasmic tegumented capsids and HCMV particles (Chen et al., 1999; Trus et al., 1999). In SCMV cytoplasmic capsids, two tegument densities were found to be associated with each triplex. In contrast, three densities were shown to be attached to each triplex of the nucleocapsid of the HCMV particles (Fig. 3.6(e)). It is conceivable that the extra tegument densities observed in HCMV structure may represent those that were loosely associated with the capsids and probably lost during the purification of the SCMV capsids.

Based on their relative abundance and close association with the nucleocapsids, two CMV tegument proteins, UL32 and UL82, have been proposed to constitute the majority of the observed tegument material that attach to the capsids (Chen et al., 1999; Trus et al., 1999). UL82, which encodes a transcriptional activator (Liu and Stinski, 1992), has a molecular weight of ∼70 kDa, similar to the estimated molecule mass of the capsomer-capping tegument protein densities. UL32 has been suggested to be involved in the transport of DNA-containing capsids through nuclear membrane during envelopment or in the stabilization of capsids in the cytoplasm (Meyer et al., 1997). In recent experiments, CMV virion particles were subjected to different chemical conditions, which do not disrupt the integrity of the nucleocapsids, to selectively remove the components not tightly associated with the capsids. These experiments showed that most of the known tegument proteins, including UL99 and UL83, are removed, but UL32 is not affected (Yu, X., Lee, M., Lo, P., Liu, F., and Zhou, Z. H., unpublished results). Thus, these results further suggest that UL99 and UL83 are loosely and distantly associated with capsids and that UL32 is in close proximity and possibly involved in direct interactions with the capsids.

Structure and assembly of viral envelope

The envelope contains most, if not all, of the virion glycoproteins. Each of the herpesviruses encodes a set of 20–80 glycoproteins, very few of which are highly conserved among all the herpesviruses (Kieff and Rickinson, 2001; Mocarski and Courcelle, 2001; Roizman and Knipe, 2001). For example, HSV-1 encodes at least 20 glycoproteins, 11 of which are found in the virions (Roizman and Knipe, 2001). HCMV potentially encodes more than 75 membrane-associated proteins, at least 15 of which are found in the virions (Mocarski and Courcelle, 2001). The exact organization of viral surface glycoproteins in the envelope is not completely understood. Virion glycoproteins are found to aggregate into complexes on the surface of the virion. For example, HCMV glycoproteins gH, gL, and gO are associated to form a heterotrimeric envelope glycoprotein complex (Gibson, 1996; Mocarski and Courcelle, 2001). These proteins may form their complexes in the cellular membrane compartment before trafficking to the viral envelope. However, it remains possible that further higher-order complexes are assembled after these protein components are delivered in the viral envelope membrane.

In addition to the viral encoded glycoproteins, the envelope also contains numerous host proteins or constituents. For example, host proteins associated with HCMV envelope include β2-microglobumin, CD55 and CD59, and annexin Ⅱ (Grundy et al., 1987a, b; Wright et al., 1995). These molecules may participate in the induction of host cellular responses. It is conceivable that these host proteins, in combination with the viral encoded G protein-coupled receptors associated with the HCMV envelop, play an important role in modulating host cell response during initial virus attachment, as observed in recent studies (Compton et al., 2003; Zhu et al., 1998).

Herpesvirus envelopment is believed to take place initially at the inner nuclear membrane, and then further proceeds with an envelopment/de-envelopment process that allows the capsid to cross the double nuclear membranes and other cytoplasmic membrane structures (Gibson, 1996; Steven and Spear, 1997). Cytoplasmic envelopment of HSV-1 and CMV capsids can also take place in endosomes as well as Golgi networks (Eggers et al., 1992). Thus, it is not surprising that the envelope contains diverse lipid components that are associated with different parts of the cytoplasmic membrane system in addition to the nuclear membrane. These components include the phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol (Roby and Gibson, 1986). Whether these lipid components may be important in maintaining the integrity of the viral envelope has not been determined and their functional role in stabilizing virion structure is unknown.

The envelopment process appears to be not affected in the absence of a mature capsid since noninfectious enveloped particles and dense bodies can be produced and contain similar envelop contents. In the case of HCMV, pp65 constitutes at least 90% of the dense body protein mass (Gibson, 1996). It is conceivable that this protein may contain signals that promote its own envelopment. There are presumably interactions between proteins of the tegument and envelope that promote the envelopment process. Indeed, recent results indicate that HCMV UL99, a tegument protein, facilitates the cytoplasmic envelopment of the nucleocapsid (Sanchez et al., 2000; Silva et al., 2003). Two HSV-1 membrane proteins, UL34 and UL31, are also implicated to be essential for viral envelopment (Reynolds et al., 2001). Further studies on the organization of the proteins in these particles and their potential interactions with envelope components will provide insight into the process of their assembly.

Other constituents in the virions

Recent studies indicated that viral mRNAs were found in HSV-1 and HCMV virions (Bresnahan and Shenk, 2000; Sciortino et al., 2001). These mRNAs appear to be packaged selectively into the infectious virion particles. They have been proposed to function to facilitate the initiation of viral infection upon viral entry. It is unknown whether these virion mRNAs play a role in maintaining the integrity of the virion structure, as ribosomal RNAs provide the backbone for ribosome assembly.

Depending on the approach of how the virions are prepared and the quality of the preparations being analyzed, numerous host constituents, including lipids, polyamines, and cellular enzyme and structural proteins, are also found to be associated with the viral particles. In particular, two of these host constituents, polyamines and actin-related protein (ARP), may play an important role in stabilizing and maintaining the intact structure of the infectious particles. Two kinds of polyamines, spermidine and spermine, have commonly been found in herpesvirus virions, including HSV-1 and HCMV (Gibson and Roizman, 1971; Gibson et al., 1984). In highly purified HSV-1 virion preparations, there are about 70 000 molecules of spermidine and 40 000 molecules of spermine per virion (Gibson and Roizman, 1971). The functions of these polyamines are believed to provide positive charges to neutralize the highly negatively charged viral DNA genome during the genome replication and packaging. This hypothesis is consistent with the observations that none of the herpesvirus capsid proteins are highly positive charged and addition of arginine facilitates capsid assembly and virion production (Mark and Kaplan, 1971). Spermidine appears to be in the tegument while spermine is localized in the nucleocapsid. It is estimated that the spermine contained in the virion has the capacity to neutralize about 40% of the DNA phosphate, consistent with its role in stabilizing the packed genomic DNA in the nucleocapsid core.

In analyzing highly purified HCMV virions as well as noninfectious enveloped particles and dense bodies, Baldick and Shenk first reported the presence of a substantial amount of a cellular actin-related protein (ARP) in the tegument compartment (Baldick and Shenk, 1996). The exact localization of the ARP is currently unknown, and preliminary studies using stepwise chemical treatment of HCMV virion for removal of different parts of the particles have suggested that ARP is localized in the tegument space distant from the nucleocapsid (Yu, X., Lee, M., Lo, P., Liu, F., and Zhou, Z. H., unpublished results). Based on their roles for providing cytoskeleton and maintaining cellular structure and morphology, it is conceivable that actin-related proteins stabilize the tegument structure. Meanwhile, some ARPs have been implicated in participating dynein-driven microtubule transport system (Lees-Miller et al., 1992; Schroer et al., 1994). Given the fact that viral capsid trafficking from cytoplasm to the nuclear pore complex is driven by the dynein-microtubule system (Dohner et al., 2002; Sodeik et al., 1997), it is possible that these ARPs are specifically incorporated into the teguments and facilitate the transport of the viral particles from the nucleus to the cytoplasmic membrane during viral envelopment and to the nucleus during post-penetration. Further studies are needed to completely elucidate the function of these proteins in assembly and maintenance of the virus structure.

Acknowledgments

We thank NIH for financial support; Pierrette Lo for assistance in preparing Figs. 3.1(b), 4; and Dr. Sarah Butcher for providing an HCMV capsid map.

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