NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

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

Cover of Human Herpesviruses

Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

Show details

Chapter 20Maturation and egress

.

Department of Pediatrics, University of Alabama at Birmingham, AL, USA

Introduction

The assembly of betaherpesviruses, specifically cytomegaloviruses, is a topic of considerable interest to virologists and structural biologists. These viruses are among the largest and most complex animal viruses and encode a large number of proteins. Some clinical isolates of human cytomegalovirus (HCMV) have been predicted to contain as many as 250 ORFs (Chee et al., 1990; Murphy et al., 2003), while other authors have suggested that the coding capacity of HCMV may actually be on the order of 165 ORFs (see Chapter 14). Although the number of virus-encoded proteins that are incorporated into the infectious particle is unknown, estimates from several laboratories suggest that it could approach 100 proteins (Varnum et al., 2004). In addition, the particle also contains an unknown number of host cell proteins, some that may have functional significance in the replicative cycle of these viruses (Varnum et al., 2004). Thus, the complexity of virus assembly rivals that of some cellular organelles. Furthermore, CMVs do not arrest host cell protein synthesis even at late phases of replication as do the alphaherpesviruses and therefore during their assembly can either compete with host cell protein synthetic and targeting pathways or more likely, express viral specific functions that modulate host cellular pathways to optimize viral protein synthesis and transport. Identification of these virus-specified host cell modifications together with their interactions with virion proteins will aid in the understanding of the assembly of this virus. Similar approaches have provided a reasonably detailed view of the assembly pathways of bacteriophages and eukaryotic RNA viruses, including HIV-1, and these studies have in some instances served as templates for investigations of DNA virus assembly. However, herpesviruses encode vastly more virion proteins compared to RNA viruses, and assembly takes place in both nuclear and cytoplasmic compartments. Definitive studies of capsid assembly have been carried out in alphaherpesviruses and have been extended to include in vitro, cell free assembly of the herpes simplex viral (HSV) capsid (Newcomb et al., 1994). Many of the capsid proteins of CMVs and other betaherpesviruses share extensive structural and functional homology with HSV capsid proteins and cryoelectron microscopic analysis of the HCMV capsid suggests similar, but not identical, capsid structure (Butcher et al., 1998; Chen et al., 1999; Trus et al., 1999; Zhou et al., 1999, 2000). The most structurally diverse region of the herpesvirus virion appears to be the tegument which is composed of a great many betaherpesvirus- and CMV-unique proteins. Although proteins with homologous functions localized to this region of the virus can be readily identified for many different herpesviruses, only a limited number of these proteins exhibit significant structural homologies (Mocarski and Tan Courcelle, C., 2001). Despite these similarities, a large number of structural proteins appear to be unique for each subfamily of herpesviruses and in some cases, such as HCMV, several tegument proteins have no counterparts in alphaherpesviruses (Mocarski and Tan Courcelle, C., 2001). Together, these findings suggest that, although common themes likely exist for herpesvirus assembly, it is almost certain that distinct aspects of the assembly pathway of each of these viruses will be identified and these differences in the assembly of different herpes viruses could point to key features of the infectious cycle of these viruses.

Early studies of the assembly of alphaherpesviruses including HSV suggested that virion envelopment occurred at the nuclear membrane (Campadelli-Fiume et al., 1991; Johnson and Spear, 1982). This concept was extended to other herpesviruses including HCMV, even though early studies utilizing electron microscopy suggested major differences between the morphogenesis of these two viruses, both within the infected cell and in the overall appearance of extracellular virions (Smith and DeHarven, E., 1973). Subsequent studies of alphaherpesviruses provided evidence for nuclear envelopment–de-envelopment followed by envelopment at cytoplasmic membranes (Granzow et al., 1997; Jones et al., 1988; Whealy et al., 1991). These reports provided clear evidence that perinuclear virions contained a different subset of tegument and envelope proteins than mature virions, findings that support acquisition and then loss of an immature envelope by perinuclear virions (Brack et al., 1999; Granzow et al., 1997, 2001; Klupp et al., 2000). These results have been further supported by more recent studies of viruses with deletion mutations in genes encoding both virion envelope and tegument proteins (Brack et al., 1999; Fuchs et al., 2002; Kopp et al., 2003). The elegant studies of pseudorabies virus (PRV) have provided definitive evidence of cytoplasmic virion envelopment and served as a guide for studies of the envelopment of β-herpesviruses (Mettenleiter, 2002). The envelopment of CMVs, specifically HCMV, also appears to take place within a cytoplasmic compartment based on studies of tegument protein trafficking and incorporation into the virion particle (Britt et al., 2004; Sanchez et al., 2000a; Silva et al., 2003). Major gaps remain in the understanding of capsid tegumentation, capsid egress from the nucleus, and cytoplasmic assembly of the mature, infectious HCMV particle. The development of bacterial artificial chromosome (BAC) technology that allows the maintenance of HCMV as infectious clones coupled with the application of techniques of recombinant prokaryotic genetic manipulation has accelerated experimental studies in HCMV assembly (Adler et al., 2003; Borst et al., 1999; Messerle et al., 2000; Smith and Enquist, L. W., 1999). Together with other technologies such as mass spectrometry and cryoelectron microscopy, current studies should provide more definitive understanding of this complex process. In the following review, the focus will be on the assembly of HCMV as a model for betaherpesvirus assembly because the majority of studies have been carried out in systems utilizing HCMV or the related virus, murine CMV (MCMV). When possible, studies of other betaherpesvirus will be discussed.

Assembly of the capsid

Structural studies of the HSV capsid have indicated that structural and functional protein homologues of HSV capsid proteins are present in the capsid of HCMV, HHV-6, and HHV-7 (Table 20.1). Cryoelectron microscopic analysis of the capsids of HCMV and HSV have revealed near identical structures, albeit with slight differences in the floor of the capsid, perhaps because of the larger size of the HCMV genome (Butcher et al., 1998; Chen et al., 1999; Trus et al., 1999; Zhou et al., 2000). Because of this structural conservation, it has been assumed that the assembly of the HCMV capsid as well as capsids of other β-herpesviruses follow a very similar assembly pathway as that of HSV (Steven et al., 1997; Grunewald et al., 2003). The size of the HCMV particle is approximately 2000 angstroms. The nucleocapsid is approximately 1300 angstroms and is of iscosahedral T = 16 symmetry (Butcher et al., 1998; Chen et al., 1999). As noted in Table 20.1, there are six identified protein components of the mature HCMV capsid present in the infectious particle (Gibson, 1996). Interestingly, the capsid proteins from both HSV and HCMV share considerable structural and biochemical characteristics and, in the case of MCP, even express cross-reactive antibody binding sites (Rudolph et al., 1990).

Table 20.1. Proteins of the capsid of β-herpesviruses: comparison with HSV.

Table 20.1

Proteins of the capsid of β-herpesviruses: comparison with HSV.

The HCMV capsid is composed of 162 capsomeres consisting of 150 hexons and 12 pentons (Butcher et al., 1998; Chen et al., 1999). The most abundant protein components of the capsid is the major capsid protein (MCP, UL86) and the smallest capsid protein (SCP, UL48–49), with 960 and 900 copies respectively present in each capsid. Two copies of the minor capsid protein (MnCP, UL85; triplex dimer or TR-2) combined with a single copy of the minor capsid binding protein (MnCP-bp; triplex monomer or TR-1) UL46 form the triplexes that are located between adjacent pentons and hexons (Fig. 20.1) (Butcher et al., 1998; Chen et al., 1999; Gibson, 1996; Gibson et al., 1996; Trus et al., 1999). The capsid pentons and hexons are assembled entirely from the major capsid protein. Each hexon is decorated on each vertex with the smallest capsid protein (Yu et al., 2005). The smallest capsid protein of HSV, Vp26, has been shown to be dispensable for capsid assembly in cell culture, whereas deletion of the HCMV homologue results in the loss of infectivity, presumably by preventing the assembly of infectious virions (Borst et al., 2001). A cartoon depicting the assembly pathway is detailed in Fig. 20.1.

Fig. 20.1. Model of HCMV encapsidation.

Fig. 20.1

Model of HCMV encapsidation. (1) The capsid proteins MCP, SCP, and products of the UL80a and UL80.5 orf that contain a MCP binding domain are thought to interact in the cytoplasm and are then translocated into the nucleus of the infected cell. The MnCP (more...)

Protein interactions and capsid assembly

As noted above, evidence from transmission electron microscopy and cryo-EM, combined with the conservation of capsid protein function, suggests that the capsid assembly of HCMV and other betaherpesviruses closely follows that of HSV (Beaudet-Miller et al., 1996; Gibson, 1996, Gibson et al., 1996, 1990, 1993; Welch et al., 1991a,b; Wood et al., 1997; Grunewald et al., 2003). Yet several unique features of the HCMV capsid such as the role of the smallest capsid protein in production of infectious virions suggest that there are differences in the assembly of capsids of these two viruses. Although the atomic structure of HCMV capsid proteins has not been determined, several reports have characterized some of the more relevant protein–protein interactions that appear to be required for HCMV capsid assembly. The identity of participating cellular proteins and in many cases the temporal order of protein–protein interactions that lead to capsid formation remain undefined. The template for HCMV capsid assembly can be derived from the pioneering studies of HSV capsid assembly from several laboratories (Heymann et al., 2003; Newcomb et al., 1994, 1996, 1999, 2001; Oien et al., 1997; Tatman et al., 1994; Zhou et al., 2000).

The initial steps of capsid assembly involve formation of a scaffold for the generation of the capsid subunit and the pre-capsid structures, protein interactions between the MCP and UL80a and UL80.5, and possible interactions between MCP and SCP. The scaffolding protein(s) of HCMV has been identified as gene products of UL80a and UL80.5 ORFs (Welch et al., 1991a, b). These genes are organized as nested, in-frame 3-coterminal genes that give rise to four transcripts and four gene products with common carboxyl termini (Welch et al., 1991a). The largest transcript is derived from UL80a and encodes the proteinase precursor which includes the HCMV proteinase in the amino terminal half of the precursor and the assembly protein in the carboxyl terminal half (Welch et al., 1991a,b). Self-cleavage of UL80a by the proteinase function leads to generation of the assemblin and a COOH terminal fragment that overlaps with UL80.5 (Welch et al., 1991b). The role of this protein in capsid assembly appears to be analogous to that reported for the HSV scaffolding protein, VP24 (Liu and Stinski, 1992b; Preston et al., 1992, 1994; Tatman et al., 1994). A second protein, the assembly protein, is the product of UL80.5, a nested ORF collinear with the carboxyl terminus of UL80a. The proteins encoded by the longest ORF, UL80a, and the UL80.5 ORF both express a conserved domain (CCD) at the extreme carboxyl terminus that has been shown to interact with the HCMVMCP (Gibson, 1996; Wood et al., 1997). This interaction is required for nuclear translocation of the MCP (and presumably SCP) because the MCP lack a nuclear localization signal and its mass of 150 kDa would exclude it from passive nuclear entry. Consistent with this prediction is the finding that expression of MCP in the absence of other viral proteins results in cytoplasmic localization (Beaudet-Miller et al., 1996; Lai and Britt, W. J., 2003; Nicholson et al., 1994). Because a conserved functional nuclear localization signal is present in the C-terminal domain of UL80.5 as well as some forms of UL80a, interaction between these proteins and MCP results in the nuclear translocation of MCP (Beaudet-Miller et al., 1996) as depicted in Fig. 20.1. An amino-terminal conserved domain (ACD) can be found in the amino terminal domain of UL80.5 and the corresponding cleavage product of UL80a. This domain promotes self-interaction and has been suggested to lead to the generation of UL80a and UL80.5 multimers (Beaudet-Miller et al., 1996; Gibson, 1996; Wood et al., 1997). The self-interaction, together with interactions with MCP, is thought to catalyze the association of MCP into multimers leading to the formation of intranuclear hexons and pentons (Fig. 20.1) (Gibson, 1996). Interestingly, the SCP has also been shown to interact with the MCP in the absence of other viral proteins (Lai and Britt, W. J., 2003). The sequences of SCP that are responsible for this interaction have been mapped but the functional significance of this interaction has not been studied in the context of replicating virus (Lai and Britt, W. J., 2003). The interaction between these two abundant and essential components of the capsid raises several important questions, including if the observed interaction between the SCP and MCP is critical for structural events leading to capsid assembly or for a downstream event in virion assembly such as capsid/tegument interactions required for capsid tegumentation and virion assembly. Interactions between the products of the UL85 and UL46 have also been demonstrated by two-hybrid systems (Gibson et al., 1996). This interaction is required for the formation of the triplexes of the capsid and presumably results in a conformational change that allows their positioning between capsomeres. The interacting sequences of this set of capsid proteins have not been unequivocally mapped, perhaps as a result of the limited solubility of the protein products of UL46 and their aggregation by heating (Gibson et al., 1996).

Capsid maturation and DNA packaging

Once the immature shell of the capsid is formed, viral DNA must be packaged into this immature capsid or precapsid to generate a mature nucleocapsid. In studies of HSV the packaging of unit length viral DNA takes place in a well described pathway in which capsid maturation and DNA packaging appear to be coupled (Heymann et al., 2003; Steven and Spear, 1997). However, capsid assembly and most aspects of capsid maturation can take place in the absence of viral DNA as evidenced by HSV capsid formation in a virus-free and in an in vitro cell free system (Newcomb et al., 1994; Tatman et al., 1994). The final step in HCMV capsid maturation, based on models of HSV capsid assembly, involve proteolytic cleavage of the carboxyl terminal MCP binding domain (M domain) of UL80.5 and M domain containing forms of UL80a in the shells of the immature B-capsids and including any full length UL80a that may be present in these precapsid forms (Gibson, 1996). The loss of the UL80 encoded scaffolding structures appears to be coupled to viral DNA packaging (Gibson, 1996; Lee et al., 1988). Unit length viral DNA enters the maturing capsid presumably through an asymmetric site of the capsid structure that based on the HSV model, contains a portal protein. In the case of HCMV, the portal protein has been proposed to be encoded by the UL104 product (Komazin et al., 2004). Entering viral DNA is thought to lead to extrusion of the cleavage products of the UL80 from the virion capsid (Gibson, 1996). It is likely that fragments of UL80a or 80.5 remain in the capsid; however, their role in the maintenance of the structure of the virion capsid is unclear.

The packaging of viral DNA is mediated through virus-encoded protein recognition of two conserved sequence motifs, the pac-1 and pac-2 sequences, that are located in the a sequence at each end of the viral genome (Mocarski and Tan Courcelle C., 2001). The virus proteins mediating packaging comprise the viral terminase complex that consists of at least two proteins, the products of the UL56 and UL89 orfs, that function together with the UL104 portal protein (Bogner et al., 1998; Giesen et al., 2000; Scheffczik et al., 2002; Scholz et al., 2003). The product of UL56 is a 130 kDa protein that has been shown to bind to AT rich sequences in the pac sequences and also to have nuclease activity, both activities that suggest its role in packaging as well as cleavage of viral DNA (Bogner et al., 1998; Scholz et al., 2003). More recently, studies from this same laboratory have suggested that the product of the UL89 ORF, a 75 kDa protein, is actually responsible for the DNA cleavage activity that has been assigned to the terminase complex (Scheffczik et al., 2002). Interestingly, the activity of several members of a group of antiviral drugs that have in common a benzimidazole core structure has been shown to map to UL89 and UL56 (Krosky et al., 1998). Although these two proteins are essential for the recognition and packaging of unit length viral DNA, several unexpected findings in studies of the mechanism of action of the benzimidazole antiviral drugs, including maribavir, have suggested that other virus-encoded proteins could be present in this cleavage-packaging complex.

Nuclear tegumentation and nuclear egress

Reports from several laboratories in the early 1990s resolved several inconsistencies in the literature of alphaherpesvirus envelopment. These studies lead to a model in which virion capsids are initially enveloped at the nuclear envelope and following a de-envelopment step at the outer nuclear membrane are delivered to the cytoplasm for final envelopment at cytoplasmic membranes (Brack et al., 1999; Jones and Grose, C., 1988; Skeppner et al., 2001; Wang et al., 2000; Whealy et al., 1991; Zhu et al., 1995). These studies of capsid egress from the nucleus of HSV (or PRV, VZV) infected cells have been illuminating, but differences in cellular tropism, replication, virion protein composition and virion morphology between betaherpesviruses and alphaherpesviruses suggest that virion assembly distal to the nuclear events of encapsidation could be significantly different. Furthermore, more recent studies have demonstrated that a significant number of tegument proteins encoded by HCMV lack sequence homologues in HSV, and in some cases virion tegument proteins of one betaherpesvirus (HCMV) do not have homologues in other betaherpesviruses, including other cytomegaloviruses. Finally, the changes in the size and morphology of the nucleus that are observed in HCMV infected primary fibroblasts are characteristic of the cytopathic effect of this virus and distinct from changes in cells infected with HSV or PRV. As a result, investigators studying the assembly of HCMV and other betaherpesviruses have postulated models for nuclear egress and envelopment that could be specific to this group of viruses.

Perhaps the most poorly understood aspect of the assembly of herpesviruses is the tegumentation of virion particle. In addition to the undefined number of proteins in the tegument, it remains to be determined whether the essential function of individual tegument proteins is regulatory such as pp71 (UL82), interference with host responses such as pp65 (UL83), structural such as pp28 (UL99), or in some cases multifunctional (Baldick et al., 1997; Browne et al., 2003; Kalejta and Shenk, T., 2003; Liu and Stinski, 1992a; Silva et al., 2003). Thus, genetic deletions affecting ORFs encoding tegument proteins that result in loss of infectivity could be ascribed to several possible mechanisms other than a block in the assembly of an infectious particle secondary to the loss of an essential structural protein. Furthermore, the trafficking of nuclear tegument proteins that eventually are incorporated into the infectious particle remains almost completely unstudied. Examples of the different distribution of HCMV tegument proteins are illustrated in Table 20.2. The most striking aspect of the differing cellular localization of this subset of tegument proteins is that virus assembly presumably requires organization of the tegument to insure ordered incorporation of tegument proteins as well as maintenance of essential protein–protein interactions.

Table 20.2. Cellular localization of a subset of HCMV tegument proteins.

Table 20.2

Cellular localization of a subset of HCMV tegument proteins.

This can be readily explained by a radial distribution of tegument proteins in the particle and by the sequential addition of these proteins during nucleocapsid transit from the nucleus through cellular compartments that are used for final assembly. However, cryoelectron microscopic analysis of the virion structure, although consistent with the sequential acquisition of tegument proteins, has not provided definitive evidence for such a pathway leading to tegument assembly (Chen et al., 1999; Trus et al., 1999). Thus, the assembly of the tegument layer of HCMV remains unclear and may involve nuclear tegumentation and de-tegumentation as has been proposed for PRV (Fuchs et al., 2002; Granzow et al., 1997, 2001). Studies of the alphaherpesvirus PRV have demonstrated that nuclear viral capsids are essentially free of detectable tegument proteins and acquire tegument entirely in the cytoplasm (Granzow et al., 1997, 2001). From a combination of elegant electron microscopic analyses and studies of viral deletion mutants, Mittenleiter and colleagues have proposed compelling arguments that the PRV capsid interacts with non-structural virus-encoded nuclear proteins that facilitate capsid envelopment and de-envelopment at the nuclear envelope (Fig. 20.2) (Mettenleiter, 2002). The virus-encoded proteins UL31 and UL34 of PRV are thought to be essential for nuclear egress and have been shown to be localized to the nucleus of infected cells but cannot be detected in the virion (Fuchs et al., 2002). The viral and/or cellular proteins that participate in these nuclear fusion events, other than UL31 and UL 34, have not been identified. Although this model of PRV nuclear egress is consistent with a number of observations from these and other investigators, other examples of tegument protein trafficking fail to fit precisely with this model. An example is the HSV tegument protein Vp22 (UL49) that has been shown localized to the nucleus of the infected cell and cannot be detected on immature perinuclear capsids, yet it is eventually incorporated into the virion as a tegument protein, presumably during cytoplasmic tegumentation (del Rio et al., 2002). Early electron microscopic findings indicated that the cytoplasmic forms of HCMV contained a considerably thicker tegument as compared to HSV (and presumably PRV) and it is well known that numerous HCMV nuclear tegument proteins are present in the virion. Thus, it is likely that nuclear egress of the HCMV capsid is more complex than the models proposed for alphaherpesviruses. This HCMV pathway of nuclear egress could involve nuclear tegumentation, de-tegumentation, and a final tegumentation that would incorporate virion tegument proteins that localize to the nucleus, or even more simply, both nuclear and cytoplasmic tegumentation.

Fig. 20.2. Two models of nuclear egress of the HCMV capsid.

Fig. 20.2

Two models of nuclear egress of the HCMV capsid. The structure of the bi-leaflet nuclear envelope and underlying nuclear lamina is shown. Nuclear pore complexes are depicted as cylinders. Focal accumulation of intranuclear capsids adjacent to inner nuclear (more...)

Early studies of HCMV egress noted that virion structural proteins could be localized to the nuclear matrix of virus infected cells (Sanchez et al., 1998). The nuclear matrix of eurkaryotic cells can be viewed as the cytoskeleton of the nucleus. This nuclear structure has been shown to be a site of transcription and DNA replication, including for DNA viruses (Pombo et al., 1994; Schirmbeck et al., 1989). Major components of the nuclear matrix include the nuclear lamins, a group of cellular proteins that represent the intermediate filaments of the nucleus (Stuurman et al., 1998). Nuclear lamins are known to play an important role in the integrity of the nuclear envelope and have been shown to link the nuclear matrix with the inner membrane of the nuclear envelope through their interactions with a number of integral membrane proteins in the inner nuclear membrane, thus creating what is referred to as the nuclear lamina. The finding of a specific interaction of HCMV virion tegument proteins with the nuclear matrix, and in particular lamin B, raised the possibility that this interaction could represent a pathway of nuclear egress of tegumented capsids (Sanchez et al., 1998). Studies have demonstrated that nuclear lamin phosphorylation and dephosphorylation can be coupled with loss of lamin structure and, in some cases, the disruption of the nuclear envelope (Steen et al., 2000; Stuurman et al., 1998). Lamin phosphorylation has been demonstrated in HCMV infected cells and it was initially suggested that either an intrinsic kinase activity of an HCMV encoded protein or a cellular kinase activity localized to regions adjacent to the nuclear envelope could account for phosphorylation of nuclear lamins (Muranyi et al., 2002; Radsak et al., 1991). The focal loss of nuclear envelope integrity has been suggested to contribute to the nuclear egress of the HCMV capsid (Gallina et al., 1999; Sanchez et al., 1998). However, it should be noted that loss of lamin structure alone cannot account for loss of nuclear membrane integrity.

Consistent with proposed mechanisms of egress of HCMV that include a focal loss of nuclear membrane integrity have been studies of the Vpr protein of HIV-1. Expression of this virus-encoded protein has been shown to be associated with the focal loss of nuclear membrane integrity and mixing of cytoplasmic and nuclear proteins (de Noronha et al., 2001). A recent study utilizing murine CMV as a model system reported similar findings by demonstrating that two non-structural nuclear proteins encoded by m50 and m53 could localize host protein kinase C activity to an area adjacent to the nuclear envelope (Muranyi et al., 2002). The redistribution of protein kinase C activity resulted in the phosphorylation of lamins A and C at levels that could be associated with loss of structural integrity of the inner nuclear membrane (Muranyi et al., 2002). The m50 and m53 are sequence homologues of the alphaherpesvirus UL34 and UL31, respectively, and are believed to represent the functional homologues of these proteins. Similar to the alphaherpesvirus UL34 protein, the m50 product is a type Ⅱ membrane protein that contains motifs with limited homology with known integral membrane proteins of the nuclear envelope (Muranyi et al., 2002). Because both m50 and m53 encode essential functions for virus replication, this finding suggests that nuclear egress of CMVs could require focal disruption of the nuclear lamins leading to the loss of the integrity of the inner nuclear membrane. The homologous proteins of HCMV, pUL50 and ppUL53, have also been shown to be essential for virus replication and interestingly, ppUL53 can be detected in the virion tegument, suggesting that it has an additional role in virus assembly other than its proposed function in facilitating nuclear egress (Dal Monte et al., 2002). Studies of ppUL53 trafficking indicate that, in the absence of other virus-encoded proteins, it is expressed only in the nucleus but that it can be detected in both the nucleus and the virus assembly compartment in virus infected cells late in infection (Dal Monte et al., 2002) (W. Britt, unpublished data). Thus, in contrast to findings in the PRV, one of the essential nuclear virus-encoded proteins that is thought to be required for modification of the nuclear envelope is also found within the tegument of the mature virion. It is unclear if HCMV pUL50 is present in the virion tegument. Expression of the abundant HCMV virion tegument protein pp65 (UL83) is also restricted to the nucleus of infected cells secondary to a functional bipartite nuclear localization signal until late in infection when it is distributed in the cytoplasm and can be detected predominantly in the assembly compartment (Sanchez et al., 2000a). These findings would indicate that either HCMV virions are partially tegumented in the nucleus or that nuclear tegument proteins have trafficking pathways to sites of tegument assembly in the cytoplasm.

Other models of nuclear egress have been proposed yet insufficient data is available to determine their validity. In studies of HHV-7, Frenkel and co-workers described an intranuclear, cytoplasmic vacuole or invagination that appeared to be a site of nuclear budding of the mature capsid (Roffman et al., 1990). This structure designated a tegusome and was identified by electron microscopic analysis of infected cells (Roffman et al., 1990). This structure provided an explanation consistent with the incorporation of virus-encoded nuclear proteins into the tegument of the mature particle. Similar structures have not been identified in other betaherpesviruses.

A pathway of egress of herpesvirus capsids from nucleus that unifies available data has yet to be described. However, it is clear that these viruses have devoted at least some of their genomic information to encode proteins that can either directly modify key structures of the nuclear envelope or recruit cellular functions to the nuclear membrane. These findings would suggest that immature capsids (and possibly tegumented capsids) could exit the nucleus through focal herniations of nuclear membrane as proposed for the function of Vpr of HIV. Alternatively, translocation of the capsid across the nuclear envelope into the cytoplasm of the infected cell could require a membrane fusion event, a mechanism that is favored by the bulk of the literature describing alphaherpesvirus assembly.

Cytoplasmic tegumentation and envelopment

Final tegumentation and envelopment of the infectious betaherpesvirus particle appear to occur exclusively in the cytoplasm of the infected cell. Studies that have described the trafficking of virion tegument and envelope proteins have led to several proposed models of cytoplasmic assembly of the infectious virion. Two studies of HCMV utilizing cryoelectron microscopy indicated that the tegument layer adjacent to the capsid exhibited aspects of iscosahedral symmetry and, from estimates of the mass of the presumed tegument protein that occupied this layer, one group of investigators argued that the protein adjacent to the capsid was pp150 (UL32) (Trus et al., 1999); however, the identity of the protein was not pursued either biochemically or immunologically. The suggestion that the tegument protein pp150 was adjacent to the capsid raised the possibility that this virion protein was acquired during nuclear egress of the capsid. The distribution of pp150 within infected cells was initially proposed to be nuclear and cytoplasmic, although a subsequent study that employed a larger number of viral and cell markers suggested that pp150 was expressed only in the cytoplasm of infected cells (Hensel et al., 1995; Sanchez et al., 2000a). In this latter study, pp150 was used to define an isolable cellular compartment that was designated as the assembly compartment (Sanchez et al., 2000a). This compartment was shown to be localized to a juxtanuclear site that was in close proximity to the microtubular organizing center (MTOC) of infected cells (Sanchez et al., 2000a). Subsequent studies from other laboratories have also identified this cytoplasmic structure (Dal Monte et al., 2002; Silva et al., 2003). A number of both nuclear and cytoplasmic localized tegument proteins have been shown to localize to the assembly compartment, including many of the proteins listed in Tables 20.2 and 20.3. Isolation of this compartment by centrifugation of cell lysates over density gradients allowed the identification of both tegument and envelope proteins, including processed glycoprotein B (gB, UL55) within in this compartment, consistent with this compartment being a site of virus envelopment and assembly. The trafficking and accumulation of virion proteins and subviral structures to this site is not understood; however, recent studies of tegument protein trafficking coupled with a greater understanding of glycoprotein trafficking are beginning to suggest model pathways for its formation within infected cells and may represent a cytoplasmic inclusion identified in early studies of HCMV morphogenesis.

Table 20.3. HCMV envelope glycoproteins.

Table 20.3

HCMV envelope glycoproteins.

Tegument protein trafficking and incorporation into the particle

Although it is likely that understanding the trafficking of virion tegument proteins will be key to understanding the assembly of the infectious particle, little is known about the localization of these proteins to the assembly compartment. One structural tegument protein for which some features of its intracellular trafficking are known is pp28 (UL99). Studies demonstrated that this small (191 amino acid) myristylated protein is membrane associated and when expressed in the absence of other viral proteins, is retained in the ER/Golgi intermediate compartment (ERGIC) (Sanchez et al., 2000b). In virus infected cells, pp28 is transported to the assembly compartment, where it localizes with envelope glycoproteins and with other tegument proteins including pp150 (Sanchez et al., 2000a; Silva et al., 2003). Recombinant viruses in which the gene encoding UL99 has been deleted, the reading frame has been interrupted, or the protein mistargeted in the infected cell by deletion of the myristylation site are non-infectious and non-enveloped particles were observed in the cytoplasm of cells infected with the mutant pp28 deletion virus (Britt et al., 2004; Jones and Lee, 2004; Silva et al., 2003). Interestingly, the virion assembly compartment was formed in cells infected with the UL99 deletion mutant, indicating that this protein was not required for trafficking of other tegument and presumably envelope proteins to this cytoplasmic compartment. The trafficking of other tegument proteins, including nuclear tegument proteins, to the assembly compartment has not been studied in sufficient detail to allow investigators to develop a model of HCMV tegument assembly.

Envelope glycoprotein trafficking and envelopment of the particle

Similar to the uncertainties that surround the structure and composition of the tegument, the envelope of HCMV remains poorly defined. Although the analysis of the coding sequence of HCMVs suggest that as many as 50 ORFs could encode proteins with N-linked carbohydrate modification, to date, some eight experimentally defined virus specific glycoproteins have been shown to be present in the virion envelope (Table 20.3). At least six of these have been shown to exist as disulfide-linked oligomers (gB, gH/gL/gO; gM/gN) in the virion, a characteristic of CMVs that at first glance appears unique in the herpesvirus family. Formation of these disulfide-linked oligomers takes place in the ER prior to transport of these complexes through the secretory pathway. When expressed in the absence of other viral proteins, glycoproteins (gB) or complexes of glycoproteins (gH/gL/gO; gM/gN) listed in Table 20.3, with the exception of gpTRL10, traffic to Golgi and post-Golgi compartments.

Many of these glycoproteins have been shown to contain well-described signals within their cytoplasmic domains that enable them to utilize the cellular secretory pathway for intracellular trafficking. Examples of these signals include phosphorylated amino acid residues and acidic amino acid clusters that are recognized by cellular adaptor proteins including PACS-1 that localize proteins to the TGN, tyrosine and di-leucine signals that promote glycoprotein retrieval from the cell surface and possibly from other cellular membranes through their interactions with cellular adaptor proteins, and likely other motifs that facilitate interactions with tegument proteins (Crump et al., 2003; Jarvis et al., 2002, Tugizov et al., 1999). Although the role of these various signals on individual glycoproteins has been shown in many cases to function in intracellular trafficking as predicted based on studies of similar signals on other viral glycoproteins, it remains unclear what role these signals play in the assembly and infectivity of the mature virion. As an example, it has been argued that gB in the envelope of infectious virus was derived from cell surface gB retrieved from the cell surface by endocytosis and targeted to the TGN, a presumed site of virus assembly (Radsak et al., 1996; Tugizov et al., 1999). Yet recent studies have shown that mutation of this targeting signal in gB has no effect on the phenotype of the mutant virus when compared to wild-type virus (Jarvis et al., 2002). This latter finding raises several possible interpretations such as a redundancy of targeting signals in the cytoplasmic domain of HCMV virion glycoproteins or that additional intracellular pathways are operative in cells infected with this virus that enable the virion glycoproteins to localize in the assembly compartment. However, it is important to note that several of these glycoproteins have very conventional targeting motifs that are conserved in their cytoplasmic domains, a finding that suggests that well-described cellular pathways of viral glycoprotein localization are utilized during virion assembly. A complete discussion of the intracellular trafficking of these proteins is beyond the scope of this section but, because of the potential cooperativity between proteins and redundancy of function, many of the observations made in the study of individual HCMV glycoproteins in isolation from virus infection could be of limited value in definition of their role in assembly and function in the mature virion. The development of recombinant systems that enable investigators to insert specific mutations should help address these aspects of HCMV envelopment.

The mechanisms leading to final envelopment of the infectious HCMV or other betaherpesvirus particles have not been described. In fact, localizing the virion tegument proteins and envelope glycoproteins to an isolable cellular compartment suggests that envelopment takes place after tegumentation of the nucleocapsid. The recent findings that described unenveloped particles in the cytoplasm of cells infected with the UL99 (pp28) deletion virus supports a working model of envelopment that includes budding of a tegumented capsid through a membrane that contains viral envelope glycoproteins (Silva et al., 2003). The source of the membrane structure and the mechanisms that localize the large number of glycoproteins to this single membrane have not been well described but recent studies of the trafficking of HCMVUL33 and UL27 have suggested that virions were wrapped in membrane tubules as well as budding into multivesicular bodies, structures connected with the endosomal system (Fraile-Ramos et al., 2001). The location of these membranous structures in a juxtanuclear compartment and the differentiation of this compartment from Golgi and TGN are consistent with previous studies of the virus assembly compartment identified in virus infected cells (Fraile-Ramos et al., 2001; Sanchez et al., 2000a; Homman-Londiyi et al., 2003). Previous studies also suggested that HCMV was enveloped in an endosomal compartment (Tooze et al., 1993). Other enveloped viruses, most notably retroviruses, have been shown to utilize a pathway that includes budding into late endosomal compartments during their assembly (Amara et al., 2003; Pelchen-Matthews et al., 2003). If such cellular structures are ultimately shown to be a site of HCMV envelopment (budding), it could follow that HCMV exits the infected cell by a similar exocytic pathway (Gould et al., 2003).

References

  • Adler H., Messerle M., Koszinowski U. H. Cloning of herpesviral genomes as bacterial artificial chromosomes. Rev. Med. Virol. 2003;13(2):111–121. [PubMed: 12627394]
  • Amara A., Littman D. R. After Hrs with HIV. J. Cell Biol. 2003;162(3):371–375. [PMC free article: PMC2172700] [PubMed: 12900390]
  • Baldick C. J. Jr, Marchini A., Patterson C. E., Shenk T. Human cytomegalovirus tegument protein pp71 (ppUL82) enhances the infectivity of viral DNA and accelerates the infectious cycle. J. Virol. 1997;71(6):4400–4408. [PMC free article: PMC191658] [PubMed: 9151830]
  • Beaudet-Miller M., Zhang R., Durkin J., Gibson W., Kwong A. D., Hong Z. Virus-specific interaction between the human cytomegalovirus major capsid protein and the C terminus of the assembly protein precursor. J. Virol. 1996;70(11):8081–8088. [PMC free article: PMC190882] [PubMed: 8892933]
  • Bogner E., Radsak K., Stinski M. F. The gene product of human cytomegalovirus open reading frame UL56 binds the pac motif and has specific nuclease activity. J. Virol. 1998;72(3):2259–2264. [PMC free article: PMC109523] [PubMed: 9499084]
  • Borst E. M., Hahn G., Koszinowski U. H., Messerle M. Cloning of the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome in Escherichia coli: a new approach for construction of HCMV mutants. J. Virol. 1999;73(10):8320–8329. [PMC free article: PMC112849] [PubMed: 10482582]
  • Borst E. M., Mathys S., Wagner M., Muranyi W., Messerle M. Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth. J. Virol. 2001;75(3):1450–1458. [PMC free article: PMC114051] [PubMed: 11152518]
  • Brack A. R., Dijkstra J. M., Granzow H., Klupp B. G., Mettenleiter T. C. Inhibition of virion maturation by simultaneous deletion of glycoproteins E, Ⅰ, and M of pseudorabies virus. J. Virol. 1999;73(7):5364–5372. [PMC free article: PMC112592] [PubMed: 10364283]
  • Britt W., Jarvis M., Seo J. Y., Drummond D., Nelson J. Rapid genetic engineering of human cytomegalovirus using a lambda phage based linear recombination system: demonstration that pp28 (UL99) is essential for production of infectious virus. J. Virol. 2004;78(1):539–543. [PMC free article: PMC303398] [PubMed: 14671136]
  • Browne E. P., Shenk T. Human cytomegalovirus UL83-coded pp65 virion protein inhibits antiviral gene expression in infected cells. Proc. Natl Acad. Sci. USA. 2003;100(20):11439–11444. [PMC free article: PMC208776] [PubMed: 12972646]
  • Butcher S. J., Aitken J., Mitchell J., Gowen B., Dargan D. J. Structure of the human cytomegalovirus B capsid by electron cryomicroscopy and Image reconstruction. J. Struct. Biol. 1998;124(1):70–76. [PubMed: 9931275]
  • Campadelli-Fiume G., Farabegoli F., Gaeta, Roizman B. Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1. J. Virol. 1991;65(3):1589–1595. [PMC free article: PMC239943] [PubMed: 1847476]
  • Chee M. S., Bankier A. T., Beck S., et al. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr. Top. Microbiol. Immunol. 1990;154:125–170. [PubMed: 2161319]
  • Chen D. H., Jiang H., Lee M., Liu F., Zhou Z. H. Three-dimensional visualization of tegument/capsid interactions in the intact human cytomegalovirus. Virology. 1999;260:10–16. [PubMed: 10405351]
  • Crump C. M., Hung C. H., Thomas L., Wan L., Thomas G. Role of PACS-1 in trafficking of human cytomegalovirus glycoprotein B and virus production. J. Virol. 2003;77(20):11105–11113. [PMC free article: PMC224974] [PubMed: 14512558]
  • Monte, Pignatelli S., Zini N., et al. Analysis of intracellular and intraviral localization of the human cytomegalovirus UL53 protein. J. Gen. Virol. 2002;83(5):1005–1012. [PubMed: 11961254]
  • Noronha, Sherman M. P., Lin H. W., et al. Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.[see comment] Science. 2001;294(5544):1105–1108. [PubMed: 11691994]
  • Rio, Werner H. C., Enquist L. W. The pseudorabies virus VP22 homologue (UL49) is dispensable for virus growth in vitro and has no effect on virulence and neuronal spread in rodents. J. Virol. 2002;76(2):774–782. [PMC free article: PMC136827] [PubMed: 11752167]
  • Dominguez G., Dambaugh T. R., Stamey F. R., Dewhurst S., Inoue N., Pellett P. E. Human herpesvirus 6B genome sequence: coding content and comparison with human herpesvirus 6A. J. Virol. 1999;73(10):8040–8052. [PMC free article: PMC112820] [PubMed: 10482553]
  • Fraile-Ramos A., Kledal T. N., Pelchen-Matthews A., Bowers K., Schwartz T. W., Marsh M. The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Mol. Biol. Cell. 2001;12(6):1737–1749. [PMC free article: PMC37337] [PubMed: 11408581]
  • Fuchs W., Klupp B. G., Granzow H., Osterrieder N., Mettenleiter T. C. The interacting UL31 and UL34 gene products of pseudorabies virus are involved in egress from the host-cell nucleus and represent components of primary enveloped but not mature virions. J. Virol. 2002;76(1):364–378. [PMC free article: PMC135715] [PubMed: 11739701]
  • Gallina A., Simoncini L., Garbelli S., et al. Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein. J. Virol. 1999;73(2):1468–1478. [PMC free article: PMC103972] [PubMed: 9882353]
  • Gibson W. 1996Structure and assembly of the virion Intervirology 39(5–6), 389–400. [PubMed: 9130048]
  • Gibson W., Marcy A. I., Comolli J. C., Lee J. Identification of precursor to cytomegalovirus capsid assembly protein and evidence that processing results in loss of its carboxy-terminal end. J. Virol. 1990;64:1241–1249. [PMC free article: PMC249239] [PubMed: 2154607]
  • Michelson, PlotkinGibson, W., McNally, L. M., Welch, A. R. et al1993). Cytomegalovirus maturational proteinase: site-directed mutagenesis used to probe enzymatic and substrate domains. In Multidisciplinary Approach to Understanding Cytomegalovirus Disease ed. , pp. 21–25. Amsterdam: Elsevier;
  • Gibson W., Baxter M. K., Clopper K. S. Cytomegalovirus “missing” capsid protein identified as heat-aggregable product of human cytomegalovirus UL46. J. Virol. 1996;70(11):7454–7461. [PMC free article: PMC190812] [PubMed: 8892863]
  • Giesen K., Radsak K., Bogner E. The potential terminase subunit of human cytomegalovirus, pUL56, is translocated into the nucleus by its own nuclear localization signal and interacts with importin alpha. J. Gen. Virol. 2000;81(9):2231–2244. [PubMed: 10950981]
  • Gompels U. A., Nicholas J., Lawrence G., et al. The DNA sequence of human herpesvirus 6: structure, coding content, and genome evolution. Virology. 1995;209:29–51. [PubMed: 7747482]
  • Gould S. J., Booth A. M., Hildreth J. E. The Trojan exosome hypothesis. Proc. Natl Acad. Sci. USA. 2003;100(19):10592–10597. [PMC free article: PMC196848] [PubMed: 12947040]
  • Granzow H., Weiland F., Jons A., Klupp B. G., Karger A., Mettenleiter T. C. Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment. J. Virol. 1997;71(3):2072–2082. [PMC free article: PMC191296] [PubMed: 9032339]
  • Granzow H., Klupp B. G., Fuchs W., Veits J., Osterrieder N., Mettenleiter T. C. Egress of alphaherpesviruses: comparative ultrastructural study. J. Virol. 2001;75(8):3675–3684. [PMC free article: PMC114859] [PubMed: 11264357]
  • Grunewald K., Desai P., Winkler D. C., et al. Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science. 2003;302:1396–1398. [PubMed: 14631040]
  • Hensel G., Meyer H., Gartner S., Brand G., Kern H. F. Nuclear localization of the human cytomegalovirus tegument protein pp150 (ppUL32) J. Gen. Virol. 1995;76:1591–1601. [PubMed: 9049366]
  • Heymann J. B., Cheng N., Newcomb W. W., Trus B. L., Brown J. C., Steven A. C. Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy [comment] Nat. Struct. Biol. 2003;10(5):334–341. [PubMed: 12704429]
  • Homman-Loudiyi M., Hultenby K., Britt W., Soderberg-Naucler C. Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase Ⅱ J. Virol. 2003;77:3191–3203. [PMC free article: PMC149787] [PubMed: 12584343]
  • Jarvis M. A., Fish K. N., Soderberg-Naucler C., et al. Retrieval of human cytomegalovirus glycoprotein B from cell surface is not required for virus envelopment in astrocytoma cells. J. Virol. 2002;76(10):5147–5155. [PMC free article: PMC136176] [PubMed: 11967330]
  • Jarvis M. A., Jones T. R., Drummond D. D., et al. Phosphorylation of human cytomegalovirus glycoprotein B (gB) at the acidic cluster casein kinase 2 site (Ser900) is required for localization of gB to the trans-Golgi network and efficient virus replication. J. Virol. 2004;78(1):285–293. [PMC free article: PMC303410] [PubMed: 14671110]
  • Johnson D. C., Spear P. G. Monesin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and egress of virions from infected cells. J. Virol. 1982;43:1102–1112. [PMC free article: PMC256222] [PubMed: 6292453]
  • Jones F., Grose C. Role of cytoplasmic vacuoles in varicella-zoster virus glycoprotein trafficking and virion envelopment. J. Virol. 1988;62(8):2701–2711. [PMC free article: PMC253703] [PubMed: 2839696]
  • Jones T. R., Lee S. W. An acidic cluster of human cytomegalovirus UL99 tegument protein is required for trafficking and function. J. Virol. 2004;78:1488–1502. [PMC free article: PMC321399] [PubMed: 14722304]
  • Kalejta R. F., Shenk T. The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle. J. Virol. 2003;77(6):3451–3459. [PMC free article: PMC149542] [PubMed: 12610120]
  • Klupp B. G., Granzow H., Mettenleiter T. C. Primary envelopment of pseudorabies virus at the nuclear membrane requires the UL34 gene product. J. Virol. 2000;74(21):10063–10073. [PMC free article: PMC102045] [PubMed: 11024135]
  • Komazin G., Townsend L. B., Drach J. C. Role of a mutation in human cytomegalovirus gene UL104 in resistance to benzimidazole ribonucleosides. J. Virol. 2004;78(2):710–715. [PMC free article: PMC368810] [PubMed: 14694102]
  • Kopp M., Granzow H., Fuchs W., et al. The pseudorabies virus UL11 protein is a virion component involved in secondary envelopment in the cytoplasm. J. Virol. 2003;77(9):5339–5351. [PMC free article: PMC153988] [PubMed: 12692236]
  • Krosky P. M., Underwood M. R., Turk S. R., et al. Resistance of human cytomegalovirus to benzimidazole ribonucleosides maps to two open reading frames: UL89 and UL56. J. Virol. 1998;72(6):4721–4728. [PMC free article: PMC110001] [PubMed: 9573236]
  • Lai L., Britt W. J. The interaction between the major capsid protein and the smallest capsid protein of human cytomegalovirus is dependent on two linear sequences in the smallest capsid protein. J. Virol. 2003;77(4):2730–2735. [PMC free article: PMC141075] [PubMed: 12552013]
  • Lee J. Y., Irmiere A., Gibson W. Primate cytomegalovirus assembly: evidence that DNA packaging occurs subsequent to B capsid assembly. Virology. 1988;167(1):87–96. [PubMed: 2847426]
  • Liu B., Stinski M. F. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements. J. Virol. 1992;66:4434–4444. [PMC free article: PMC241251] [PubMed: 1318413]
  • Liu F., Roizman B. Differentiation of multiple domains in the herpes simplex virus 1 protease encoded by the UL26 gene. Proce. Natl Acad. Sci. USA. 1992;89(6):2076–2080. [PMC free article: PMC48599] [PubMed: 1312713]
  • Messerle M., Hahn G., Brune W., Koszinowski U. H. Cytomegalovirus bacterial artificial chromosomes: a new herpesvirus vector approach. Adv. Virus Res. 2000;55:463–478. [PubMed: 11050952]
  • Mettenleiter T. C. Herpesvirus assembly and egress. J. Virol. 2002;76(4):1537–1547. [PMC free article: PMC135924] [PubMed: 11799148]
  • Mocarski E. S., Courcelle, D. M. K. A. P. M. Howley Vol. 2. 2001). Cytomegaloviruses and their replication. 4th ed. In Fields Virology 4th edn., ed. , pp. 2629–2673. Philadelphia: Lippincott, Williams and Wilkins;
  • Mori Y., Yang X., Akkapaiboon P., Okuno T., Yamanishi K. Human herpesvirus 6 variant A glycoprotein H-glycoprotein L-glycoprotein Q complex associates with human CD46. J. Virol. 2003;77(8):4992–4999. [PMC free article: PMC152135] [PubMed: 12663806]
  • Muranyi W., Haas J., Wagner M., Krohne G., Koszinowski U. H. Cytomegalovirus recruitment of cellular kinases to dissolve the nuclear lamina.[comment] Science. 2002;297(5582):854–857. [PubMed: 12161659]
  • Murphy E., Yu D., Grimwood J., et al. Coding capacity of laboratory and clinical strains of human cytomegalovirus. Proc. Natl Acad. Sci. USA. 2003;100(25):14976–14981. [PMC free article: PMC299866] [PubMed: 14657367]
  • Newcomb W. W., Homa F. L., Thomsen D. R., Ye Z., Brown J. C. Cell-free assembly of the herpes simplex virus capsid. J. Virol. 1994;68:6059–6063. [PMC free article: PMC237013] [PubMed: 8057482]
  • Newcomb W. W., Homa F. L., Thomsen D. R., et al. Assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation. J. Mol. Biol. 1996;263(3):432–446. [PubMed: 8918599]
  • Newcomb W. W., Homa F. L., Thomsen D. R., et al. Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. J. Virol. 1999;73(5):4239–4250. [PMC free article: PMC104203] [PubMed: 10196320]
  • Newcomb W. W., Homa F. L., Thomsen D. R., Brown J. C. In vitro assembly of the herpes simplex virus procapsid: formation of small procapsids at reduced scaffolding protein concentration. J. Struct. Biol. 2001;133(1):23–31. [PubMed: 11356061]
  • Nicholas J. Determination and analysis of the complete nucleotide sequence of human herpesvirus. J. Virol. 1996;70(9):5975–5989. [PMC free article: PMC190618] [PubMed: 8709220]
  • Nicholson P., Addison C., Cross A. M., Kennard J., Preston V. G., Rixon F. J. Localization of the herpes simplex virus type 1 major capsid protein VP5 to the cell nucleus requires the abundant scaffolding protein VP22a. J. Gen. Virol. 1994;75(5):1091–1099. [PubMed: 8176370]
  • Oien N. L., Thomsen D. R., Wathen M. W., Newcomb W. W., Brown J. C., Homa F. L. Assembly of herpes simplex virus capsids using the human cytomegalovirus scaffold protein: critical role of the C terminus. J. Virol. 1997;71(2):1281–1291. [PMC free article: PMC191183] [PubMed: 8995652]
  • Pelchen-Matthews A., Kramer B., Marsh M. Infectious HIV-1 assembles in late endosomes in primary macrophages. J. Cell Biol. 2003;162(3):443–455. [PMC free article: PMC2172706] [PubMed: 12885763]
  • Pombo A., Ferreira J., Bridge E., Carmo-Fonseca M. Adenovirus replication and transcription sites are spatially separated in the nucleus of infected cells. EMBO J. 1994;13(21):5075–5085. [PMC free article: PMC395454] [PubMed: 7957073]
  • Preston V. G., Rixon F. J., McDougall I. M., McGregor M., al-Kobaisi M. F. Processing of the herpes simplex virus assembly protein ICP35 near its carboxy terminal end requires the product of the whole of the UL26 reading frame. Virology. 1992;186(1):87–98. [PubMed: 1309284]
  • Preston V. G., al-Kobaisi M. F., McDougall I. M., Rixon F. J. The herpes simplex virus gene UL26 proteinase in the presence of the UL26.5 gene product promotes the formation of scaffold-like structures. J. Gen. Virol. 1994;75(9):2355–2366. [PubMed: 8077934]
  • Radsak K. D., Brucher K. H., Georgatos S. D. Focal nuclear envelope lesions and specific nuclear lamin A/C dephosphorylation during infection with human cytomegalovirus. Eur. J. Cell Biol. 1991;54:299–304. [PubMed: 1652444]
  • Radsak K., Eickmann M., Mockenhaupt T., et al. Retrieval of human cytomegalovirus glycoprotein B from the infected cell surface for virus envelopment. Arch. Virol. 1996;141:557–572. [PubMed: 8645095]
  • Roffman E., Albert J. P., Goff J. P., Frenkel N. Putative site for the acquisition of human herpesvirus 6 virion tegument. J. Virol. 1990;64(12):6308–6313. [PMC free article: PMC248811] [PubMed: 2173796]
  • Rudolph S. A., Kuhn J. E., Korn K., Braun R. W., Jahn G. Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1. J. Gen. Virol. 1990;71:2023–2031. [PubMed: 2170571]
  • Sanchez V., Angeletti P. C., Engler J. A., Britt W. J. Localization of human cytomegalovirus structural proteins to the nuclear matrix of infected human fibroblasts. J. Virol. 1998;72:3321–3329. [PMC free article: PMC109810] [PubMed: 9525659]
  • Sanchez V., Greis K. D., Sztul E., Britt W. J. Accumulation of virion tegument and envelope proteins in a stable cytoplasmic compartment during human cytomegalovirus replication. Characterization of a potential site of virus assembly. J. Virol. 2000a;74:975–986. [PMC free article: PMC111618] [PubMed: 10623760]
  • Sanchez V., Sztul E., Britt W. J. Human cytomegalovirus pp28 (UL99) localizes to a cytoplasmic compartment which overlaps the endoplasmic reticulum-golgi-intermediate compartment. J. Virol. 2000b;74(8):3842–3851. [PMC free article: PMC111892] [PubMed: 10729158]
  • Scheffczik H., Savva C. G., Holzenburg A., Kolesnikova L., Bogner E. The terminase subunits pUL56 and pUL89 of human cytomegalovirus are DNA-metabolizing proteins with toroidal structure. Nucl. Acids Res. 2002;30(7):1695–1703. [PMC free article: PMC101837] [PubMed: 11917032]
  • Schirmbeck R., Deppert W. Nuclear subcompartmentalization of simian virus 40 large T antigen: evidence for in vivo regulation of biochemical activities. J. Virol. 1989;63(5):2308–2316. [PMC free article: PMC250649] [PubMed: 2539520]
  • Scholz B., Rechter S., Drach J. C., Townsend L. B., Bogner E. Identification of the ATP-binding site in the terminase subunit pUL56 of human cytomegalovirus. Nucl. Acids Res. 2003;31(5):1426–1433. [PMC free article: PMC149822] [PubMed: 12595550]
  • Silva M. C., Yu Q. C., Enquist L., Shenk T. Human cytomegalovirus UL99-encoded pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J. Virol. 2003;77(19):10594–10605. [PMC free article: PMC228509] [PubMed: 12970444]
  • Skeppner J. N., Browne H., Minson A. Herpes simplex virus nucleocapsids mature to progeny virus by an envelopment–deenvelopment–reenvelopment pathway. J. Virol. 2001;75(12):5697–5702. [PMC free article: PMC114284] [PubMed: 11356979]
  • Smith G. A., Enquist L. W. Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus. J. Virol. 1999;73(8):6405–6414. [PMC free article: PMC112720] [PubMed: 10400733]
  • Smith J. D., DeHarven E. Herpes simplex virus and human cytomegalovirus replication in WI-38 cells. I. Sequence of viral replication. J. Virol. 1973;12:919–930. [PMC free article: PMC356711] [PubMed: 4359959]
  • Steen R. L., Martins S. B., Tasken K., Collas P. Recruitment of protein phosphatase 1 to the nuclear envelope by A-kinase anchoring protein AKAP149 is a prerequisite for nuclear lamina assembly. J. Cell Biol. 2000;150(6):1251–1262. [PMC free article: PMC2150688] [PubMed: 10995432]
  • Chiu, Burnett, GarceaSteven, A. C. and Spear, P. G. (1997). Herpes capsid assembly and envelopment. In Structural Biology of Viruses ed. , pp. 312–351. New York: Oxford University Press;
  • Stuurman N., Heins S., Aebi U. 1998Nuclear lamins: their structure, assembly, and interactions J. Struct. Biol. 122(1–2), 42–66. [PubMed: 9724605]
  • Tatman J. D., Preston V. G., Nicholson P., Elliott R. M., Rixon F. J. Assembly of herpes simplex virus type 1 capsids using a panel of recombinant baculoviruses. J. Gen. Virol. 1994;75(5):1101–1113. [PubMed: 8176371]
  • Tooze J., Hollinshead M., Reis B., Radsak K., Kern H. Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes. Eur. J. Cell. Biol. 1993;601(1):163–178. [PubMed: 8385018]
  • Trus B. L., Gibson W., Cheng N., Steven A. C. Capsid structure of simian cytomegalovirus from cryoelectron microscopy: evidence for tegument attachment sites [erratum appears in J. Virol. 1999 May;73(5):4530] J. Virol. 1999;73(3):2181–2192. [PMC free article: PMC104463] [PubMed: 9971801]
  • Tugizov S., Maidji E., Xiao J., Pereira L. An acidic cluster in the cytosolic domain of human cytomegalovirus glycoprotein B is a signal for endocytosis from the plasma membrane. J. Virol. 1999;73(10):8677–8688. [PMC free article: PMC112888] [PubMed: 10482621]
  • Varnum S. M., Streblow D. N., Monroe M. E., et al. Identification of proteins in human cytomegalovirus (HCMV) particles: the HCMV proteome. J. Virol. 2004;78:10960–10966. [PMC free article: PMC521840] [PubMed: 15452216]
  • Wang Z. H., Gershon M. D., Lungu O., Zhu Z., Gershon A. A. Trafficking of varicella-zoster virus glycoprotein gI: T(338)-dependent retention in the trans-Golgi network, secretion, and mannose 6-phosphate-inhibitable uptake of the ectodomain. J. Virol. 2000;74(14):6600–6613. [PMC free article: PMC112170] [PubMed: 10864674]
  • Welch A. R., McNally L. M., Gibson W. Cytomegalovirus assembly protein nested gene family: four 3′-coterminal transcripts encode four in-frame, overlapping proteins. J. Virol. 1991a;65(8):4091–4100. [PMC free article: PMC248841] [PubMed: 1649317]
  • Welch A. R., Woods A. S., McNally L. M., Cotter R. J., Gibson W. A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site. Proc. Natl Acad. Sci. USA. 1991b;88:10792–10796. [PMC free article: PMC53017] [PubMed: 1961747]
  • Whealy M. E., Card J. P., Meade R. P., Robbins A. K., Enquist L. W. Effect of Brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress. J. Virol. 1991;65:1066–1081. [PMC free article: PMC239872] [PubMed: 1847436]
  • Wood L. J., Baxter M. K., Plafker S. M., Gibson W. Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains. J. Virol. 1997;71(1):179–190. [PMC free article: PMC191038] [PubMed: 8985337]
  • Yu X., Shah S., Atanasov I., et al. Three-dimensional localization of the smallest capsid protein in the human cytomegalovirus capsid. J. Virol. 2005;79(2):1327–1332. [PMC free article: PMC538561] [PubMed: 15613360]
  • Zhu Z., Gershon M. D., Hao Y., Ambron R. T., Gabel C. A., Gershon A. A. Envelopment of varicella-zoster virus: targeting of viral gylcoproteins to the trans-Golgi network. J. Virol. 1995;69:7951–7959. [PMC free article: PMC189740] [PubMed: 7494308]
  • Zhou Z. H., Chen D. H., Jakana J., Rixon F. J., Chiu W. Visualization of tegument-capsid interactions and DNA in intact herpes simplex virus type 1 virions. J. Virol. 1999;73(4):3210–3218. [PMC free article: PMC104084] [PubMed: 10074174]
  • Zhou Z. H., Dougherty M., Jakana J., He J., Rixon F. J., Chiu W. Seeing the herpesvirus capsid at 8.5 A. Science. 2000;288(5467):877–880. [PubMed: 10797014]
Copyright © Cambridge University Press 2007.
Bookshelf ID: NBK47422PMID: 21348107

Views

  • PubReader
  • Print View
  • Cite this Page

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...