Taxonomic Structure of the Family
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Virions of herpesviruses have complex and characteristic structures consisting of both symmetrical and non-symmetrical components ( Homa and Brown, 1997; Steven and Spear, 1997). The spherical virion comprises core, capsid, tegument and envelope. The core consists of the viral genome which is packaged as a single, linear, dsDNA molecule into a preformed capsid. DNA is packed in a liquid-crystalline array which fills the entire internal volume of the capsid. The mature capsid is a T = 16 icosahedron. In Human herpesvirus 1 (HHV-1) the 16 nm thick protein shell has an external diameter of 125 nm. The 12 pentons and 150 hexons are composed primarily of 5 and 6 copies respectively of the same protein and are joined by masses, termed triplexes, which are made of two smaller proteins present in a 2:1 ratio. Capsids assemble by co-condensation around a protein scaffold to form a procapsid in which the subunits are weakly connected. Proteolytic cleavage of the scaffolding protein triggers loss of scaffold and reorganisation of the shell into the characteristic capsid form. The structure of the tegument is poorly defined with no evidence of symmetry. It contains many proteins, not all of which are required for formation of virions. Some individual tegument proteins can vary markedly in abundance. Enveloped tegument structures lacking capsids can assemble and are released from cells along with virions. The envelope is a lipid bilayer which is intimately associated with the outer surface of the tegument. It contains a number (at least 10 in HHV-1) of different integral viral glycoproteins (Fig. 1).
Physicochemical and Physical Properties
The mass of the HHV-1 virion is about 13 10-16 g of which DNA comprises about 10%. The mass of a full capsid is about 5 10-16 g. The buoyant density of virions in CsCl is 1.22-1.28 g/cm3. The stability of different herpesviruses varies considerably, but they are generally unstable to desiccation and low pH. Infectivity is destroyed by lipid solvents and detergents.
The genomes are composed of linear dsDNA ranging from 125 to more than 240 kbp in size and from 32 to 75% in G+C content. Those genomes examined in sufficient detail contain a single nucleotide extension at the 3 ends and no terminal protein has been identified. The arrangement of reiterated sequences, either at the termini or internally, results in a number of different genome structures, as illustrated in Figure 2, and in the existence of isomers due to recombination between terminal and inverted internal reiterations.
In Figure 2, structure 1 shows a unique sequence flanked by a direct repeat which may be larger than 10 kbp (Human herpesvirus 6; HHV-6) or as short as 30 bp (Murid herpesvirus 1; MuHV-1). Structure 2 also contains a single unique sequence but in this case it is flanked by a variable number of repeated sequences at each terminus (e.g., Human herpesvirus 8; HHV-8). Structure 3 contains different elements at each terminus which are present internally in inverted orientation. The genome is thus divided into two unique regions (one ‘long’ and one ‘short’) which are flanked by inverted repeats. The repeated sequence flanking the long unique sequence is very short (88 bp in Human herpesvirus 3; HHV-3). Homologous recombination results in inversion of the short unique region and genome populations are therefore composed predominantly of two isomers (e.g., HHV-3). Structure 4 is the most complex. Like structure 3 it contains long and short unique regions but in this case both are flanked by large inverted repeat sequences and in consequence homologous recombination inverts both unique sequences resulting in the formation of four isomers each of which is equimolar in virion populations (e.g., HHV-1). In addition, structure 4 genomes contain a short terminal redundancy which is present internally in inverse orientation. The different isomers of type 3 and 4 genomes appear to be functionally equivalent. It should be stressed that Figure 2 is a simplified depiction of herpesvirus genome structures. Many herpesviruses contain large repetitive elements within the genome unrelated to those found at the termini (e.g., Human herpesvirus 4; HHV-4) and a more complex set of structures can be catalogued if these are included. Particular genome structures are associated with certain herpesvirus taxa. Thus the presence of multiple repeated elements at both termini (structure 2) is associated with the Gammaherpesvirinae (though not all members have this structure) while structure 3 is associated with members of the genus Varicellovirus. Distantly related viruses may, however, have equivalent genome structures which have presumably evolved independently (e.g., HHV-1, Human herpesvirus 5; HHV-5).
The polypeptide composition of the mature virion varies greatly among different herpesviruses. HHV-1 is the best studied and more than thirty different polypeptides have been identified, though others doubtless remain to be found. The mature capsid is composed of six proteins, while the tegument contains at least 15 different polypeptides, many of which are dispensable in vitro and are not therefore required for virion morphogenesis. The viral envelope contains at least ten (and in some cases many more) integral membrane proteins, a subset of which is required for adsorption and penetration of the host cell.
The lipid composition of few herpesvirus envelopes has been reported. The composition of the HHV-1 envelope is reported to resemble the composition of Golgi membranes more closely than that of other cellular membranes.
The virion envelopes contain multiple proteins that carry N-linked and O-linked glycans. Secreted virions contain complex glycans while a proportion of intracellular virions contains N-linked glycans of the immature high mannose type.
Genome Organization and Replication
The number of ORFs contained within herpesvirus genomes ranges from about 70 to more than 200. Among the viruses of mammals and birds a subset of about 40 genes is conserved, arranged into seven gene blocks. These gene blocks have different orders and orientations in different herpesvirus subfamilies but genes within a block maintain order and transcriptional polarity (Fig. 3). The conserved genes encode capsid proteins, components of the DNA replication complex, nucleotide modifying enzymes, membrane proteins and tegument proteins, reinforcing the view that despite their genetic diversity these viruses share common features in many aspects of their replication strategies. The herpesviruses of fish and amphibians are very distant from those of mammals and birds and analysis of the sequences of these viruses has not revealed common coding sequences. The classification of these viruses as herpesviruses is based on morphology of the virion rather than on genetic content.
Given the genetic diversity of the Herpesviridae it is probable that the details of their replication strategy will vary, perhaps substantially. What follows is, therefore, a brief description based on well-studied members of the family and, in particular, on HHV-1: for more detailed descriptions see Ward and Roizman, 1994 or Davison and Clements, 1997. Adsorption and penetration involve the interaction of multiple virion envelope proteins with multiple cell surface receptors. Entry takes place at the cell surface by fusion of the envelope with the plasma membrane. The nucleocapsid is transported to the nuclear pore by unknown mechanisms while tegument proteins, many of whose functions are unknown, are thought to modify cellular metabolism. In HHV-1 one tegument protein (the UL41 gene product) acts in the cytoplasm to inhibit host protein synthesis while another (the UL48 gene product) is a transcription factor which enters the nucleus and activates immediate early viral genes. In permissive cells entry of the genome into the nucleus is followed by a transcriptional cascade. Immediate early (alpha) genes, which are distinct among different subfamilies, regulate subsequent gene expression by transcriptional and post-transcriptional mechanisms. Early (beta) genes encode the DNA replication complex and a variety of enzymes and proteins involved in modifying host cell metabolism, while the structural proteins of the virus are encoded primarily by late (gamma) genes. Transcription is directed by host RNA polymerase II.
Viral DNA synthesis occurs from one or more origins of replication by a rolling circle mechanism. Replication of HHV-1 DNA requires seven gene products comprising an origin-binding protein, a ssDNA binding protein, a DNA polymerase composed of two subunits and a helicase-primase complex composed of three gene products. Homologues of all but the origin binding protein have been identified in all subfamilies of herpesviruses. Newly synthesized DNA is packaged from the concatemer into pre-formed immature capsids within the nucleus. Immature capsids contain a core of scaffolding proteins which are expelled by proteolytic cleavage during maturation. The subsequent steps in morphogenesis of the secreted enveloped virion are uncertain. Nucleocapsids are observed budding through the inner nuclear membrane into the perinuclear space and the resulting enveloped virions may then be transported by exocytosis to the cell surface. An alternative view is that the enveloped particles, in the perinuclear space become ‘de-enveloped’ by fusion with the outer nuclear membrane and that the resulting nucleocapsids are re-enveloped in a Golgi or post-Golgi compartment. Current evidence does not distinguish the alternatives. Only a subset of herpesvirus genes is required to achieve this basic replication cycle in vitro. Almost half the genes of HHV-1 are found to be dispensable for in vitro culture; the products of these genes ‘fine tune’ the replication cycle or are required for survival in vivo. See 4.
The alternative to the productive cycle, and consequent cell death, is latent infection. The establishment and maintenance of the latent state is not thoroughly understood but the weight of evidence favours a ‘default’ mechanism in which failure of immediate early gene expression leads to the maintenance of the input genome as a circular episomal element. Changes in the transcription factor milieu of the latently infected cell, due to external stimuli or cell differentiation, lead to immediate early gene expression and entry into the productive cycle.
Infected hosts produce antibodies to a wide variety of structural and non-structural virus antigens. Some of the envelope glycoproteins are particularly immunogenic and are the targets for neutralizing antibodies. Cross-neutralization is observed only between closely related viruses within a single genus.
The range of host species is very wide. It is probable that all vertebrates carry multiple herpesvirus species and a herpesvirus has been identified in molluscs. As a general rule the natural host range of individual viruses is highly restricted and most herpesviruses are thought to have evolved in association with single host species, though occasional transfer to other species can occur in nature. In experimental animal systems the host range varies considerably: some members of the Alphaherpesvirinae can infect a wide variety of animal species while members of the Betaherpesvirinae and Gammaherpesvirinae exhibit a very restricted experimental host range. The in vitro host range also varies considerably, though the same general rule holds true: members of the Alphaherpesvirinae will often infect a variety of cells of different species in vitro whereas members of the Betaherpesvirinae and Gammaherpesvirinae exhibit greater restriction. The basis of host restriction both in vivo and in vitro is poorly understood. In a few instances (e.g., HHV-4) cell surface receptors play an important part in determining host range but more commonly (e.g., HHV-5) the virion is capable of entering a wide variety of cells but intracellular factors determine susceptibility.
Natural transmission routes range from highly contagious aerosol spread (HHV-3) to intimate oral contact (HHV-4) or sexual transmission (HHV-2). Vector mediated transmission has not been reported.
Herpesviruses are highly adapted to their hosts and severe infection is usually observed only in the very young, the foetus, the immunosuppressed or following infection of an alternative host. Most herpesviruses establish a systemic infection, a cell-associated viraemia being detectable during primary infection. Some members of the Simplexvirus genus appear to be an exception to the rule: in the normal host infection is limited to epithelium at the inoculation site and to sensory nerves innervating the site. A variety of immune evasion mechanisms have been identified in different viruses including the evasion of complement, antibody, MHC class I presentation and NK cell killing, but the key to survival of herpesviruses is their ability to establish life-long latent infection, a feature which is assumed to be the hallmark of all herpesviruses. The cell type responsible for harboring the latent virus has been established in relatively few instances. Nevertheless, the picture that emerges is that members of the Alphaherpesvirinae establish latent infection in neurones, members of the Betaherpesvirinae establish latent infection in cells of the monocyte series and members of the Gammaherpesvirinae establish latent infection in lymphocytes. It should be emphasized, however, that this general picture is based on a very limited number of examples and that there are reports of latent infection at other sites.
List of Species Demarcation Criteria in the Family
Related herpesviruses are classified as distinct species if (1) their nucleotide sequences differ in a readily assayable and distinctive manner across the entire genome and (2) they occupy different ecological niches by virtue of their distinct epidemiology and pathogenesis or their distinct natural hosts. A paradigm is provided by the two serotypes HHV-1, and HHV-2, which differ in their sequence throughout the genome, tend to infect different epithelial surfaces and exhibit distinct epidemiological characteristics. These two viruses recombine readily in culture, but despite the fact that they can infect the same sites in the host, no recombinants have been isolated in nature and the two viruses appear to have evolved independently for millions of years.
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