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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.
Introduction
Viral early genes are defined by two criteria: they require prior de novo synthesis of viral immediate-early (IE) and cellular proteins for their transcription, and this expression is insensitive to inhibitors of viral DNA synthesis such as phosphonoformate, ganciclovir, cidofovir, and phosphonoacetate. Close inspection of the kinetics of synthesis of this class of genes reveals multiple subgroups (for review, see Fortunato and Spector, 1999). The earliest of the early gene transcripts appear and accumulate to their highest levels by 8 hours postinfection (h p.i.) (e.g., the HCMV 2.2 kb family of transcripts – UL112–113), while the latest of the early transcripts cannot be detected until just prior to the onset of viral DNA replication (e.g., the HCMV 2.7 kb major early transcript – β2.7 or TRL4) and accumulate to highest levels much later during infection when viral DNA replication is allowed to proceed. (Mocarski and Courcelle, 2001) Levels of a third subgroup increase at late times (e.g., the abundant HCMV 1.2 kb RNA – TRL7) and are partially blocked by inhibitors of viral DNA synthesis. This subgroup may be further divided into genes that are referred to as early–late or leaky–late.
This review will describe the viral factors and cellular environment required for the expression of the viral early genes, the function of the early genes with respect to viral replication, and the subversion of host cellular processes and modulation of host immune responses that are associated with the expression of these genes. Selected examples of early genes will be used to illustrate different mechanisms controlling this complex class of genes. The focus is on human cytomegalovirus (HCMV) and its replication in fibroblasts, the most extensively studied betaherpesvirus. A separate section at the end of the review is devoted to human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7).
The host cell range of HCMV permissivity in vivo is broad. The major targets of infection are cell types such as epithelial and endothelial cells as well as peripheral blood leukocytes in the myelomonocytic lineage. Infection also extends to specialized parenchymal cells such as neurons and astrocytes in the brain and retina, smooth muscle cells, and hepatocytes (Sinzger and Jahn, 1996). Infection is restricted, however, when the virus is grown in tissue culture cells. Primary human fibroblasts have been used for virus isolation as well as for most studies on the cellular and molecular biology of HCMV. Fresh clinical isolates of this virus retain the ability to replicate in cultured epithelial cells and macrophages. This is not a property of laboratory propagated strains. It should be kept in mind that the rules governing early gene expression and the functional importance of the gene products might be quite different in other cell types than in fibroblasts.
Identification of HCMV early genes
The initial identification of HCMV early genes was based on studies that measured the rate of accumulation of viral transcripts. Pulse-labeled whole cell, nuclear, or cytoplasmic RNA was hybridized to cloned subgenomic fragments or viral DNA cleaved with restriction endonucleases and fractionated by gel electrophoresis (DeMarchi, 1981; DeMarchi, 1983; DeMarchi et al., 1980; McDonough and Spector, 1983; Wathen and Stinski, 1982; Wathen et al., 1981). In complementary experiments, the size and abundance of various RNAs were determined by hybridization of radiolabeled viral DNA or cloned subgenomic fragments to dot blots or Northern blots of RNA isolated at various times in the infection (DeMarchi, 1983; McDonough and Spector, 1983; Wathen and Stinski, 1982). Two important points emerged from these studies. First, there is no clustering of RNA transcripts in the genome according to their temporal expression. Furthermore, some HCMV early expression appeared to be subject to post-transcriptional controls that governed either the transport of RNA from the nucleus to the cytoplasm or the stability of the RNA in the cytoplasm. These early studies provided information on the relative abundance, size, and temporal expression of about 30% of the RNAs (Chambers et al., 1999; Mocarski and Courcelle, 2001) and served as the framework for more detailed analysis of individual transcription units.
More recently, additional studies have used PCR analysis, gene arrays, and large-scale mutagenesis to classify each of the predicted HCMV ORFs as IE, early, or late and to characterize these as essential or dispensable for growth in tissue culture cells (Chambers et al., 1999; Dunn et al., 2003; Yu et al., 2003). In one study (Chambers et al., 1999), infected cell RNA was hybridized to a DNA microarray carrying oligonucleotides corresponding to the majority of the strain AD169 ORFs and four ORFs present in the Towne strain. IE transcripts were isolated from cells infected in the presence of the protein synthesis inhibitor cycloheximide and harvested at 13 h p.i., while cells treated with ganciclovir for 72 h were the source of RNAs transcribed with early kinetics. Late RNA was isolated from untreated cells at 72 h p.i., and differential sensitivity to ganciclovir was the basis for further characterization of these transcripts as early–late or late. The expression of selected transcripts not previously characterized was confirmed by further hybridization analysis. In agreement with the initial studies, there was no clear correlation of kinetic class with location or polarity of the ORF in the genome, although the majority of the US region ORFs were expressed with early kinetics.
HCMV-mediated changes in the cellular environment prior to early gene expression
Examination of the viral growth cycle reveals intricate interactions between HCMV and the host cellular machinery that optimize the environment for viral replication and prevent recognition of the infected cell by the immune system. The details of these events are discussed in depth in Chapters 16, 19, 20, 21, 58, and 59. Because early gene products are important in many aspects of infection, a brief summary is provided here.
Events triggered by the binding of the virus to the host cell
The initial contact of the virus with the cell membrane triggers physiological changes and activates signaling pathways that in many ways resemble the interferon response or the second messenger-type response that occurs during regulation via hormones and growth factors (for review, see Chapter 16 and Fortunato et al., 2000). For example, there is hydrolysis of phosphatidylinositol(4,5)-biphosphate, stimulation of arachidonic acid metabolism, a transient influx of calcium, upregulation of the transcription factors Sp1 and NF-κB, and activation of the mitogen activated protein kinases (ERK1/2 and pp38) and the phosphatidylinositol 3-kinase pathways (Albrecht et al., 1991; Johnson et al. 2000, 2001a,b; Rodems and Spector, 1998). Also associated with this immediate activation of the host cell is the induction of a subset of RNAs encoding genes induced by alpha or beta interferon (IFN-α/β) in uninfected cells (Boyle et al., 1999; Browne et al., 2001; Navarro et al., 1998; Zhu et al., 1997, 1998). The HCMV-associated induction occurs in the absence of any detectable IFN-α/β or de novo protein synthesis and appears to require simply the exposure of the cell to virions, non-infectious enveloped particles, or dense bodies. The up-regulation of the interferon-responsive genes, however, is tempered by the viral matrix protein pp65 as well as by one or more viral gene products or cellular factors that are expressed during the first few hours of the infection (Abate et al., 2004; Browne and Shenk, 2003; Browne et al., 2001). It has recently been shown that the IE1 and IE2 genes encode at least two of the proteins responsible for the downregulation of this IFN-α/β-mediated signaling response. IE2 86 kDa protein blocks the production of IFN-β and several chemokines (Taylor and Bresnahan, 2005; Taylor and Bresnahan, 2006), while IE1 72 acts further downstream to block type Ⅰ interferon-mediated signaling and the induction of multiple interferon-responsive genes (Paulus et al., 2006).
ND10 are sites of genome deposition and IE transcription
Upon infection of cells with a variety of DNA viruses, the morphology of nuclear structures referred to variously as ND10 (nuclear domain 10), PODs (PML oncogenic domains), or PML bodies is rapidly altered (for review, see Chapter 17). While infection with adenovirus 5 (Ad5) results in these punctate structures acquiring a reticular appearance, both HCMV and herpes simplex virus type 1 (HSV-1) infections disrupt ND10 sites and disperse associated proteins. In uninfected cells, a number of proteins including the growth suppressors promyelocytic leukemia protein (PML), Sp100, HP1, and Daxx are localized to ND10 domains, and many of these are interferon inducible. As is the case for Ad5 and HSV-1, a subset of HCMV genomes are deposited at ND10 sites immediately following infection, and it is these genomes that provide the template for early transcription (Ishov and Maul, 1996; Ishov et al., 1997). Transcripts produced at these sites are consequently in close proximity to spliceosome assembly factor SC35 domains, which may further aid in rapid expression of IE genes following infection.
Once the major HCMV IE proteins, IE1 72 and IE2 86, are expressed during the infection, these first localize to ND10 sites. While the punctate pattern of IE2 86 expression persists for a longer time, between 3 and 6 h p.i. both IE1 72 and ND10-associated proteins, including PML, become completely dispersed throughout the nucleus (Ahn and Hayward, 1997; Ishov et al., 1997; Kelly et al., 1995; Korioth et al., 1996). Further studies involving either transfection of IE1 or IE2 expression vectors or infection with a recombinant virus unable to express IE1 72 indicate that IE2 86 protein is able to localize to ND10 sites in the absence of IE1 72, but disruption does not occur (Ahn et al., 1998a; Ahn and Hayward, 1997; Ishov et al., 1997). IE1 72 is required for disruption of the ND10 sites, but since the IE1 deletion mutant virus replicates well under high multiplicity conditions, this event appears not to be required for the infection to proceed. Modification of IE1 by SUMO-1 is also not required for HCMV-mediated ND10 dispersal or for viral replication, although a virus with a mutation in the IE1 sumoylation site grows more slowly (Lee et al., 2004; Nevels et al., 2004; Xu et al., 2001). The observation that IE1 abrogates sumoylation of PML and Sp100 suggests that this is at least one element of the mechanism by which ND10 associated proteins are dispersed during the infection (Muller and Dejean, 1999).
A growing body of evidence suggests that even after IE1 72 has caused dispersal of ND10 sites, these locations remain important for viral replication. Between 3 and 8 hours p.i., aggregates of cyclin dependent kinase (cdk) 9 and cdk 7 appear in the nuclei of HCMV-infected cells. Input viral genomes and IE1 and IE2 proteins are also present at some, but not all, of these sites (Tamrakar et al., 2005). When cells are infected with the IE1 72 deletion mutant virus, ND10 structures are maintained and the cdk 9 aggregates can be visualized at the periphery of the PML-containing ND10 sites. In addition, the UL112–113 early gene proteins appear to colocalize with IE2 86 at the periphery of the original ND10 sites beginning about 6 h p.i., and these nucleate viral DNA replication compartments that form later during infection (Ahn et al., 1999a).
Finally, a series of studies suggest that an interaction between the HCMV tegument protein pp71 and the ND10-associated Daxx is the basis of a mechanism by which early events in the viral life cycle are initiated at ND10 sites (Hofmann et al., 2002; Ishov et al., 2002; Marshall et al., 2002). Work using recombinant viruses has shown that the Daxx-binding ability of pp71 is required for efficient HCMV replication and IE gene expression during low, but not high, multiplicity infections (Cantrell and Bresnahan, 2005; Cantrell and Bresnahan, 2006; Saffert and Kalejta, 2006). When Daxx expression is reduced, the activity of the major IE promoter increases in transient transfection assays. Knockdown of Daxx expression also alleviates the reduced IE gene expression observed in pp71 deletion mutant virus-infected cells (Cantrell and Bresnahan, 2006; Preston and Nicholl, 2006). These findings are further explained by the observation that pp71 is required for the proteasome-mediated degradation of Daxx that begins 2 h p.i. in HCMV-infected cells. This degradation is required for efficient IE gene expression and is thought to increase gene activity by eliminating Daxx-mediated histone deacetylase recruitment to promoters (Saffert and Kalejta, 2006).
Inhibition of apoptosis
Two viral IE proteins from the UL36–38 region of the genome serve to prevent the host cell from undergoing apoptosis (for review see Chapter 21). One (vMIA) is the protein product of UL37 exon 1, and the second (vICA) is encoded by UL36. vMIA, which is essential for viral replication, travels from the endoplasmic reticulum (ER) to the Golgi and finally to mitochondria. Its expression in transiently transfected HeLa cells blocks apoptosis induced by either anti-Fas antibody plus cycloheximide or by TNF-α, and in stably transfected HeLa clones, it appears to act at a stage between activation of caspase 8 and cytochrome c release into the cytoplasm (Goldmacher et al., 1999; McCormick et al., 2003b). Recently, it was found that vMIA sequesters the pro-apoptotic protein Bax in the mitochondria, thus suppressing mitochondrial permeabilization (Arnoult et al., 2004). The half-life and localization pattern of vICA in infected cells vary depending on the strain of virus used in the infection (Patterson and Shenk, 1999). Like vMIA, vICA is involved in preventing apoptosis in infected cells, but it acts further upstream to block cleavage of pro-caspase 8 and its subsequent activation (Skaletskaya et al., 2001). Either protein appears to be dispensable for growth in culture so long as the other is retained. Many laboratory-adapted viral strains express non-functional UL36 (Skaletskaya et al., 2001). The high degree of UL36 and UL37 exon 1 conservation across the cytomegalovirus family indicates that both contribute critical functions to replication of the virus in the host organism (McCormick et al., 2003a; McCormick et al., 2005).
Functions of viral early genes
Many early gene products are required for successful viral replication. In libraries of HCMV BACs constructed to disrupt each unique ORF (Dunn et al., 2003; Yu et al., 2003) 41–45 of the ORFs examined appear essential for replication in fibroblasts; 117 are not required but when deleted, give rise to phenotypes ranging from growth like wild type to severe impairment of viral replication. Interestingly, some of these dispensable ORFs (UL24, UL64, and US29) are required for viral growth in cell types other than fibroblasts. In addition, four of the mutants with non-essential genes deleted (UL10, UL16, US16, and US19) grow significantly better than the wild type in cell types other than fibroblasts. Early gene products constitute a significant proportion of essential loci, given that 23 to 25 essential and augmenting genes were characterized as early or early–late in the study by Chambers et al. (1999).
Most of the viral early genes function in one of two ways. A subset of the early products required for growth in tissue culture are directly involved in viral DNA synthesis, cleavage and packaging of the viral genome, and assembly of the virus particles (see Chapters 19 and 20). A second group of genes functions to create a cellular and extracellular environment that is optimal for viral gene expression and replication, either by modulating factors involved in the control of cellular DNA synthesis or by altering the host organism’s immune response to the virus.
Genes involved directly in viral replication
The majority of the proteins required for synthesis and processing of the viral DNA are expressed with early kinetics, as are many of the factors involved in the initial stages of viral particle assembly. In conjunction with some of the IE and late viral proteins, these products provide the central functions of the viral life cycle.
Initial studies on the replication of the viral DNA identified 11 loci required for origin of lytic replication (oriLyt)-dependent DNA replication (Pari and Anders, 1993; Pari et al., 1993) (Chapter 19). These were conducted as complementation assays in which cloned fragments of the HCMV genome were tested for their ability to support replication of a vector containing oriLyt sequences. Six of the required genes are predicted to function directly in DNA replication and are homologous to factors required for herpes simplex virus type 1 (HSV-1) DNA replication. The products of these genes are pUL54, the viral DNA polymerase; ppUL44, the polymerase processivity factor; ppUL57, a single-stranded DNA binding protein; and three proteins that comprise a helicase-primase complex: pUL70, pUL102, and pUL105. Each of these genes is expressed with early or delayed early kinetics (Chambers et al., 1999; Smith and Pari, 1995). Two additional early loci, UL112–113 and UL84, were identified in the complementation assays and are required together with UL122–123, IRS/TRS1, and UL36–38, three IE loci with regulatory functions that are discussed elsewhere in this chapter.
The functions of several viral early proteins have been closely examined in subsequent work. Viable viruses with point mutations in the UL54 gene, selected on the basis of reduced sensitivity to ganciclovir and cidofovir, tend to grow more slowly than parental virus (Cihlar et al., 1998; Smith et al., 1997). The DNA polymerase processivity factor encoded by UL44 forms a complex with the viral polymerase and binds to double-stranded DNA, thereby stabilizing interactions with the template (Ertl and Powell, 1992; Hwang et al., 2000; Weiland et al., 1994). Recently, residues in the C-terminus of the polymerase have been shown to be required for the ppUL44–pUL54 interaction (Loregian et al., 2004; Loregian et al., 2003). The function of the UL57 gene product has not been examined directly, but by analogy to its homologous HSV-1 counterpart ICP8, this protein is predicted to bind the single-stranded DNA unwound by the helicase–primase complex (Kiehl et al., 2003). The UL102 and UL105 genes have been characterized (Smith et al., 1996; Smith and Pari, 1995), and biochemical studies demonstrating interactions between their protein products and the product of the UL70 gene further support the idea that, as in HSV-1, these three factors function together as the HCMV helicase-primase (McMahon and Anders, 2002).
The product of the UL84 gene is essential for viral DNA synthesis and productive infection (Dunn et al., 2003; Xu et al., 2003, 2004; Yu et al., 2003). The protein localizes to replication centers in the nuclei of infected cells (Lischka et al., 2003; Xu et al., 2002), interacts with IE2 86 (Spector and Tevethia, 1994), and can promote oriLyt-dependent DNA replication when core replication proteins from Epstein-Barr virus are supplied (Sarisky and Hayward, 1996). Recent studies with a BAC defective for the expression of UL84 suggest that it may regulate some of the functions of IE2 86 as well as contribute to the early formation of the replication centers. Interpretation of the results with this mutant BAC, however, is complicated by the observation that the UL84 protein provided in trans does not complement viral growth (Xu et al., 2004). There is also evidence that the UL112–113 proteins localize to viral replication centers early in their formation and may play a role in the recruitment of additional factors to these sites (Ahn et al., 1999b; Iwayama et al., 1994; Penfold and Mocarski, 1997).
UL114 is an early gene that, although not identified in these complementation assays, appears to contribute to efficient replication of viral DNA (Courcelle et al., 2001; Prichard et al., 1996). The UL114-encoded uracil-DNA glycosylase is not strictly required for growth in fibroblasts, but a mutant lacking this gene is delayed in the initiation of DNA replication.
Following synthesis, viral DNA is cleaved into genome-length segments and packaged into preformed capsids (Chapter 19). Early proteins involved are introduced only briefly here. Four early–late products (major capsid protein, minor capsid protein, minor capsid binding protein, and small capsid protein) contribute to capsid formation and are the products of the UL86, UL85, UL46, and UL48.5 genes, respectively (Gibson, 1996). Capsid formation also relies on the assemblin precursor UL80.5 and the proteinase precursor UL80a. UL89 and UL56 early gene products play roles in DNA cleavage (Buerger et al., 2001; Krosky et al., 2003; Underwood et al., 1998), and by homology to HSV-1 proteins at least four HCMV products, most expressed with early kinetics, are predicted to be involved in packaging cleaved DNA into progeny capsids. These four proteins are encoded by the HCMV UL51, UL52, UL77, and UL104 genes and are predicted to have functions including transport of the capsids to sites of DNA packaging and formation of a structure through which DNA enters the capsid. Recently, it has been shown that the TRS1 protein also may be involved in packaging at a step that occurs after the cleavage of the DNA (Adamo et al., 2004).
Preparing the cell for viral DNA replication
In a cell that is permissive for the viral infection, the expression of the early genes is associated with a cascade of events that results in the stimulation of host cell genes, particularly those encoding proteins that are required for host cell DNA synthesis and proliferation. Early studies revealed a marked increase in the levels of the enzymes ornithine decarboxylase, thymidine kinase, DNA polymerase alpha, and dihydrofolate reductase following HCMV infection (Boldogh et al., 1991; Estes and Huang, 1977; Hirai and Watanabe, 1976; Isom, 1979; Wade et al., 1992). More recent DNA microarray analyses show that the viral infection leads to upregulation of multiple DNA synthesis and cell cycle genes at the level of transcription (Browne et al., 2001). In part, this may follow HCMV-induced hyperphosphorylation of the retinoblastoma family of proteins (Jault et al., 1995; McElroy et al., 2000), which likely releases the inhibition that these proteins confer to the E2F/DP transcription factors that regulate the transcription of many of these same genes (Dyson, 1998). The tumor suppressor protein p53 is stabilized in HCMV-infected cells and is sequestered in viral replication centers (Fortunato and Spector, 1998; Jault et al., 1995). Other proteins that are sequestered in the viral replication centers are PCNA and RPA, which are both essential for the elongation phase of host cell DNA synthesis and may play some role in viral DNA synthesis (Dittmer and Mocarski, 1997; Jault et al., 1995).
HCMV also induces elevated levels of cyclin E and cyclin B and their associated kinase activities (Bresnahan et al., 1996; Jault et al., 1995; McElroy et al., 2000; Salvant et al., 1998; Sanchez et al., 2003). Cyclin E transcription is induced, and this up-regulation requires the expression of viral early genes (McElroy et al., 2000; Salvant et al., 1998). In contrast, multiple posttranscriptional pathways are used in the activation of Cdk1/cyclin B1 complexes (Sanchez et al., 2003). The accumulation of the cyclin B1 subunit is the result of increased synthesis and reduced degradation of the protein via the ubiquitin-proteasome pathway. In addition, the active catalytic subunit of the complex, Cdk1, accumulates in virus-infected cells. This is due partially to the down-regulation of the expression and activity of the Cdk1 inhibitory kinases Myt1 and Wee1 and the accumulation of the Cdc25 phosphatases that remove the inhibitory phosphates from Cdk1. Modulation of these pathways appears to require at least some early gene expression (Sanchez et al., 2003).
During this early period in the infection, HCMV also inhibits selective host cell functions, presumably to ensure that viral replication is favored over that of the host. These events lead the cell to a fully “activated” state, but it is clear that the virus primes the cell for its own DNA replication at the host’s expense and has sufficiently dysregulated the cell cycle and signaling pathways to ensure that cellular DNA synthesis and cell division is blocked (Bresnahan et al., 1996; Dittmer and Mocarski, 1997; Jault et al., 1995; Lu and Shenk, 1996; Salvant et al., 1998). In contrast to the activation of cyclins E and B, the expression of cyclin A and its associated kinase activity is inhibited by infection with HCMV (Jault et al., 1995). Although the failure to induce cyclin A in the virus-infected cells probably plays a role in the blockage of cellular DNA synthesis, it has recently been found that viral early gene products also affect key steps in this process prior to the requirement for cyclin A. Briefly, DNA synthesis in eukaryotic cells is precisely regulated such that genomic DNA doubles only once during each cell cycle (Diffley, 2001; Fujita, 1999; Lei and Tye, 2001). The first step involves the assembly of prereplication complexes (pre-RC) at the replication origins. The origin recognition complex (Orc), a multisubunit complex, binds to the origins of cellular DNA replication and remains bound during most of the cell cycle (Quintana and Dutta, 1999; Tatsumi et al., 2000; Vashee et al., 2001). Cdc6 and Cdt1 are recruited to the complex and facilitate the loading of the family of six Mcm proteins on to DNA (Maiorano et al., 2000; Nishitani et al., 2000, 2001; Rialland et al., 2002). Cdt1 itself is regulated by a protein called geminin that normally accumulates during S-phase and ensures that each origin is used only once. Analysis of this process has revealed that there is a delay in the expression of the Mcm proteins in infected cells. The greatest effect is observed with Mcm5, whose levels remain low until after 32 h p.i. The loading of the Mcm proteins onto the DNA pre-RC complex is also defective in the virus-infected cells and is associated with the premature accumulation of geminin (Biswas et al., 2003; Wiebusch et al., 2003b). Interestingly, as is the case with cyclin B, the increased levels of geminin results from decreased levels of proteasome-mediated degradation (J. W. Choi and D. H. Spector, unpublished results).
Although there is evidence from transient expression systems that the IE1 and IE2 proteins and the virion constituent proteins UL69 and UL82 contribute to the virus-mediated alteration in cell growth control (Bresnahan et al., 1998; Castillo et al., 2000; Kalejta et al., 2003; Kalejta and Shenk, 2003; Lu and Shenk, 1999; Murphy et al., 2000; Sinclair et al., 2000; Song and Stinski, 2002; Wiebusch et al., 2003a; Wiebusch and Hagemeier, 1999, 2001), studies in the context of viral infection show that early gene products are also required (McElroy et al., 2000; Sanchez et al., 2003). A challenge remains to determine which viral genes are involved and elucidate the mechanisms governing their activity. Given the large number of early genes, most of which have not yet been studied, the task is not trivial.
Modulation of host immune responses
In addition to subversion of the intracellular machinery, HCMV needs to deal early in the infection with its survival in its human host and evasion of the immune response. This topic is discussed in detail in Chapters 58 and 59, and so is only briefly summarized here (for other reviews, see (Mocarski, 2002, 2004)). Optimization of in vivo pathogenesis and viral persistence is accomplished by effects on intracellular processes, the release of soluble factors, and regulation of cellular receptors that are involved in modulation of innate, inflammatory, and adaptive immune responses. The number of viral genes that are known to play a role in these processes is still small, and all of these have proven to be dispensable for productive infection in tissue culture. The general consensus, however, is that the large block of “non-essential” early genes in the US region whose functions have yet to be determined are key players in viral pathogenesis.
One well-studied mechanism of viral immune evasion involves interference with MHC class Ⅰ antigen presentation by at least four gene products (US2, US3, US6, and US11) (Ploegh, 1998) (Chapter 58). At early times in the infection, HCMV also disarms the interferon-mediated branch of the host’s antiviral defense. The cells become refractory to IFN-α/β-mediated stimulation of MHC class Ⅰ, IRF-1, MxA, and 2′–5′-oligoadenylate synthetase gene expression, transcription factor activation, and signaling (Miller et al., 1999). Viral genes that have been implicated in these events include UL83 and TRS1/IRS1 (Abate et al., 2004; Browne and Shenk, 2003; Child et al., 2004). In addition, there is repression of IFN-γ-mediated signal transduction, and thus cells do not respond to the presence of IFN-γ by upregulating the expression of MHC class Ⅱ genes (Miller et al., 1998; Sedmak et al., 1994). The viral US2, and possibly US3, proteins can also downregulate HLA-DRα and DMα, two proteins that are involved in MHC class Ⅱ antigen presentation (Tomazin et al., 1999).
Another mechanism that HCMV uses to interfere with immune surveillance is modulation of extracellular host factors (i.e., interleukins and chemokines) that are involved in inflammatory reactions and function to activate and recruit T cells, NK cells, neutrophils, and monocytes to the sites of infection. HCMV also encodes several chemokines, cytokines, and receptors that likely play a role in the inflammatory response and in the dissemination of the virus. One factor, cmvIL-10 (UL111A), is a functional homologue of IL-10 that has been proposed to downregulate macrophage and T cell responses and hence have an anti-inflammatory role (Kotenko et al., 2000; Spencer et al., 2002). Another is a functional alpha chemokine, vCXCL-1 (UL146), that attracts neutrophils via CXCR2 (Penfold et al., 1999). HCMV also encodes four glycoproteins with homology to G-protein-coupled seven-TM receptor proteins (UL33, UL78, US27, and US28) (Chee et al., 1990a). One of these (US28) serves as a specific receptor for the CX3C chemokine fractalkine and can also bind many other CC chemokines (Gao and Murphy, 1994; Kuhn et al., 1995; Mizoue et al., 2001). The observation that expression of US28 in vascular smooth muscle cells causes the cells to exhibit enhanced migration towards inflammatory cytokines has led to the hypothesis that US28 may not only facilitate viral dissemination, but also contribute to the progression of vascular diseases. (Streblow et al., 1999).
Transactivating functions of the major IE proteins
Since viral immediate–early (IE) gene products are the primary regulators of HCMV early gene expression, it is important to introduce these proteins and their functions. This topic is covered in detail in Chapters 17, and only the salient points are discussed here. The main sites of IE transcription are the UL122–123 (major immediate early, MIE), UL36–38, US3, and IRS1/TRS1 open reading frames (Fig. 18.1). The predominant and best-characterized members of this group are the products of the major IE region: the IE1 72 and IE2 86 kDa proteins and related products.
Structure and function of the IE1 72 and IE2 86 kDa proteins
The tight control and remarkably strong transactivating capacity of IE1 and IE2 proteins have caused significant effort to be directed towards understanding how they control the progression of the infection. A single, five-exon transcript from the major IE region is differentially spliced to give two predominant products the IE1 72 kDa protein (exons 1–4) and IE2 86 kDa protein (exons 1–3 and 5). Translation of each mRNA initiates in exon 2, and the two proteins share 85 amino acids (aa) at their amino termini (Stenberg et al., 1984, 1985; Stinski et al., 1983). IE1 72 has modest transactivating effects, including the ability to transactivate the major immediate early promoter (MIEP). Both the regions unique to IE1 and to IE2 encode additional, minor transcripts, some of which are cell-type specific (Awasthi et al., 2004; Jenkins et al., 1994; Kerry et al., 1995; Puchtler and Stamminger, 1991; Shirakata et al., 2002; Stenberg et al., 1989).
IE2 86 is thought to transactivate and repress transcription via protein-protein and protein-DNA interactions. IE2 86 binds to itself, to the UL84 gene product, and to a number of cellular proteins. These host factors include components of the basal transcription complex TBP, TFIIB, and multiple TBP-associated factors (TAFs), Rb, p53, and transcription factors including Sp1, Tef-1, c-Jun, JunB, ATF-2, NF-κB, protein kinase A-phosphorylated delta CREB, p300, CBP, P/CAF, Nil-2A, CHD-1, Egr-1, and UBF (Bonin and McDougall, 1997; Bryant et al., 2000; Caswell et al., 1993; Chiou et al., 1993; Choi et al., 1995; Fortunato et al., 1997; Furnari et al., 1993; Gebert et al., 1997; Hagemeier et al., 1992, 1994; Jupp et al., 1993; Lang et al., 1995; Lukac et al., 1994, 1997; Schwartz et al., 1994, 1996; Scully et al., 1995; Sommer et al., 1994; Spector and Tevethia, 1994; Speir et al., 1994; Wara-Aswapati et al., 1999; Wu et al., 1998; Yoo et al., 1996) (F. Ruchti and D. H. Spector, unpublished results). While interactions between IE1 72 and cellular proteins including p107 (Johnson et al., 1999) have also been demonstrated, the IE1 product does not bind to DNA. IE2 86 binds to specific DNA sequences through interactions that are thought to involve the minor groove (Lang and Stamminger, 1994; Waheed et al., 1998), a notable example being its site-specific binding to the 14 bp cis-repression signal (crs) located between the TATA box and transcription start site in the major immediate–early promoter (MIEP). It has been shown that this interaction with DNA is the mechanism by which IE2 86 negatively regulates its own transcription (Cherrington et al., 1991; Huang and Stinski, 1995; Lang and Stamminger, 1994; Liu et al., 1991; Macias and Stinski, 1993; Pizzorno and Hayward, 1990). In addition, IE2 86 binds to similar 14 bp sites upstream of the TATA box in early promoters including the UL112–113 (2.2kb RNA), TRL7 (1.2 kb RNA), and UL4 promoters (Arlt et al., 1994; Chang et al., 1989; Huang and Stinski, 1995; Schwartz et al., 1994; Scully et al., 1995).
Multiple studies have aimed to define motifs and domains of IE2 86 that are required for both protein–protein and protein–DNA interactions as well as to identify amino acids that are likely to be post-translationally modified. The ability of IE2 86 to interact with other proteins maps broadly to the region not shared with IE1 72, amino acids 86–542 (Chiou et al., 1993; Sommer et al., 1994). A subset of this region, aa 388–542, is required for IE2 86 dimerization (Ahn et al., 1998b; Chiou et al., 1993; Furnari et al., 1993). The DNA-binding capability of IE2, which controls regulation of early promoters and autoregulation, is also the result of sequences present in the C-terminal half of the protein between residues 290–579 (Chiou et al., 1993; Lang and Stamminger, 1993; Schwartz et al., 1994). Regions spanning the full length of the protein appear to be important for IE2 86 transactivation of promoters, including HCMV early promoters, with the critical regions located between aa 1–98 and 170–579 (Hermiston et al., 1990; Malone et al., 1990; Pizzorno et al., 1991; Scully et al., 1995; Sommer et al., 1994; Stenberg et al., 1990; Yeung et al., 1993). Activation of different viral promoters may require different IE2 86 domains, such as the requirement of sequences from aa 26–85 and aa 290–579 to transactivate the UL112–113 promoter and the additional requirement for aa 86–135 for activation of the 1.2 kb RNA promoter (Scully et al., 1995; Sommer et al., 1994).
The extensive posttranslational modifications of the major IE products suggest that they may be important for the functions of the proteins. Both IE1 72 and IE2 86 are phosphorylated and modified by sumoylation (Ahn et al., 2001; Heider et al., 2002a, b; Hofmann et al., 2000; Spengler et al., 2002; Xu et al., 2001). Although a virus with a mutation in the IE1 sumoylation site grows slightly more slowly than the wild type, mutation of the IE2 sumoylation sites has no effect on viral replication (Lee and Ahn, 2004; Nevels et al., 2004). In vitro and in vivo studies show that IE2 86 is phosphorylated on multiple residues (Harel and Alwine, 1998). When the consensus MAP kinase motifs at amino acids 27, 144, 233–234, and 555 are mutated to alanine, some of the resulting proteins have a stronger capacity to transactivate in transient expression assays than wild type IE2 86. However, in the context of the viral genome, these mutations have no effect on viral replication in fibroblasts (Heider et al., 2002b). In contrast, mutations of the multiple serines in the region between amino acids 258 and 275 have complex effects on viral growth, with some mutations accelerating and others inhibiting the infection (Barrasa et al., 2005). The fact that these major differences in growth rate are associated with only modest effects of the mutations on the transactivation function of IE2 86 in transient expression assays underscores the difficulty of extrapolating results from transient expression assays to events that occur in the context of viral infection.
In vitro and transient expression assays demonstrating the transactivating functions of the major IE proteins
Numerous studies have shown that the major IE proteins function together and separately to activate their own promoter as well as a wide range of heterologous viral and cellular promoters. Studies have been conducted primarily using transient transfection of effector plasmids expressing IE proteins and target plasmids expressing reporters driven by a range of viral promoters. In particular, these include the 1.2 and 2.7 kb RNA and UL112–113 (2.2 kb RNA) early promoters and sequences driving expression of genes involved in viral DNA replication (Colberg-Poley et al., 1992; Klucher et al., 1993; Schwartz et al., 1994; Scully et al., 1995). The results of these studies indicate that IE2 86 makes the greatest contribution to activation and in some cases, increases the level of reporter expression 40- to 80-fold over expression in the absence of IE1 72 or IE2 86. IE1 72 alone is a relatively weak transactivator, and only affects a limited number of promoters that have been tested. The transient assay function that best accounts for the growth defects exhibited by mutant viruses lacking IE1 72 is cooperation with IE2 86 in the activation of early promoters (Gawn and Greaves, 2002; Greaves and Mocarski, 1998).
Mutational analysis of the major IE products in the viral genome
The above studies laid the groundwork for elucidating the critical domains and functions of the major IE products. The recent studies that have used recombinant viruses with mutations in the UL122–123 ORFs, however, are more biologically relevant. A human fibroblast cell line expressing IE1 72 allowed the propagation of a mutant virus lacking UL123 exon 4 that was unable to express full-length IE1 72 (Mocarski et al., 1996). While our laboratory and others have attempted the construction of a similar cell line expressing the IE2 86 protein, to date none has been isolated. In the absence of a complementing cell line, it is difficult to propagate recombinant viruses with mutations in essential genes like IE2 86; however, the advent of bacterial artificial chromosomes (BACs) as vectors for the cloning of herpesvirus genomes has largely allowed this problem to be circumvented (for review see Adler et al., 2003). Since the majority of the viral genome is present in the BAC, mutations can be made and characterized entirely in bacteria regardless of the viability of the resulting virus. Reconstitution of virus from the clone is achieved by transfecting the altered genome and a construct expressing pp71 into cells permissive for HCMV infection (Baldick et al., 1997).
Several groups have since used this approach to construct HCMV IE2 86 mutants. A recombinant virus with most of the unique region of the IE2 gene (ORFUL122) deleted is defective in early gene expression and does not produce infectious progeny, providing additional evidence that IE2 86 is required for the activation of early genes and for viral replication (Marchini et al., 2001). Members of our group generated a viable mutant with a deletion spanning IE2 86 residues 136–290 and showed that this virus expresses IE and early genes and replicates its DNA comparably to the wild type but is delayed in expression of a subset of late genes (Sanchez et al., 2002).
Smaller mutations introduced into the IE2 86 gene in the viral genome have also been used to define specific regions of the protein required for the activation of early promoters. Members of our laboratory have constructed recombinants with internal deletions of amino acids 356–359, 427–435, or 505–511 (White et al., 2004). These mutations were selected on the basis of the IE2 86 domain mapping and functional studies discussed above and are located in the C-terminal region important for protein–protein interactions and DNA binding. Each deletion results in a non-viable virus. The IE2 86Δ356–359 mutation removes amino acids implicated in the activation of the UL112–113 and UL54 promoters (Stenberg et al., 1990) and results in a clone that is able to support limited early gene expression but not replication of viral DNA. The IE2 86Δ427–435 and IE2 86Δ505–511 mutations disrupt the zinc finger and helix-loop-helix motifs present in the protein, and the resulting recombinants do not support early gene expression. All are defective in crs-mediated autorepression of the major IE promoter, although the degree of this defect varies with the mutant. Similarly, a temperature-sensitive IE2 86 mutant virus that contains the point mutation C509G (C510G in AD169) is able to transactivate the UL112–113 promoter at 32.5℃, but not at 39.5℃ (Heider et al., 2002a). This mutant also exhibits increased transcription from immediate early loci consistent with a defect in autoregulation.
The use of other recombinant viruses has helped elucidate the contributions of IE2 86 to host cell cycle dysregulation during HCMV infection. A virus with a deletion of the majority of exon 3 of the major IE region expresses altered forms of both IE1 72 and IE2 86 proteins and is viable, but severely growth impaired (White and Spector, 2005). It is defective both in the activation of viral early promoters and in altering the expression of certain cellular proteins including cyclin E. Neither of these defects can be fully complemented by growth in the presence of wild type IE1 72 protein. C-terminal sequences of IE2 86 are also required for host cell cycle dysregulation, as infection with a virus carrying a glutamine-to-arginine point mutation at aa 548 does not arrest the host cell cycle and does allow host DNA replication to proceed (Petrik et al., 2006).
Fewer HCMV mutants with disruptions in the IE1 72 coding region have been isolated and characterized, but an existing mutant constructed by deleting exon 4 of the major IE region gives important information about the role of this protein in regulation of the infection. The recombinant is viable, exhibiting minimal growth defects in cells infected at a high multiplicity but exhibits striking replication deficiency at low MOIs (Gawn and Greaves, 2002; Greaves and Mocarski, 1998; Mocarski et al., 1996). Fibroblasts infected with 0.4 pfu/cell of IE1 mutant virus express IE2 86 about as frequently, and to similar levels, as wild-type infected cells, but by immunostaining, deletion mutant-infected cells express delayed early proteins, including ppUL44, ppUL57, and pUL69, much less frequently or to lower levels than wild-type infected cells. This effect on protein expression is apparent at the transcriptional level as well, with decreased accumulation of delayed early RNAs in cells infected at low multiplicity with the mutant. UL112–113 expression is supported to an intermediate degree, with fewer mutant-infected cells staining positively for these proteins than for IE2 86, but more than stained positive for ppUL44. Apparently, while high levels of IE2 86 or another factor are able to compensate for the loss of IE1 72 during a high multiplicity infection, efficient activation of early genes in cells infected at low multiplicity requires IE1 72. A further study used constructs expressing wild type or mutant forms of IE1 72 to complement the IE1 deletion mutant virus in trans (Reinhardt et al., 2005). This work showed that aa 476–491 tether IE1 72 to chromatin but are not required for complementation, while aa 421–475 comprise an acidic domain necessary for complementation and restoration of wild type titers during low multiplicity growth.
Additional immediate early proteins have regulatory roles
In addition to UL122–123, the UL36–38 and IRS1/TRS1 families and the US3 locus encode IE proteins with regulatory functions. Knockout mutants constructed to date suggest that either UL37 exon 1 or UL36 are necessary for progression of the infection (Blankenship and Shenk, 2002; Borst et al., 1999; Dunn et al., 2003; Patterson and Shenk, 1999; Yu et al., 2003; McCormick et al., 2005). The IRS1 and TRS1 ORFs encode three proteins: two expressed from a promoter located in the repeated region flanking the US segment of the genome with an ORF continuing into the unique region, and a third, smaller, protein designated pIRS1263 which is expressed from an internal promoter in the unique region of the IRS1 gene (Romanowski and Shenk, 1997). In transient expression assays, either the IRS1 or TRS1 protein can complement HCMV origin-dependent DNA replication and modestly upregulate transcription from the MIEP or cooperate with IE1 72 and IE2 86 in activation of other viral promoters (Pari and Anders, 1993; Romanowski and Shenk, 1997). None of these three gene products is essential for viral replication in tissue culture, since recombinant viruses lacking the unique regions of the IRS or TRS1 open reading frames are viable (Blankenship and Shenk, 2002; Jones and Muzithras, 1992). The mutant lacking the IRS1 products grows normally, whereas the TRS1 deletion mutant exhibits a multiplicity-dependent growth phenotype. The amount of virus released from cells infected with the TRS1 deletion mutant at a low MOI is reduced drastically compared to that released from wild-type infected cells, but cells infected at a high MOI produce only slightly less virus than the wild type. TRS1 mutant virus replication proceeds normally through DNA replication, and appears to be defective in packaging the viral DNA at a step that occurs after the cleavage of the DNA (Adamo et al., 2004). Two studies have investigated TRS1 and IRS1 protein function using recombinant vaccinia or herpes simplex type 1 viruses that lack the normal ability to block host cell protein synthesis shutoff upon infection (Child et al., 2004; Cassady, 2005; Hakki and Geballe, 2005). TRS1 and IRS1 proteins were able to restore this ability in both cases, either after transient expression in the vaccinia virus-infected cell or from the HSV-1 recombinant. TRS1 protein also binds dsRNA via an N-terminal domain distinct from the C-terminal region that contributes to the evasion of the shutoff of translation (Hakki and Geballe, 2005).
The UL36–38 gene products were also identified in the study that identified eleven loci required for complementation of DNA replication (Pari and Anders, 1993), and are individually dispensable for growth in culture (Dunn et al., 2003; Yu et al., 2003; McCormick et al., 2005). Four of five transcripts from this region are expressed with IE kinetics, three from the UL37 promoter and one coding for the UL36 product (Fig. 18.1) (Chee et al., 1990b; Goldmacher et al., 1999; Kouzarides et al., 1988). The 3.4 kb spliced transcript from the UL37 promoter is present only at IE times and encodes an integral membrane glycoprotein, gpUL37 (Kouzarides et al., 1988; Tenney and Colberg-Poley, 1991a,b). An alternative splice of this transcript generates the 3.2 kb mRNA coding for gpUL37M(Goldmacher et al., 1999; Colberg-Poley et al., 2000), and these proteins traffic from the ER to mitochondria. While gpUL37 is able to transactivate the hsp70 promoter in transient assays, and UL37 exon 2 or exon 3 sequences are required for this activity, deletion of these exons does not affect the ability of the virus to replicate in culture (Borst et al., 1999; Colberg-Poley et al., 1998; Goldmacher et al., 1999; Tenney et al., 1993; Zhang et al., 1996). The conservation of the UL37 exon 1 sequence in clinical isolates and high-passage laboratory strains, as well as in other primate CMVs, and in rodent CMVs (McCormick et al., 2003b, 2005) suggest that the anti-apoptotic functions of vMIA homologues are important in response to the stress of infection. In transient transfection assays, a construct expressing UL37 exon 1 can also activate the UL54, UL44, and other early viral promoters. Although this activation is observed when constructs expressing IE1 72 and IE2 86 are used in this assay (Colberg-Poley et al., 1998), adding UL37 exon 1 and IE1/IE2 expression vectors together results in a synergistic activation effect. The final IE transcript from the UL36–38 region is the UL36 RNA, which encodes a protein (vICA) that is also involved in preventing apoptosis in infected cells (Skaletskaya et al., 2001). The fifth transcript from this region is the product of the UL38 gene and is expressed with early kinetics.
A third region transcribed with IE kinetics is the US3 gene, which specifies at least three alternatively spliced RNAs coding for related proteins. It is the target of complex positive and negative regulatory control (Biegalke, 1995, 1997, 1998, 1999; Chan et al., 1996; Thrower et al., 1996) and like much of UL36–38, is dispensable for growth in culture (Jones and Muzithras, 1992). Proteins generated from this region help the virus to evade the host immune response by keeping MHC class Ⅰ molecules retained in the ER and unable to traffic to the plasma membrane (Ahn et al., 1996; Jones et al., 1996). US3 transcripts are abundant during the first three hours of infection and decrease by five h p.i. Transcription is regulated by a combination of elements located near the promoter including a silencer, enhancer, and transcriptional repressive element (tre) which shares sequence similarity with the crs element involved in control of major IE region expression (Biegalke, 1998; Bullock et al., 2001, 2002; Chan et al., 1996; Lashmit et al., 1998; Thrower et al., 1996). The product of the UL34 gene binds the tre element and represses transcription of US3 (LaPierre and Biegalke, 2001). The US3 proteins seem to possess limited intrinsic transactivating capability, as they have only been shown to induce the cellular hsp70 promoter in transient assays (Colberg-Poley et al., 1992; Tenney et al., 1993; Zhang et al., 1996).
UL112–113 transcription is differentially controlled at early and late times
Many of our current views on the regulation of early gene expression are derived from studies on the UL112–113 region of the HCMV genome (Fig. 18.2). The UL112–113 ORFs encode a family of phosphoproteins of 84, 50, 43, and 34 kDa that share a common amino terminal domain. The initial evidence for the importance of these gene products came from the studies of Pari and Anders, who identified the locus encoding them as one of the eleven required for the replication of a plasmid containing the HCMV origin of DNA replication oriLyt (Pari and Anders, 1993). As noted above, the UL112–113 proteins were found to be among the first to colocalize with IE2 86 at the periphery of the original ND10 sites and appeared to form the initial nucleation sites for subsequent viral DNA replication (Ahn et al., 1999a). Transient expression assays also have shown that they can cooperate with the UL36–38 and IRS1/TRS1 ORFs to augment the stimulation of several early gene promoters by the IE1 and IE2 proteins. Although the recent studies with mutant recombinant viruses that do not express these proteins indicate that the gene products are not absolutely essential for the viral infection, the resulting viruses are severely debilitated in their ability to replicate (Dunn et al., 2003; Yu et al., 2003). The molecular and cellular mechanisms underlying their function, however, are still unknown.
Transcription from the UL112–113 locus begins as early as 8 h p.i. and continues for the duration of the infection, although the relative abundance of members of the family changes as the infection progresses (Staprans et al., 1988; Staprans and Spector, 1986). Two spliced RNAs of 2.1 and 2.2 kb are expressed by 8 h p.i. and encode 50 kDa and 43 kDa phosphoproteins, respectively (Staprans et al., 1988; Staprans and Spector, 1986; Wright and Spector, 1989; Wright et al., 1988). These transcripts are coterminal at both 5′ and 3′ ends and share identical 5′ and internal exons, but use different splice acceptor sites in the 3′ exon to generate the two species, which encode proteins with different carboxyl termini. Later in the infection, transcription from the early start site decreases and initiation of transcription occurs at a site further upstream at nt −62. Two RNAs of 2.5 and 2.65 kb also increase in abundance as the infection proceeds, with the 2.5 kb transcript having spliced out only the first intron and encoding an 84 kDa phosphoprotein and the unspliced 2.65 kb transcript specifying a 34 kDa phosphoprotein (Staprans and Spector, 1986; Wright and Spector, 1989; Wright et al., 1988).
In initial studies, transiently transfected UL112–113 promoter-CAT reporter constructs were used to determine that the region located at −113 to −59 relative to the transcription start site is required for activation of this promoter during the infection. These assays utilized 5′ and internal promoter deletion mutants to show that these sequences were required for activation by IE2 86; this region also contained one of four binding sites for IE2 86, at −113 to −85 (Schwartz et al., 1994; Staprans et al., 1988). Further work indicated that although this IE2 86 binding site contributes to full activation of the UL112–113 promoter, it is in fact sequences between −84 and −59 that are strictly required for full activation of this promoter by IE2 86 (Arlt et al., 1994; Schwartz et al., 1996). A consensus ATF/CREB site is located between nt −71 to −66, suggesting that a member of the ATF/CREB family of transcription factors contributes significantly to IE2 86-mediated promoter activation (Staprans et al., 1988). Additional mutational analyses of the region indicate that this contribution is modulated by interactions with factors bound to other regions of the promoter.
Further work used a series of gel shift analyses to establish that CREB is the major ATF-related protein in uninfected U373 MG cells that binds to this site (Schwartz et al., 1996). Three bands were observed following incubation of wild-type UL112–113 promoter sequences from −84 to −59 with nuclear extracts from U373 MG cells, and two of these were reduced or eliminated when wild type sequences from −72 to −61 were mutated. A complex comigrating with one of the bands also formed when a DNA fragment containing a consensus ATF/CREB site was used as the probe instead of the UL112–113 promoter. The majority of this consensus probe-protein complex was supershifted by the addition of anti-CREB antibody, as was most of the corresponding band in the UL112–113 promoter-protein complexes. The other two bands were less affected by the addition of anti-CREB antibody, but also were not supershifted in the presence of either anti-ATF-2 or anti-ATF-4.
Subsequent studies from our laboratory have used recombinant viruses to define the promoter elements controlling UL112–113 expression in the infected cell (Rodems et al., 1998). In these viruses, a cassette containing the UL112–113 promoter driving expression of the CAT reporter was inserted between the US9 and US10 loci in the viral genome. Reporter expression from this ectopic location authentically reproduced the kinetics of UL112–113 expression, in particular the switch from the +1 to the −62 transcription start site late in the infection (Staprans and Spector, 1986). The family of viruses was constructed based on the observations discussed above and comprised recombinants containing the wild type UL112–113 promoter or one of a series of IE2 86 and/or ATF/CREB binding site mutations in the promoter. As in transient assays, deletion of the ATF/CREB site in the virus resulted in a severe reduction in reporter activity early in the infection, but by 72 h p.i. wild type and ATF/CREB deletion mutants differed in reporter activity by less than twofold. Deletion of the ATF/CREB site also resulted in a shift of the late transcription start site downstream by the number of residues deleted. Consistent with data from transient transfection assays, when the IE2 86 binding site between −113 and −85 was deleted, the level of transcription from the mutant promoter was reduced to half of wild-type promoter levels at early times. Later in the infection, this level was not sustained, and extracts from IE2 86 binding site mutant-infected cells exhibited 15-fold less CAT activity than extracts from wild-type promoter virus-infected cells. These results support a model in which transcription from the UL112–113 promoter is differentially controlled at early and late times postinfection, with the ATF/CREB site providing significant regulatory control at early times and little to none at late times. The IE2 86 binding site, in contrast, modulates UL112–113 transcription at early times but is even more important for the maintenance of elevated transcript levels late in the infection.
Further mutational analysis of the UL112–113 promoter was used to define the sequences between −113 and −85 involved in the control of late transcription. Insertion of 5- or 10-nt sequences into the promoter and a corresponding shift in the late transcription start site suggest that sequences in this region direct late transcription from the UL112–113 promoter with distance-dependent characteristics (D. Kim and D. H. Spector, unpublished results). This complex control of transcription from the UL112–113 region reinforces the idea that, while overall expression of a given viral gene may appear to change little as the infection progresses, this steady-state level is achieved through a series of temporally distinct regulatory mechanisms.
In addition to the above controls operating at the level of transcription, analysis of the pattern of expression of the four RNAs and their corresponding proteins revealed that there were additional mechanisms being used to regulate the level of the proteins (Wright and Spector, 1989). The high levels of the 43 kDa UL112–113 protein at early times correlated well with the abundance of the 2.2 kb RNA at this time. The kinetics of synthesis of the 84- and 34-kDa proteins also correlated well with those of their corresponding RNAs. In contrast, the level of the 2.1 kb RNA was only slightly lower than that of the 2.2 kb RNA at all times during the infection, but the 50 kDa protein did not accumulate until later in the infection. Interestingly, the level of the 50 kDa protein was most sensitive to inhibition of viral DNA replication, suggesting that its accumulation at late times might be coupled to ongoing viral DNA replication. The mechanism for this post-transcriptional regulation is still unknown. It does not appear to be related to stability of the full-length protein, as in pulse-chase experiments at early times in the infection, the 50 kDa protein showed similar kinetics of decay as the 43 kDa protein. Other possibilities are that there is some block to efficient translation of the RNA or that the protein is transiently unstable during translation.
Multiple cis-acting sequences regulate UL54 expression
UL54, the ORF encoding the HCMV DNA polymerase, is a prototypical early–late gene whose regulation has been studied both in the context of the viral genome and in numerous transient expression and in vitro assays (Fig. 18.3). Seven transcripts resulting from initiation at four sites and polyadenylation at two sites in the UL54–57 region are expressed from this cluster of genes. The first RNA transcripts containing UL54 sequences are detectable as early as 8–12 h p.i., but in contrast to the UL112–113 family of transcripts, their level increases significantly at later times (Smuda et al., 1997).
Initial transient expression studies on the regulation of the UL54 promoter indicated that IE2 86 was required for its activation and that other IE and early products including IE1 72, TRS/IRS1, and the UL112–113 proteins cooperated to further activate transcription from the promoter (Kerry et al., 1996). These experiments also identified promoter elements required for activation in transient and in vitro assays, defining the minimal polymerase promoter as the region from −128 to +20 relative to the transcription start site and demonstrating that an 8 bp inverted repeat element at −53 to −45, IR1, was required for activation of the polymerase promoter by viral IE proteins in transient transfection assays (Kerry et al., 1994, 1996). A second copy of the IR1 element is present at −225, but did not contribute significantly to activation of the polymerase promoter in these assays (Kerry et al., 1996). IR1 was found to bind cellular factors found in nuclear extracts prepared from infected cells. If the IR1 element was mutated, the cellular factors failed to bind and activation of the promoter by IE proteins was decreased threefold (Kerry et al., 1994). A pair of studies identified the transcription factor Sp1 as one of the cellular proteins that could bind the IR1 element. In one study (Luu and Flores, 1997), scanning mutagenesis of the promoter from −270 to +200 confirmed the requirement of the −54 to −43 region for IE mediated activation of the promoter. Using a 30 bp DNA probe and uninfected HeLa cell extracts, the authors demonstrated that Sp1 bound to this region. In the other study (Wu et al., 1998), a shorter IR1-containing DNA probe also bound Sp1 in extracts of U373 cells overexpressing IE2 86, but not in parental U373MG cells or in HeLa cells. The complex could be supershifted by the addition of antibody specific to IE2 86, but the presence of IE2 86 in this complex did not seem to require DNA binding activity since addition of an unlabeled DNA probe containing the crs did not reduce complex formation. In addition, an Sp1 binding oligonucleotide competed away this complex in the IE2 86-expressing U373 cells. Based on these results, the authors proposed that an inhibitory factor present in HeLa cells prevents the Sp1-UL54 promoter interaction from occurring. Numerous differences in experimental design prevent further direct comparison of these studies, and the role of Sp1 in control of UL54 expression in infected cells has not yet been examined.
Another regulatory domain in the UL54 promoter was localized to between nt −88 to −80 (Kerry et al., 1994, 1997). A 40 bp DNA probe from this region bound nuclear proteins from infected human fibroblasts, with DNA binding activity particularly strong in 48–72 h p.i. extracts and weaker at earlier times or when DNA replication was inhibited by the addition of phosphonoacetate (PAA). Supershift analyses confirmed that one protein present in this complex is ATF-1. Since recombinant ATF-1/DNA complexes migrate differently than those present in infected cell nuclear extracts, it is possible that an additional protein is involved in this interaction or that ATF-1 is differentially modified during the infection.
Analysis of the UL54 promoter in the context of the viral genome began with the characterization of this promoter driving a CAT reporter in a construct inserted between ORFs US9 and US10 (Kohler et al., 1994). Subsequent studies used similar recombinant viruses to better define the role of the IR1 element in UL54 promoter activation. A family of viruses was constructed in which either the full-length polymerase promoter (−425 to +20), the full length promoter with a mutation in the IR1 site, or the minimal activation domain (−128 to +20) drove expression of the CAT reporter (Kerry et al., 1996). Based on CAT activity and RNA levels by Northern blot, UL54 promoter activity at early times was three- to fourfold lower than the wild-type when the IR1 element was mutated. In contrast, deleting the upstream promoter region and including only the minimal promoter resulted in a slight increase in promoter activity. This was consistent with the increase in promoter activity observed in transient assays when the −425 to −128 region was deleted, but the effect was smaller in the context of the viral genome. At late times, the IR1 element appears to be less important to the activation of this promoter, since the virus carrying the IR1 mutation exhibited only a slight reduction in both RNA levels and CAT activity by 72 to 96 h p.i. The ATF site in the UL54 promoter was similarly examined by constructing recombinant viruses (Kerry et al., 1997). In contrast to the IR1 element, the ATF binding site appears to control UL54 promoter activity both at early and late times p.i. Mutation of the ATF site in a UL54 promoter-CAT reporter virus resulted in five- to sixfold decreases in both mRNA expression and CAT activity over a range of times, from 24–96 h p.i.
Finally, recent work has attempted to use regulation of the UL54 promoter to understand the species specificity of HCMV (Garcia-Ramirez et al., 2001). These experiments examined the activity of the HCMV polymerase promoter following HCMV infection of murine NIH 3T3 cells transiently or stably transfected with a yeast artificial chromosome (YAC) clone containing much of the HCMV genome. The authors found that mutating the IR1 element in this context reduced reporter activity and interpreted these results to invoke similar cellular factors in both 3T3 and human cells, suggesting that species specific cellular factors do not underlie activation of the UL54 promoter. Given the many differences between murine and human fibroblasts, it is difficult to put these findings in context. A complete characterization of the UL54 promoter-reporter YAC construct in human cells to understand the consequences of promoter mutations in the natural context might help understand any work across species barriers.
UL4 expression is controlled at the transcriptional and translational levels
UL4 is an example of an early gene whose expression is regulated by unique mechanisms operating at both the transcriptional and translational levels (Fig. 18.4). Three unspliced transcripts are expressed from the UL4 locus, with 1.4 and 1.5 kb RNAs present at early times and a 1.7 kb product detectable later. These encode an incompletely characterized membrane glycoprotein, gp48, which is a virion component that is completely dispensable for viral growth in tissue culture (Hobom et al., 2000).
Transcriptional control of the UL4 gene was first analyzed in a series of transient transfection assays demonstrating that the UL4 promoter is responsive to IE2 86 and that there are two cis-acting sites in the UL4 promoter upstream of the TATA box (Chang et al., 1989; Huang et al., 1994). The first site, a CCAAT box with inverted dyad symmetry between −88 and −98 relative to the transcription start site, appeared to be a positive regulatory element that could bind the transcription factor NF-Y in vitro (Huang et al., 1994). Gel shift analyses demonstrated the formation of two complexes that were inhibited by the addition of a competing oligonucleotide containing the NF-Y target sequence or supershifted by the addition of antibody to NF-Y. Neither could be supershifted by the addition of IE2 86-specific antibody. One complex was present in both uninfected and infected cells; the second was only detected in infected cells. When phosphatase was added, the second complex decreased in abundance while the first increased, suggesting that NF-Y may be differentially phosphorylated in HCMV-infected cells.
A second regulatory region located between −169 and −139 in the UL4 promoter, site 2, bound a cellular factor in vitro, as indicated by DNA footprinting and gel shift assays, and negatively influenced transcription (Huang et al., 1994; Huang and Stinski, 1995). The importance of IE2 86 in the activation of this promoter was suggested by the following observations: first, the negative effect on the promoter was relieved when an IE2 86 expression vector was cotransfected with the UL4 promoter – CAT reporter construct; second, a truncated version of IE2 86 containing amino acids 290 to 579 bound site 2 in vitro (Huang and Stinski, 1995). These results led to the hypothesis that IE2 86 binds to a specific negative regulatory region (which is 65% homologous to the crs element in the major IE promoter) and in the process displaces a bound cellular factor, thus allowing activation of the UL4 promoter. Mutation of the putative zinc finger region of IE2 86 resulted in a protein that could no longer interact with the promoter, lending further support to this idea.
Recent studies examining transcriptional control of the UL4 promoter in the context of the viral genome confirm some, but not all, of these initial findings and demonstrate important differences between control of viral gene expression in transient assays and in the infected cell. A series of HCMV recombinants containing the CAT reporter cassette driven by a wild type or mutant UL4 promoter inserted into the viral genome between US7 and US12 were derived (Chen and Stinski, 2000). In this ectopic location, the CAT transcript was expressed with kinetics much like those of the native UL4 transcript, but to slightly lower overall levels. Here, a mutation in the UL4 promoter NF-Y binding site did not alter CAT transcript levels, leading to the conclusion that in the virus, the NF-Y binding site contributes little or nothing to control of UL4 transcription. In contrast, site 2 mutants displayed altered reporter expression. Site 2 contains two putative (but non-consensus) IE2 86 binding sites and a predicted Elk-1 binding site that together appeared to be required for maximal expression in the context of viral infection (Chen and Stinski, 2000). In a later study, however, disruption of the Elk-1 site alone was sufficient for the effect, indicating that it is likely Elk-1 or a related cellular factor and not the IE2 86 binding sites that play the critical regulatory role (Chen and Stinski, 2002). Elk-1 binding to this site is supported by EMSA data in which a consensus Elk-1 site competes for cellular factor binding and an Elk-1 antibody partially supershifts the UL4 promoter-cellular factor complex (Chen and Stinski, 2000).
Transcriptional upregulation of UL4 expression has been used to demonstrate the importance of the MAPK/ERK and p38 MAPK pathways in HCMV replication. Peak activation of these signal transduction cascades occur at 4 and 8 h p.i., respectively, and appears to be important to the progression of productive infection via efficient expression of early and late genes (Johnson et al., 2001a; Rodems and Spector, 1998). In one study (Chen and Stinski, 2002), the requirement for these pathways was analyzed in the context of UL4 transcription using the MAPK/ERK kinase (MEK) inhibitor UO126 and the p38 MAPK inhibitor FHPI. Cells infected with one of the recombinant viruses expressing the CAT gene from wild-type or mutant UL4 promoters were treated with an inhibitor and assayed for CAT activity. Strikingly, inhibition of MEK with the chemical inhibitor UO126 caused UL4 promoter activity to drop by 70%–80% for all promoters tested, with approximately equivalent loss of expression when the wild-type UL4 promoter was used as well as when site 2 or the NF-Y or Elk-1 binding sites were disrupted. The effect of the inhibitor FHPI was very similar, with no effect on UL4 promoter constructs when used at a low concentration and a 50%–80% reduction of both wild type and mutant promoter activity at a higher concentration. These data suggest that the UL4 promoter is responsive to both MAPK/ERK and p38 MAPK pathways and that the minimal responsive element is the TATA, not one of the other transcription factor binding sites that have been analyzed. This effect, however, was less prominent when endogenous UL4 RNA expression was examined, and the possibility of general inhibition of viral transcription or downregulation of other upstream factors in the presence of these inhibitors has not yet been addressed.
Novel post-transcriptional controls also distinguish the regulation of UL4 expression. Three small ORFs are located in the UL4 transcript upstream of the gp48 transcription start site. The largest of these, uORF2, encodes a 22 aa product which acts in cis to repress translation of the authentic UL4 transcript (Degnin et al., 1993). Interestingly, this repression is amino acid- (but not nucleotide-) sequence dependent and is particularly sensitive to mutations in codons near the C-terminus of uORF2. In transient transfection-superinfection assays, missense mutation or deletion of the terminal amino acid, a proline, resulted in significantly elevated expression of reporter protein while the level of UL4 promoter-driven transcript remained unchanged. Changes in the N-terminal leader sequence also had an effect, with missense mutations at codons 7 and 8 reducing the ability of uORF2 to block translation. A further series of modified primer extension assays showed that ribosomes are stalled on the RNA at the uORF2 termination codon. These data have led to the following hypothesis regarding the mechanism of inhibition of downstream translation by uORF2. A ribosome translates through the uORF2 sequence until it reaches the final proline codon. Here, the nascent uORF2 peptide remains covalently linked to the peptidyl-tRNA and bound to the ribosome on the transcript. A possible interaction between this complex and the release factor eRF1, mediated by C-terminal prolines at positions 21 and 22 in the uORF2 peptide and a GGQ motif in eRF1, stabilizes the intermediate and prevents hydrolysis of the peptidyl-tRNA bond (Cao and Geballe, 1996, 1998; Janzen et al., 2002). Ribosomes stall before they are able to reach the gp48 translation start site, resulting in uORF2-dependent repression of gp48 translation.
The above model is supported by results from recombinant viruses as well. When the uORF2 initiation codon in the UL4 region was mutated, there was a significant up-regulation of gp48 protein expression by the mutant relative to the wild-type virus (Alderete et al., 2001). In vivo, ribosomes were also stalled at the uORF2 termination codon in wild-type but not mutant-infected cells. The relatively normal expression of other early proteins indicates that it is not a general upregulation of translation in mutant-infected cells that accounts for this phenotype. Curiously, it appears that there is a slight delay in the accumulation of the UL4 transcript in mutant infected cells, with the UL4 RNA detectable by Northern blot 24 h p.i. in cells infected with the parental wild-type virus, but not until 48 h p.i. in uORF2 mutant-infected cells.
Since neither loss of nor overexpression of UL4 appears detrimental to viral replication in cultured fibroblasts (Alderete et al., 2001; Hobom et al., 2000), it seems likely that this complex regulation is important in the infected host where gp48 may have a cell type-specific role. Control of its transcription and translation remain interesting illustrations of the diverse methods used by the virus to regulate gene expression.
Human herpesviruses 6 and 7
Human herpesvirus 6 is distinguished by its ability to grow in T-lymphocytes. There are two variants: HHV-6A (GS and U1102-like isolates) and HHV-6B (Z-29 and HST-like isolates). Although the variants have approximately 90% nucleotide sequence identity and share many properties, they differ with respect to their epidemiology, cell tropism, interaction with the host cell, and in vivo pathogenesis. HHV-6B infection is very common in early childhood and is associated with exanthem subitum (roseola infantum), a mild illness that lasts only a few days (Yamanishi et al., 1988). HHV-7 is biologically similar to the two HHV-6 variants and is less frequently associated with this illness (Dominguez et al., 1999; Isegawa et al., 1999; Megaw et al., 1998; Nicholas, 1996; Tanaka et al., 1994). The DNA genomes of HHV-6 and HHV-7 are smaller than that of HCMV, with the length 159 kbp for HHV-6A, 161–170 kbp for HHV-6B, and 145–153 kbp for HHV-7 DNA. The genome of all of these viruses consists of a long unique segment bounded by direct repeats (Dominguez et al., 1999; Gompels et al., 1995; Isegawa et al., 1999; Lindquester and Pellett, 1991; Martin et al., 1991b; Megaw et al., 1998; Nicholas, 1996)). In general, the genes of these viruses are collinear with those of human cytomegalovirus UL region (see Chapter 14).
Analysis of the molecular biology of HHV-6 and HHV-7 has been limited by poor growth and low yields in cultured cells. Thus, the elucidation of the regulation of gene expression is not well advanced and has relied almost exclusively on transient assays that are known to be relatively poor predictors of behavior in the context of viral infection. The overall pattern of gene expression (IE, early, and late) resembles that of HCMV and other herpesviruses, but the assignment of many of the individual genes to a particular class is still tentative and has been influenced by the sensitivity of the assay applied, the specific strain of virus, and cell line used in the analysis. The reader is referred to Chapters 42 and 43 as well as the following papers for the details: Menegazzi et al. (1999); Mirandola et al. (1998); Oster and Hollsberg (2002); Rapp et al. (2000). For the purposes of this review, only HHV-6 IE genes that appear to have some regulatory functions and two early genes whose regulation has been studied in greatest depth – the HHV-6 DNA polymerase (U38) and the DNA polymerase processivity factor (U27) (Fig. 18.5) – will be discussed.
HHV-6 IE gene products with regulatory activities
The HHV-6 regulatory proteins that have been studied are encoded by the IE-A and IE-B loci, DR7, U3, U27, and U94. The IE-A and IE-B loci of both HHV-6 and HHV-7 are the major sites of IE gene expression (Fig. 18.5). The IE-A region (U86 to U90) is collinear with the major immediate early region of HCMV that encodes the IE1 and IE2 proteins (UL122–123), and the IE-B region (U16 to U19) is collinear with the HCMVIE ORFs UL36 to UL38 (see Chapter 17).
These putative viral regulatory proteins have only been studied with promoters from cellular genes and genes from heterologous viruses. Because HHV-6 has been proposed to be a cofactor in the progression of AIDS, many of these studies have used the HIV-1 LTR promoter (for review, see Lusso and Gallo, 1995). The HIV-1 LTR has been found to be activated by regions of the HHV-6A genome corresponding to the IE-B locus (Chen et al., 1994; Garzino-Demo et al., 1996; Geng et al., 1992; Horvat et al., 1991), the IE-A locus (Gravel et al., 2003; Martin et al., 1991a; Papanikolaou et al., 2002; Stanton et al., 2002), DR7 (Kashanchi et al., 1994; Thompson et al., 1994a), U3 (Mori et al., 1998), and U27 (Zhou et al., 1994). The IE-A locus also transactivates the CD4 promoter (Flamand et al., 1998) and adenovirus E3 and E4 promoters (Martin et al., 1991a), and the IE-B locus activates HPV 16 and 18 promoters (Chen et al., 1994). Some of the regulatory proteins also appear to have negative effects. For example, U94 negatively regulates HIV-1 and H-ras promoters (Araujo et al., 1995), and DR7 negatively regulates promoters that are responsive to p53 (Kashanchi et al., 1997).
IE-A
HHV-6A and HHV-6B exhibit a different organization in the IE-A region (Dominguez et al., 1999; Isegawa et al., 1999). The HHV-6A IE-A locus consists of two genetic units that are referred to as IE1 and IE2 (Chapter 17). Although this region is collinear with the HCMV major IE locus UL123 (IE1 72) and UL122 (IE2 86), it shares no nucleotide or protein sequence homology. The IE1 region encodes a 3.5 kb transcript that consists of 4 small 5′ exons and a large 3′ exon (Fig. 18.5) (Schiewe et al., 1994). Translation begins in exon 3, yielding a major protein of approximately 150 to 170 kDa that contains ORFs U90 and U89 (Papanikolaou et al., 2002). The corresponding region of HHV-6B shares only 62% identity at the amino acid level and encodes a major IE protein of approximately 150 kDa (Gravel et al., 2002; Takeda et al., 1996). The proteins are phosphorylated and conjugated to the ubiquitin-like protein SUMO-1 (Chang and Balachandran, 1991; Gravel et al., 2002). The sequence divergence of the HHV-6A and HHV-6B IE1 protein is reflected in their function in that the HHV-6A IE1 protein is a stronger activator of heterologous promoters in transient expression assays than the corresponding HHV-6B protein (Flamand et al., 1998; Gravel et al., 2002; Martin et al., 1991a).
Both HHV-6A and HHV-6B IE1 proteins are able to traffic to the ND10 sites (PODs). However, in contrast to other herpesviruses, interaction of the ND10 domains with IE1 does not lead to their dispersal, and IE1 maintains a stable interaction with ND10 throughout the infection (Gravel et al., 2002; Stanton et al., 2002). By immunostaining, the highest number of individual IE1 bodies can be detected at 12 h p.i., when they begin to coalesce into 1–3 larger bodies. It is likely that other viral proteins are needed to generate the larger bodies, since when the IE1 proteins were individually expressed, they colocalized with PML and SUMO-1 but the ND10 sites did not coalesce. Thus as is the case for HCMV, dispersal of ND10 domains is not a prerequisite for productive infection.
Recently, a full-length cDNA encoding the HHV-6A IE2 protein was isolated (Gravel et al., 2003); it contains ORFs U90 and U86/87, the positional homologues of the HCMVORF encoding IE2 86 (Fig. 18.5). The 5.5 kb IE2 mRNA is expressed in the absence of de novo protein synthesis with kinetics that are somewhat delayed relative to the IE1 transcript. The IE2 mRNA is also less abundant, but it continues to increase throughout the infection while the IE1 mRNA reaches maximal levels by 12 h p.i. At later times, larger transcripts that have not been characterized and may initiate from upstream sites appear. The processed IE2 mRNA consists of 5 exons; 4 small exons located upstream of U89 are shared with IE1 and the fifth exon corresponds to the IE2 specific ORF U86/87. The sequence of the HHV-6A IE2 transcript diverges from that encoded by the HHV-6B variant, with only 64% identity at the amino acid level. The HHV-6A IE2 protein is also 167 amino acids shorter. By Western blot analysis with an antibody specific for the IE2 region, it appears that the major IE2 protein is approximately 220 kDa. At later times in the infection, additional smaller proteins of 100, 85 and 55 kDa have also been observed (Gravel et al., 2003; Papanikolaou et al., 2002). Analogous to the HCMVIE2 86 protein, the full-length HHV-6A IE2 protein functions in transient expression assays as a promiscuous activator of multiple promoters, including minimal promoters containing only a TATA box (Gravel et al., 2003; Papanikolaou et al., 2002). Recently, deletion analysis showed that both the N- and C-terminal domains of this protein are required for full function (Tomoiu et al., 2006). Interestingly, the HHV-6A IE2 protein does not appear to downregulate its own promoter, possibly because it does not contain the HCMV cis-repression signal.
IE-B
The HHV-6 IE-B region consists of the ORFs U16, U17, U18, and U19 and is collinear with the HCMV region containing the UL36–38 ORFs. As shown for the HCMVUL36–38 region, transcription from the HHV-6 IE-B region is complex (Flebbe-Rehwaldt et al., 2000; Mirandola et al., 1998). In HHV-6A (GS strain), the U17 and U16 ORFs are positional homologues of HCMVUL36 and yield a spliced IE RNA (Fig. 18.5) (Flebbe-Rehwaldt et al., 2000; Mirandola et al., 1998). HCMVUL36 also consists of 2 exons, with exon 2 corresponding to HHV-6A U16 (Tenney and Colberg-Poley, 1991a, b). In addition to the HHV-6 transcripts that contain the U17/U16 ORFs, there are also transcripts that include the U16 and U15 ORFs (U15 is unique to HHV-6). The HHV-6A and HHV-6B U17/U16 RNAs first appear at IE times and are maintained throughout the infection. The other U16 containing transcripts are expressed primarily as early RNAs, although a low level of transcription can be detected at IE times.
The HHV-6 U18 and U19 ORFs are transcribed as multiply spliced early RNAs. These are positional homologues of the HCMVUL37 and UL38 genes, respectively, but may not be functional homologues. With the caveat that all of the functional assays have been performed with transient assays and plasmid constructs, the ORFs do not correspond to their HCMV homologues with respect to their potential role as transactivators. In HHV-6, the ORFs that can transactivate the HIV-1 LTR promoter are U17/U16, not U18/U19 (Chen et al., 1994; Garzino-Demo et al., 1996; Geng et al., 1992; Horvat et al., 1991). UL37 exon 1, which has no apparent homologue in HHV-6 or HHV-7, serves as a cell death suppressor and transactivator of HCMV early gene promoters in transient assays (Colberg-Poley et al., 1998; Patterson and Shenk, 1999; Goldmacher et al., 1999).
U94
U94 is encoded by HHV-6A and HHV-6B and is a homologue of the adenovirus associated virus 2 (AAV-2) rep gene (Thomson et al., 1991). Although not conserved in primate CMVs, a homologue is found in rat CMV (Vink et al., 2000). In AAV-2, the rep68 gene encodes a site-specific ATP-dependent endonuclease and helicase that is involved in the site-specific integration of AAV into chromosome 19. U94 can serve as a helper for AAV-2 replication and can complement an AAV-2 virus with a mutation in the rep gene (Thomson et al., 1994). The role of the U94 protein is yet to be elucidated, but it may play a role in latency and modulation of the infection. Relevant to this are the data showing that cell lines expressing HHV-6B U94 cannot be infected by HHV-6A and that the U94 transcript can be detected during latency in PBMC (Rotola et al., 1998). U94 was also found to suppress H-ras and BPV-1 transformation (Araujo et al., 1997) and transcription from H-ras and HIV-1 LTR promoters (Araujo et al., 1995). HHV-7 does not encode a U94 homologue.
cis-acting sequences within HHV-6 early promoters
DNA polymerase (U38)
Analogous to HCMV, the HHV-6 DNA polymerase gene has been used as a prototypical early gene for examining cis-acting regulatory sequences on the promoter (Fig. 18.6). Based on the position of the RNA start site, standard transient expression assays with promoter-CAT reporter genes have been used to identify and characterize the major regulatory region for transcription (Agulnick et al., 1994). The data showed that expression of CAT from a construct containing sequences −524 to +115 relative to the transcription start site was below the limit of detection in uninfected HSB-2 human T cells, but was highly up-regulated in infected cells. By mutational analysis, the cis-acting sequences for activation were localized to the sequences between −78 and +13. The major regulatory element within this region was a consensus ATF/CREB binding site located at nt −77 to −70. Further support for this domain serving a regulatory role was the observation that in gel shift assays, this site bound to two protein complexes in both infected and uninfected nuclear extracts. The ATF/CREB site, however, is not required for activation of this promoter by the HHV-6A IE2 protein in transient assays (Tomoiu et al., 2006).
The U38 promoter does not contain a consensus TATA element in the −30 region, although there are several AT-rich domains located at positions −48 to −43, −29 to −24, and −11 to −6. When point mutations were introduced into each of the domains individually, there was no effect on the promoter, leading the authors to conclude that the promoter is TATA-less (Agulnick et al., 1994). However, the start site for transcription was not identified for any of the mutants in the transient expression assays, and thus the possibility that the elements might be able to compensate for one another, albeit with a change in start site, cannot be excluded. As has been the case for all herpesvirus promoters, conclusions cannot be drawn from transient assays alone and each promoter must be assessed in the context of the viral genome. It would seem likely that the ATF/CREB site is the major regulatory element, as this sequence is located in a similar position in the promoter of the HCMVDNA polymerase gene and its function has been confirmed in the context of the HCMV genome (Kerry et al., 1994, 1996, 1997).
DNA polymerase processivity factor (U27)
U27 encodes a 41 kDa nuclear phosphoprotein that is the homologue of the HCMVUL44 gene encoding DNA polymerase processivity factor (Fig. 18.7) (Agulnick et al., 1993; Chang and Balachandran, 1991). The U27 region actually includes two ORFs, ORF A and ORF B. ORF A corresponds to a 41 kDa protein, and the downstream ORF B could encode a 27 kDa protein (Zhou et al., 1997). The transcription pattern of this gene is similar for both the variant A HHV-6 GS strain and the variant B HHV-6 Z29 strain. At least five early–late unspliced RNA species ranging in size from 1.2 to 2.7 kb (1.2, 1.5, 1.8, 2.3, and 2.7 kb) map to this locus, with the 2.3 kb RNA being the most abundant (Agulnick et al., 1993; Zhou et al., 1997). Four of the RNAs (2.7, 2.3, 1.8, and 1.2 kb RNAs) are 3′ co-terminal. The 1.5 kb RNA utilizes the same start site as the 2.3 kb RNA but terminates upstream of the other four RNAs. The 2.3 kb and 1.5 kb RNAs have the potential to encode the p41 protein in ORF A, and the 1.2 kb RNA could specify a protein of about 17 kDa within ORF B. Although the 2.7 kb RNA includes ORF A, the presence of several AUGs before ORF A makes it unlikely that it encodes p41. Likewise, the 1.8 kb RNA has several AUGs with short ORFs before ORF B, but it could be translated into a truncated version of ORF A.
Regulation of the promoter for the 1.5 kb and 2.3 kb U27 RNAs was studied for the HHV-6A GS strain in HSB-2 human T-cell line (Thompson et al., 1994b). The transcription start site, which is 48 bp upstream of the translation initiation codon AUG, is preceded by a TATA sequence starting at nt −33 relative to the RNA start site. Using mutant promoter-CAT constructs in transient expression assays, an essential regulatory element that was activated in the infected cells was localized between nt −73 and −52. Within this region were a putative binding site for the transcription factor C/EBP (CAAT enhancer-binding protein) and two other repeat sequences. The activity of this site was both distance and orientation dependent relative to the TATA sequence.
Mobility shift assays indicated that there were four complexes that bound to this region. The two that were present in both uninfected and infected cells (C1 and C2) did not appear to contain C/EBP factors. Point mutations within these sites that eliminated binding also inactivated the promoter. Two of the binding complexes were only present in uninfected cells (C3 and C4) and they could be competed by an oligonucleotide containing a consensus C/EBP site. The construction and use of a HHV-6 BAC will greatly facilitate further studies on the regulation of this and other promoters during the viral life cycle.
Conclusions
The studies presented here highlight the central role that early gene expression plays in the viral life cycle. It is the middleman in the relay race leading to the production of infectious virus. Events precipitated by the initial contact of the virus with the host cell and the synthesis of IE gene products set the stage for early gene expression. The products of these early genes not only provide the component parts for the factories devoted to viral DNA synthesis, cleavage and packaging of the viral genome, and assembly of the virus particles, but also serve to commandeer the host cell machinery and signaling pathways to create a cellular environment that is optimal for viral gene expression and DNA replication. Although dispensable for growth in tissue culture, a cadre of the early genes must also establish a blockade to the host’s immune response and prepare routes of escape for viral dissemination. Analogous to the control of host cell genes, viral early gene expression is regulated at multiple levels by mechanisms operating at the initiation of transcription, RNA processing and transport, translation, and mRNA and protein stability. The development of BACs as vectors for the cloning of herpesvirus genomes has revolutionized the field so that the function of viral genes and the regulation of their expression can be studied in the biologically relevant context of the viral infection. These techniques coupled with the rapidly moving fields of genomics and proteomics will greatly enhance our ability to elucidate the cellular and molecular mechanisms governing the interaction of the virus with its host.
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- Introduction
- Identification of HCMV early genes
- HCMV-mediated changes in the cellular environment prior to early gene expression
- Functions of viral early genes
- Transactivating functions of the major IE proteins
- Additional immediate early proteins have regulatory roles
- UL112–113 transcription is differentially controlled at early and late times
- Multiple cis-acting sequences regulate UL54 expression
- UL4 expression is controlled at the transcriptional and translational levels
- Human herpesviruses 6 and 7
- Conclusions
- References
- Review Coordination of late gene transcription of human cytomegalovirus with viral DNA synthesis: recombinant viruses as potential therapeutic vaccine candidates.[Expert Opin Ther Targets. 2013]Review Coordination of late gene transcription of human cytomegalovirus with viral DNA synthesis: recombinant viruses as potential therapeutic vaccine candidates.Isomura H, Stinski MF. Expert Opin Ther Targets. 2013 Feb; 17(2):157-66. Epub 2012 Dec 12.
- Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production.[J Virol. 2004]Cyclin-dependent kinase activity is required at early times for accurate processing and accumulation of the human cytomegalovirus UL122-123 and UL37 immediate-early transcripts and at later times for virus production.Sanchez V, McElroy AK, Yen J, Tamrakar S, Clark CL, Schwartz RA, Spector DH. J Virol. 2004 Oct; 78(20):11219-32.
- Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection.[Virology. 2000]Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection.Ahn JH, Hayward GS. Virology. 2000 Aug 15; 274(1):39-55.
- Structure of transcripts and proteins encoded by U79-80 of human herpesvirus 6 and its subcellular localization in infected cells.[Virology. 2000]Structure of transcripts and proteins encoded by U79-80 of human herpesvirus 6 and its subcellular localization in infected cells.Taniguchi T, Shimamoto T, Isegawa Y, Kondo K, Yamanishi K. Virology. 2000 Jun 5; 271(2):307-20.
- The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle.[J Gen Virol. 1997]The human cytomegalovirus glycoprotein B gene (ORF UL55) is expressed early in the infectious cycle.Smuda C, Bogner E, Radsak K. J Gen Virol. 1997 Aug; 78 ( Pt 8):1981-92.
- Early viral gene expression and function - Human HerpesvirusesEarly viral gene expression and function - Human Herpesviruses
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