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

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Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis.

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Chapter 17Immediate–early viral gene regulation and function

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1 and 2.

1 Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
2 Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

Introduction

Betaherpesviruses such as human cytomegalovirus (HCMV), human herpesvirus-6A and 6B (HHV-6), and human herpesvirus-7 (HHV-7) replicate more slowly than alphaherpesviruses, are highly species-specific for infection, and establish latency in progenitor cells of the bone marrow and monocytes of the blood. HCMV has been the prototype of the betaherpesviruses for studies of gene expression and regulation. In cell culture, HCMV strains have been adapted to preferentially infect and replicate in fibroblasts. However, low passage isolates replicate well in other cell types, such as endothelial cells, macrophages and dendritic cells. In the host, HCMV replicates in macrophages, dendritic cells, colonic and retinal pigmented epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, neuronal cells, glial cells, hepatocytes, and trophoblasts (Fish et al., 1995, 1996; Hertel et al., 2003; Ibanez et al., 1991; Lathey and Spector, 1991; Maidji et al., 2002; Schmidbauer et al., 1989; Sinzger et al., 1993, 1995, 1996). In contrast, HHV-6 and HHV-7 infect CD4+ lymphocytes (Takahashi et al., 1989) as well as monocyte/macrophages. Although HCMV can be transferred into and out of polymorphonuclear leukocytes via cell-to-cell contact, these cells do not permit viral replication (Grundy et al., 1998; Sinclair and Sissons, 1996; Sinzger and Jahn, 1996).

Various animal betaherpesviruses have been used as models for HCMV infection. CMVs infecting seven different mammalian hosts (humans, chimpanzees, African green monkeys, rhesus macaques, guinea pigs, rats and mice) have been investigated in some level of detail. Murine CMV (MCMV) infection of mice has been the most widely used animal model. MCMV tissue tropism, virulence, latency, and reactivation exhibit similarities to those of HCMV infections (Hudson, 1979; Jordan, 1983) and many important insights have emerged from this model despite the fact that rodent and primate CMVs are evolutionarily distant relatives. Between 75 and 80 open reading frames (ORFs) of MCMV have significant sequence homology to the predicted ORFs of HCMV (Chee et al., 1990; Davison et al., 2003; Murphy et al., 2003a,b; Rawlinson et al., 1996). Rhesus CMV (RhCMV) and chimpanzee CMV (CCMV) have approximately 138 and 166 ORFs with significant homology to HCMV, respectively (Davison et al., 2003; Hanson et al., 2003). Current estimates suggest that HCMV has at least 165 genes, but estimates of over 190 genes have been reported, depending on the method of prediction. HCMV has 45 essential genes and 15 of them have an unknown function. The majority of the non-essential genes also have unknown functions (Dunn et al., 2003). For replication in human fibroblast, 68 ORFs are completely dispensable (Dunn et al., 2003). Some of the viral genes that are dispensable for replication in human fibroblast cells are required for replication in human microvascular endothelial cells or in retinal pigment epithelial cells (Dunn et al., 2003). Although many of the HCMV genes are dispensable for viral growth in cell culture, studies with MCMV or RhCMV suggest that many dispensable genes are important for modulating the virus–host interaction. Approximately two-thirds of the genes of HHV-6 are collinear with the unique long (UL) region of the HCMV genome (Chee et al., 1990; Gompels et al., 1995; Neipel et al., 1991).

This chapter will review viral replication events, with emphasis on expression and function of the betaherpesvirus immediate early (IE) genes. Understanding betaherpesvirus replication is important for the development of better strategies to prevent and treat virus-induced disease.

Betaherpesvirus immediate early genes

The IE genes are the first viral genes transcribed after infection, and their transcription does not require de novo viral protein synthesis. These gene products optimize the cell for viral gene expression and replication. Figure 17.1 depicts the location of the IE gene loci on the HCMV and HHV-6 genomes. The HCMV long (L) genomic component encodes two betaherpesvirus-conserved transcription loci, the major IE genes (IE-A locus) and the IE-B locus as well as UL119-115 locus. The UL119–115 genes are not known to be involved in regulation of cellular or viral gene expression, and are not discussed in this chapter. The HCMV short (S) genomic component encodes US3 and the TRS1/IRS1 IE genes. HHV-6 lacks a region analogous to the S component of HCMV and so does not encode homologues of these HCMV genes.

Fig. 17.1. Diagram of the immediate early (IE) genes of human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6).

Fig. 17.1

Diagram of the immediate early (IE) genes of human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6). The genes of the unique long (UL) components are colinear and designated alpha numerically. The unique short (US) component containing the TRS1/IRS1 (more...)

The IE-A locus of HCMV encodes the major immediate early (MIE) genes and is collinear with a homologous region in HHV-6. In both viruses there is a strong transcriptional enhancer between two divergent genes (Fig. 17.1), with regulatory genes transcribed in the leftward direction designated IE1 and IE2. The function of the viral genes transcribed to the right is unknown. These viral genes and their enhancers have a major effect on productive viral replication for all betaherpesviruses where they have been studied and, consequently, they will be the focus of a majority of this chapter. For both MCMV (m128) and HCMV (UL127), the rightward transcribed viral genes in this locus are dispensable for replication in cell culture.

The IE-B locus is located in the L-component of the HCMV genome and exhibits homologous protein coding regions to HHV-6 (Flebbe-Rehwaldt et al., 2000; Nicholas and Martin, 1994) (Fig. 17.1), as well as to other betaherpesviruses. The HCMV UL36–38 genes in the IE-B locus encode proteins that are required for viral growth. There are at least five transcripts from three different promoters in this region (Colberg-Poley, 1996). The products of the HCMV UL36–37 gene locus (e.g., UL37 exon 1, gpUL37, gpUL37m, and UL36) are expressed with IE kinetics and presumably serve to quickly thwart the cellular anti-viral response of apoptosis (see Chapter 21). The UL37 exon 1 unspliced RNA is abundant at IE times and remains abundant until late times after infection (Su et al., 2003). HCMV oriLyt-mediated DNA replication assays are also enhanced by the UL36–38 gene products (Pari et al., 1993). The HHV-6 U15–17 genes are multiply spliced in a pattern reminiscent of UL36–38 and have regulatory activities. For example, U16/17 spliced gene product activates expression from the HIV LTR promoter (Geng et al., 1992).

The US3 gene is transcribed at IE times to yield three alternatively spliced transcripts (Colberg-Poley, 1996). IE gene expression is controlled at multiple levels. Upstream of the US3 promoter is two sequence repeats designated R2 and R1 (Weston, 1988). R2 is a NF-κB containing enhancer that promotes a high level of US3 transcription. R1 has 19 repetitions of a 5’-TRTCG-3’ pentanucleotide arranged as direct repeats, inverted repeats, and variably spaced single pentanucleotides. Although R1 was reported to silence expression from the US3 promoter in transient transfection assays (Chan et al., 1996; Thrower et al., 1996), R1 enhances expression of the flanking US3 and US6 genes by an unknown mechanism when assayed in the context of viral infection (Bullock et al., 2001, 2002). The US3 and US6 viral gene products disrupt the process of cellular HLA presentation of viral antigens at the cell surface and, consequently, they likely contribute to evasion of the host immune response (see Chapter 62). Between the start site of transcription and the TATA box of the US3 promoter is a cis-acting repressor element that binds the viral protein encoded by the essential HCMV UL34 gene (Dunn et al., 2003; LaPierre and Biegalke, 2001). Mutation of the cis-acting element causes a high level of expression of the US3 gene at early and late times after infection (Lashmit et al., 1998). The positive regulation of US3 gene expression may trap viral antigens introduced into the cell upon virus entry or expressed de novo. The negative regulation may prevent the toxic consequences of continued trapping of cellular HLA molecules on the membranes of the endoplasmic reticulum. US3 expression is repressed at late times after infection when the viral proteins encoded by US2, US6, and US11 contribute to immune evasion (see Chapter 62).

Because their transcription initiates in repeats flanking the US region, the TRS1/IRS1 genes are controlled by identical promoter elements. This arrangement results in proteins with identical N-terminal domains and divergent C-terminal domains, given the 3′-end, of their transcripts arise from different unique sequences in the US region (Fig. 17.1). TRS1 and IRS1 proteins are packaged into the virion as tegument components and, therefore, these viral proteins are introduced into cells in advance of IE gene expression (Romanowski et al., 1997). Viruses lacking IRS1 replicate normally. However, mutation of TRS1 together with IRS diminishes the yield of infectious virus and the mutant viral particles sediment abnormally in gradients, suggesting defective viral assembly (Blankenship and Shenk, 2002). In transient assays, TRS1/IRS1 gene products activate expression of early viral promoters in cooperation with the viral MIE gene products (Romanowski and Shenk, 1997) and are necessary components for HCMV oriLyt-dependent DNA replication (Pari et al., 1993). An amino terminal truncated IRS1 gene product (pIRS1263) controlled by a downstream promoter in the unique region antagonizes the activation function of the IRS1 and TRS1 viral proteins (Romanowski and Shenk, 1997). Recent work has suggested that both TRS1 and IRS1 encode functions that evade interferon response in HCMV-infected cells (Child et al., 2004). These HCMV gene products appear to be RNA-binding proteins that prevent activation of host cell protein kinase R (PKR) pathways, thereby averting shut off of protein synthesis (Hakki and Geballe, 2005). It remains possible that all of the activities previously ascribed to these IE proteins are due to this central control of the cellular response to infection as has been found for PKR inhibitors encoded by other DNA viruses.

Betaherpesvirus transcriptional enhancers upstream of the MIE genes

Transient transfection and transgenic mouse experiments with the HCMV enhancer-containing MIE promoter driving expression of an indicator gene suggest that the activity of this enhancer is influenced by the cell type and the state of cell differentiation (Baskar et al., 1996a,b). However, additional transgenic mice studies yielded different data failing to show such correlation expression patterns. During infection, viral IE gene expression is first affected by attachment and entry requirements, and then, by cellular signal transduction pathways induced by the virus or other external stimuli. Serum and virion components increase MIE promoter activity at low multiplicity of infection (MOI). The relative importance of individual enhancer cis-acting sites may vary depending on the type of cell and external stimulus. In addition, it is not known which cis-acting elements in the MIE enhancer are important in a given cell type. For example, the NF-κB sites may be important in the hepatocytes under conditions of inflammation, when pro-inflammatory factors activate the transcription factor NF-κB (Prosch et al., 1995). The function of the MIE enhancers of betaherpesviruses is important to understand because the enhancers likely play a pivotal role in regulating viral latency, reactivation, and pathogenesis.

Figure 17.2 compares the known consensus binding sites for eukaryotic transcription factors in enhancer elements upstream of the MIE genes of human, chimpanzee, simian, murine, and rat (England and Maastricht strains) CMVs. Uncharacterized cis-acting elements may emerge from further studies seeking an impact on viral replication. CMV MIE enhancer elements have an array of cis-acting sites that bind cellular transcription factors (Angulo et al., 1996, 1998a; Beisser et al., 1998; Chang et al., 1990; Davison et al., 2003; Dorsch-Hasler et al., 1985; Meier and Stinski, 1996; Sandford and Burns, 1996; Stinski, 1999; Thomsen et al., 1984; Vink et al., 2000; Meier and Stinski, 2006). Many of these binding sites are repetitive, but their arrangement and numbers vary among the different species-specific viruses. HCMV, ChCMV, SCMV, and MCMV share common regulatory elements that are functional binding sites for NF-κB, CREB/ATF, AP-1, and RAR-RXR (Angulo et al., 1995, 1996, 1998a). RCMV (England strain) is unusual in that it contains fewer cis-acting elements than other viruses. The HCMV enhancer also has functional binding sites for serum response factor, Elk-1, Sp-1, CAAT/enhancer binding protein and gamma-interferon activating sequence (Meier and Stinski, 1996; Netterwald et al., 2005). Binding sites for other cellular transcription activators are also present in this enhancer, but their significance is unknown. In transient transfection assays, the different cis-acting elements act individually or cooperatively to attract the RNA polymerase Ⅱ transcription initiation complex to the MIE promoter (Hunninghake et al., 1989).

Fig. 17.2. Comparison of the enhancers of cytomegaloviruses.

Fig. 17.2

Comparison of the enhancers of cytomegaloviruses. The known consensus binding sites of cellular transcription factors in the major immediate early enhancers are designated.

The betaherpesvirus MIE enhancer elements are considered important because they may also serve as a target for reactivation from latency. The original hypothesis was that the MIE enhancer contributed to a quick and robust expression of the MIE genes, and evidence supporting this remains strong when expression of viruses carrying mutations in this region are studied in cell culture (Meier and Pruessner, 2000). Substitution of the enhancer of HCMV with the MCMV IE enhancer generates a virus with reduced expression and growth in culture (Isomura and Stinski, 2003), whereas subsitution of the MCMV with the HCMV MIE enhancer generates a virus that can replicate in culture or in mice (Grzimek et al., 1999) with characteristics that indicated a role for the enhancer in increasing the number of cells expressing IE gene products in specific infected host tissues.

Figure 17.3 compares the enhancers of HCMV and HHV-6. Although these viruses share little common sequence, the enhancers are positioned similarly between divergent promoters upstream of the IE1 and IE2 genes (Lashmit et al., 2004; Lundquist et al., 1999; Takemoto et al., 2001). The HHV-6 enhancer is characterized by repeat elements of 104 to 107-bp which contain polyomavirus enhancer A binding protein (PEA3) sites, NF-κB binding sites, some AP-2 binding sites (not identified in Fig. 17.3). The only obvious common elements shared by the two enhancers are NF-κB sites. The HHV-6 enhancer cannot substitute for the HCMV enhancer (H. Isomura and M. F. Stinski, unpublished data). While the HCMV enhancer influences IE1 and IE2 gene transcription, the HHV-6 enhancer has an effect on the divergent IE U95 gene transcription and is only speculated to have an effect on transcription of the IE1 and IE2 genes (Takemoto et al., 2001). The HCMV MIE region has two repressor elements, one that binds IE2 proteins located immediately upstream of the IE1/IE2 transcription start site that likely represses gene expression when IE2 levels rise at early times of infection (Cherrington et al., 1991; Liu et al., 1991; Pizzorno and Hayward, 1990) and one that binds a cellular protein immediately upstream of the TATA box of the divergent UL127 gene and blocks expression throughout infection (Angulo et al., 2000b; Lashmit et al., 2004; Lundquist et al., 1999). Additionally, silencing of the MIE enhancer appears to occur through a variety of cellular transcription factors in certain cell types, particularly when the cells are undifferentiated. Murine CMV has a similar arrangement of enhancer, MIE gene and divergent gene, but transcriptional repression signals have not been found. As far as has been determined, the IE1 and IE2 homologues are important in replication but the divergent genes of the betaherpesviruses are non-essential for replication in cell culture and their function is not known. The U94 gene of HHV-6, of interest because it is similar to the rep gene of adeno-associated virus (AAV), is located in the intron of the viral U95 gene and is transcribed in the opposite direction of U95 (Takemoto et al., 2001). The AAV rep gene product is a DNA binding protein with ATPase and helicase activity and is required for AAV DNA replication. The HHV-6 U94 gene product is found in very low abundance during productive HHV-6 infection but shares some of these properties (see Chapter 47).

Fig. 17.3. Comparison between the HCMV major immediate early (MIE) and the HHV-6 R3 enhancer.

Fig. 17.3

Comparison between the HCMV major immediate early (MIE) and the HHV-6 R3 enhancer. Viral genes and promoter/ transcription start sites are designated by an arrow. The HCMV also has a unique region and a modulator discussed in the text. The various transcription (more...)

Function of the betaherpesvirus major immediate–early enhancers

Thus far, two approaches have been used to study the function of betaherpesvirus MIE enhancers during viral infection: (ⅰ) mutation of enhancer components, and (ⅱ) substitution of the enhancer from one species with that from a different species. Mutational analysis in the context of the viral genome demonstrated that the HCMV enhancer has two functional components referred to as the distal enhancer (−580 to −300) and the proximal enhancer (−300 to −39) relative to the transcription start site (+1) (Fig. 17.3). The distal enhancer is dispensable at high MOIs in cultured cells but has a significant effect on the efficiency of viral replication at low MOIs (Meier and Pruessner, 2000). Without the distal enhancer, the virus has a small plaque phenotype. The distal enhancer is composed of multiple cis-acting elements. Deletion of −300 to −347 or −347 to −579 has little to no effect on MIE promoter-dependent transcription, but deletion of the entire region (−580 to −300) has a significant effect (Meier et al., 2002). The distal enhancer functions in cis and is orientation-independent relative to the transcription start site. The hypothetical ORFs in the distal enhancer are not important for viral replication in cell culture because insertion of stop codons at −300 or −345 had no effect on IE gene expression or virus titer (Meier et al., 2002). The proximal enhancer upstream of −39 also determines the efficiency of virus replication in cell culture (Isomura et al., 2004). Deletion of the proximal enhancer affects IE and early viral gene expression, viral DNA synthesis, and the rate of viral growth (Isomura et al., 2004). Which elements in the distal or proximal enhancer are required for virus replication in various cell types is currently unknown. Despite the commonality of NF-κB sites in betaherpesvirus enhancers, the minimal enhancer element for HCMV appears to be an Sp-1 binding site (Isomura et al., 2004, 2005).

Mutation of the CREB/ATF binding sites in the entire enhancer had little to no effect on HCMV replication in human fibroblast (HF) or in NTera2-derived neuronal cells at high or low MOIs (Keller et al., 2003). Likewise, mutation of the NF-κB sites in the entire enhancer had little to no effect on viral replication at high or low MOIs in HF cells (Benedict et al., 2004). Since MCMV and HCMV replicate efficiently in cells where the NF-κB activation pathway has been inhibited (Benedict et al., 2004; Melnychuk et al., 2003), the NF-κB transcription factor may not always be necessary for replication in cell culture. The requirement for particular HCMV MIE enhancer elements has not yet been assessed using viral mutants.

Enhancer substitution experiments have also demonstrated that CMV MIE enhancers affect viral replication. Recombinant with the rat CMV (England) MIE enhancer substituted with the MCMV MIE enhancer was deficient in replication in rat fibroblasts and in the infected rat, with greatly reduced levels of viral replication in the salivary glands (Sandford and Burns, 1996). While recombinant HCMV with the MIE enhancer substituted by the SCMV (Colburn) MIE enhancer replicated as well as wildtype virus (H. Isomura and M. F. Stinski, unpublished data), recombinant HCMV with the enhancer substituted with the MCMV enhancer replicated slower and to lower levels in HF cells (Isomura and Stinski, 2003). Consistent with this, the plaques of the enhancer substituted HCMV recombinant virus had a small plaque phenotype. When a recombinant MCMV substituted with the HCMV MIE enhancer was made, the recombinant MCMV replicated in mouse fibroblast (Angulo et al., 1998b) and in mouse liver at levels similar to wild-type virus, but there was a decrease in the infection at other sites in the mouse (Grzimek et al., 2001). While the MCMV enhancer is not essential for replication in cultured murine fibroblasts at high MOI, this region is required for cytopathic effects in culture and disease in the mouse (Angulo et al., 1998b). Taken together, these observations suggest that CMV enhancers are not always functionally equivalent and suggest one role they play is to optimize the efficiency of viral replication in various cell types with which the virus normally interacts. The cis-acting elements in the betaherpesvirus enhancers have evolved over millions of years for each of the species-specific viruses.

Silencing of the immediate-early genes

Betaherpesvirus MIE genes are regulated in a cell type- and differentiation-dependent manner. The viral genomes may be organized into a nucleosome-array like latent genomes of herpesviruses in other subfamilies (Deshmane and Fraser, 1989; Dyson and Farrell, 1985). Conditionally permissive cell lines have been used to investigate silencing and reactivation of HCMV. In the undifferentiated cell, the MIE enhancer-containing promoter is silent, and the cells are non-permissive for viral replication. In the differentiated cell, the MIE enhancer-containing promoter is active, and the cells are permissive for viral replication. For example, HCMV fails to replicate after penetration into NTera2 cells, an undifferentiated embryonic carcinoma line (Gonczol et al., 1984). This postentry block corresponds to silencing of the MIE promoter-dependent transcription (LaFemina and Hayward, 1986; Meier, 2001; Nelson and Groudine, 1986). Inactivity of the MIE promoter appears to be a feature of natural HCMV latency (Taylor-Wiedeman et al., 1994; Kondo et al., 1994, 1996; Slobedman and Mocarski, 1999). The MIE promoter of MCMV is generally inactive during viral latency, except in a rare subset of cells where spontaneous reactivation appears to be occurring (Grzimek et al., 2001; Hummel et al., 2001; Koffron et al., 1998; Kurz et al., 1999; Kurz and Reddehase, 1999).

Transient transfection studies identified the HCMV 21-bp-repeats, the unique region, and the modulator as cis-acting sites that confer repression of transcription in the undifferentiated monocytic THP-1 and embryonal NTera2 cell lines (Fig. 17.3) (Huang et al., 1996; Kothari et al., 1991; Liu et al., 1994; Nelson et al., 1987; Shelbourn et al., 1989; Sinclair et al., 1992). Three copies of the 21-bp-repeats are located in the distal MIE enhancer, whereas the unique region and modulator forms the 5′-extent of the MIE regulatory region. The following cellular repressors of transcription have also been proposed to act through one or more elements in the modulator, the unique region, or the enhancer: silencing binding protein (SBP) (Thrower et al., 1996), modulator recognition factor (Huang et al., 1996), PDX1 (Chao et al., 2004), Yin Yang-1 (YY1) (Liu et al., 1994), methylated DNA-binding protein (Zhang et al., 1995), growth factor independence-1 (Gfi-1) (Zweidler-McKay et al., 1996), and the ETS2-repressor factor (ERF) (Wright et al., 2002). While eliminating any one of these sets of negative cis-acting elements increases the MIE promoter activity in transient transfection experiments, their selective removal from the HCMV genome results in a completely different outcome. Silencing in the context of the viral genome is not alleviated by removal of the 21-bp-repeats, the modulator, the unique region, or both 21-bp-repeats and modulator in either undifferentiated monocytic THP-1 or embryonal NTera2 cells (Meier, 2001). Site-specific mutation of the Gfi-1 sites has no effect in undifferentiated monocytic THP-1 cells (R. Schnetzer and M. F. Stinski, unpublished data). Thus, silencing occurs in the context of viral infection, and its regulatory mechanism differs quantitatively from that observed in transfected cells. It remains possible that redundancy of negative cis-acting elements explains these differences, but studies have not yet provided any evidence of this. Nonetheless, the findings suggest that the HCMV MIE promoter becomes silenced in undifferentiated cells, but this process depends on factors that remain to be identified.

In the embryonal NTera2 cell culture model, a portion of quiescent HCMV genomes have a super-coiled structure (Meier, 2001), which appears similar to CMV latency in blood monocytes of healthy subjects (Bolovan-Fritts et al., 1999). The super-coiled structure would be expected to package into nucleosomes as is the case for gammaherpesviruses. The HCMV MIE enhancer of the quiescent viral genomes is inactive even when positive-acting transcription factors NF-κB and RAR-RXR are activated (Meier, 2001). However, inhibition of cellular histone deacetylase (HDAC) reactivates transcription from the MIE promoter. These data suggest that betaherpesvirus MIE promoter silencing may involve HDAC-based modification of viral chromatin. Murphy et al. (2002) showed that the silent HCMV MIE promoter is associated with less acetylated histone H4 as compared to an active MIE promoter. Acetylated H4 was also less abundant on silent MIE promoters in experimentally infected blood monocytes compared to active MIE promoters in permissive monocyte-derived macrophages (Murphy et al., 2002). In addition, the cellular HP1 protein, which selectively binds methylated histone H3 at lysine 9 in cellular heterochromatin (Jenuwein and Allis, 2001), is preferentially associated with the repressed MIE promoters (Murphy et al., 2002). While MCMV MIE promoter reactivation can also be achieved in latently infected murine tissue (Hummel et al., 2003), direct evidence for CMV DNA silencing via chromatin components is lacking at this time. The combined findings imply that chromatin may condense on the betaherpesvirus genomes in non-permissive and undifferentiated cells to restrict MIE promoter activity.

Reactivation of the immediate–early genes

Reactivation of betaherpesviruses is observed commonly in the setting of immunosuppression, particularly where allogeneic stimulation and proinflammatory cytokines are present and stimulate monocyte differentiation (Cook et al., 2002; Fietze et al., 1994; Hahn et al., 1998; Hummel et al., 2001; Mutimer et al., 1997; Soderberg-Naucler et al., 1997b; Soderberg-Naucler et al., 2001). Proinflammatory cytokines, such as those released during allogeneic transplantation, AIDS, sepsis, or myelosuppressive chemotherapy, induce the MIE promoter-dependent transcription.

For MCMV, the effect of MIE promoter reactivation can be dampened by a mechanism that prohibits the production of the alternatively spliced ie3 RNA (Grzimek et al., 2001; Kurz et al., 1999), which is the functional equivalent of the HCMV IE2 gene. HCMV MIE promoter reactivation may also be subjected to further regulation in infected monocytes, as certain stimuli only reactivate production of spliced RNA for IE1, but not IE2 (Taylor-Wiedeman et al., 1994). Stimuli sufficient to induce differentiation of these cells into a monocyte-derived macrophage or dendritic cell phenotype, enables completion of the HCMV reactivation program (Soderberg-Naucler et al., 1997b 2001).

The molecular mechanisms that trigger and sustain CMV reactivation are largely unknown. Transgenic mice and transient transfection experiments implicate NF-κB and CREB/ATF as important mediators in stimulus-induced MIE promoter reactivation (Hummel et al., 2001; Prosch et al., 1995; Stein et al., 1993). TNF-α potently induces NF-κB activity and reactivates MCMV’s MIE promoter in latently infected lung tissue, but is not alone sufficient to sustain the reactivation process (Hummel et al., 2001). HCMV reactivation from monocytes in cell culture induced by allogeneic stimulation does not require TNF-α, but instead, depends on the combination of interferon-γ and other unidentified factors (Soderberg-Naucler et al., 2001). The mechanism(s) by which these cytokines or other factors stimulate HCMV reactivation is unknown. In the embryonal NTera2 cell model, forskolin stimulation of the cyclic AMP signaling pathway and inhibition of HDAC induce HCMV MIE promoter reactivation. The reactivation is dependent on CREB/ATF-binding sites within the enhancer (M. Keller and J. Meier, unpublished data). Taken together, it appears that betaherpesvirus reactivation from latency may entail multiple regulatory mechanisms for orchestrating both derepression and activation of the viral IE gene expression.

Betaherpesvirus major immediate–early genes

All betaherpesviruses have CpG dinucleotide suppression in the major immediate early (MIE) locus. The significance of CpG dinucleotide suppression is unknown, but it suggests that these genes are regulated by host methylation in a certain setting such as latency. Downstream of the betaherpesvirus enhancer-containing MIE promoter are two regulatory genes designated IE1 and IE2 in HCMV and HHV-6 as well as most other betaherpesviruses. Importantly, in MCMV, they are designated ie1 and ie3, respectively. In all betaherpesviruses, multiple gene products are encoded by these two genes through differential mRNA spicing and promoter usage throughout infection. For the CMVs, the single MIE promoter directs transcription of three small exons that are spliced alternatively to either exon 4 or exon 5 of the precursor IE RNA followed by cleavage and polyadenylation (Fig. 17.4) (Nicholas, 1994 Stenberg et al., 1984, 1985). An initiation codon in exon 2 and a termination codon in either exon 4 or exon 5 gives rise to the major proteins encoded by IE1 and IE2, respectively, at IE times (Fig. 17.4). In HHV-6, the first four exons of a precursor RNA are spliced alternatively to either exon 5 or exon 6 (Fig. 17.4) (Nicholas, 1994; Schiewe et al., 1994). An initiation codon in exon 3 and a termination codon in either exon 5 or exon 6 give rise to the proteins encoded by IE1 and IE2, respectively. The viral proteins encoded by HCMV are designated according to their apparent molecular weight and have amino acids in common at the amino terminus, with the exception of a late viral protein, designated L40 that arises from a unique promoter element within the exon 5 region (Fig. 17.4). HHV-6 also generates multiple forms of the IE1 and IE2 proteins by alternate splicing between and within exons. The HHV-6 MIE proteins are also designated according to their apparent molecular weight (Fig. 17.4) (Papanikolaou et al., 2002).

Fig. 17.4. Comparison of the IE1 and IE2 genes and their isomers for HCMV and HHV-6.

Fig. 17.4

Comparison of the IE1 and IE2 genes and their isomers for HCMV and HHV-6. The exons with ORFs for the IE1 and IE2 genes are designated as open boxes. The isomers of the IE1 and IE2 genes are designated according to apparent molecular weight. A HCMV late (more...)

Functions of the major immediate–early viral proteins

While it is assumed that the proteins encoded by betaherpesvirus IE1 and IE2 genes have similar functions, these proteins exhibit dramatic evolutionary divergence in amino acid sequence. These differences may have evolved to meet the regulatory needs for broadly different species-specific and cell-specific herpesviruses represented by this subgroup. The functions of the major HCMV IE1 and IE2 gene products, IE72 and IE86, have been investigated extensively, and minor products IE38, IE55, and IE18 isomers have received less attention. During the two hours after HCMV infection of HFs, the mRNA for the IE2 gene is expressed predominantly, and this is followed by a period when the mRNA for the IE1 gene predominates (Stamminger et al., 1991). In contrast, the expression of HHV-6 IE1 precedes that of IE2 (Papanikolaou et al., 2002). Relative to the mRNAs for HCMV IE72 and IE86 proteins, the amount of mRNA for IE55 or IE38 is low in fibroblasts. The mRNA for IE18 is lower in HFs than in macrophages (Stenberg, 1996). The IE2 protein of HCMV negatively autoregulates the expression of the IE1 and IE2 genes, but there is no evidence to date that HHV-6 IE2 protein negatively autoregulates (Cherrington et al., 1991; Liu et al., 1991; Pizzorno and Hayward, 1990). The IE86 protein of HCMV binds to the minor groove of the MIE promoter that contains a cis-repression sequence (crs) between −13 and −1 relative to the transcription start site (+1) (Lang and Stamminger, 1994). The late 40 kDa protein (L40) of the IE2 gene can also bind to the crs and negatively autoregulate transcription of the MIE genes (Plachter et al., 1993; Puchtler and Stamminger, 1991; Stenberg et al., 1989). While the IE1 and IE2 gene products are discussed separately below, they seem to work synergistically in executing their functions during viral infection.

The IE1 proteins

The IE1 proteins of CMVs have only a few regions of homology and a characteristic acidic acid residue cluster towards the C-termini. The IE1 genes of human and MCMV are dispensable for viral replication at high MOIs (1 to 5 PFU/cell). When the IE1 gene is deleted, the efficiency of viral replication is reduced at low MOIs (0.001 to 0.05 PFU/cell) (Greaves and Mocarski, 1998; Mocarski et al., 1996). After infection at low MOI with recombinant HCMV containing an IE1 gene deletion, there are insufficient levels of early viral gene expression required for viral DNA replication (Gawn and Greaves, 2002). At high MOIs, virion-associated proteins present in infectious and non-infectious particles may compensate for the absence of functional IE1.

After synthesis in the cytoplasm, IE72 is transported to the nucleus and targeted to nuclear bodies known as promyelocytic leukemia (PML) oncogenic domains (PODs) or nuclear domain 10 (ND10). The viral protein is modified by conjugation of SUMO-1 or SUMO-2 (small ubiquitin-like modifier) at lysine residue 450. The apparent molecular weight of IE72 following modification is approximately 92 kDa (Spengler et al., 2002; Xu et al., 2001). Although sumoylation may be important for the efficiency of viral replication, a recombinant virus with lysine residue 450 mutated is replication competent in HFs (Lee et al., 2004). Recombinant virus with lysine residue 450 mutated expresses lower levels of IE2 RNA and IE86 protein (Nevels et al., 2004). With or without SUMO conjugation, IE72 displaces the ND10 presumably by binding to its associated proteins, such as PML, SP100, and hDaxx. The central hydrophobic region of IE72 binds to PML (Lee et al., 2004) and inhibits the accumulation of sumoylated forms of PML. A PML-associated transcriptional repressor, HDAC-2, is inactivated (Ahn and Hayward, 1997; Ahn et al., 1998a; Wilkinson et al., 1998) and, consequently, the basal transcription initiation complex is activated (Muller and Dejean, 1999; Tang and Maul, 2003; Xu et al., 2001). Early after infection, the viral DNA and transcripts are detected in the nucleus, and RNA polymerase and mRNA spliceosome assembly factors are juxtaposed to the ND10 (Ishov et al., 1997). HHV-6 IE1 protein is also localized to ND10 and modified by SUMO-1 at lysine residue 802, but the IE1 protein does not dispense PML (Gravel et al., 2002). The reason for this significant difference in the function of these betaherpesvirus IE1 proteins is not known; however, disruption of ND10 is not a requisite for HCMV replication. The multiple functions of the betaherpesvirus IE1 proteins are not fully understood. One function of the HCMV IE72 protein may be to counter the innate immune response in cells by down-regulation of virus-induced interferon-like response (Singh and Compton, 2004) and another may block apoptosis (Zhu et al., 1995), promoting conditions for viral replication in the host cell.

IE38 (IE19) is reported to be an HCMV IE1 gene product that lacks amino acids 88 to 404 of IE72 (Fig. 17.4). A radioactive probe to the 5 end of IE1 detected a cDNA of 0.65 kb that could code for IE38 (Shirakata et al., 2002). An antibody to a peptide between amino acids 383 and 420 detected both IE72 and IE38 (Kerry et al., 1995). Others have not detected IE38 and suggest it may represent an N-terminal cleavage product of IE72 (Awasthi et al., 2004). Transient transfection experiments suggested that IE38 functions synergistically with IE72 (Shirakata et al., 2002). Whether or not these functions of IE38 occur in the virus-infected cell remains to be determined.

It is unclear how IE72 (or IE38) activates cellular promoters, but an association of IE72 with TATA box-associated factors (TAFs) and transcription factors (Sp-1, E2F-1, CTF-1) has been proposed (Hayhurst et al., 1995; Lukac et al., 1997; Margolis et al., 1995). In transient transfection experiments, IE72 moderately activates the viral MIE promoter and the cellular DNA polymerase α, c-fos, c-myc and dihydrofolate reductase promoters (Cherrington and Mocarski, 1989; Hagemeier et al., 1992b; Sambucetti et al., 1989; Wade et al., 1992). The mechanism by which IE72 protein activates promoters may be related to inhibition of HDAC-2 activity (Tang and Maul, 2003).

It has been proposed that HCMV IE72 has intrinsic protein kinase activity (Pajovic et al., 1997). The related protein, IE38 does not have the proposed protein kinase domain. The Rb family members p107 and p130, the E2F transcription factor family members, and PML are phosphorylated by the IE72 protein (Ippolito et al., 2003; Pajovic et al., 1997). Phosphorylation of the Rb family members would dissociate the repressor from E2F and activate the E2F cellular transcription factor. The leucine zipper region of IE72 can bind the N-terminus of p107, alleviate the repressive effect of p107 and, consequently, activate cyclin dependent kinase-2 (cdk2)/cyclin E activity (Zhang et al., 2003). Therefore, IE72 induces cycle cell progression, but the effect of the viral protein is more demonstrable in a p53 negative cell than in a p53 positive cell (Castillo et al., 2000). An active p53 pathway should increase levels of p21 cyclin-dependent kinase inhibitor even in the presence of activated E2F transcription factors, but p21 is decreased in the HCMV-infected cell (Chen et al., 2001; Noris et al., 2002). HCMV infection affects p53 because the cellular protein is sequestered in the nucleus in viral replication centers (Fortunato and Spector, 1998).

The IE2 proteins

The betaherpesvirus proteins encoded by the IE2 gene exhibit amino acid similarity across their C-terminal regions. The HCMV IE2 gene and its functional homologue in MCMV (ie3) are absolutely essential for the cascade of viral gene expression (Angulo et al., 2000a; Marchini et al., 2001). Recombinant viruses with deletion of the HCMV IE2 gene or the MCMV ie3 gene are unable to activate early viral gene expression, and, consequently, viral DNA synthesis and late gene expression are also affected. The betaherpesvirus IE2 proteins have N- and/or C-terminal domains that activate viral gene expression and are considered a master regulator of productive infection. The IE2 gene of HCMV has been studied in detail. It regulates activation of transcription from viral and cellular promoters, negatively auto-regulates the MIE promoter, and induces cell cycle progression. Figure 17.5 is a diagram of the functional domains of the HCMV IE2 protein IE86. IE86 is also targeted to ND10 in the nucleus and modified by conjugation with SUMO-1 or SUMO-2 at lysine (K) residues 175 or 180. The apparent molecular weight of the modified protein is 105 kDa (Ahn et al., 2001; Hofmann et al., 2000); however, sumoylation of IE86 is not required for viral growth (Lee and Ahn, 2004).

Fig. 17.5. Domains of HCMV IE86 encoded by the IE2 gene.

Fig. 17.5

Domains of HCMV IE86 encoded by the IE2 gene. The domains are demarcated according to amino acid residue. Transcription activation domains (AD) are located at the amino and carboxyl termini. There are serine-rich (S-rich) domains and two nuclear localization (more...)

Unlike IE72, IE86 does not disperse ND10. However, IE86 remains adjacent to ND10 where a portion of the input viral DNA is also located (Ishov et al., 1997). Modification of IE86 by SUMO may facilitate interaction with the basal transcription machinery and/or with cellular or viral transcription factors. In transient transfection experiments, IE86 mutated at lysine residues 175 and 180 autoregulates the MIE promoter, but fails to efficiently activate early viral promoters (Hofmann et al., 2000). A recombinant virus with deletion of amino acid residues 136 to 290 still down-regulates transcription from the MIE promoter, but the mutant exhibited lower levels of late gene (UL83 and UL99) expression (Sanchez et al., 2002). Late gene expression is also disrupted by deletion of four amino acids from 356 to 359 (White et al., 2004). This mutation would disrupt a region where IE86 interacts with the basal transcription factors TFIIB and TBP (Fig. 17.5). IE86 has a serine-rich region from amino acids residues 258 to 275. Mutation of the serine residues from 258 to 264 or 266 to 269 delays viral growth, but mutation from 271 to 275 accelerates viral growth (Barrasa et al., 2005). These serine residues are in a region of the protein that can bind TBP (Fig. 17.5). IE86 is also modified by phosphorylation at amino acid residues T27, S144, T233, and S234 (Harel and Alwine, 1998) (Fig. 17.5). Differences in phosphorylation may have significant effects on the function of the viral protein.

Data indicate that mutation of amino acids between 427 to 435 and 505 to 511 caused a loss of auto-regulation of the MIE promoter by IE86 (White et al., 2004). Mutation of histidines 446 and 452 generates a protein that cannot bind to the crs and negatively autoregulates the MIE promoter in an in vitro transcription assay (Macias and Stinski, 1993). Therefore, the carboxyl region of the IE86 protein is critical for auto-regulation of the MIE promoter (Fig. 17.5). Autoregulation of the MIE promoter by the IE86 protein at early times is likely due to the blockage of RNA polymerase Ⅱ and transcription initiation factors at the transcription start site (Macias et al., 1996). At late times after infection the IE86 protein was found to be associated with repressive chromatin (Reeves et al., 2006). With murine CMV, the M112/113 gene product co-localizes with and binds ie3 protein (the equivalent of human CMV IE86) to inhibit the repressive effect on the MIE promoter and promotes continued MIE gene expression (Tang et al., 2005).

In transient transfection assays, multiple regions of IE86 are reported to be important for promoter activation. Two activation domains (AD) are located at amino acid 25 to 85 and 544 to 579 (Fig. 17.5). Amino acid residues 1 to 98, 169 to 194, 175 to 180, and multiple regions in the carboxyl terminal half of the protein are also important for viral promoter activation (Malone et al., 1990; Pizzorno et al., 1991; Sommer et al., 1994; Stenberg, 1996; Yeung et al., 1993). Other important regions of IE86 are amino acid residues 388 to 542, and 463 to 513, which encompass a dimerization region and a helix–loop–helix region, respectively (Ahn et al., 1998b; Macias et al., 1996; Macias and Stinski, 1993; Waheed et al., 1998). There is a core region between amino acids 450 and 544. Mutations in this region affect most of the activities of this protein (Fig. 17.5) (Asmar et al., 2004).

IE86 interacts with a wide variety of cellular transcription factors (Bryant et al., 2000; Lang et al., 1995; Lukac et al., 1994; Yoo et al., 1996). These factors include TBP, TFIIB, and TAF4 as well as histone acetyl-transferase (Bryant et al., 2000; Caswell et al., 1993; Fortunato and Spector, 1999; Hagemeier et al., 1992a; Lukac and Alwine, 1997; Spector, 1996). IE86 may serve as a link between various upstream sequence-specific DNA binding regulators of transcription and the basal transcription initiation complex. The protein also interacts with an early viral protein encoded by the UL84 gene of HCMV (Colletti et al., 2004; Samaniego et al., 1994; Spector and Tevethia, 1994). Over-expression of UL84 prior to viral infection will antagonize activation of early viral promoters by IE86 protein (Gebert et al., 1997). The UL84 gene is essential for viral DNA synthesis and growth (Xu et al., 2004b). The UL84 protein interacts with itself and with the IE86 protein which is essential for oriLyt-dependent viral DNA synthesis (Colletti et al., 2004; Sarisky and Hayward, 1996). DNA synthesis is initiated by the activation of a oriLyt bidirectional promoter by IE86 and UL84 viral proteins (Xu et al., 2004a). The other HCMV IE genes, IE1, UL36–38, and IRS1/TRS1, have a stimulatory effect on ori-Lyt-mediated DNA replication in HF cells (Anders and McCue, 1996).

The IE86 protein also interacts with cellular proteins that control cell cycle progression. Several types of biological assays indicate that IE86 physically binds Rb (Fortunato et al., 1997; Hagemeier et al., 1994; Sommer et al., 1994). The release of the cellular E2F transcription factor from Rb, as a consequence of this interaction, is considered to be one of the key HCMV mechanisms for induction of cell cycle progression. As many as 4-fold more serum-starved glioblastoma U373 or 293T cells are induced into S phase by wild type IE86 compared to a mutant protein (Murphy et al., 2000). IE86 blocks cell division by arresting p53 wild type cells at G1/S (Murphy et al., 2000; Song and Stinski, 2002; Wiebusch and Hagemeier, 1999; Wiebusch et al., 2003). In p53 mutant U373 cells or p53 null Saos-2 cells, IE86 does not inhibit cell cycle progression at G1/S (Song and Stinski, 2005). These cells synthesize cellular DNA and cell cycle progression stops at the S phase for U373 cells or the G2/M-phase for Saos-2 cells (Murphy et al., 2000; Song and Stinski, 2005). In p53 null Saos-2 cells, a block in cell cycle progression at the G2/M-phase by the IE86 protein correlates with an aberrant increase in cyclin B and cdk1 levels (Song and Stinski, 2002). IE86 likely prepares the cell for DNA synthesis by activating cellular genes that regulate the cell cycle, enzymes for DNA precursor synthesis, and proteins for initiation of cellular DNA synthesis (Song and Stinski, 2002). For example, production of mRNAs for cyclin E, cdk-2, E2F-1, DNA polymerase α, and MCMs is significantly increased by IE86 (Song and Stinski, 2002). Preparation for DNA synthesis is critical for the virus because CMVs typically infect terminally differentiated cells in the Go/G1 phase of the cell cycle when the pool of dideoxynucleotide triphosphates and biosynthetic enzyme levels are low. HCMVIE86 is an unusual regulatory protein in comparison to regulatory proteins of adenovirus, SV40, or papilloma DNA viruses because it pushes the p53 wild type cell from Go/G1 to the G1/S transition, yet blocks further cell cycle progression (Murphy et al., 2000; Wiebusch and Hagemeier, 2001). Both of the IE 72 and IE86 proteins stabilize p53, which is associated with phosphorylation of p53 at serine residue 15 (Castillo et al., 2005; Song and Stinski, 2005). The IE86 protein induces the degradation of cellular mdm2 and thereby prevents ubiquitination and proteasome degradation of p53 (Zhang et al., 2006). Recombinant virus with a mutant IE86 protein that fails to block cell cycle progression at the G1/S interface and allows for cellular DNA synthesis, replicates slowly relative to wild type virus (Petrik et al., 2006). The IE86 protein may block rather than promote apoptosis (Zhu et al., 1995).

One of the functions of cellular p53 tumor suppressor protein is to ensure termination of cells that have lost the ability to regulate growth. Even though the HCMV IE86 protein binds to p53 (Bonin and McDougall, 1997; Speir et al., 1994), p53 is not inactivated by the IE86 protein and, as a result, cdk inhibitor p21 increases in relative amount (Shen et al., 2004; Song and Stinski, 2002). However, in the HCMV infected cell the levels of p21 decrease (Chen et al., 2001; Noris et al., 2002). In the p53 wild type HF cell, IE86 induces senescence that is manifested in continued cellular metabolism, production of plasminogen activator inhibitor type Ⅰ, and neutral β-galactosidase activity (Noris et al., 2002).

While the viral IE86 protein acts by a different mechanism to activate cellular gene expression, it acts to favor virus survival by inhibiting cellular beta-interferon, cytokine, and pro-inflammatory chemokine expression by an unknown mechanism (Taylor and Bresnahan, 2005, 2006).

Factors that stimulate betaherpesvirus immediate–early gene expression

Cellular signal transduction events

Infection of HF cells with HCMV triggers activation of multiple signal transduction pathways. There is an activation of the phosphatidylinositol 3-kinase (PI3K), the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and the p38 MAPK signal transduction pathways (Johnson et al., 2000, 2001; Rodems and Spector, 1998). Inhibitors of these signal transduction pathways suppress HCMV replication, which implies that the signal transduction pathways are necessary for efficient viral replication. The PI3K pathway is activated by virion components immediately upon entry. An activated PI3K pathway induces production of secondary messengers like phosphotidyl inositol and diacylglycerol (Albrecht et al., 1990, 1991; Wang et al., 2003) and increases in cyclic AMP, GMP, and intracellular stores of calcium. The MAPK/ERK and p38 MAPK pathways are activated early after infection and sustained until late times (Johnson et al., 2000; Rodems and Spector, 1998). The PI3K, MAPK/ERK, and p38 MAPK pathways have major effects on the activation of a variety of cellular transcription factors. This activation often results from phosphorylation of the transcription factors and, consequently, up-regulates both cellular and viral gene expression. HCMV early promoters are suppressed by inhibitors of signal transduction pathways (Chen and Stinski, 2002). Therefore, activation of signal transduction pathways is an important step in the efficient replication of betaherpesviruses.

Prostaglandins and reactive oxygen species (ROS) also serve as secondary messengers that elicit multiple responses in betaherpesvirus-infected cells (Zhu et al., 2002). Virions of HCMV up-regulate cellular cyclooxygenase-2 (cCOX-2) (Zhu et al., 2002). The rhesus CMV encodes a viral COX-2 (vCOX-2), which is required for efficient viral replication in endothelial cells (Hanson et al., 2003; Rue et al., 2004). Prostaglandin E2, a product of COX-2, activates the HCMV MIE enhancer-containing promoter (Kline et al., 1998). HCMV infection generates ROS partly through a COX-2-dependent pathway. ROS also activates the HCMV MIE promoter and augments viral replication. Inhibition of cCOX-2 or scavenging of ROS decreases HCMV IE gene expression and viral replication (Speir et al., 1998; Zhu et al., 2002). Treatment with prostaglandin E2 reverses the inhibitory effects on HCMV replication.

Lastly, proinflammatory cytokines induce various signaling pathways that stimulate MIE gene expression and viral replication. This outcome is observed, for example, in HCMV-infected macrophages or granulocyte–macrophage progenitors treated with tumor necrosis factor-α or interferon-γ (Soderberg-Naucler et al., 1997a; Hahn et al., 1998).

Virion components

A wide variety of virion components are involved in activating betaherpesvirus IE gene expression. Engagement of HCMV or viral glycoprotein gB with epidermal growth factor receptor activates the PI3K-mediated signaling pathway (Wang et al., 2003). HCMV glycoproteins also have a role in stimulating the release of pro-inflammatory cytokines mediated by the CD14 and Toll-like receptor 2 molecules on the cell surface (Compton et al., 2003). These cytokines may act on signaling pathways, which in turn, activate cellular transcription factors.

HHV-6 U54 is similar in amino acid sequence to HCMV UL82. The HCMV UL82 gene and the UL35 gene, which encode viral tegument proteins, have a profound positive impact on the efficiency of viral replication (Bresnahan and Shenk, 2000; Dunn et al., 2003; Schierling et al., 2004). These viral tegument proteins are transported to the nucleus of the infected cell where they are targeted to the ND10 (Schierling et al., 2004). UL82 (pp71) protein interacts with a cellular repressor of transcription, hDaxx, and may disrupt or inactivate the hDaxx-histone deacetylase (HDAC) complex (Cantrell and Bresnahan, 2006; Hensel et al., 1996; Hofmann et al., 2002; Ishov et al., 1997, 2002). The viral pp71 tegument protein induces proteasome dependent degradation of hDaxx and thereby neutralizes an intrinsic immune defense mechanism of the cell (Saffert and Kalejta, 2006). In cells deficient in hDaxx, UL82 protein is not targeted to the ND10. The viral MIE promoter and early promoters are activated by UL82 protein (Baldick et al., 1997; Bresnahan and Shenk, 2000; Liu and Stinski, 1992). Heterologous promoters, like herpes simplex virus IE promoters, are also activated by UL82 protein (Homer et al., 1999). The mechanism involves interaction with cellular repressors of transcription and reduction of HDAC activity. The UL82 protein also has effects on cell cycle progression. It stimulates quiescent cells to re-enter the cell cycle and accelerates cells through G1(Kalejta and Shenk, 2003a). The UL82 protein binds to members of the retinoblastoma (Rb) protein family and induces proteosome-dependent Rb family degradation (Kalejta and Shenk, 2002, 2003b). A release of the Rb family of repressors from E2F responsive cellular promoters increases cellular cyclins, cdks, and biosynthetic enzymes for DNA synthesis. UL82 protein increases the infectivity of HCMV in HF cells (Baldick et al., 1997).

HHV-6 U42 is the homologue of HCMV UL69. Deletion of the HCMV UL69 gene, which also encodes a tegument protein, causes a delay in viral DNA replication and late gene expression (Lu Hayashi et al., 2000). At low MOI, virus mutated in the UL69 gene replicate slower and take longer to reach peak levels of infectious virus (Lu Hayashi et al., 2000). The UL69 protein is transported to the nucleus where it may interact with proteins that regulate chromatin structure such as hSPT6 (Winkler et al., 1994). The UL69 protein enhances transcription from the viral MIE promoter in transient transfection assays (Winkler et al., 1994). It also affects cell cycle progression by blocking G1/S transition (Lu Hayashi et al., 2000).

Members of the betaherpesvirus family also have structural homologues of seven transmembrane G-protein-coupled receptors (GPCRs) (Chee et al., 1990; Gompels et al., 1995; Gruijthuijsen et al., 2002; Nicholas, 1996; Rawlinson et al., 1996; Vink et al., 2000; Waldhoer et al., 2002). Recombinant viruses with deletions of the GPCR genes replicate less efficiently in certain cell types. For example, recombinant murine or rat CMV with M33, R33, or M78 genes deleted replicate less efficiently in macrophages and are unable to disseminate to the salivary gland of the animal (Beisser et al., 1999; Davis-Poynter et al., 1997). Several of the viral GPCRs have been shown to initiate ligand-independent constitutive signaling through the G protein phospholipase C (PLC) pathway. Human and rhesus CMV US28 GPCRs induce intracellular signaling following binding of ligands such as fractalkine and CC chemokines. Although HCMV has a sequence homologue (UL33), US28 has been proposed to act in a manner similar to MCMV M33 in that both result in activation of the PLC pathway and activation of CREB and NF-κB. Betaherpesvirus GPCRs are postulated to have a role in the early re-programming of the host cell to favor viral replication. Their activation of cellular transcription factors may enhance IE and early gene transcription.

Infection and dysregulation of the cell cycle by betaherpesviruses

Although betaherpesviruses encode a viral DNA polymerase, processivity factor, single-stranded DNA binding protein and helicase/primase complex for viral DNA synthesis, the viruses depend on many host cell enzymes for this process. The betaherpesviruses do not encode many of the enzymes for synthesis of DNA precursors. Permissive HF cells are typically in the G0/G1 phase of the cell cycle and have low amounts of dideoxynucleotide triphosphates and biosynthetic enzymes of DNA precursors at the time of infection. Infection of mouse fibroblasts by MCMV or HF cells by HCMV stimulates production of cellular enzymes of DNA precursor synthesis, e.g., thymidylate synthetase, ribonucleotide reductase, deoxycytidylate deaminase, and dihydrofolate reductase (Gribaudo et al., 2000; Hertel and Mocarski, 2004; Song and Stinski, 2002). Upon HCMV infection, there is a 30-fold increase in the size of the thymidylate triphosphate pool (Biron et al., 1986). In addition, cells are primed for cellular DNA synthesis when the virus induces production or activities of select cellular cyclins and cdks, which are required for cell cycle progression. For example, cyclin E and cdk2, which are necessary for the G1/S transition, are highly activated after HCMV infection of HF cells (Bresnahan et al., 1997; Jault et al., 1995; McElroy et al., 2000; Salvant et al., 1998). In contrast, cyclin D, which is necessary for G1 phase, and cyclin A, which is necessary for the S phase, are not induced by HCMV in HF cells (Zhu et al., 1998). Betaherpesviruses appear to activate the cell differently from that of mitogenic stimulation where the cyclins are activated in a cascade fashion.

The cdks phosphorylate Rb family members and activate the E2F family of transcription factors (Nevins, 1992; Weinberg, 1995). E2F expression is increased after HCMV infection (Salvant et al., 1998; Song and Stinski, 2002), which, along with cyclin E, comprises a feed-forward loop allowing amplification of signals that promote cell cycle progression from G1 to S phase. However, with HCMV-infected HF cells, the majority of the cells are blocked at the G1/S transition point (Bresnahan et al., 1996; Dittmer and Mocarski, 1997; Lu and Shenk, 1996). Progression into S phase interferes with efficient HCMV replication because there is little to no IE gene expression during S phase (Fortunato et al., 2002; Salvant et al., 1998). Cellular proteins might be involved in suppressing IE gene expression during S phase, because an inhibitor of cellular proteosome activity allows for a higher level of IE gene expression (Fortunato et al., 2002). In general, the G1/S compartment of the cell cycle is the most favorable environment for betaherpesvirus gene expression, but the S phase is unfavorable.

An increase in cyclins and cdk activity is likely important for HCMV because cdk inhibitors like roscovitine and olomoucine affect splicing of the IE mRNAs (Sanchez et al., 2004) and inhibit infectious virus production (Bresnahan et al., 1997). In addition, inhibitors of cellular enzymes involved in DNA precursor synthesis (e.g., thymidylate synthetase and deoxycytidylate deaminase) can block HCMV and MCMV replication (Gribaudo et al., 2000). The combination of inactivation of the repressor proteins such as Rb and the activation of proteins involved in movement of the cell cycle such as cyclin E and cdk2 induce an environment most favorable for betaherpesvirus DNA replication. IE proteins of betaherpesviruses play an important role in this stage of the viral replication cycle.

Summary

Betaherpesviruses infect multiple cell types in the host. In general, the productive infection ensues in cells that are terminally differentiated and in the Go/G1 phase of the cell cycle. Expression of the IE genes is tightly regulated because these gene products are required for viral replication and have a potential detrimental impact on viral latency. The virions contain components that stimulate both cellular and viral genes. Viral glycoproteins, tegument proteins, and GPCRs can stimulate signal transduction pathways that activate cellular transcription factors and enhance the efficiency of viral replication. The MIE gene products directly activate early viral genes and further stimulate the cell for efficient early and late viral gene expression. Both viral tegument proteins and the MIE proteins cause dysregulation of the cell cycle. The cell is pushed from the G0/G1 phase to the G1/S transition point, with concomitant activation of cellular proteins for DNA precursor synthesis, cell cycle progression, and DNA initiation and synthesis. In the HCMV-infected cell, the level of phospho-serine15-p53 is increased, which stabilizes p53. In p53 wild type cells, the cell cycle stops at the G1/S transition. The HCMV UL69 tegument protein also prevents transition into S phase. Replication of betaherpesviruses appears to be best in a cell that progresses to the G1/S transition point, but is prevented from entering the S phase. The betaherpesviruses include both human and animal pathogens for which there are no vaccines, and the available antiviral therapies are fraught with limited efficacy and high rates of adverse effects. A better understanding of betaherpesvirus replication and pathogenesis is needed for developing novel strategies to prevent disease by these opportunists.

Acknowledgments

Because of page and reference limitations, the authors regret that they were unable to acknowledge all important details and contributions. We thank members of the Stinski laboratory and anonymous reviewers for helpful comments on the manuscript. Our work is supported by grants AI-13562 (MFS) and AI-40130 (JLM) from the National Institutes of Health and a grant from the Department of Veterans Affairs (JLM).

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Image 9780521827140psl_fig009
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