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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 25Reactivation and lytic replication of EBV

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University of Wisconsin, Madison, WI, USA

Viral pathogenesis

The lytic form of EBV infection is required for the production of progeny virus, and is thus essential for cell-to-cell spread of the virus, as well as transmission from host to host. Unfortunately, there is currently no cell culture system in vitro that is permissive for efficient primary lytic EBV infection. Although a recent report suggests that allowing the virus to first attach to the surface of primary B cells greatly facilitates EBV infection of epithelial cells in vitro (Shannon-Lowe et al., 2006), even this system of infection still does not result in efficient horizontal spread of virus from cell to cell. Thus, lytic EBV infection in vitro has been studied by reactivating the lytic form of infection from latently infected cell lines using a variety of inducing agents, including phorbol esters, butyrate, calcium ionophores, and B-cell receptor stimulation.

During primary infection, EBV probably initially infects oral epithelial cells in a lytic form, and then subsequently infects B-cells, where the virus usually assumes one of the latent forms of infection. In contrast to alpha and beta herpesviruses, which cause human diseases during the lytic form of viral infection but are essentially innocuous while in the latent form of infection, most illnesses attributable to EBV infection are associated with the latent forms of infection. During primary EBV infection, some individuals, particularly adolescents, develop the syndrome infectious mononucleosis (IM) approximately 1 month after being infected (Cohen, 2000; Jenson, 2000). The EBV -positive B-cells in patients with IM contain primarily the latent form(s) of infection, and the symptoms associated with this illness are attributable to the onset of a vigorous cytotoxic T-cell response against the virally infected B-cells (Cohen, 1999; Jenson, 2000; Andersson et al., 1987; Cohen, 2000). Lytic EBV infection (in either oral epithelial cells or B-cells) presumably precedes the onset of clinical IM, as IM patients have an extremely robust CD 8 T-cell response directed against lytic viral protein epitopes (Steven et al., 1997; Hislop et al., 2002). Nevertheless, it has been difficult to document primary lytic EBV infection in patients, presumably due to the long asymptomatic incubation period for IM (Cohen, 1999, 2000).

Following recovery from IM, it is often difficult or impossible to find any lytically-infected cells in immunocompetent individuals (Herrmann et al., 2002), although the preponderance of evidence suggests that reactivated lytic EBV infection most commonly occurs in tonsillar plasma cells as well as in tonsillar B-cells (Laichalk et al., 2005; Pegtel et al., 2004; Niedobitek et al., 1997, 2000; Faulkner et al., 2000). Nevertheless, the only human disease that is unequivocally due to lytic EBV infection, oral hairy leukoplakia (OHL), occurs in epithelial cells on the lateral aspect of the tongue (Lau et al., 1993; Walling et al., 2001, 2003b; Greenspan et al., 1985; Resnick et al., 1990). This hyperproliferative lesion is observed only in highly immunocompromised patients and is easily treated by antiviral agents such as acyclovir that inhibit the lytic form of EBV infection. Interestingly, the lytic (but not latent) form of EBV infection was also recently found in the malignant breast epithelial cells, as well as normal breast cells, in some cases of breast cancer (Huang et al., 2003), suggesting that the breast may also be a site for lytic EBV replication.

The lytic form of EBV infection is clearly required for transmission of the virus from host to host, and thus is an essential aspect of viral pathogenesis. The saliva from immunocompetent hosts often contains infectious EBV (Shu et al., 1992; Lucht et al., 1995; Ikuta et al., 2000; Ling et al., 2003; Walling et al., 2003a), indicating that lytically infected cells in or near the oral cavity must exist, even if they are difficult to detect in the presence of a vigorous cytotoxic T-cell response (Tao et al., 1995). Thus it is not surprising that the great majority (90% or more) of the human population ultimately becomes infected with this virus. EBV has also been reported to be present in both male and female genital secretions, suggesting that this virus could in some instances be sexually transmitted (Israele et al., 1991; Sixbey et al., 1986).

Activation of lytic EBV infection

Lytic viral gene cascade

EBV lytic genes are expressed in a temporally regulated manner. In many cell lines the two immediate–early genes, BZLF 1 and BRLF 1, are the first viral genes expressed following lytic induction stimuli, and both BZLF 1 and BRLF 1 transcription occurs even in the presence of protein synthesis inhibitors (Biggin et al., 1987; Flemington et al., 1991; Takada and Ono, 1989). In some cell lines, such as the Burkitt lymphoma line, Akata, BZLF 1 expression may precede BRLF 1 expression (Yuan et al., 2006). Once activated, the IE gene products function as transcription factors and together activate transcription of the early viral genes. Early viral genes are defined as genes that are transcribed prior to lytic viral replication (and thus are not inhibited by viral replication inhibitors), but are not transcribed in the presence of protein synthesis inhibitors. Early viral genes encode the viral replication proteins, including the virally encoded DNA polymerase. Following viral replication, the late viral genes are transcribed; late gene transcription is repressed by viral replication inhibitors. Many late genes encode structural proteins that make up the virion particle.

Stimuli that induce lytic EBV infection

In the human host, it is likely that differentiation of B cells into plasma following B-cell receptor stimulation by antigen (Laichalk and Thorley-Lawson, 2005) as well as differentiation in epithelial cells, both activate the lytic form of EBV infection (Tovey et al., 1978; Young et al., 1991). In cell culture systems in vitro, agents such as the phorbol ester, 12-0-tetradecanoyl phorbol-13-acetate (TPA), sodium butyrate, and calcium ionophores are commonly used to induce the lytic form of EBV infection (Faggioni et al., 1986; zur Hausen et al., 1978). Ultimately, what each of these various lytic EBV inducing stimuli shares is the ability to activate transcription of the two EBV IE genes, BZLF 1 and BRLF 1, from the latent viral genome. Lytic induction stimuli induce BZLF 1 and BRLF 1 transcription with similar kinetics (Biggin et al., 1987; Flemington et al., 1991; Takada and Ono, 1989), and many stimuli activate both the BZLF 1 and BRLF 1 promoters in EBV -negative cells (Zalani et al., 1995; Shimizu and Takada, 1993; Feng et al., 2004; Flemington and Speck, 1990d). Thus, other than possibly Akata cells, there is no firm evidence that one IE promoter is more important than the other for mediating lytic induction, and it may be that simultaneous activation of both promoters is required for efficient lytic induction. Once made, the BZLF 1 and BRLF 1 proteins function as transcription factors which activate their own promoters, as well as one another’s promoters (Liu and Speck, 2003; Adamson et al., 2000; Flemington et al., 1991; Ragoczy and Miller, 2001; Sinclair et al., 1991; Speck et al., 1997; Zalani et al., 1996), and thus greatly amplify the inducing effect of the initial lytic stimulus.

Of the various methods used in the lab to induce lytic EBV gene transcription, engagement of the B-cell receptor may be the most physiologic. B-cell receptor activation is accomplished using antibody directed against human IgG or IgM, depending upon which immunoglobulin is produced by the B-cell line. Akata Burkitt lymphoma cells are particularly responsive to this treatment, with up to 50% of cells converting to the lytic form of infection in a synchronous manner. Viral IE gene expression occurs very rapidly (30 minutes or less) after B-cell receptor engagement, followed a few hours later by early gene transcription (Takada and Ono, 1989; Mellinghoff et al., 1991; Flemington et al., 1991). Induction of lytic EBV infection by B-cell receptor engagement requires the activation of calcium-dependent signaling pathways (Chatila et al., 1997; Liu et al., 1997b), as well as the activation of numerous other signaling pathways (Adamson et al., 2000; Bryant and Farrell, 2002; Darr et al., 2001; Iwakiri and Takada, 2004; Mellinghoff et al., 1991). The potential mechanism(s) leading from B-cell receptor engagement to EBV IE gene activation are outlined in Fig. 25.1. The LMP -2A viral latency protein helps to maintain viral latency by preventing the ability of antigen to activate B-cell receptor stimulated signaling pathways (Miller et al., 1994a,b, 1995).

Fig. 25.1. Pathways leading to EBV reactivation in the host cell.

Fig. 25.1

Pathways leading to EBV reactivation in the host cell. Signal transduction pathways that are activated by B-cell receptor (BCR) engagement at the surface of the B-cell, or by phorbol ester (TPA) treatment of cells, are indicated. Promoter motifs in the (more...)

Certain cytokines, particularly TGF -beta, can also induce lytic viral infection in a subset of Burkitt lymphoma lines in vitro, and could potentially reactivate EBV in vivo (Fahmi et al., 2000; Adler et al., 2002). The interaction between CD 4 T cells and EBV -infected B cells has also been reported to induce lytic infection (Fu and Cannon, 2000). Finally, there is increasing evidence that severe host cell stress in response to many different toxic stimuli (including chemotherapy and irradiation) can induce lytic EBV infection (Feng et al., 2002a, 2004; Westphal et al., 2000; Roychowdhury et al., 2003).

In oral epithelial cells, lytic EBV infection is normally confined to differentiated cells. In OHL lesions, the EBV genome, and expression of lytic viral proteins, are only found in the more differentiated epithelial cell layers (Greenspan et al., 1985; Young et al., 1991). In vitro, differentiation of at least some EBV -positive epithelial lines induces the lytic form of EBV infection (Karimi et al., 1995; Li et al., 1992).

Many of the stimuli used to activate lytic EBV infection in vitro share the ability to activate a variety of signal transduction pathways, including PI 3 kinase, p38 kinase, ERK kinase, and protein kinase C, and these kinases have been shown to be essential for induction of lytic EBV transcription by a number of different stimuli (Hayakawa et al., 2003; Mansouri et al., 2003; Fahmi et al., 2000; Fan and Chambers, 2001; Dent et al., 2003; Iwakiri and Takada, 2004; Furukawa et al., 2003; Ionescu et al., 2003; Gao et al., 2001; Satoh et al., 1999; Mellinghoff et al., 1991). In the case of anti-immunoglobulin treatment, activation of calcium-dependent signaling pathways also plays an essential role in lytic viral induction. Two different calcium-dependent proteins activated by B-cell receptor engagement, the calcium/calmodulin-dependent phosphatase, calcineurin, and the calcium/calmodulin-dependent protein kinase type IV /Gr, are required for lytic EBV induction by anti-IgG in Akata cells (Goldfeld et al., 1995; Chatila et al., 1997). Cyclosporin, an immunosuppressive drug that inhibits calcineurin, prevents anti-IgG induction of lytic EBV (Bryant and Farrell, 2002; Goldfeld et al., 1995). The relative importance of each particular signal transduction pathway may vary somewhat depending upon the nature of the stimulus and the cell type.

Organization of the IE gene region of EBV

The organization of the various transcripts and proteins derived from the EBV IE gene region is shown in Fig. 25.2. The immediate–early transcripts that encode BZLF 1 and BRLF 1 are derived from two different immediate–early promoters, Zp and Rp. The BRLF 1 protein is encoded by messages initiating from Rp. These transcripts are bicistronic and could potentially make both the BRLF 1 and BZLF 1 gene products (Manet et al., 1989). There is some evidence that translation of the BZLF 1 protein from the Rp-derived transcripts occurs in vivo, and that the BRLF 1 protein is required for this process (Chang et al., 1998; Chang and Liu, 2001). However, the great majority of BZLF 1 protein is probably derived from the BZLF 1 transcript that initiates from the Zp promoter. A spliced message which contains parts of both BRLF 1 and BZLF 1 (referred to as RAZ), initiated from Rp, is transcribed later in infection and its gene product may serve as a negative regulator of BZLF 1 transcriptional function (Furnari et al., 1994; Manet et al., 1989; Segouffin et al., 1996). The BRRF 1 transcript, derived from the BRRF 1 promoter and encoded by the opposite strand of the BRLF 1 intron (Manet et al., 1989; Segouffin-Cariou et al., 2000), produces an early protein that was recently shown to be a transcriptional activator (Hong et al., 2004) important for efficient lytic EBV gene expression under certain circumstances.

Fig. 25.2. Transcription of the EBV immediate-early gene region.

Fig. 25.2

Transcription of the EBV immediate-early gene region. The location of the two immediate-early (IE) genes, BZLF 1 and BRLF 1, the two IE promoters, Zp and Rp, and the BRRF 1 early gene and promoter is shown. Rp directs transcription of a bicistronic message (more...)

Initial steps in viral reactivation

The promoters driving BZLF 1 and BRLF 1 transcription, Zp and Rp, are inactive in B-cells containing the latent form of EBV infection (Flemington et al., 1991; Zalani et al., 1992; Biggin et al., 1987; Takada and Ono, 1989; Mellinghoff et al., 1991). Epigenetic modifications such as DNA methylation and histone deacetylation likely contribute to inhibition of IE gene transcription in the context of the intact viral genome (Szyf et al., 1985; Paulson and Speck, 1999; Nonkwelo and Long, 1993; Falk and Ernberg, 1999; Ambinder et al., 1999; Paulson et al., 2002; Bhende et al., 2004; Jenkins et al., 2000; Gruffat et al., 2002b). Nevertheless, even “naked” DNA reporter gene constructs driven by the Zp and Rp promoters are essentially inactive in many EBV -negative B-cell lines (Feng et al., 2004; Kenney et al., 1989b; Sinclair et al., 1991; Zalani et al., 1995), but can be activated by lytic inducing stimuli such as TPA and B-cell receptor stimulation. Thus, the inactivity of the Zp and Rp promoters in unstimulated B cells must reflect the lack of trans-acting transcription factors, which positively regulate the two IE promoters, and/or the relative excess of cellular factors which negatively regulate the two IE promoters.

Cellular factors which activate Zp

Cellular transcription factors that positively and negatively regulate the Zp and Rp promoters are shown in Figs. 25.3 and 25.4. Regulation of the Zp promoter has been much more extensively studied than regulation of the Rp promoter. The region of Zp between –233 and +12 contains the cis-acting sequences required for Zp activation by lytic-inducing agents. Two types of cis-acting motifs, the “ZI” and “ZII” motifs, are critical for activation of Zp by a variety of different lytic-inducing stimuli. The four ZI motifs (ZIA, ZIB, ZIC, and ZID) are AT -rich sequences that have dual roles as both negative, as well as positive, regulators of Zp transcription (Borras et al., 1996; Daibata et al., 1994, Flemington and Speck, 1990a,b; Binne et al., 2002). In the absence of lytic-inducing agents, these elements down-regulate constitutive Zp activity in B cells. However, the ZI elements are also essential for Zp activation by a variety of different inducing stimuli, including TPA, anti-immunoglobulin, calcium ionophores, and chemotherapy (Feng et al., 2004; Binne et al., 2002). Three of the four ZI motifs (ZIA, ZIB, ZID) bind to the transcription factor, MEF 2D (Liu et al., 1997b). ZIA, ZIC and ZID also bind weakly to Sp1 and Sp3 (Liu et al., 1997a). This dual role of the ZI motifs as both negative, as well as positive, regulators of Zp transcription may reflect a preferential interaction of MEF 2D with histone deacetylating complexes during viral latency (hence acting to inhibit Zp transcription), which is switched to a preferential interaction with acetylating complexes by lytic inducing stimuli (Gruffat et al., 2002b). The phosphorylation status of MEF 2D may play an essential role in determining if it represses, versus activates, Zp transcription. Interestingly, calcium/calmodulin-dependent protein kinase type IV /Gr indirectly activates MEF 2D by preventing the association between MEF 2D and HDAC proteins, whereas MAP kinases directly activate MEF 2D by phosphorylating the transcriptional activator domain and enhancing its function (McKinsey et al., 2000). Nevertheless, phosphorylation of other sites in MEF 2D may inhibit its function (Li et al., 2001). Cross-linking the B-cell receptor in Akata Burkitt lymphoma cells results in rapid dephosphorylation of the MEF 2D in a cyclosporine-sensitive manner, suggesting that calcineurin is involved in dephosphorylating (and perhaps activating) MEF2D (Bryant and Farrell, 2002).

Fig. 25.3. Regulation of the EBV immediate–early BZLF 1 promoter.

Fig. 25.3

Regulation of the EBV immediate–early BZLF 1 promoter. Regulatory motifs in the BZLF 1 (Zp) promoter are shown. Cellular and viral proteins known to bind to each motif are indicated.

Fig. 25.4. Regulation of the EBV immediate–early BRLF 1 promoter.

Fig. 25.4

Regulation of the EBV immediate–early BRLF 1 promoter. Regulatory motifs in the BRLF 1 (Rp) promoter are shown. Cellular and viral proteins known to bind to each motif are indicated.

The “ZII” motif is also essential for induction of BZLF 1 transcription by most stimuli (Binne et al., 2002; Flamand and Menezes, 1996; Feng et al., 2004; Flemington and Speck, 1990d; Daibata et al., 1994; Chatila et al., 1997). The “ZII” motif is a slightly atypical CREB -responsive element (CRE) that binds CREB, ATF -1, the ATF -2/c-jun heterodimer, and possibly the c-jun/c-fos (“AP1”) heterodimer as well (Flamand and Menezes, 1996; Flemington and Speck, 1990d; Liu et al., 1998; Adamson et al., 2000; Wang et al., 1997). The cellular factors which bind to CRE motifs are constitutively expressed in many cell lines, but cannot function as efficient transcriptional activators unless they are phosphorylated over specific residues by certain kinases. The c-jun N-terminal kinase (JNK) is the major activator of c-jun transcriptional function, while the stress Map kinase p38 phosphorylates and activates ATF -2. Many of the stimuli that induce lytic EBV infection in vitro (including B-cell receptor engagement, BRLF 1-mediated induction, and certain chemotherapy agents) are known to activate the p38 and JNK kinases, and conversely, inhibitors of p38 and JNK kinases reduce the effectiveness of a number of lytic-inducing stimuli (Adamson et al., 2000; Feng et al., 2002a, 2004). Thus, the activated (phosphorylated) form of an ATF -2/c-jun heterodimer appears to be important for activation of BZLF 1 transcription by a number of different stimuli.

Lytic induction may also be mediated through ATF -1 or CREB binding to the ZII motif (Wang et al., 1997), as the activated (phosphorylated) forms of these transcription factors activate Zp in reporter gene assays. Consistent with a role for ATF - 1 and CREB in at least some cell types, activation of the Zp promoter induced by epithelial cell differentiation is associated with increased binding of ATF -1 and CREB to the Zp CRE motif (Karimi et al., 1995; MacCallum et al., 1999). In addition, engagement of the B-cell receptor results in calcium mobilization and activation of the calcium/calmodulin-dependent protein kinase type IV /Gr, which phosphorylates and activates CREB (Matthews et al., 1994). Both ATF -1 and CREB phosphorylation are observed transiently following anti-IgG treatment of Akata cells (Bryant and Farrell, 2002).

The BZLF 1 promoter sequences –233 to –229 bind to Smad3/Smad4, which mediate signaling by the TGF -beta cytokine, and this region of the promoter is required for the TGF -beta activation of Zp that occurs in certain BL lines (Fahmi et al., 2000; Liang et al., 2002). In addition, TGF -beta activation of Zp requires the ZII motif (Liang et al., 2002). C-jun bound to the ZII motif interacts directly with Smad3/4 bound to the Smad binding site in Zp, perhaps explaining the need for both elements (Liang et al., 2002).

BZLF1 autoregulation

There are two ZRE sites in Zp (ZIIIA and ZIIIB), and BZLF 1 activates Zp transcription in reporter gene assays (Binne et al., 2002; Flemington and Speck, 1990a). The Zp promoter (in the context of a stable oriP-containing episomal vector) cannot be efficiently activated by anti-IgG signaling in Akata cells when the ZIIIA site of Zp is deleted (Binne et al., 2002). These results suggest that the ability of Z to autoactivate its own transcription may be an essential component for induction of lytic EBV infection. However, it has been suggested that the ZIIIA site in Zp may also bind cellular factors required for induction of BZLF 1 transcription by anti-Ig prior to the onset of BZLF 1 binding to this site. Since BZLF 1 activates TGF -beta transcription (Cayrol and Flemington, 1995), and TGF -beta activates the BZLF 1 promoter in at least some cell lines, the TGF -beta pathway may serve as another potential mechanism by which BZLF 1 activates its own promoter. Nevertheless, whether BZLF 1 actually activates the Zp promoter in the context of the intact genome during viral reactivation remains controversial. A number of published reports suggest that it may not (Binne et al., 2002; Zalani et al., 1996; Le Roux et al., 1996).

Negative regulatory elements in Zp

Negative regulation of BZLF 1 transcription may play a critical role in promoting viral latency in B cells. A number of different negative regulatory elements in the BZLF 1 promoter have been identified. Of particular importance is the “ZV” motif located near the TATA box that binds to the zinc-finger protein, ZEB (Kraus et al., 2001, 2003). Deletion of this site significantly enhances activation of Zp induced by B-cell receptor engagement in Akata cells (Binne et al., 2002). Binding sites for the cellular YY -1 transcription factor have also been mapped (the “ZIV” elements), and shown to function as negative regulators of BZLF 1 transcription (Montalvo et al., 1991, 1995). A cis-acting sequence, ZIIR, which overlaps the ZII motif, also reportedly inhibits Zp (Liu et al., 1998), although the cellular factor(s) binding to this repressor have not been identified. The cellular protein, smubp-2, binds to Zp ZI motifs and reportedly functions as a negative regulator, although it is not yet clear whether this negative regulation is mediated by direct binding of smubp-2 to Zp or acts more generally to disrupt formation of a stable TBP -TFIIA-DNA complex (Zhang et al., 1999b). Finally, a series of “H-box” elements, that are similar to E-box motifs and bind to the transcription factor E2–2, have been reported to function as negative regulators of Zp in B-cells, but positive regulators in epithelial cells (Thomas et al., 2003).

Regulation of Rp

Less is known about the regulation of the BRLF 1 promoter (Rp) than the BZLF 1 promoter. Both the BZLF 1 and BRLF 1 gene products activate this promoter in EBV -negative cells (Sinclair et al., 1991; Ragoczy and Miller, 2001; Zalani et al., 1992), and the combination of BZLF 1 and BRLF 1 together is more effective than either protein alone (Liu and Speck, 2003). The Rp promoter, like Zp, is activated by B-cell receptor stimulation, phorbol ester agents, and chemotherapy agents (Zalani et al., 1995; Sinclair et al., 1991; Feng et al., 2004). There are two EGR -1 (Zif-268) binding motifs in Rp, and activation of Rp by TPA and chemotherapy requires these sites in at least some cell types (Zalani et al., 1995; Feng et al., 2004). Phorbol esters, chemotherapy, and B-cell receptor engagement have all been shown to increase the level of cellular EGR -1, suggesting a common mechanism by which these inducing agents might activate BRLF 1 transcription. There are also several Sp1 sites in Rp (Zalani et al., 1992), which are required for constitutive promoter activity as well as efficient autoactivation of Rp by its own gene product (Ragoczy and Miller, 2001; Liu and Speck, 2003). A binding site for NF 1 has been reported to be a positive regulatory element in HeLa cells but not lymphoid cells (Glaser et al., 1998). There are at least three BZLF 1 binding sites (ZREs) in Rp, and BZLF 1 activation of Rp is mediated by direct binding of BZLF 1 to these ZRE sites (Sinclair et al., 1991; Liu and Speck, 2003; Bhende et al., 2004). Like Zp, the Rp promoter also contains binding sites for the negative regulators, YY -1 and ZEB. The two YY -1 binding motifs function as negative regulators (Zalani et al., 1997) of Rp; the role of the ZEB site, if any, has not yet been defined. Potential ZI -like and ZII -like motifs in Rp exist, but their function has not been studied.

Mechanisms by which TPA and butyrate activate IE gene transcription

TPA treatment induces lytic EBV gene transcription in some cell lines, and this effect is at least partially mediated through TPA activation of protein kinase C (Gao et al., 2001; Gradoville et al., 2002). Why PKC activation leads to IE gene transcription is not totally clear, but it has been shown to require both the ZI and ZII motifs in Zp (Flemington and Speck, 1990d). In the case of the ZII motif, this may reflect the ability of TPA to activate the stress map kinases (p38 and c-jun N-terminal kinase) in a PKC -dependent manner (Grab et al., 2004; Krappmann et al., 2001), thus leading to phosphorylation of the ATF -2 and c-jun transcription factors which bind to the ZII site. TPA increases the level of EGR -1 in cells (Krappmann et al., 2001), and the Rp EGR -1 binding sites are required for TPA activation of this promoter (Zalani et al., 1995).

Agents that increase histone acetylation by inhibiting histone deacetylase (HDAC) activity, including sodium butyrate, trichostatin A, and valproic acid (Davie, 2003), also induce lytic EBV gene transcription in some cell lines. These agents presumably act by increasing the histone acetylation state of the two viral IE gene promoters (Jenkins et al., 2000). Butyrate-responsive cellular genes (only 2% of all genes), like Zp and Rp, often have Sp1/Sp3 binding sites (Davie, 2003). Sp1 and Sp3 interact with HDAC proteins, as does MEF 2D. Thus, in the absence of HDAC inhibitors, Sp1/Sp3 and MEF 2D bound to the ZI motifs in Zp may act to inhibit Zp transcription by tethering HDAC complexes to the promoter, whereas in the presence of HDAC inhibitors these transcription factors instead interact with histone acetylases and activate Zp (Gruffat et al., 2002b; Davie, 2003). Unlike the inducing effect of TPA, which requires PKC, the inducing effect of histone deacetylase inhibitors is PKC -independent (Gradoville et al., 2002). HDAC inhibitors alone are insufficient to induce lytic EBV infection in many cell lines (Gradoville et al., 2002), presumably because additional trans-acting factors are required to activate BZLF 1 or BRLF 1 transcription, and the combination of TPA and sodium butyrate is thus required for efficient induction of lytic viral gene expression in many cell lines.

Host cell and viral factors which influence stringency of viral latency

Of note, even in the most susceptible cell lines (generally BL lines), fewer than 50% of cells ever enter the lytic form of infection even when a combination of inducing agents is used. Why a portion of cells always remain in the latent form of viral infection (Gradoville et al., 1990), while others switch to the lytic form of infection, is not currently well understood. In general, we have found that lymphoblastoid cell lines (LCLs) that have been extensively passaged are much more resistant to a variety of different inducing agents than newly derived lines, which often contain a portion of cells in the lytic form of infection even in the absence of inducing agents. In contrast to extensively passaged LCL s, BL lines often retain the ability to respond to one or more lytic-inducing agents.

The tendency of certain cell lines to remain tightly latent even in the face of multiple different inducing agents may be due to either viral and/or cellular factors. Viral factors that promote EBV latency include LMP -2A expression (since LMP -2A inhibits B-cell receptor mediated activation of Zp and Rp), as well as epigenetic modifications of Zp and Rp (including DNA methylation and/or chromatin deacetylation) (Gradoville et al., 1990, 2002; Paulson and Speck, 1999; Nonkwelo and Long, 1993; Szyf et al., 1985). Complete resistance to lytic inducing agents occasionally reflects an integrated viral genome. In some cell lines, such as the Raji BL line, inducing agents cause an abortive lytic infection, with expression of IE and early genes but no lytic viral replication, due to the deletion of one or more essential viral replication genes. In contrast, cell lines containing a defective rearranged form of the EBV genome, dHet, as well as wild-type virus, often have particularly high levels of lytic viral replication (Taylor et al., 1989). This is because the defective rearranged viral genome contains the BZLF 1 gene product under the control of the constitutively active EBNA -2 latency promoter, Wp (Countryman et al., 1987; Rooney et al., 1988; Grogan et al., 1987). There is some evidence that similar defective rearranged viral genomes occur during natural infection in humans (Gan et al., 2002; Walling et al., 1992).

Cellular factors contributing to EBV latency include the activated (nuclear) form of NF -KB, which directly interacts with BZLF 1 and inhibits its transcriptional function (Morrison and Kenney, 2004, Gutsch et al., 1994). Likewise, the retinoic acid receptor also directly interacts with BZLF 1 and inhibits its function (Sista et al., 1993). Nitric oxide also potently down-regulates the lytic form of EBV infection (Gao et al., 1999; Kawanishi, 1995). The cellular level of ZEB, which binds to the ZV motif and inhibits Zp activity (Krauss et al., 2001, 2003), presumably also influences the stringency of EBV latency.

EBV immediate-early proteins

Introduction

Transcription of the BZLF 1 and BRLF 1 genes results in expression of the BZLF 1 (also known at ZEBRA, Z, EB 1 and Zta) and BRLF 1 (R, Rta) proteins. Both BZLF 1 and BRLF 1 are transcription factors, and high-level expression of either BZLF 1 or BRLF 1 (under the control of a strong heterologous promoter) is sufficient to induce the switch from the latent to lytic form of EBV infection in some latently infected cell lines (Chevallier-Greco et al., 1986; Countryman and Miller, 1985; Rooney et al., 1989; Takada et al., 1986; Zalani et al., 1996; Westphal et al., 1999; Ragoczy et al., 1998). However, in many cell lines, BZLF 1 is much more effective than BRLF 1 for inducing lytic EBV gene expression, and in some cell lines (such as Raji cells and some lymphoblastoid lines) only BZLF 1, and not BRLF 1, can disrupt viral latency (Ragoczy and Miller, 1999; Zalani et al., 1996; Hong et al., 2004). Thus, activation of BZLF 1 expression (and hence regulation of the Zp promoter) may be relatively more important in vivo for inducing lytic EBV infection than activation of BRLF 1 expression. In contrast, only the BRLF 1 homologue (and not the BZLF 1 homologue) can induce lytic infection in latently infected KSHV cell lines.

BZLF1 transcriptional effects

BZLF1 is a homologue to c-jun and c-fos, and binds as a homodimer to AP -1 like motifs (including the consensus AP -1 site) known as Z-responsive elements (ZREs) (Chang et al., 1990; Packham et al., 1990; Lieberman et al., 1990; Lieberman and Berk, 1990; Farrell et al., 1989; Flemington and Speck, 1990a). BZLF 1 transcriptionally activates immediate-early, and early, lytic EBV promoters (Urier et al., 1989; Lieberman et al., 1989; Rooney et al., 1989; Kenney et al., 1989b; Holley-Guthrie et al., 1990; Zetterberg et al., 2002). As shown in Fig. 25.5, the amino-terminus of BZLF 1 encodes the transactivator domain (Flemington et al., 1992; Deng et al., 2001), as well as a region required for replication but not transcription (Sarisky et al., 1996). DNA binding is mediated through a domain that is highly homologous to the basic DNA binding domains of c-jun and c-fos (Flemington et al., 1994; Farrell et al., 1989). Homodimerization is mediated through a bZIP domain in the carboxy-terminal portion of the protein (Flemington and Speck, 1990c; Kouzarides et al., 1991). BZLF 1 does not heterodimerize efficiently with either c-fos or c-jun, but heterodimerizes with another cellular bZIP protein, C/EBP-alpha (Wu et al., 2003), and can activate at least some promoters through C/EBP-alpha binding sites. The crystal structure of BZLF 1 was recently published (Petosa et al., 2006) and indicates that the bZIP domain in BZLF 1 is somewhat unusual in that the carboxy-terminal region of the protein is also required to form a stable dimer.

Fig. 25.5. EBV BZLF 1 Immediate–early protein.

Fig. 25.5

EBV BZLF 1 Immediate–early protein. Domains in the BZLF 1 protein that mediate dimerization, DNA -binding and transactivation functions are shown, as well as certain phosphorylation and sumo-1 modification sites.

BZLF1 activation of early lytic EBV promoters is generally mediated through direct binding of BZLF 1 to ZRE motifs within the promoters (Flemington and Speck, 1990a; Urier et al., 1989). BZLF 1 may also activate certain cellular promoters through a non-DNA binding mechanism (Flemington et al., 1994). In general, at least two ZRE sites are required for efficient activation of early viral promoters (Carey et al., 1992), and these sites are usually located within a few hundred basepairs of the transcriptional start site. Once bound to DNA, the ability of BZLF 1 to interact directly with histone acetylating complexes (including CBP and p300) results in acetylation of chromatin, converting it to a conformation favorable for transcription (Adamson and Kenney, 1999; Chen et al., 2001a; Deng et al., 2003; Zerby et al., 1999). BZLF 1 also interacts directly with a number of basic transcription factors, including TFIID and TFIIA (Chi and Carey, 1993; Chi et al., 1995; Lieberman and Berk, 1991, 1994; Lieberman et al., 1997; Mikaelian et al., 1993).

In the context of the intact viral genome, all evidence to date suggests that BZLF 1 activation of the BRLF 1 IE promoter precedes activation of the early lytic promoters, and that both the BZLF 1 and BRLF 1 gene products are required for activation of most early lytic genes (Feederle et al., 2000). BZLF 1 may bind to the atypical ZRE sites in the BRLF 1 promoter in a somewhat different manner (or perhaps conformation) than it binds to the consensus AP -1 site (El-Guindy et al., 2002). This point is perhaps most clearly indicated by the phenotype of a mutant BZLF 1 protein in which serine 186 in the basic DNA binding domain is altered to alanine (the residue encoded by the analogous region in c-jun and c-fos). The Z(S186A) mutant cannot bind efficiently to either of the two ZRE sites in the BRLF 1 promoter, although it binds efficiently to the consensus AP -1 site and a variety of ZRE sites within early EBV gene promoters (Adamson and Kenney, 1998; Francis et al., 1997). When transfected into latently infected cells, Z(S186A) cannot induce BRLF 1 transcription, and consequently is completely defective for inducing early lytic EBV gene transcription, but its lytic defect is rescued by co-transfection with a BRLF 1 expression vector (Francis et al., 1999; Adamson and Kenney, 1998). Serine residue 186 is phosphorylated by PKC in vitro, but whether this phosphorylation actually occurs in vivo remains controversial (El-Guindy et al., 2002; Baumann et al., 1998; Gradoville et al., 2002; Daibata et al., 1992). In lytically-infected B95–8 cells, BZLF 1 is phosphorylated at residues Thr 14, Ser167, Ser173 and Ser186, and may be weakly phosphorylated at additional residues (EI-Guindy et al., 2004, 2006). Phosphorylation of BZLF 1 residues Ser167 and Ser173 by casein Kinase Ⅱ, while not required for Z activation of early lytic genes, is required for efficient viral replication and modulates the ability of BRLF 1 to regulate late gene transcription (EI-Guindy and Miller, 2004).

BZLF1 activation of methylated ZRE motifs

The EBV genome is highly methylated during the latent form of viral infection, and DNA methylation of promoters generally acts as a potent inhibitor of cellular gene transcription. However, BZLF 1 was recently shown to preferentially bind to the methylated vs. unmethylated, forms of two ZRE sites in Rp (Bhende et al., 2004). BZLF 1 binding to the ZRE -2 site in Rp, which contains the sequence TGAGCGA, is much enhanced when the cytosine in this motif is methylated, and a previously unrecognized ZRE site in Rp, ZRE -3, which contains the sequence TTCGCGA, can only be bound by BZLF 1 in the methylated form. Furthermore, BZLF 1 preferentially activates the methylated form of the BRLF 1 promoter in reporter-gene assays, and preferentially induces lytic EBV transcription from a methylated versus unmethylated, viral genome (Bhende et al., 2004). Thus, BZLF 1 is the first example of a transcription factor that preferentially activates the methylated form of a downstream target gene. This unexpected ability of BZLF 1 to activate methylated lytic viral promoters reveals a novel mechanism by which EBV circumvents the inhibitory effects of viral genome methylation.

BZLF1 activation of cellular genes

Not surprisingly, BZLF 1 also transcriptionally activates certain cellular genes, some of which may be important for EBV pathogenesis. The cellular genes known to be activated by BZLF 1 include TGF -beta (Cayrol and Flemington, 1995), c-fos (Flemington and Speck, 1990b), the tyrosine kinase TKT (Lu et al., 2000), matrix metalloproteinases 1 and 9 (Lu et al., 2003; Yoshizaki et al., 1999), and cellular IL -10 (Mahot et al., 2003). BZLF 1 activation of the immunosuppressive cytokines, TGF -beta and IL -10, could potentially dampen the host immune response during the lytic form of virus infection, whereas induction of the matrix metalloproteinases could potentially enhance metastasis of EBV -positive tumors cells expressing BZLF 1. In addition, cellular IL -10 is a potent B-cell growth factor, suggesting a mechanism by which lytic EBV gene expression in a small percentage of cells could promote B-cell malignancies in a paracrine manner.

BZLF1 replication function

In addition to its essential role as a transcription factor, BZLF 1 also plays a direct role in lytic viral replication. BZLF 1 binds directly to a number of ZRE sites in the lytic origin of replication, oriLyt, and this binding is required for oriLyt replication (Fixman et al., 1992, 1995; Hammerschmidt and Sugden, 1988; Schepers et al., 1993a,b). Furthermore, a BZLF 1 mutant altered at residues 12/13 is transcriptionally competent, but completely defective for mediating viral replication (Sarisky et al., 1996). BZLF 1 also interacts directly with some of the core viral replication proteins (Zhang et al., 1996; Takagi et al., 1991; Gao et al., 1998). Together, these results suggest that BZLF 1 acts as an essential oriLyt binding protein during lytic EBV replication, and that this binding may promote formation of the initial replication complex.

The BZLF 1-knockout virus is less efficient in promoting lymphoproliferative disease in SCID mice

The phenotype of a BZLF 1-deleted EBV has been recently described (Feederle et al., 2000) in 293 cells and primary B cells. As expected, this mutant cannot undergo the lytic form of EBV replication unless the BZLF 1 gene product is expressed in trans. In 293 cells infected with the BZLF 1-knockout virus, expression of the BZLF 1 gene product induces expression of the IE protein, BRLF 1, as well as the complete complement of early and late lytic genes. In contrast, expression of the BRLF 1 gene product in 293 cells infected with BZLF 1-knockout virus does not result in expression of the majority of early or late genes. The BZLF 1-knockout virus is not reported to be defective in immortalizing B cells. The phenotype of the BZLF 1-knockout virus confirms that both BZLF 1 and BRLF 1 transcriptional functions are required for the induction of many (but not all) lytic EBV genes in the context of the intact viral genome.

Surprisingly, however, recent findings suggest that early-passage lymphoblastoid cell lines (LCLs) derived from either BZLF1 -deleted, or BRLF 1-deleted, viruses are less efficient than lines derived using wild-type virus in regard to their ability to form lymphoproliferative disease in SCID mice (Hong et al., 2005a,b). LCLs containing the BZLF 1-deleted virus secrets less of the two B-cell growth factors, cellular IL -6 and cellular IL -10, and less of the potent angiogenesis factor, VEGF, than LCLs from the same donor containing wild-type EBV. These results suggest that a small number of lytically infected cells may contribute to the growth of some EBV -associated tumors in vivo through the release of paracrine growth factors or angiogenesis factors.

BZLF1 effects on the host cell environment

In addition to its essential roles as a transcription factor and viral replication protein, BZLF 1 alters the host cell environment in numerous different ways that presumably act together to enhance the efficiency of lytic viral replication. As the first viral protein expressed during lytic reactivation (and primary lytic infection), BZLF 1 is ideally situated to protect the virus from a variety of different host defenses, including cellular apoptosis and the host innate immunity, and to regulate the host cell cycle.

BZLF1 cell cycle effects

There is increasing evidence that herpesviruses usurp the host cell cycle control mechanisms to assure adequate substrates for lytic viral DNA replication. However, the cell cycle effects of BZLF 1 appear to be cell-type dependent. BZLF 1 produces a profound G1/S block in some cell types, including primary fibroblasts (Rodriguez et al., 1999, 2001a; Cayrol and Flemington, 1995, 1996a,b; Mauser et al., 2002b; Wu et al., 2003). In cell types susceptible to this G1/S block, BZLF 1 decreases expression of cyclin A and c-myc (Mauser et al., 2002b; Rodriguez et al., 2001b), and increases p21 expression (Wu et al., 2003; Cayrol and Flemington, 1996b). BZLF 1 activates p21 expression through a C/EBP-alpha binding site in the p21 promoter, an effect that involves the direct interaction between BZLF 1 and C/EBP-alpha (Wu et al., 2003). In other cell types, such as HeLa cells, BZLF 1 induces both a G2 and mitotic block (Mauser et al., 2002a; Cayrol and Flemington, 1996a). The G2 block results from decreased cyclin B, while the mitotic block is associated with a defect in chromosome condensation (Mauser et al., 2002a).

Nevertheless, the cell types in which BZLF 1 induces cell cycle blocks are either not normally infected by EBV (fibroblasts), and/or are likely to be deficient in normal cell cycle regulation controls (tumor cells). In sharp contrast to the results in fibroblasts and tumor cell lines, in telomerase-immortalized, as well as primary, keratinocytes, BZLF 1 actually enhances expression of a number of S-phase dependent cellular proteins, and increases the level of E2F-1, cyclin E and cyclin A (Mauser et al., 2002b). Likewise, inducible BZLF 1 expression in the EBV -immortalized marmoset B-cell line, B95–8, results in enhanced activity of cyclin-dependent kinases, although cellular DNA replication is blocked (Kudoh et al., 2003). Most importantly, agents that inhibit the activity of cyclin-dependent kinases also inhibit lytic EBV gene expression (Kudoh et al., 2004), although it is not currently understood why cyclin-dependent kinases are required for efficient lytic EBV replication. These results suggest that a “pseudo-lateG1/S-phase” environment, in which certain late G1/S-phase restricted cellular proteins are expressed, but cellular DNA does not actually replicate, may be the ideal host cell environment for lytic EBV replication. Inhibition of cellular DNA replication presumably decreases competition between the virus and host cell DNA for limiting substrates involved in DNA replication, while the expression of certain G1/S-phase restricted cellular proteins, such as E2F-1, may be required for viral replication.

BZLF1 effects on p53

Activation of p53 in host cells serves as an important host defense mechanism, since p53 induces cellular apoptosis and hence limits viral replication. Not surprisingly, therefore, many viruses, including herpesviruses, encode proteins that inhibit various aspects of p53 function. The effects of BZLF 1 on p53 in the host cell are quite complex. Somewhat paradoxically, in some (but not all) cell types, the presence of BZLF 1 results in a rather dramatic increase in the level of total p53, and induces a number of post-translation modifications of p53 (including a series of activating phosphorylations and acetylations) that are usually associated with enhanced p53 transcriptional function (Mauser et al., 2002c). BZLF 1 also increases the amount of p53 binding in some cell types (Mauser et al., 2002c). This activation of p53 that occurs in BZLF 1-expressing cells may represent an attempt by the host cell to limit EBV replication. Nevertheless, the majority of evidence suggests that BZLF 1 quite efficiently inhibits p53 transcriptional function (Zhang et al., 1994; Mauser et al., 2002c). BZLF 1 inhibition of p53 may be due in part to the previously observed direct interaction between the BZLF 1 and p53 proteins (Zhang et al., 1994). In addition, BZLF 1 significantly reduces the level of the basic transcription factor, TATA -binding protein (TBP), in host cells, and restoration of this protein partially reverses the ability of BZLF 1 to inhibit p53 transcriptional function (Mauser et al., 2002c). Finally, as discussed below, BZLF 1-mediated dispersion of nuclear PML bodies in host cells may also decrease p53 function, since optimal p53 transcriptional function requires PML bodies. In any event, given that p53 is an important cellular mediator of apoptosis, the ability of BZLF 1 to inhibit p53 function likely plays a crucial role in protecting the virus from apoptosis during the earliest timepoints of lytic infection.

BZLF1 dispersion of PML bodies

Promyelocytic leukemia (PML) bodies, also known as nuclear domain 10 (ND-10) bodies, are nuclear structures which contain a number of different cellular proteins, including CREB -binding protein (CBP), Sp100, Rb, Daxx, ISG 20, and the small ubiquitin-related modifier, sumo-1 (Bernardi and Pandolfi, 2003; Salomoni and Pandolfi, 2002). Only the PML protein is absolutely essential for formation of PML bodies, and the PML protein must be covalently modified by sumo-1 in order to form these bodies. Although the exact function(s) of PML bodies is somewhat mysterious, the fact that the formation of these bodies is dramatically enhanced by both type Ⅰ and type Ⅱ interferons, and that a number of different viruses encode proteins capable of dispersing PML bodies, suggests that these bodies have an antiviral function (Chee et al., 2003; Bernardi and Pandolfi, 2003; Salomoni and Pandolfi, 2002). PML bodies have been proposed to be important for certain types of apoptosis, MHC class Ⅰ presentation, efficient acetylation of p53, interferon effects, and for the stability and function of an important cellular DNA repair complex, Mre11/Rad50/NBS1 (Bernardi and Pandolfi, 2003; Salomoni and Pandolfi, 2002; Chee et al., 2003). As each of these proposed functions would be expected to reduce viral replication, it is perhaps not surprising that many viruses attempt to inhibit the formation of PML bodies.

High level BZLF 1 expression in EBV -negative cells is sufficient to disperse PML bodies, and this effect is correlated with the ability of BZLF 1 to inhibit sumo-1 modification of the PML protein (Bell et al., 2000; Adamson and Kenney, 2001). BZLF 1 itself is efficiently modified by sumo-1 over lysine 12, and the ability of BZLF 1 to inhibit PML protein sumo-1 modification may be at least partially due to competition between BZLF 1 and PML for limiting amounts of sumo-1 in the host cell (Adamson and Kenney, 2001). A BZLF 1 mutant altered at residues 12 and 13 is transcriptionally competent, but defective in mediating lytic replication (Sarisky et al., 1996). Thus, sumo-1 modification of BZLF 1 may be primarily important for its replicative, rather than transcriptional, function.

In the context of the intact virus, lytic EBV infection in cells results in the release of proteins such as Sp100 and Daxx from ND 10 bodies, followed by the release of PML (Bell et al., 2000). Lytic viral replication commences after dispersion of the Sp100 and Daxx proteins, but prior to the onset of PML dispersion, and lytic viral replication complexes are often closely associated with dispersed PML protein aggregates (Bell et al., 2000). These findings suggest that reorganized PML complexes may play a role in promoting lytic EBV replication.

BZLF1 effects on the host immune response

BZLF1 plays a key role in attenuating the host immune response to lytic viral infection. PML bodies are required for an efficient antiviral effect of interferon alpha in herpes simplex virus 1 infection (Chee et al., 2003), suggesting that BZLF 1-mediated dispersion of PML bodies may protect lytic EBV infection from interferon alpha. BZLF 1 also strongly inhibits transcription of the gene encoding an essential component of the interferon gamma receptor, and thereby abrogates interferon gamma signaling in host cells (Morrison et al., 2001). This ability of BZLF 1 to inhibit interferon gamma signaling is likely important not only for protecting the virus from the immunostimulatory effects of interferon gamma (including induction of MHC class Ⅱ and IRF -1), but may also be required for epithelial cell-derived virus to efficiently infect B cells. Virus produced in cells expressing MHC class Ⅱ, which would normally be induced by gamma interferon in infected epithelial cells if BZLF 1 did not prevent this, preferentially infects epithelial cells, whereas virus produced in cells not expressing MHC class Ⅱ preferentially infects B cells (Borza and Hutt-Fletcher, 2002). In addition to preventing interferon gamma signaling, BZLF 1 was recently shown to directly interact with, and inhibit the function of, IRF 7 (Hahn et al., 2005). AS IRF 7 augments production of type Ⅰ interferons, the interaction between BZLF 1 and IRF 7 no doubt helps the virus to attenuate the anti-viral effects of interferon alpha and beta in lytically infected cells.

Tumor necrosis factor alpha (TNF-alpha) is another important antiviral cytokine inhibited by BZLF 1 expression in host cells. TNF -alpha not only activates expression of a number of important inflammatory genes (through its effects on NF -KB), but also induces cellular apoptosis. As TNF -alpha production is an immediate, and important component of the host immune response to viral infection, not surprisingly many viruses encode proteins that limit the effects of TNF -alpha in the infected host cell. In the case of lytic EBV infection, BZLF 1 dramatically inhibits the activity of the promoter for the gene encoding the major TNF-alpha receptor (TNF-R1) (Morrison et al., 2004). Since TNF -R1 is a fairly short-lived protein, this reduction in TNF -R1 transcription results in dramatically decreased expression of the TNF -R1 protein. Thus, TNF -alpha cannot activate transcription of important downstream target genes such as ICAM -1 in BZLF 1-expressing cells, and is also unable to induce cellular apoptosis (Morrison et al., 2004). The exact mechanism(s) by which BZLF 1 inhibits transcription of the genes encoding the TNF -R1 and interferon gamma receptors has not yet been defined.

Complex interactions between BZLF 1 and the NF -KB transcription factor are also involved in inhibiting expression of many different NF -KB dependent cellular genes involved in the host immune response. BZLF 1 interacts directly with the p65 component of NF -KB, and this interaction inhibits the transcriptional function of BZLF 1 (Gutsch et al., 1994; Hong et al., 1997). However, recent evidence suggests that BZLF 1 also potently inhibits NF -KB-dependent activation of promoters (Keating et al., 2002; Morrison and Kenney, 2004), and decreases NF -KB binding to promoters in the context of the intact cellular genome (Morrison and Kenney, 2004). Thus, the IL -1 cytokine cannot activate NF -KB-responsive cellular genes in the presence of BZLF 1, even though the upstream components of the IL -1 signaling pathway appear to be unaffected by BZLF 1 (Morrison and Kenney, 2004). Somewhat paradoxically, however, since one of the NF -KB responsive genes inhibited by BZLF 1 is I-kappa B (IK-B) (Morrison and Kenney, 2004), and the IK -B protein normally acts to retain NF -KB in the cytoplasm in an inactive form, BZLF 1-expressing cells actually have a very high level of nuclear NF -KB (Morrison and Kenney, 2004). This BZLF 1-mediated translocation of NF -KB into the nucleus may act to negatively regulate BZLF 1 transcriptional function and hence promote viral latency in situations where BZLF 1 expression is limiting relative to the amount of nuclear NF-KB.

Finally, BZLF 1 also regulates cellular cytokine expression in ways that would be anticipated to protect the virus. As discussed previously, BZLF 1 stimulates expression of both TGF -beta, and IL -10 (Cayrol and Flemington, 1995). Both TGF -beta and IL -10 have immunomodulatory effects that would be expected to attenuate the cytotoxic T cell response directed against the virus. In addition, BZLF 1 inhibits expression of MHC class Ⅰ on cells (Keating et al., 2002; Mahot et al., 2003). This latter effect may be at least partially mediated through BZLF 1 effects on NF -KB, although other pathways appear to be involved, as well (Keating et al., 2002). The immunomodulatory effects of BZLF 1 are summarized in Fig. 25.6.

Fig. 25.6. Inhibitory effects of BZLF 1 on host immunity.

Fig. 25.6

Inhibitory effects of BZLF 1 on host immunity. BZLF 1 activates transcription of the immunosuppressive cytokines, TGF -beta and IL -10, while decreasing transcription of the receptors for interferon gamma and TNF -alpha. BZLF 1 inhibits the transcriptional (more...)

The role of BRLF 1 in lytic induction

Expression of the EBV immediate–early protein, BRLF 1, also induces lytic EBV infection in a subset of latently infected cell lines (Feederle et al., 2000; Ragoczy et al., 1998; Zalani et al., 1996; Westphal et al., 1999). Interestingly, the BRLF 1 homologue in KSHV (ORF50) is the major inducer of lytic infection for this virus. Even in cell lines (such as the Raji Burkitt lymphoma line) where BRLF 1 expression by itself is not sufficient to disrupt viral latency, it is clear that BRLF 1 expression is required (in concert with BZLF 1) to activate many early lytic genes, including BMRF 1 (Feederle et al., 2000; Ragoczy and Miller, 1999). Consistent with this, the BRLF 1 gene product is essential for lytic viral replication (Feederle et al., 2000). Like BZLF 1, BRLF 1 also binds directly to the EBV oriLyt (Gruffat and Sergeant, 1994; Hammerschmidt and Sugden, 1988). In contrast to the importance of certain oriLyt ZRE sites, the BRLF 1 binding sites in oriLyt are not absolutely essential for oriLyt replication, at least in plasmid-based replication assays (Fixman et al., 1992).

The primarily nuclear BRLF 1 gene product is a transcriptional activator that contains an amino-terminus DNA binding domain and homodimerization domain (Manet et al., 1991) and a carboxy-terminal transcriptional activation domain (Hardwick et al., 1988, 1992)(Fig. 25.7). The transcriptional activator domain of BRLF 1 interacts directly with TBP and TFIIB (Manet et al., 1993). BRLF 1 also interacts directly with the histone acetylase, CREB -binding protein (CBP) (Swenson et al., 2001). BRLF 1 activates lytic EBV gene promoters through at least two different mechanisms. BRLF1 binds directly to a GC -rich motif (consensus GGCCN 7GTGGTG) which is present in the promoters of at least three early EBV genes, SM (formerly called BMLF 1), BHRF 1, and BMRF 1 (Gruffat et al., 1990; Gruffat et al., 1992; Gruffat and Sergeant, 1994; Kenney et al., 1989a; Quinlivan et al., 1993). In the case of the BHRF 1 and SM promoters, the BRLF 1-binding motifs function as powerful enhancer elements in the presence of the BRLF 1 protein (Kenney et al., 1989a; Cox et al., 1990; Chevallier-Greco et al., 1989). In the case of the BMRF 1 promoter, BRLF 1 by itself induces little activation, but cooperates with BZLF 1 to produce efficient activation of this promoter (Holley-Guthrie et al., 1990; Quinlivan et al., 1993).

Fig. 25.7. EBV BRLF 1 immediate–early protein.

Fig. 25.7

EBV BRLF 1 immediate–early protein. Domains in the BRLF 1 protein that mediate dimerization, DNA -binding and trans-activation functions are shown.

In contrast, BRLF 1 activates its own promoter (Rp) and the BZLF 1 promoter (Zp) through mechanisms that do not involve direct DNA binding of BRLF 1 to these promoters. BRLF 1 stimulation of its own promoter requires the Rp Sp1 motifs (Ragoczy and Miller, 2001; Liu and Speck, 2003), although it is not yet known exactly how (or if) BRLF 1 regulates cellular factors binding to the Sp1 motif. BRLF 1 stimulation of the Zp promoter requires the ZII motif, and is likely mediated by BRLF 1 activation of the c-jun and ATF -2 transcription factors (Adamson et al., 2000). BRLF 1 expression in cells activates the stress Map kinases (p38 and c-jun N-terminal kinase) (Adamson et al., 2000), as well as PI 3 kinase (Darr et al., 2001), and inhibition of either p38 stress Map kinase, or PI 3 kinase, activity abolishes the ability of BRLF 1 to activate BZLF 1 transcription, or disrupt viral latency (Adamson et al., 2000; Darr et al., 2001). BRLF 1 stimulation of the viral DNA polymerase promoter is also mediated through an indirect mechanism, involving USF and E2F-1 binding motifs (Liu et al., 1996).

The inability of BRLF 1 expression by itself to induce fully lytic gene expression in certain cell lines, such as Raji, primarily reflects the inability of BRLF 1 to activate BZLF 1 transcription in these cell lines (Zalani et al., 1996). In Raji cells, BRLF 1 by itself efficiently activates an early promoter (SM promoter) that is directly bound by BRLF 1, but cannot activate Zp, or an early viral promoter, BMRF 1, which requires the combination of BZLF 1 and BRLF 1 for activation (Ragoczy and Miller, 1999). Why BRLF 1 induces BZLF 1 transcription in some latently infected cell lines, but not others, is not currently understood.

BRLF1-knockout virus phenotype

A BRLF 1-deleted knockout virus has been made and its phenotype studied in 293 cells as well as primary B cells (Feederle et al., 2000). As is the case for the BZLF 1- knockout virus, the BRLF 1-knockout virus immortalizes primary B cells with efficiency similar to wild-type virus, but early-passage B cell lines obtained with BRLF 1-deleted virus are impaired for producing lymphoproliferative disease in SCID mice (Feederle et al., 2000; Hong et al., 2005a,b). In 293 cells, the BRLF 1-knockout virus is unable to enter the lytic form of infection, or express the BZLF 1 immediate–early gene, unless the BRLF 1 gene product is supplied in trans. These results confirm that the BRLF 1 gene product is an important and essential activator of the Zp IE promoter, and that many early viral promoters require both BZLF 1 and BRLF 1 functions for activation in the context of the intact viral genome. As discussed later, this particular BRLF 1-knockout virus was subsequently discovered to be unable to express an early EBV gene product, BRRF 1. The phenotype of the “BRLF1-knockout” virus in certain cell types is also partially due to the loss of BRRF 1 expression, which cooperates with BRLF 1 to activate transcription of BZLF 1 (Hong et al., 2004). Nevertheless, it is clear that the BRLF 1 gene product is essential for fully lytic EBV gene expression, as well as lytic viral replication, in all cell lines tested to date (Feederle et al., 2000; Hong et al., 2004).

There is emerging evidence that BRLF 1 may be able to activate transcription of a subset of late viral genes even in the absence of viral replication. In 293 cells containing the BZLF 1-knockout virus, BRLF 1 induces expression of a subset of “late” genes, although the virus cannot replicate due to the lack of BZLF 1 expression (Feederle et al., 2000). BRLF 1 also activates some late genes in Raji cells, in which the viral genome is unable to replicate (Ragoczy and Miller, 1999). The exact mechanism by which BRLF 1 activates late genes has not been well defined. Assuming that BRLF 1 also activates certain late genes when expressed at a physiologic level in the context of the intact viral genome, the regulation of EBV late gene transcription may be fundamentally different from that of late viral genes in the alpha herpesviruses. The ability of BRLF 1 to activate certain viral late genes is modulated by the BZLF 1 protein (EI-Guindy and Miller, 2004).

BRLF1 activation of the cellular fatty acid synthase gene

Recent evidence suggests that BRLF 1 activation of the cellular gene, fatty acid synthase (FAS), may be an essential component in BRLF 1-mediated induction of lytic EBV infection (Li et al., 2004). The FAS enzyme is required for the synthesis of many different lipids, including palmitate, and high-level expression of FAS is normally restricted to fat cells and liver cells. BRLF 1 robustly activates FAS gene expression in host cells, and this effect is lost in the presence of p38 kinase inhibitors. Of potential therapeutic interest, agents known to specifically inhibit the FAS enzyme (cerulenin and C75) also inhibit the ability of transfected BRLF 1 to induce the lytic form of EBV gene expression, including induction of BZLF 1 transcription (Li et al., 2004). In contrast, FAS inhibitors do not affect the ability of transfected BZLF 1 (driven by a strong heterologous promoter) to induce lytic EBV gene expression. These results suggest cellular FAS activity is required for BRLF 1 activation of the BZLF 1 promoter (Zp). Exactly why this is the case remains unknown, but may reflect the fact that palmitoylation of proteins is often required for entry of these proteins into lipid rafts, and the ability of these proteins to initiate signal transduction cascades. Since FAS inhibitors also repress anti-IgG induction of lytic EBV in Akata cells, and the constitutively lytic EBV infection which occurs in AGS (gastric carcinoma) cells (Li et al., 2004), they could potentially be developed as a completely novel approach for inhibiting the earliest aspects of lytic EBV infection in patients.

BRLF1 cell cycle effects

Like BZLF 1, BRLF 1 also profoundly affects the regulation of the host cell cycle. BRLF 1 expression results in an increased number of cells in S-phase in both primary human fibroblasts and HeLa cells, and this effect is associated with dramatically increased E2F-1 expression (Swenson et al., 1999). In addition, since BRLF 1 activates the promoter of the viral DNA polymerase gene through an E2F-1 site (rather than a direct binding mechanism) (Liu et al., 1996), the ability of BRLF 1 to increase the level of E2F-1 in the host cell may be required for efficient transcription of the viral DNA polymerase (and consequently efficient lytic viral replication). BRLF 1 also interacts directly with the tumor suppressor protein, Rb (Zacny et al., 1998). Since Rb plays an essential role in regulating the host cell cycle, the interaction between Rb and BRLF 1 may be a mechanism by which BRLF 1 activates cell cycle progression.

Early lytic EBV gene regulation

The expression of the two immediate–early proteins, BZLF 1 and BRLF 1, allows the subsequent expression of viral early genes. In the context of the intact viral genome, transcriptional activation of many early promoters, such as BMRF 1, requires that both the BZLF 1 and the BRLF 1 gene products be expressed (Feederle et al., 2000; Cox et al., 1990). The most extensively studied early lytic EBV promoter is the BMRF 1 gene promoter. This promoter contains two BZLF 1 binding motifs, as well as one BRLF 1 binding site (Holley-Guthrie et al., 1990; Quinlivan et al., 1993). In reporter gene assays in EBV -negative cells, BZLF 1 alone efficiently activates the BMRF 1 promoter (an effect which requires that both of the ZRE sites be present), whereas the BRLF 1 gene product by itself at most modestly activates the BMRF 1 promoter. In some cell types, but not others, the combination of BZLF 1 and BRLF 1 together is significantly more effective than BZLF 1 alone at activating BMRF 1 reporter gene plasmids (Holley-Guthrie et al., 1990). In cells containing the latent form of EBV infection, all data to date suggest that the combination of both BZLF 1 and BRLF 1 is required for significant activation of BMRF 1 transcription, regardless of cell type (Adamson and Kenney, 1998; Feederle et al., 2000; Ragoczy and Miller, 1999; Zalani et al., 1996).

Nevertheless, the relative importance of the BZLF 1 versus BRLF 1 gene products for activation of early gene transcription in the context of the intact viral genome appears to be promoter-specific. The early SM promoter, which contains at least one BZLF 1 binding motif, as well as a BRLF 1 binding site, can be activated by transfected BRLF 1 in a cell line (Raji) where BRLF 1cannot induce BZLF 1 expression from the endogenous viral genome (Ragoczy and Miller, 1999; Swenson et al., 2001). Thus the SM early promoter can be activated by BRLF 1 alone, even in the context of the intact viral genome. Two other early promoters, BHLF 1 and BHRF 1, which are divergent promoters contained within the lytic EBV origin of replication, oriLyt, have been extensively studied in vitro, but much less so in the context of the intact virus. There are two strong BRLF 1 binding sites located between the BHLF 1 and BHRF 1 promoters in oriLyt (Gruffat and Sergeant, 1994); in reporter gene assays, the BHRF 1 promoter appears to be more responsive to BRLF 1 than the BHLF 1 promoter (Hardwick et al., 1988; Cox et al., 1990). Both the BHLF 1 and BHRF 1 promoters have two or more BZLF 1 sites and are activated by BZLF 1 alone in reporter gene assays (Hardwick et al., 1988). The relative importance of BZLF 1 vs. BRLF 1 for BHLF 1 vs. BHRF 1 transcriptional activation in the context of the intact virus has not been well studied.

Early lytic EBV gene products

Replication proteins

Many of the early lytic EBV gene products are directly involved in mediating the lytic form of viral replication. The six core viral replication proteins which mediate lytic viral replication include the catalytic component of the viral DNA polymerase (BALF5), the DNA polymerase processivity factor (BMRF1), the single-stranded DNA binding protein (BALF2), helicase (BBLF4), primase (BSLF1), and primase-associated protein (BBLF2/3)(Fixman et al., 1992). In addition, some early EBV genes encode enzymes involved in deoxynucleotide metabolism, including the viral thymidine kinase gene (BXLF1) (Littler et al., 1986), the viral dUTPase gene (Fleischmann et al., 2002) and viral ribonucleotide reductase (BORF2 and BARF 1). From the results of studies performed on the analogous genes in HSV -1, it is likely that these early EBV gene products are required for efficient production of nucleotide substrates in non-replicating cells. In addition, the EBV -encoded thymidine kinase (TK) function may be required for the therapeutic effect of the antiviral drug, acyclovir, as the EBV TK is a deoxypyrimidine kinase that phosphorylates a broad range of nucleoside analogues (such as acyclovir) not recognized efficiently by the cellular TK (Littler and Arrand, 1988).

Transcription factors

There are at least two viral transcription factors encoded by early lytic EBV genes. Interestingly, the BMRF 1 gene product (Cho et al., 1985) functions not only as the viral DNA polymerase-processivity factor (Tsurumi et al., 1993, 1994), but also as a transcription factor (Zhang et al., 1997, 1999a). BMRF 1 activates transcription of the oriLyt promoter, BHLF 1, and the BMRF 1-responsive region of this promoter is contained within a GC -rich domain (the downstream essential domain) that is also essential in cis for oriLyt replication (Zhang et al., 1997). The exact mechanism by which BMRF 1 activates oriLyt transcription is not yet known. The transcriptional function of BMRF 1 requires its carboxy-terminal domain (Zhang et al., 1999a), which is not required for DNA polymerase-processivity function in vitro (Kiehl and Dorsky, 1991; 1995).

Another early lytic viral transcription factor is the BRRF 1 gene product (Hong et al., 2004). The BRRF 1 gene is encoded by the opposite strand of the first intron of the BRLF 1 immediate–early gene (Segouffin-Cariou et al., 2000). BRRF 1 enhances the transcriptional function of c-jun, and activates the BZLF 1 immediate–early promoter through the CRE (“ZII”) motif (Hong et al., 2004). BRRF 1 activation of c-jun transcriptional function is associated with increased c-jun phosphorylation, although the precise mechanism for this effect is not yet known. Although BRRF 1 expression alone is not sufficient to activate BZLF 1 transcription from the latent viral genome, BRRF 1 function is required for efficient BRLF 1-mediated activation of Zp in some latently infected cell lines (Hong et al., 2004). A knockout virus originally thought to specifically delete the BRLF 1 gene product (Feederle et al., 2000) was subsequently shown to be defective in BRRF 1 transcription, and rescue of the fully lytic phenotype of this virus requires both the BRLF 1 and BRRF 1 gene products in trans (Hong et al., 2004). KSHV contains a gene that is homologous to BRRF 1 in the analogous position of the viral genome and has a similar function (Gonzalez et al., 2005).

SM: a protein that regulates RNA transport and stability

In contrast to the latent and IE genes of EBV, the early and late genes often contain no introns. RNA derived from intronless genes is often unstable in cells. The early lytic gene product, SM (previously known as BMLF 1) (Cook et al., 1994), is an RNA -binding protein which plays an important role in increasing the stability of intronless lytic viral transcripts, as well as promoting the transport of such messages from the nucleus to the cytoplasm (Kenney et al., 1989c; Gruffat et al., 2002a; Hiriart et al., 2003a,b; Boyle et al., 1999, 2002; Buisson et al., 1989, 1999; Chen et al., 2001b; Farjot et al., 2000; Ruvolo et al., 1998, 2001, 2004; Semmes et al., 1998). The SM protein thus acts to create a cellular environment in which viral lytic (intronless) messages are preferentially expressed over cellular (intron-containing) messages. Recent studies of an SM -knockout virus confirm that the SM gene product is essential for efficient viral replication and virion production (Gruffat et al., 2002a). Another recently described function of the SM protein is its ability to inhibit PKR activation (Poppers et al., 2003), which appears to be distinct from its effect on RNA. PKR inhibition by SM would presumably allow the virus to escape the inhibitory effect of PKR activation on protein translation. In contrast, SM actually enhances the expression of STAT 1 (Ruvolo et al., 2003); it is as yet unclear how this activation benefits the virus.

Proteins that inhibit cellular apoptosis and immune evasion

EBV also encodes an early lytic gene product, BHRF 1, which inhibits cellular apoptosis. BHRF 1 is a homologue of the cellular anti-apoptosis gene, BCL -2, and like BCL -2 inhibits apoptosis in response to a number of different stimuli (Henderson et al., 1993; Tarodi et al., 1994). The ability of BHRF 1 to prevent cellular apoptosis very likely increases the efficiency of lytic EBV viral replication by preventing the death of the host cell prior to the completion of replication.

The BARF 1 early gene encodes a soluble receptor for colony-stimulating factor 1 (Strockbine et al., 1998). BARF 1 inhibits the differentiating and proliferative effects of this cytokine on monocytes/macrophages (Strockbine et al., 1998), and decreases alpha-interferon secretion from monocytes/macrophages (Cohen and Lekstrom, 1999). Thus, BARF 1 may be important for protecting the virus from antiviral effects mediated through monocytes and macrophages.

A viral kinase

The BGLF 4 early gene encodes a viral kinase (EBV-PK) which is homologous to the UL 97 gene product of cytomegalovirus. In addition to autophosphorylation, at least two other EBV proteins, BMRF 1 and the EBNA Leader protein, are phosphorylated by the BGLF 4 kinase (Kato et al., 2001, 2003; Chen et al., 2000; Gershburg and Pagano, 2002), although the effect of this phosphorylation on BMRF 1 and EBNA Leader protein function is not currently defined. In addition, like the CMV (UL97) and HSV (UL13) homologues, BGLF 4 also phosphorylates the cellular protein, translation elongation factor 1 delta (Kato et al., 2001). To date the kinase motifs recognized by the BGLF 4 and homologous proteins encoded by CMV and HSV appear to be similar to cdc2 kinase motifs (Kawaguchi et al., 2003). Although the exact function of the BGLF 4 kinase during lytic EBV replication has not yet been clearly defined, as is the case with the CMV (but not HSV) homologue, BGLF 4 expression in cells results in phosphorylation of the antiviral nucleoside analogue, ganciclovir, converting it to the active form (Marschall et al., 2002). Thus, ganciclovir (as well as acyclovir) can be used to inhibit lytic EBV replication.

Viral replication

Lytic EBV replication probably occurs through a rolling-circle mechanism and involves the formation of head-to-tail concatamers of the genome. Lytic EBV replication requires the lytic origin of replication (oriLyt) in cis, and the viral core replication proteins (BALF5, BMRF 1, BBLF 2, BBLF 4, BSLF 1, and BBLF 2/3) in trans (Fixman et al., 1992, 1995). Each of these core replication proteins is absolutely essential for the lytic form of replication, and a number of these core replication proteins directly interact with one another, presumably allowing formation of a large replication initiation complex (Daibata and Sairenji, 1993; Fujii et al., 2000; Gao et al., 1998; Liao et al., 2001). In addition, the BZLF 1 IE protein is absolutely required for replication, even when the core replication proteins are expressed under strong constitutive heterologous promoters. BZLF 1 interacts directly with the core replication proteins, BMRF 1 and BBLF 4 (Zhang et al., 1996; Liao et al., 2001).

EBV DNA polymerase activity is mediated by the catalytic component of the enzyme (BALF5) in conjunction with the polymerase processivity factor (BMRF1). The catalytic component of the polymerase also has 3′ -to 5′ proofreading exonuclease activity (Tsurumi et al., 1993). The EBV polymerase, in contrast to cellular polymerase, is active in high-salt (100 mM ammonium sulfate) (Tsurumi et al., 1993).

There are usually two copies of oriLyt in the EBV genome, although viral strains that contain only one copy of oriLyt (such as B95–8) seem to replicate equally well. The minimal oriLyt contains the divergent promoters of two EBV early genes, BHRF 1 and BHLF 1, as well as binding sites for the two EBV immediate-early proteins, BZLF 1 and BRLF 1 (Hammerschmidt and Sugden, 1988) (Fig. 25.8). Two domains of oriLyt are absolutely essential for replication. The “upstream” essential domain contains two binding sites for BZLF 1, and BZLF 1 binding to these sites is required for replication (Schepers et al., 1993a,b, 1996). The “downstream” essential domain is a GC -rich sequence bound by the Sp1, Sp3 and ZBP -89 cellular transcription factors (Baumann et al., 1999; Gruffat et al., 1995; Schepers et al., 1993b), and this region also mediates BMRF 1 transcriptional effects (Zhang et al., 1997). ZBP -89 binding appears to be particularly important for oriLyt replication, and ZBP -89 over-expression in cells enhances replication of an oriLyt containing plasmid (Baumann et al., 1999). In addition, the ability of the downstream essential domain to form a triple helix DNA structure may be required for oriLyt replication (Portes-Sentis et al., 1997).

Fig. 25.8. Cis-acting elements of oriLyt.

Fig. 25.8

Cis-acting elements of oriLyt. OriLyt overlaps the divergent promoters of two early genes, BHLF 1 and BHRF 1. The locations of BZLF 1 (ZRE) and BRLF 1 (RRE) binding sites, as well as the binding sites of cellular transcription factors ZBP -89, Sp1, and (more...)

Once replication is completed, the virus is clipped within the terminal repeat region of the genome into a linear, unit-length genome and then packaged into virion particles. The EBV terminal repeats are sufficient to allow packaging of plasmids in cis (Zimmermann and Hammerschmidt, 1995); however, the viral proteins that mediate the cleavage and packaging functions have not been identified.

Late viral gene regulation

Late EBV genes are traditionally defined as genes that are expressed after the onset of viral replication; expression of late genes is inhibited by agents that prevent viral DNA replication. Relatively little is known about the regulation of late EBV promoters. Unlike the early viral promoters, the late promoters do not usually contain BZLF 1 or BRLF 1 binding sites. Assuming that EBV late viral gene promoters are regulated in a similar manner as the late viral gene promoters in herpes simplex virus, it would be anticipated that EBV late promoters are primarily activated in cis by viral replication, and that this effect requires only a small region of the upstream promoter sequences. However, recent studies using the BZLF 1- and BRLF 1- knockout viruses have indicated that the requirement for viral replication may not be as absolute for late gene expression in gamma herpesviruses as it appears to be the case for alpha herpesviruses. For example, expression of the BRLF 1 immediate-early protein in cells containing the BZLF 1-knockout virus induces expression of a subset of “late” genes in the absence of viral replication (Feederle et al., 2000). BRLF 1 can also activate some late genes in Raji cells in the absence of viral replication (Ragoczy and Miller, 1999). In reporter gene assays, certain late promoters are also activated in a replication-independent manner (Serio et al., 1997, 1998).

Late viral proteins

Many late genes encode structural viral proteins, including the nucleocapsid proteins that make up the virion particle. The viral glycoproteins which mediate EBV binding and fusion to the cellular receptor and co-receptor (gp350/220, gp85, gp42, gp25) are also encoded by late genes and are further discussed in Chapter 23. In addition, EBV encodes at least one late viral gene product, vIL-10 (BCLF1), that is likely important for protecting the virus from the host immune response. The vIL-10 gene product is a homologue of cellular IL -10 (Hsu et al., 1990) and shares its ability to potently repress the cytotoxic T-cell response. Therefore, secretion of viral IL -10 from lytically infected cells would be expected to protect the virus from this response (Salek-Ardakani et al., 2002). In addition, as both cellular and viral IL -10 function as B-cell growth factors, the lytically infected pool of EBV -positive B cells could potentially support the growth of the latently infected pool through a paracrine mechanism involving the release of viral IL -10 (Miyazaki et al., 1993; Stuart et al., 1995; Rousset et al., 1992). An EBV mutant virus deleted in the viral IL -10 gene was not found to be defective in B-cell transformation in vitro or lymphoma formation in mice (Swaminathan et al., 1993); however, the lack of an obvious phenotype in this mutant virus may reflect the finding that BZLF 1 induces cellular IL -10, and that the functions of cellular and viral IL -10 are redundant. Perhaps not surprisingly, one or more late viral gene products also appear to inhibit apoptosis, suggesting that prevention of cellular apoptosis is important for efficient EBV infection throughout the lytic replication cycle (Inman et al., 2001). A late viral protein which has homology to the anti-apoptotic cellular Bcl-2 protein is encoded by the BALF 1 gene, but whether this protein inhibits or activates apoptosis remains controversial (Marshall et al., 1999; Bellows et al., 2002).

Viral assembly and egress

At the end of the lytic replication cycle, the structural proteins involved in initiation of infection must be reassembled into the mature virion. Current models of herpesvirus assembly and egress propose that capsids are first built around a scaffold in the nucleus, that the scaffold is then lost to make room for packaging of the genome, and that the completed nucleocapsids, associated with at least some of the tegument proteins, bud through the inner nuclear membrane into the perinuclear space, acquiring a first envelope in the process. De-enveloped nucleocapsids are then delivered to the cytoplasm by fusion with the outer nuclear membrane or that of the endoplasmic reticulum. The final envelope and additional or different tegument proteins are acquired during rebudding into a cytoplasmic compartment, probably the trans-golgi network, that puts the virus back into a later stage of the exocytic pathway for release by exocytosis (for review see Mettenleiter, 2002). This model has been developed primarily from the study of alphaherpesviruses. However, the probability that EBV follows a similar envelopment, de-envelopment, reenvelopment pathway is supported by observations of the B-958 strain of EBV where higher levels of glycoprotein gB are seen in the nuclear membrane of cells producing virus than in mature enveloped virions and there is a reverse distribution of the major virion glycoprotein gp350/220 (Gong and Kieff, 1990). This is, in turn, dependent on a second soluble nuclear protein, BFLF 2 (Gonella et al., 2005; Lake and Hutt-Fletcher, 2004). These two proteins are conserved throughout the herpesvirus family and appear to have similar functions in each. In addition, however, the EBV glycoprotein gB also plays a role in nuclear egress (Herrold et al., 1995). This function is not shared by the gB homologs of the alpha and betaherpesviruses. One possibility is that gB, known to be essential for glycoprotein-mediated cell fusion (Haan et al., 2001), is required for the fusion of the first virus envelope with the outer nuclear membrane. Acquisition of the second and final envelope requires a complex of glycoproteins gN and gM (Lake and Hutt-Fletcher, 2000), but little more is known about the process aside from the provocative finding that loss of the transforming protein LMP 1 severely impairs virus release (Ahsan et al., 2005). These final and critical stages in EBV replication remain poorly understood.

Treatment of lytic EBV infection

Lytic EBV infection is inhibited by both acyclovir and ganciclovir (Lin and Machida, 1988; Datta et al., 1980; Lin et al., 1987; Meerbach et al., 1998). The nucleoside analogues, acyclovir and ganciclovir, are phosphorylated to their active forms in lytically infected cells, presumably due to the effects of the two viral kinases, EBV thymidine kinase and BGLF 4 (Lin et al., 1986; Moore et al., 2001; Marschall et al., 2002). As acyclovir is less toxic than ganciclovir in patients, acyclovir is the preferred agent for treating the one disease that is definitely due to lytic EBV infection, oral hairy leukoplakia. The antiviral drug foscarnet, which inhibits the viral DNA polymerases of all known herpesviruses and effectively inhibits lytic EBV replication in vitro (Datta and Hood, 1981), could also theoretically be used to treat lytic EBV infection in patients. However, as foscarnet is quite toxic, and there is no evidence to date that EBV strains resistant to acyclovir or ganciclovir are a clinical problem, foscarnet is not currently used to treat lytic EBV infection in patients. There is no convincing evidence that inhibition of lytic EBV infection in immunocompetent patients with infectious mononucleosis shortens or ameliorates this illness (Torre and Tambini, 1999). Whether acyclovir or ganciclovir treatment is useful in treating early polyclonal EBV -associated lymphoproliferative disease in post-transplant patients is somewhat controversial (Cohen, 2000; Oertel et al., 1999). As lytic EBV gene products are not currently thought to be required for immortalization and growth of B cells in vitro (Feederle et al., 2000), it is not clear that preventing this form of infection in vivo would slow the growth of EBV -immortalized B cells in patients. There is more convincing evidence suggesting that anti-viral prophylaxis reduces the subsequent development of EBV -associated lymphoproliferative disease in transplant recipients (Farmer et al., 2002; Malouf et al., 2002; Green et al., 2001; Fong et al., 2000; McDiarmid et al., 1998; Darenkov et al., 1997). In this case, inhibition of lytic EBV infection presumably reduces the pool of latently infected B cells, thereby decreasing the probability that one or more of these cells eventually becomes malignant.

Lytic induction as a strategy for treating EBV -positive tumors

Finally, the purposeful induction of lytic EBV infection in EBV -positive tumor cells is increasingly being explored as a potential way to selectively kill EBV -infected tumor cells (Gutierrez et al., 1996; Israel and Kenney, 2003; Westphal et al., 1999). Theoretically, EBV -positive tumors containing the latent forms of viral infection could be switched to the lytic form of infection by either inducing expression of the EBV immediate–early genes from the endogenous viral genome in tumors, or by using gene delivery methods to express either of the EBV immediate–early proteins in tumor cells. When adenovirus vectors expressing either the BZLF 1 or BRLF 1 gene products are injected directly into EBV -positive nasopharyngeal carcinoma tumors grown in nude mice, tumor growth is inhibited, whereas control adenovirus vectors have no effect (Feng et al., 2002b). In addition, certain cytotoxic therapies, including some chemotherapy agents and gamma irradiation, have been found to induce lytic EBV gene transcription in at least a portion of tumor cells (Feng et al., 2002a, 2004; Roychowdhury et al., 2003; Westphal et al., 2000). Lytic induction by cytotoxic agents is mediated through activation of the two EBV immediate-early promoters and requires the PI 3 kinase, p38 kinase, and MAP kinase pathways (Feng et al., 2002a, 2004). Specific transcription factor binding sites in both the BZLF 1 promoter (ZI and ZII), and the BRLF 1 promoter (EGR-1) are also required (Feng et al., 2004). Agents that inhibit histone deacetylases (such as butyrate compounds) have also been shown to enhance the amount of lytic EBV infection in some mouse tumor models (Westphal et al., 2000).

Lytic induction strategies are most effective for inhibiting tumor growth when combined with the antiviral drug, ganciclovir (Mentzer et al., 1998; Feng et al., 2002a, 2004; Faller et al., 2001; Roychowdhury et al., 2003; Westphal et al., 2000). In cells containing the lytic (but not latent) type of EBV infection, virally encoded kinases (BGLF4 and the viral thymidine kinase) are expressed which phosphorylate the nucleoside analogue, ganciclovir, converting it to its active cytotoxic form (Moore et al., 2001). Phosphorylated ganciclovir inhibits not only viral DNA replication, but also inhibits the host cell DNA replication, and is thus cytotoxic. Furthermore, phosphorylated ganciclovir can be transferred into nearby cells that are unable to phosphorylate ganciclovir (i.e., tumor cells with latent EBV infection), and thus induce “bystander” killing. As chemotherapy and irradiation induce lytic infection in only a portion of tumor cells, the combination of these agents with ganciclovir is much more effective than either agent alone for treating EBV -positive tumors in mouse models (Feng et al., 2002a, 2004). Whether ganciclovir will be effective in combination with lytic induction strategies for treating EBV -positive human tumors is currently being investigated (Faller et al., 2001; Mentzer et al., 2001).

Unresolved issues for the future

The discovery in 1985 that BZLF 1 activates lytic EBV expression in latently infected cells was a true milestone in our understanding of lytic EBV gene regulation. Since then, many further advances have been made. However, many questions remain. For example, numerous EBV -encoded microRNAs were recently discovered, including microRNAs which overlap the lytic BHRF 1 (anti-apoptosis) and BALF 5 (viral DNA polymerase) genes (Shen and Goodman, 2004; Pfeffer et al., 2004) as well as multiple microRNAs located in the introns of the latent BART gene (Cai et al., 2006). Since microRNAs generally promote gene silencing by targeting homologous mRNAs for degradation, the EBV -encoded microRNAs may serve to promote the latent form of viral infection. However, at this point relatively little is known regarding the regulation of microRNA formation during EBV infection, and whether these microRNAs do indeed regulate viral gene expression. Furthermore, will EBV, like a growing list of other viruses, encode a mechanism for preventing the effect of host genome-derived, antiviral microRNAs?

We still know very little about the nature of the proteins comprising the viral tegument. Does EBV, like other herpesviruses, have a viral tegument protein which serves to transcriptionally activate the viral IE promoters during primary lytic infection? The availability of modern proteomic techniques should allow us to determine the cellular and viral protein composition of the virion, as well as how lytic EBV infection affects cellular proteins. A proteomic analysis of the virion particle was recently published (Johannsen et al., 2004)

It remains unclear which viral proteins confer sensitivity to commonly used antiviral drugs, including acyclovir and ganciclovir. Are these drugs activated primarily by the viral thymidine kinase, or the viral protein kinase (BGLF4)? Alternatively, is the combination of both of these viral proteins required for nucleoside analogue activation in EBV -infected cells? Can we develop new drugs (potentially fatty acid synthase inhibitors) that would inhibit lytic EBV infection at its earliest step, i.e., expression of the viral immediate-early proteins. If we could completely prevent lytic EBV infection in patients (such as organ transplant recipients) who are highly prone to the development of EBV -associated malignancies, would this reduce the number of EBV -induced tumors?

These are only a few of the many questions that will need to be answered by future investigators. What remains certain is that EBV will remain an important and fascinating pathogen for many years to come.

Acknowledgment

Many thanks to Lindsey Hutt-Fletcher, who wrote the “Viral assembly and egress” section in this chapter.

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