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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 29Effects on apoptosis, cell cycle and transformation, and comparative aspects of EBV with other known DNA tumor viruses

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Molecular Virology Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA USA

The list of human viruses presently known to cause or to contribute to tumor development comprise four DNA viruses, Epstein–Barr virus, certain human papilloma virus subtypes, hepatitis B virus, and Kaposi sarcoma herpesvirus (HHV-8); and two RNA viruses, adult T-cell leukemia virus (HTLV-1) and hepatitis virus C. In addition, while HIV infection is not directly tumorigenic, it increases the incidence of certain tumors.

The purpose of this chapter is to consider EBV and HHV-8 in relation to the known DNA tumor viruses, with particular focus on tumorigenicity.

Viral strategy at the molecular level as a tumor risk factor

Altered genes or environmental factors are usually considered as major risk factors for tumor development. However, the strategy of certain viruses may constitute a risk factor in itself. Tumor-associated viruses in humans have a survival strategy, like other viruses, aiming to maintain, replicate and propagate their genomes, but some features of this strategy entail a risk to initiate or favor tumor development under certain circumstances. This implies that only a small minority of the infected cells enter the pathway towards a malignant tumor and even fewer succeed.

Three types of virus–host cell interactions may carry a risk

  1. Blocking of late viral functions or blocking the replicative cycle, by mutation or deletion of genetic material, e.g., due to the integration of the viral genome, as exemplified by HPV or adenovirus transformation in vitro.
  2. Infection of cells that are not fully permissive for viral replication, for species or tissue specific reasons. Permissiveness for the early but not the late functions of the viral cycle is particularly dangerous. The early viral proteins may exert continuous proliferation stimulating and/or apoptosis preventing effects. Infection of hamster or guinea pig cells with some of the human adenoviruses and SV40 infection of rodent cells may serve as examples.
  3. Latent viral persistence may subvert normal controls. This can be illustrated by EBV infection of B-lymphocyes in immunodefective hosts.

Early history: up and down

Views on the role of viruses in the etiology of cancer have been polarized between two extreme positions during the major part of the last century. The belief that viruses have nothing to do with cancer was as widespread at certain times, as the suspicion that most and perhaps all tumors are caused by virus at other times. The field started with the discovery of Peyton Rous in 1911 that chicken sarcomas could be transmitted with cell-free filtrates (Rous, 1911). The tumors arose at the site of inoculation and were of the same histological type as the original sarcoma. This created great excitement: the cancer problem was solved! The enthusiasm subsided rapidly, however, when mouse and rat tumor filtrates failed to induce tumors. In retrospect we may see this as the consequence of exaggerated expectations, hasty experiments and increasing lack of confidence. It became the prevalent view that viruses may play a role for tumors in birds, but not in mammals.

Two decades later, Richard Shope (1933) found that benign warts could be transmitted from the wild cottontail to the domestic rabbit by cell-free filtrates. This did not change the climate of opinion. The rabbit was a mammalian but warts were benign tumors, not cancers. Several important points were overlooked by outside commentators, however. The initially benign rabbit papillomas turned occasionally into carcinomas. This could be accelerated by the topical application of chemical carcinogens. The term tumor progression was originally coined by Rous to designate this transition, or, in its generalized form, the process whereby “tumors go from bad to worse.” Later, Leslie Foulds (1958) defined and extended the term. It refers to the development of tumors by multiple, stepwise changes in several “unit characteristics.” Today we see them as distinct phenotypic traits. They are individually variable and reassort independently of each other. Tumor progression can therefore proceed along several alternative pathways and each tumor becomes individually unique from the biological point of view.

The early work on Shope papilloma was also interesting from the immunologist’s point of view. The virally induced warts that did not progress to carcinoma were rejected simultaneously by a systemically acting host response, mediated by lymphocytes, rather than by antibodies. This was the first example of a tumor rejection response that targeted virally encoded proteins in DNA virus transformed cells.

In the 1930s, John Bittner (1936) discovered the milk factor, later called the mouse mammary tumor virus (MMTV). This discovery did not create any major change of opinion either. This may have been due, at least in part, to the way the findings were presented and discussed. The genetically oriented mouse mammary tumor biologists proceed by careful, gradual analysis that fitted the long duration of each experiment (2 years or more). It showed that MMTV could increase the frequency of mammary cancer, but it was neither necessary nor sufficient for tumor induction. Hormonal and genetic factors modified the risk considerably. The role of MMTV as a tumor-susceptibility factor in selectively inbred high cancer strains was readily accepted, but its role as a “tumor virus” remained questionable. It was appreciated, however, that the probability of tumor development could be influenced by multiple factors, including viruses.

Up again, and how!

A major paradigmatic shift occurred in the 1950s. It was triggered by the discovery of the murine leukemia virus by Ludwik Gross (1951) and the polyoma virus by Sarah Stewart and Bernice Eddy(Stewart et al., 1958). Gross found that cell free filtrates prepared from the “spontaneous” leukemias of the high leukemic AKR strain could transmit the disease to a low leukemia strain, C3H. In contrast to many others who failed before him, Gross succeeded for three reasons: his serendipitous use of newborn, less than 24 hours old mice as recipients; his fortuitous choice of C3H, the only low leukemia strain available at the time that happened to be susceptible to the virus carried by the AKR strain, later called the Gross virus; and the dogged persistence of Ludvik Gross in an area where nobody expected positive results.

The scientific community received Gross’s first report with surprise and disbelief. This attitude prevailed for 5 years, until the originator of the AKR strain, Jacob Furth, took pains to repeat Gross’s experiments under the original conditions and with the same recipient C3H subline (Furth et al., 1956). He succeeded, in contrast to others who were less meticulous in their choice of experimental conditions. Furth’s confirmation has led to the immediate acceptance of Gross’s findings. The discovery of the polyoma virus also stemmed from Gross’s work, but in a more indirect fashion. Gross has observed occasional parotid tumors in C3H mice inoculated with AKR leukemia filtrates. He realized that they may have been induced by another virus, provisionally referred to as the parotid tumor agent.

Stewart and Eddy started out on the assumption that Gross’s leukemia virus experiments were correct. Since the virus was apparently quite weak, however, they wished to amplify it by adding the leukemia filtrates to embryonic mouse fibroblast cultures. After a few days culturing, they inoculated the filtered supernatants into newborn mice. The mice developed a wide variety of sarcomas and carcinomas, but no leukemia. Due to its ability to induce many types of tumors, the virus was named polyoma.

Classes of experimental tumor viruses

The viruses so far mentioned fall into three major categories. Rous sarcoma virus belongs to the acute or class Ⅰ RNA tumor viruses. The murine leukemia and the mammary tumor virus fall into the category of chronic or class Ⅲ RNA tumor viruses. The Shope papilloma and the polyoma virus are DNA tumor viruses.

Some interesting generalizations can be made on the basis of this and later experimental work that has identified many additional viruses in all three categories.

All experimentally derived RNA tumor viruses belong to the retrovirus family. They carry their genetic information in RNA. Following their entry into a susceptible target cell, the virally encoded reverse transcriptase rewrites their RNA into proviral DNA that can insert into cellular DNA at random. When virus production is activated again, the proviral DNA is transcribed into RNA. This is followed by viral RNA replication, the production of new viral proteins, the assembly of new viral particles, and their release by budding, but it is not accompanied by any cytopathic effect. Virus production is therefore compatible with cell proliferation.

Activation and transcription of the integrated provirus is an error-prone process. Adjacent cellular DNA may contribute to the RNA sequences carried by the viral particle. In the vast majority of the cases, this has no notable consequences, but occasionally the incorporated cellular sequence may originate from a gene whose activated product can stimulate the entry of the cell into the S-phase. Virus particles that carry such sequences may cause cell proliferation when they infect new recipient cells. The probability that this happens is very low, because every step in the process, from the integration of the virus into the “right place,” through the production of the appropriately (in frame) fused viral–cellular messages, the release and the replication of competent virus and the subsequent new infection of a susceptible cell, are all low probability events. A tumorigenic virus variant is usually generated by the purposeful and often prolonged selection for tumorigenicity This requires great persistence on the part of the investigator. Following the early discovery of the Rous sarcoma virus, it took four decades before new acute or class Ⅰ RNA tumor viruses were isolated that could induce tumors at the site of inoculation and to transform normal into tumor cells in vitro. Following the revival of viral oncology in the 1950s, some 40 such viral strains, carrying about 20 different cell derived oncogenes, as they were to be called, were isolated in rapid succession from fowl, rodent, feline and simian tumors.

class Ⅰ RNA tumor viruses are not known to play any tumorigenic role in nature. This is understandable, because most of them are defective, due to the replacement of essential viral genetic information, by the inserted cellular gene. With only some notable exceptions, they produce crippled virus particles that can only multiply in the presence of complete, but non-transforming “helper virus.”

Chronic or class Ⅲ RNA tumor viruses have no transforming activity in culture. They do not induce tumors at the site of inoculation and carry no cellular oncogenes. Insertion of the proviral DNA in the immediate neighborhood of a cellular oncogene is the most frequent mechanism whereby they contribute to the tumorigenic process. Since the proviral DNA integrates at random, the likelihood of such an insertion is low. Very high level of virus production, accompanied by viremia, is usually mandatory for tumorigenicity. This is the reason why only some mouse strains that can support virus replication and/or are deficient in their immunological responsiveness to the virus, are susceptible to the tumorigenic effect of murine leukemia virus or mammary tumor virus.

Insertion in the neighborhood of a cellular protooncogene is not the only mechanism whereby an RNA tumor virus can initiate tumor development, but other alternatives are less well documented. HTLV-1, or adult T cell leukemia (ATLV) virus, is an example of this. It is believed to stimulate the expansion of preneoplastic cell populations, paving the way to cellular changes that may be more directly involved in the tumorigenic process (Gallo et al., 1983).

In conclusion, the RNA tumor viruses have provided a wealth of information about virus-cell interactions in relation to the tumorigenic process and have led, indirectly, to the discovery of numerous cell division regulating cellular genes. They can be regarded as a model of what can happen, but they give us very little information about what does actually happen in the genesis of human tumors.

The DNA tumor viruses provide a very different picture. They belong to several unrelated virus families. In contrast to the RNA tumor viruses that can replicate in growing cells without killing them, the DNA tumor viruses kill the cells in which they replicate. Their tumorigenic activity depends therefore on the blocking of the lytic viral cycle. This may occur in cells that are non-permissive for the lytic cycle due to their species and/or tissue derivation.

The transforming genes of all DNA tumor viruses are genuine constituents of the viral genome. The number of virally encoded transforming genes varies between one (SV40), two (adeno- and papillomaviruses) and six (EBV). The virally encoded transforming proteins are immunogenic, as a rule. The challenge of viral transformation is met by the immune surveillance of the host. Cells transformed by these viruses grow usually only in immunosuppressed hosts. They represent the major part of the “opportunistic tumors” that arise exclusively or predominantly in congenitally, iatrogenically (as after organ transplantation) or virally (e.g., by HIV) immunosuppressed persons.

Inactivation of Rb and p53 is an important prelude to viral replication, as discussed below. Early after primary infection, DNA tumor viruses induce a round of DNA replication in the recipient cells. This carries the risk of malignant proliferation. The host inhibits the progressive growth of the transformed cells by its immune response, however. While the immunocompetent host rejects virus driven, proliferating cells, the virus goes into hiding. It persists in non-proliferating cells where it is not “seen” by the immune response. The example of EBV provides a particularly interesting “success story” that favors the survival of both the virus and the host. A comparison with the smaller DNA tumor viruses, in the next section highlights both parallels and contrasts.

What does the type of virus-cell interaction tell us about tumorigenic risk?

There is a fundamental difference between the small DNA tumor viruses and the herpesviruses, with regard to potentially tumorigenic interactions with their host cell. Permissiveness for the early but not for the late (lytic) steps of the viral cycle constitute the main risk for the former group. Convergent evolution has provided SV40, the transforming adenoviruses and the tumor associated papillomaviruses with the ability to inactivate two of the main tumor suppressor pathways that involve p53 and Rb, respectively. Inactivation of the same two pathways appears to be mandatory for non-virally related tumor development. As discussed elsewhere in this chapter, this impairs two of the main controlling functions that prevent the replication of cells driven by illegitimately activated oncogenes.

Inactivation of both pathways by the small DNA tumor viruses is part of the viral strategy. Both the integrating and the episomal viruses need to induce an S-phase, as already mentioned, in order to integrate or to establish the appropriate chromosomal–episomal balance, respectively. They also need to protect the activated cell from apoptosis, in order to secure their persistence.

In the natural host cell, the growth stimulating and antiapoptotic effects of these viruses have no lasting consequence, because virus production and cell lysis sets a natural endpoint.

In the case of the tumor associated herpesviruses and particularly of EBV, the possible tumorigenic contributions of the virus need to be considered in relation to the different forms of non-productive virus–cell interactions. EBV has evolved mechanisms to activate and expand its primary host cell population, the human B-lymphocyte. It is also capable of switching off its B-cell activating program and remain latent in long-lived resting memory B cells. The virus can thus use several non-lytic interaction programs, tailored to different B cell subclasses. They lead to different programs of viral expression that also differ in their ability to induce a CD8+ T-cell mediated immune response in the host that prevents the excessive proliferation of the virally transformed immunoblasts.

Unlike the small DNA tumor viruses, where tumorigenicity is favored by the structural or regulatory impairment of the lytic genes, the latent EBV-B cell interaction that occurs without any genetic defect in the virus, is a potential tumor risk in the immunodeficient host. This is due to the fact that, apart from the occasional activation of the viral cycle in EBV-carrying B cells, the interaction is largely non-lytic. In EBV-carrying immunoblastomas that arise in transplant recipients, or in certain congenitally T-cell defective patients (XLP in particular) as well as in part of the AIDS associated lymphomas (with immunoblastic morphology), the interaction of the virus with proliferating immunoblasts is comparable to or identical with the usual interaction of the virus with normal B-cells. The tumorigenic “accident” occurs at the level of the host and not at the level of the virus or the cell.

None of the other EBV-associated B-cell derived malignant lymphomas, such as BL, HL, or PEL or the unusual, EBV carrying T-cell lymphomas express the proliferation driving, blastogenic program of the virus. The virus expresses only minimalistic programs designated as latency Ⅰ or Ⅱ, in contrast to the full immunoblastic program (Ⅲ). The tumor promoting contribution of the virus must therefore be sought in other, less direct effects.

In BL, one needs to depart from the fact that the lymphoma originates in the post-GC, centroblastic or centrocytic cell, that is either resting or is on its way to a long-lived resting memory cell, but cannot leave the cycling compartment because it is driven by an Ig/myc translocation. The translocation results from a faulty recombination that occurs in the course of physiological Ig gene rearrangement. Conceivably, EBV may contribute to the emergence of the virus carrying BL clone by expanding the original population or, alternatively, or in addition, by protecting the myc-driven, apoptosis prone cell from apoptosis. (See further in the section on EBV and BL below).

Human tumor viruses

Four of the six viruses known to be involved in the causation of human cancer in a direct or a contributory capacity are DNA viruses (EBV, HPV, HBV, HHV-8) while the remaining two are RNA viruses (HTLV-1 and HCV).

As discussed below, EBV is most directly involved in the causation of immunoblastomas that arise in immunodefective persons, such as transplant recipients, certain congenital immunodefectives, and HIV-infected persons. EBV may also play a role in Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC), as indicated by the regularity of its association with these tumors, but the nature of the viral contribution is not fully understood.

Special subtypes of the human papilloma viruses are known to contribute to the genesis of cervical carcinomas and of skin tumors. Human herpesvirus no. 8 (HHV8, also called KSHV) is associated with Kaposi sarcoma, Castelman’s disease and body cavity lymphoma. Hepatitis virus type B and C contribute to the genesis of primary liver cancer. The evidence for these virus-tumor associations is epidemiological and molecular, but the relative role of the virus and of cellular changes has not been properly established.

The following two sections deal with the two human tumor associated gamma herpesviruses, Epstein–Barr virus (EBV) and, to a minor extent, Kaposi sarcoma herpesvirus (KSHV or HHV-8).

Epstein-barr virus (EBV)

EBV is the causative agent of a self-limiting lymphoproliferative disease, infectious mononucleosis. In immunodefectives, the proliferation may proceed to progressively growing immunoblastomas. Multiple viral genomes, derived from a single infectious event, are regularly found in high endemic Burkitt lymphomas (BLs) and in low differentiated or anaplastic nasopharyngeal carcinomas (NPCs). EBV is also found, although less regularly, in Hodgkin’s lymphomas, nasal T-cell lymphomas, gastric carcinomas, salivary gland tumors and leiomyosarcomas (Table 29.1).

Table 29.1. EBV-associated tumors in man (the percentage figures indicate the frequency of EBV carrying tumors).

Table 29.1

EBV-associated tumors in man (the percentage figures indicate the frequency of EBV carrying tumors).

EBV exploits B-cell specific regulatory mechanisms and signals

Normal B cell physiology is tightly regulated. The generation of B-cell receptor diversity by immunoglobulin gene rearrangements, activation of resting B cells by cognate antigen and associated molecules, expansion of the activated population, migration through germinal centers and concomitant hypermutation, generation of long-lived memory cells and differentiation into secretory subtypes, plasma cells in particular, is regulated both by internal programs and external signals, including antigens, ligands and cytokines that influence homing, proliferation, differentiation and death of the cells. EBV has a whole gamut of highly refined mechanisms that exploit normal B cell physiology. Unlike many other viruses, its strategy is not limited to the turning of its host cell into a viral protein factory with the single purpose of virus production and release to the environment, although it can switch on that mechanism when it enters the lytic cycle. Rather, the virus follows a “live and let live” principle. Its vast success in infecting all human populations and persisting in latent form over the lifetime of the host without causing disease, except by accident, testifies to the validity of this strategy, both for the virus and for the cells.

The exploitation of normal B-cell physiology by the virus manifests itself at many different points. Already the very first step, viral attachment and penetration, is based on the use of a B-cell specific surface moiety, CD21 (also called the C3d receptor). This receptor is normally involved in B-cell activation by antigen, antibody and complement complexes. Activated B-blasts secrete cytokines and lymphokines that can stimulate B cell proliferation and express the corresponding receptors (e.g., CD 23), creating an autocrine loop. Moreover, the virus encodes three membrane proteins, LMP1, 2a and 2b, of which at least two control and modulate incoming signals, that participate in Ig-receptor activation, TNF-response and programmed cell death. As discussed in more detail below, the LMPs are constitutively active multifunctional membrane proteins. LMP1 interferes with TNF-α signaling. It can replace many CD40 induced functions and activates major signaling systems in B-lymphocytes and epithelial cells, such as NFkB, JNK-kinase and one JAK/STAT-pathway. Protection from apoptosis is one of its major downstream effects. LMP2a modulates kinase signaling from membrane receptors. Most notable are the eight N-terminal phosphotyrosine motifs that interact with the Ig-receptor induced kinases lyn and syk. LMP2a has an Immunoglobulin Transactivation Motif (ITAM) – with complete homology to the corresponding Ig-receptor ITAM-motif of its gamma-chain, that binds the syk kinase in its activated, phosphorylated state. Interspersed with these motifs are a PPPPY-motif that interacts with WW-domains of the Nedd-family of E3-ubiquitine ligases (Winberg et al., 2000). It is conceivable that LMP2a may attract the kinases and is involved in their fast destruction by guiding the complex to the ubiquitine –proteasome system. Characteristically, the expression of these proteins still permits the corresponding physiological signaling pathways to operate.

For example, LMP1 can replace CD40 ligand-CD 40 signalling, but does not interfere with physiological CD40-reception. LMP2 does not completely abolish Ig-receptor signaling, although it is likely to increase the signal threshold. It may be noted that the HHV8 membrane proteins K 1 and K 15, in combination, carry many of the motifs that function in the LMPs, such as the NFkB activation site and ITAM-motifs. Their role in latency, lytic cycle and tumorigenesis remains to be elucidated.

The following section will deal with the EBV-encoded growth transformation associated proteins in some more detail.

Growth transformation associated EBV encoded proteins

Table 29.2 summarizes the information of the six nuclear proteins (EBNAs).

Table 29.2. Overview of the EBNA proteins.

Table 29.2

Overview of the EBNA proteins.

EBNA1 is encoded by the ORF/KBRF1. It is a DNA binding protein of highly variable size (60–100 kD) due to the presence of a glycine alanine repetitive sequence, inserted in the first molecule that is flanked by a highly basic domain. The C-terminal part of the protein contains a stretch of acidic amino acids. It is expressed in most EBV-carrying cells, with the possible exception of latently infected resting B-cells (Reedman & Klein, 1974; Lindahl et al., 1974; Andersson-Anvret et al., 1978; Hennessy & Kieff, 1983; Dillner et al., 1986a,b; Chen et al., 1995a,b). In all other cell types that have been studied, EBNA 1 is expressed irrespectively of the cell phenotype, level of differentiation or, in the case of lymphocytes, activation status (Niedobitek et al., 1989; Hamilton-Dutoit et al., 1991; Prevot et al., 1992; Zhou et al., 1994). It is the only latency associated EBV-encoded protein whose expression is not influenced by the cell phenotype. In somatic cell hybrids between EBV carrying immunoblasts that express the full type Ⅲ program of six nuclear and three membrane proteins and EBV negative non-B cells where all B-cell specific markers are eclipsed, EBNA1 but not EBNA 2–6 remains expressed (Contreras-Salazar et al., 1989). EBNA1 is also the only member of the EBNA-family that remains associated with the chromosomes in metaphase (Ohno et al., 1977; Jiang et al., 1991). It is randomly distributed among the chromosomes, but binds specifically to the origin of latent viral DNA replication (OriP). This binding is necessary for the maintenance of the EBV episomes, by equal distribution to the daughter cells in mitosis (Jones et al., 1989). EBNA 1 has three specific binding regions in the viral DNA, each multiple. 20 binding sites are in the family of repeats (FR), four in the dyad symmetry, and two downstream of the Q promoter (Reisman & Sugden, 1986; Sugden & Warren, 1989; Ambinder et al., 1990; Rawlins et al., 1985).

The latent replication of the viral DNA starts from ori P. EBNA1 binds to ori P as a dimer. It is composed of a flanking domain and a core domain. The flanking domain includes a helix that projects into the major DNA grove and an extended chain that travels along the minor grove. This motif is responsible for all sequence determined contacts with DNA. The core domain makes no direct contact with DNA (Polvino-Bodnar et al., 1988). The binding to chromatin is mediated via chromatin protein (ref). EBNA1 binding to the chromosomes is essential for the precise division of the replicated DNA into the two daughter cells.

Through the multiple interactions with viral DNA, EBNA 1 causes DNA looping by multimerization. This increases the complexity of its promoter regulation. Dyad symmetry controls S-phase associated viral DNA replication. EBNA 1 regulates viral promoters via its multiple binding sites. FR acts as an enhancer for the C-promoter, directing all six EBNA transcripts and the Qp elements that are negative regulators of Qp-driven EBNA1 transcription through a negative feedback loop (Bodescot et al., 1987; Sample & Kieff, 1990).

EBNA1 contains a glycine–alanine repeat of variable length that inhibits its processing through the proetasomes and the subsequent MHC class 1 association of the derived peptides, a prerequisite for recognition by CD8 positive cytotoxic T-cells (Levitskaya et al., 1995). This results in a dramatically extended half life of EBNA 1 to more than 2 weeks, and may contribute to its presence in resting B-cells without de novo synthesis.

EBNA2

EBNA2 (ORF:BYRF1) is an 82 kD phosphoprotein. It contains a 14 AA long domain that is responsible for transactivation. In contrast to EBNA1, the expression of EBNA2 is restricted to immunoblasts (Dillner et al., 1985; Hennessy & Kieff, 1985; Ernberg et al., 1986). On primary infection of B-cells it acts as a transcriptional transactivator (Rickinson et al., 1987). It is essential for the transformation of B-cells into immunoblasts and the derivation of LCLs (Cohen et al., 1989). EBNA2 defective viral substrains cannot activate B-cells. EBNA2 is the EBV encoded oncoprotein that differs most extensively between EBV types 1 and 2 (Zimber et al., 1986). Type Ⅰ EBV is a more efficient transformer of primary B lymphocytes than type 2 (Rowe et al., 1989). EBNA2 is associated with nucleoplasmic, chromatin and nuclear matrix fractions.

EBNA2 induces a variety of activation markers and other cellular proteins in B-cells, including CD23, CD21, c- fgr and c-myc. It is required for the expression of EBV encoded LMP1 and LMP2a in immunoblastic cells (Wang et al., 1987a,b, 1990; Aman et al., 1990).

The interaction of EBNA 2 with the cellular proteins p300 and CBP is critical for EBNA2 mediated transactivation, due to the intrinsic histone acetylase activities of the former and their interaction with transcription factors (Bornkamm & Hammerschmidt, 2001). CBP has been implicated in EBNA2 activation of c-myc promoter.

Even though EBNA2 is a potent activator of many cellular and viral genes, it does not bind directly to DNA. It influences the responding promoters through its interaction with RBP-Jk, PU1 and other cellular proteins. The complexes formed modify the affinity of histones for DNA. Further chromatin remodeling activity is achieved through an interaction between EBNA2 and hSFN5. Recruitment of EBNA2 to DNA is essential for the transforming activity of EBV and RBP-Jk is the most extensively studied partner. RBP-Jk functions as a downstream target of the cell surface receptor known as Notch. Notch genes encode cell surface receptors that regulate developmental processes in a wide variety of organisms. The cleaved product of Notch is targeted to the nucleus where it binds to RBP-Jk and can activate transcription, but with a lower efficiency than the intracellular part of Notch. The binding of ligand to the extracellular domain of Notch results in the cleavage of an intracellular domain. This intracellular fragment of Notch (Notch-IC) migrates to the nucleus, binds to DNA-bound RBP-Jk and converts thereby a repressor of transcription into an activator (Hsieh et al., 1996).

On the basis of these findings, EBNA2 is regarded as a constitutively active homologue of Notch. However, Notch can only partially substitute for EBNA2 in B-cell transformation experiments, probably because it does not upregulate the transcription of LMP1 or c-myc. The EBNA2 induced activation of the LMP1 promoter requires additional B lymphocyte specific factors, such as PU.1 (Johannsen et al., 1995) and RBPJK (Johannsen et al., 1996). Elements responsible for EBNA2 responsiveness have been characterized in EBV-Cp, LMP1 and LMP2 promoters and the cellular promoter for CD23. All have at least one RBP-Jk binding site.

As already mentioned, the interaction of EBNA2 with the cellular sequence specific DNA binding protein, RBP-Jk, is critical for transformation and LCL outgrowth. A sequence in EBNA2 closely mimics a corresponding sequence in the notch-receptor. Notch and EBNA2 may activate transcription from the RBP-Jk and PU.1 promoters by interacting with SKIP, a component of the HDAC2 corepressor complex. The EBNA2 domain that interacts with PU.1 includes the site that interacts with RBP-Jk. The targeting of PU.1 by the EBNA2 transactivator is an important aspect of EBV adaptation to lymphoid cells (Tamura et al., 1995).

The essential role of EBNA2 in the immortalization of B-cells is thus due to its transactivation of viral promoters (Cp, LMP1 and 2) and a variety of cellular genes associated with B-cell activation and growth, among them c-myc. Myc activation in lymphocytes induces protein synthesis and increase in cell size, D-type cyclins, cyclin E. It downregulates the inhibitors p21 and p27. The induction of c-myc is regarded as a major link between EBNA2 and the cell cycle machinery (Kaiser et al., 1999).

EBNA2 is also required to maintain the EBV driven proliferation of B-cells, as shown in the conditional LCL designated as EREB2–5, that contains an EBNA2-estrogen receptor fusion protein. The removal of estrogen from the growth medium results in cell cycle arrest and apoptosis. Early reintroduction of estrogen stimulates renewed cell cycle entry and proliferation (Kempkes et al., 1995). EBNA2 can be replaced by the constitutive expression of exogenous c-myc. The switch from the EBNA2 driven to the myc-driven state is accompanied by a phenotypic change of the LCL-like cell to a more BL-like cell, resembling dividing germinal center B-cells (Polack et al., 1996).

Paradoxically, EBNA2 can also inhibit proliferation in established BL lines. EBNA2 is a transcriptional repressor of the immunoglobulin mu-gene. In BL lines where myc is controlled by the immunoglobulin mu enhancer, EBNA2 expression results in suppression of the myc transcription from the translocated myc gene, leading to growth arrest (Jochner et al., 1996).

EBNA5 (alternative name: EBNA-LP). EBNA5 is a nuclear phosphoprotein that localizes to distinct subnuclear bodies. It is spliced from a variable number of W1-W2 exon pairs, 66 and 132 nucleotides long, respectively, forming 66 aminoacid long repeats and, from the Y1 and Y2 exons, a C-terminal 45 AA unique region. Its progenitor, the giant primary transcript originates from the W or C promoter in immunoblastic cells (and only there) (Dillner et al., 1986a,b; Wang et al., 1987a,b; Bodescot & Perricaudet, 1986). Together with EBNA2, EBNA5 is the earliest viral protein expressed in freshly infected B-cells. The two proteins can induce the entry of resting B-cells into the G1 phase. Coexpression of EBNA5 with EBNA2 enhances EBNA2 mediated transcriptional activation. EBNA5 is tightly associated with the nuclear matrix, and often accumulates in PML bodies (Pokrovskaja et al., 2001). It migrates together with various components of the proteasome dependent degradation machinery in heat shocked cells, and in cells treated with proteasome inhibitors, raising the possibility that EBNA5 participates in the regulation of specific protein degradation in the nucleus. Kashuba et al.’s experiments on EBNA5 also showed that it does not bind to Rb and p53 in the yeast two hybrid assay, but can exert an inhibitory effect on the p53-Rb axis by targeting the p53 regulator p14 ARF(Kashuba et al., 2003). The latter can bind MDM2, suppress its ability to mediate in the degradation of p53 and thereby increase the expression level of p53. It was suggested that EBNA5 participates in the elimination of the p14 ARF-HDM2- p53 complexes and thereby contributes to the downregulation of p14 ARF and p53 protein levels in EBV infected B-cells (see also Kanamori et al., 2004).

EBNA3 family

EBNA3 (ORF: BLRF3 + BERF1), EBNA4 (ORF: BERF2a + BERF2b) and EBNA6 (ORF: BERF3 + BERF4) are three large nuclear phosphoproteins in a size range of 140–180 kD. EBNA3 and EBNA6, but not EBNA4 is necessary for in vitro transformation. Individual sequences of these three EBNAs show little similarity, but they are all composed of a highly charged N-terminal half and a C-terminal half that contains numerous repeat elements. EBNA4 also contains an LxCxE motif. EBNA6 is transactivator that induces CD21, 23 and LMP1 (alternative nomenclature: EBNA3a,b,c). All three proteins are encoded by tandemly arranged genes, localized in the middle of the viral genome (Hennessy et al., 1985; Dillner et al., 1986a,b; Shimizu et al., 1988). They are all highly hydrophilic. They are stable proteins that accumulate in intranuclear clumps, sparing the nucleolus. They are believed to act as transcriptional regulators and can interact with RBP-Jk (Radkov et al., 1997; Zhao et al., 2003; Hickabottom et al., 2002). The three proteins use unrelated peptide sequences for their interaction. EBNA3 and 6, but not EBNA4, are required for B-cell transformation (Parker et al., 1996). By and large, the EBNA 3 family member, have similar but more limited effects on cellular gene expression compared to EBNA 2. EBNA4, as also EBNA3 and 6, generate highly immunogenic peptides that can associate with MHC class Ⅰ molecules. Some peptide-HLA class Ⅰ combinations can induce CD8+ CTL mediated rejection in immunocompetent hosts.

The following further information on the two members of the EBNA 3 family involved in growth transformation, EBNA 3 and 6, are of interest.

EBNA 3 (alt: EBNA 3a)

Kashuba et al. (2000, 2002) have identified two EBNA3 interacting proteins, using a two-hybrid technique. TCP-1 is part of a chaperonine complex (Kashuba et al., 1999). EBNA3 binds to the epsilon subunit of TCP-1. Kashuba et al. proposed that nascent EBNA3 is folded by the TCP-1 containing chaperon complex through its binding to the apical region of the epsilon subunit. EBNA3 may thereby receive help for its proper folding.

Kashuba et al. (2000) also found that EBNA3 interacts with p38/XAP-2. In the presence of EBNA3, the cytoplasmic p38/XAP-2 translocates to the nucleus. XAP-2 also binds to the hepatitis B virus X antigen, believed to be involved in the oncogenic effect of hepatitis B virus. XAP-2 is known to be involved in the regulation of the aryl hydrocarbon receptor (AhR) pathway. AhR is a ligand activated transcription factor, a member of the HLH transcription family.

EBNA3 was also shown to interact with a new member of the UK/UPRT (uridine kinase/uridine phosphoribasyl transferase) family (Kashuba et al., 1999). The predominantly cytoplasmic enzyme translocates to the nucleus in the presence of EBNA3. It was suggested that EBNA3 may influence the uridine salvage pathway by contributing to the increase of the nuclear UTP pool, required for active cell proliferation.

EBNA6 (alternative EBNA3c) is the only member of the EBNA3 family that has a leucine zipper (West et al., 2004). EBNA6 associates with histone deacetylase and can repress transcription through the Notch signaling pathway. EBNA6 is unique among the EBNAs in its ability to coactivate the LMP1 promoter with EBNA2 (Lin et al., 2002). EBNA6 has also a number of specific repressive effects (Touitou et al., 2001; Radkov et al., 1999). Moreover, EBNA6 associates with histone deacetylase and can repress transcription through the Notch signaling pathway (Radkov et al., 1999).

EBNA6 can also cooperate with oncogenic mutant H-ras in the immortalization and transformation of REFs (Parker et al., 2000). It can also override the suppression of this transformation by p16, by targeting the checkpoint at the G1/S transition, regulated by Rb. EBNA6 can induce aberrant nuclear division that results in multinucleated cells, polyploidy and eventually cell death (Allday review, sid. 36). All this suggests that EBNA6 may disrupt multiple cell cycle checkpoints and produce a similar phenotype as the K cyclin of KSHV (Krauer et al., 2004).

Table 29.3 summarizes some of the known interactions of the EBNAs with cellular proteins:

Table 29.3. Overview of the EBV latent membrane proteins.

Table 29.3

Overview of the EBV latent membrane proteins.

The latent membrane proteins (LMP) of EBV

In the course of infection, replication or persistence, viral gene products frequently interact with proteins that regulate signaling pathways in the host cell. This capacity to modify host cell signal transduction is particularly apparent in the control of EBV.

EBV can express three membrane proteins during latent infection, latent membrane protein (LMP) 1 and 2 A and B (LMP 2A and B) (Hennessy et al., 1984; Laux et al., 1988; Longnecker & Kieff, 1990). These proteins interfere with multiple cellular signal transduction pathways so as to modulate apoptosis and cell surface receptor signalling. They are both transmembrane proteins with six (LMP 1) or twelve (LMP 2) anchoring transmembrane domains, according to computer based structure predictions. No full crystal structures have been established. Neither one acts as a ligand-receptor, but through constitutive activation at cellular membranes (Gires et al., 1997). If there are no ligands, why are they located as membrane proteins?

The main function of both membrane proteins appears to be directed towards interaction with signalling and adaptor molecules normally regulated by cell membrane receptors. They are highly multifunctional and interact with several cellular signalling pathways. Importantly both proteins are expressed at the cell surface membrane as well as in intracellular membranes of the Golgi and endoplasmic reticulum (Lynch et al., 2002; Eliopoulos & Young, 2001). The significance of this compartmentalization is not known, since the function of the intracellularly localized LMPs has not been the subject of focused studies. Both proteins can be expressed in two forms, one full length and one shorter variant where the first (LMP 1) or the last exon (LMP2B) is excluded by alternative promoter usage or alternative splicing. The shorter variants appear structurally competent to disrupt or block the constitutive activation of the full length protein, by interfering with activation mechanism. LMP 1 is activated by aggregation to trimeres or multimeres, mediated by the transmembrane domains. The truncated protein variant lacks the C-terminus and four of the transmembrane domains. LMP 2 depends on the N-terminal tail and its phosphorylation for activation, which cannot take place with LMP 2B. Both LMP1 and 2 are expressed in latency forms Ⅱ and Ⅲ immunoblasts and derived tumors and cell lines (Rea et al., 1994). LMP2A transcripts are also expressed in resting virus carrying B-lymphocytes in healthy individuals, the reservoir of persistently latent EBV (Chen et al., 1995a,b; Qu & Rowe, 1992; Tierney et al., 1994). Both proteins are also detected in epithelial tumors of the nasopharynx (NPC) and during the early stages of oral hairy leukoplakia (Pathmanathan et al., 1995; Webster-Cyriaque & Raab-Traub, 1998). In NPC between 35 and 65% of the tumors are LMP-positive. LMP1 and 2 are expressed in a coordinate fashion Their transcription is co-regulated via a 600 bp bi-directional promoter-enhancer control element designated as the LMP-regulatory sequence (LRS). LMP 2 can only be expressed from viral episomes, since the precursor transcript passes through the terminal repeats (Table 29.3).

The short variant of LMP1 has only been demonstrated during productive, lytic virus infection where it may block the constitutive action of full length LMP 1.

Latent membrane proteins 1 (LMP1)

LMP 1 is a 356 amino acid protein with a short intracellular N-terminus and 150 aa C-terminus.

LMP1 is essential for the transformation of B lymphocytes into lymphoblastoid cell lines (Dirmeier et al., 2003). It confers a survival advantage on EBV-infected B cells by protecting them from apoptosis. This is largely due to the LMP1-induced upregulation of the anti-apoptotic protein Bcl-2 and the block of p53 mediated apoptosis by the latter (Henderson et al., 1991).

EBV-encoded LMP1 can also transform established rodent fibroblasts in vitro (Baichwal et al., 1989; Fahraeus et al., 1990). Furthermore, expression of LMP1 is correlated with a more favorable influence of treatment (chemotherapy/irradiation) on patients with NPC (Kawanishi, 2000) or Hodgkin’s lymphoma (Montalban et al., 2000). Clinical and follow-up data from 74 cases of NPC showed that LMP1 positive NPC grew faster and more expansively than LMP1 negative tumors.

LMP 1 almost completely mimicks the function of CD 40 mediated signalling and is thus functionally homologous to the TNF receptor (TNFR) family of proteins (Eliopoulos et al., 1996, 1997; Kilger et al., 1998; Zimber-Strobl et al., 1996; Lam & Sugden, 2003). LMP1 has been shown to interact with several proteins of the TNFR signaling pathway through its C-terminal activation region (CTAR) 1 and 2. Hence, LMP1 can bind TRAF (TNFR-associated factor) 1, 2 and 3, as well as TRADD (TNFR-associated death domain protein), an adaptor protein that serves to recruit caspases to the death-inducing signaling complex (DISC) of the TNFR (Mosialos et al., 1995; Devergne et al., 1996). These interactions result in the NFκB-dependent upregulation of a number of genes, including those encoding anti-apoptotic proteins such as A20 and Bcl-2 (Hatzivassiliou, 2002). Bone morphogenetic protein receptor IA-binding protein (BRAM1), a novel LMP1-interacting protein, interferes with LMP1-mediated NFκB activation and reverses the resistance of cells to TNFR-mediated apoptosis (Chung et al., 2002). Kawanishi (2000) has provided evidence that LMP1 domains CTAR1 and 2 are involved in the enhancement of TNF-induced apoptosis in epithelial cells.

It has been shown that EBV also modulates host apoptotic sensitivity by modifying the relative level of caspase-8, an initiator caspase and its competitor, FLIP (FLICE inhibitor protein (Tepper & Seldin, 1999). LMP1 may alter the ratio of caspase-8 and FLIP.

The findings of Zhang et al. (2002) suggest that the apoptotic modulation by LMP1 is stimulus dependent: tumor necrosis factor (TNF) induced apoptosis was inhibited while Fas ligation- and etoposide- induced apoptosis was potentiated. The attenuation of TNF induced apoptosis partallelled the induction of the anti-apoptotic zinc finger protein A20.

LMP 1 also induces IL 6 and IL 6-receptor expression via the JAK/STAT pathway and JNK-kinase and MAP-kinase (Eliopoulos et al., 1997; Gires et al., 1999; Kieser et al., 1997). Through its interference with a number of major signaling pathways in B-cells and epithelial cells, LMP 1 mediates deregulation of several hundred cellular proteins. LMP 1 also induces the expression of adhesion molecules such as ICAM-1 and LFA, and MHC Class I and Ⅱ (Mehl et al., 2001; Rowe et al., 1995).

LMP2 modulation of signaling in B-cells and epithelial cells

LMP2A has an intracellular N-terminal cytoplasmic region of 119 residues, which is predicted to be followed by 12 membrane-spanning regions and a short C-terminal cytoplasmic tail, also intracellular. It has been reported to aggregate into “cap-like” structures at the plasma membrane and specifically associate with lipid rafts (Dykstra et al., 2001; Higuchi et al., 2001). The C-terminal tail of LMP2A has been reported to possess a clustering signal as well (Matskova et al., 2001).

LMP2, along with EBNA1 and LMP1, is consistently detected in some latently infected B cells and EBV-associated diseases in vivo, and plays presumably important roles in vivo, related to viral replication, persistence and EBV associated diseases (Qu & Rowe, 1992; Miyashita et al., 1997). A major role of LMP2A in relation to latent EBV infection may stem from its ability to inhibit the activation of lytic EBV replication by cell-surface-mediated signal transduction (Miller et al., 1994). This may prevent lytic replication in latently infected B-cells as they circulate in the blood, bone marrow or lymphatic tissues, where they might encounter antigens, superantigens or other ligands capable of engaging B-cell receptors and activating the viral cycle. In this context it may be relevant that LMP 2A also downregulates telomerase birth in B-cells and epithelial cells (Chen et al., 2004; Scholle et al., 2000).

LMP2A’s ability to interfere with BCR signaling and to maintain viral latency may stem from protein–protein interaction motifs located within the amino-terminal tail (see Fig. 29.2). These include a YEEA (amino acid; single letter code) site that, when phosphorylated on the tyrosine residue (Y112), can serve as a binding site for the Src Homology 2 (SH2) domain of the Src family tyrosine kinase Lyn (Burkhardt et al., 1992; Miller et al., 1994; Frueling et al., 1998). In addition, LMP2A possesses an immunoreceptor tyrosine-based activation motif (ITAM) with the consensus sequence YXXI/V-X(6–8)-YXXI/V which is found in a number of immunoreceptors including the BCR, the T-cell receptor (TCR), as well as the Fc∊ receptor that binds IgE. This motif in LMP2A, when phosphorylated on tyrosines 74 and 85, provides a binding site for the dual SH2 domains of the tyrosine kinase Syk (Fruehling & Longnecker, 1997). LMP2A also possesses 2 PPPPY (PY) motifs that can bind to the WW domains of the NEDD4 family of E3 ubiquitin ligases including AIP4, NEDD4-2, and WWP2 (Winberg et al., 2000; Ikeda et al., 2000). Binding to NEDD4 proteins is abrogated by mutation of both tyrosines in the PY motifs (Y60; Y101). NEDD4 family proteins contain HECT (homologous to E6-associated protein carboxy-terminus) domains that catalyze the ubiquitination of proteins such as those associated with the WW domains and target them for degradation via either the 26S proteasome or a lysosomal pathway. LMP2A and the LMP2A-associated kinases are substrates for the NEDD4 family of proteins, suggesting that LMP2A may not only sequester tyrosine kinases away from the BCR, but may also direct them to ubiquitin-mediated pathways including degradation.

Fig. 29.1. Schematic representation of EBNA 1.

Fig. 29.1

Schematic representation of EBNA 1.

Fig. 29.3. Established major signaling pathways of LMP 1.

Fig. 29.3

Established major signaling pathways of LMP 1. Studies have revealed several functional domains of the cytoplasmic tail of LMP1 that are important for activation of transcription factor NF-kB, AP-1 and P38 through its interaction with TRAF and TRADD. These findings suggest that LMP1 mimics TNFR aggregation, which is essential for subsequent signal transduction.

Fig. 29.4. N-terminal phosphotyrosine motifs of LMP 2 a.

Fig. 29.4

N-terminal phosphotyrosine motifs of LMP 2 a.

Fig. 29.5. Functional comparison between the Epstein–Barr virus LMP2a and the Kaposi Sarcoma virus (HHV8) K1 and K15 membrane proteins.

Fig. 29.5

Functional comparison between the Epstein–Barr virus LMP2a and the Kaposi Sarcoma virus (HHV8) K1 and K15 membrane proteins.

Fig. 29.6. HHV 8 K1 membrane protein.

Fig. 29.6

HHV 8 K1 membrane protein.

Fig. 29.2. Schematic representation of EBNA 2 and its major functions.

Fig. 29.2

Schematic representation of EBNA 2 and its major functions.

Somewhat paradoxically, LMP2A has also been shown to mimic BCR signaling. When expressed as a B-lineage specific transgene in mice, it can both drive B-cell development and promote the survival of mature B cells in the absence of surface immunoglobulin (Ig) expression (Caldwell et al., 2000). Furthermore, this signal appears to be attenuated by the NEDD4 family protein, Itch, indicated by the finding that Itch −/− introduced into the LMP2A transgenic background enhanced LMP2A-mediated signaling (Ikeda et al., 2003). This suggests that LMP2A may act as a survival factor for EBV-positive B-cell tumors that have lost the expression of surface Ig, while also preventing virus reactivation, such as in HL.

LMP2A has also been shown to activate PI3 kinase and the downstream phosphorylation of Akt in epithelial cells and B cells. This may result influence cell growth and apoptosis (Swart et al., 2000).

EBV LMP2A and HHV8 K1 and K15 membrane proteins both target the BCR, but in different ways. While the LMP2A effects on cellular signal transduction have been widely studied, the functions of the HHV8 membrane proteins have been given equal attention (Choi et al., 2000; Lee et al., 2000).

The transforming HHV8 K1 transmembrane transforming protein associates spontaneously to form a trimer in the membrane, which, like LMP2A, carries ITAM motifs. K1 prevents cell surface expression of the BCR by the association of its N-terminal domain with the µ-chains of the BCR, thus preventing the CD79α and β chains from binding. The K1-BCR complex is retained in the ER. The ITAM-motif of K1 is thus available for interaction with cytoplasmic signaling proteins. It has been speculated that the ITAM motif is speculated to function by delivering growth and survival signals to the target cell (Lee et al., 2000). The K15 has a similar topology as LMP2A, with 12 transmembrane helices, but lacks the N-terminal signal transduction domain of LMP2A. It reportedly blocks BCR signaling but the mechanism is not known.

Thus, similar functional elements of function appear to have been conserved in the latent membrane protein of these two distantly related herpesviruses, although the protein structure differs.

In view of the puzzling fact that K1 appears to bind to the µ-chain of BCR in the ER, with intracellular retention of the complex as a result, it is important to determine whether its ITAM motifs bind the Syk tyrosine kinase or a different SH2-domain protein. This could answer the question how K15 can stimulate B-cell survival and proliferation.

The elucidation of the function of the gamma herpesvirus signal transduction mediators may thus allow a refined understanding of BCR signaling functions, by way of a perturbation analysis.

Immunoblastic lymphomas arise in bone marrow or organ transplant recipients (PTLD), congenital immunodeficiencies, particularly the X-linked lymphoproliferative syndrome (XLP) and in AIDS patients. In PTLD and XLP, EBV carrying immunoblasts proliferate, as in mononucleosis, but without being arrested by the immune response. Initially, the proliferation may be polyclonal but becomes eventually monoclonal. During the polyclonal phase, the progression of the disease can often be halted by viral DNA inhibitors such as acyclovir, indicating that virus release and recruitment of new virally transformed cells play a role in the initial development of the disease, but not after it has turned into a monoclonal lymphoma.

EBV carrying immunoblastomas express the full (type Ⅲ) set of the virally encoded growth transformation associated antigens. They provide the virus carrying B-cells with proliferation drive and antiapoptotic protection. They include the highly immunogenic members of the EBNA3 triad (EBNA 3,4 and 6, also called EBNA 3a,b,c), explaining why passive immunotherapy with sensitized CD8+CTLs or with unsensitized but immunocompetent, histocompatible T-cells can bring about dramatic regression even of widely disseminated tumors.

The immunoblastomas and lymphomas that arise in AIDS patients show a broader picture. Only part of them resemble the post-transplant immunoblastomas. Others are more akin to EBV carrying Burkitt lymphomas, mainly because they carry BL-type Ig/myc translocations, as discussed in more detail below, but they have a more variable cellular and viral expression phenotype. In the strict (type I) phenotype, associated with high endemic BL (only EBNA1 and the EBERs are expressed), Ig/myc translocation carrying tumors with a more immunoblastic cellular and viral (typeⅢ) expression phenotype are also found. This may be related to the well established fact that EBV-carrying type Ⅰ BL cells tend to drift towards a more immunoblastic phenotype and full (type Ⅲ) antigen expression during in vitro culturing. The selective filter that normally removes the highly immunogenic immunoblasts is, not surprisingly, impaired in immunodefectives.

EBV and Burkitt lymphoma (BL)

EBV is associated with 98% of the high endemic BLs, but only with about 20% of sporadic BLs. Both types contain essentially similar Ig/myc translocations, with only minor differences (Magrath, 1990). Only EBNA1 and the EBERs but none of the growth transformation associated EBV proteins (EBNA 2–6, LMP1–2) are expressed in BLs. The virus can therefore not be held directly responsible for the proliferation of the tumor. The latter function is attributed to the constitutive activation of the c-myc protooncogene, resulting from its juxtaposition to one of the three Ig-loci by chromosomal translocation.

Phenotypically, BL cells differ from EBV transformed or mitogen activated immunoblasts. Their markers and their V-gene mutations identify them as post–germinal center memory cells. Even in the most highly BL prone areas of Africa where malaria is rampant and where the regularly high EBV antibody levels in most young children indicate early infection and a high viral load – proven risk factors for the development of BL (Geser et al., 1982) – only a very small fraction of normal B cells (<0.1%) carry the virus. The 98% EBV positivity of the BLs must therefore mean that the presence of the virus increases the probability of lymphoma development. This is the same as to say that the virus contributes to the development of the tumor.

Falling short of the activation of cell proliferation, EBV is likely to contribute to the genesis of BL in some other way. According to one theory, apoptosis protection by the EBERs may be responsible (Takada, 2001). Another indication of a possible apoptosis protecting role has been derived from experiments with EBV negative sublines of originally EBV positive BLs that have lost the virus accidentally, or after hydroxyurea treatment. Viral loss is accompanied by decreased clonability and tumorigenicity (Komano et al., 1998). Comparison of three independently established EBV carrying BLs and their EBV –loss variants showed a marked downregulation of the tcl-1 oncogene (Kiss et al., 2003). Tcl-1 is highly expressed on both T and B cell derived leukemias. EBV reinfection has upregulated tcl-1 again in the EBV-loss variants. Since originally EBV negative BLs express tcl-1 at a high level, we have suggested that the EBV negative BLs switch on tcl-1 constitutively during their neoplastic development. In the virus carriers, EBV is responsible for the upregulation. Tcl-1 activates the apoptosis protective AKT pathway and may thus further increase the apoptotic threshold in these myc-driven and thereby apoptosis prone cells. Conceivably, EBV may also act at the pretranslocation level, by contributing a strong B-cell proliferation, driving stimulus. This would be further enhanced by malaria associated immune dysregulation and opportunistic coinfections. This could expand the target population available for the critical myc-translocation.

The absence of the virus from the EBV negative BLs is consistent with the interpretation that the virus is a contributory, but not a mandatory factor for BL development. The Ig/myc translocation is, on the other hand, regularly found in all BLs and must therefore play a more central role in the origin of the tumor.

The Ig/myc translocation carrying murine tumor prototype, plasmacytoma (MPC) develops earlier and in a higher frequency when the precursors are infected with the pre-B-cell-immortalizing Abelson virus, that carries the v-abl oncogene that has a known antiapoptotic effect.

Since myc-driven cells are highly apoptosis prone, as already mentioned, it is hardly surprising that BL cells are protected against apoptosis at several different levels. Both the Rb and p53 pathways are crippled in BLs, as a rule. In most cases, the Rb pathway is impaired by p16 promoter hypermethylation. The p53 pathway can be inactivated in at least three alternative ways: p53 mutation, ARF mutation or deletion, and MDM2 amplification (Lindströ & Wiman, 2002). In spite of this. the BL cell is still quite apoptosis prone, as indicated by its “starry sky” histology, where the “stars” are macrophages that have engulfed apoptotically generated nuclear fragments from the lymphoma cells, and also by the high chemotherapeutic sensitivity of the tumor.

The primary localization of African Burkitt lymphomas may be relevant in this context as well. It suggests that the cytokine environment associated with local tissue proliferation may favor the outgrowth of Ig/myc translocation carrying cells. Jaw tumors are frequent around the age of dentition, ovary and testis are frequent primary sites of BL in prepubertal and pubertal children. Lymphomas may arise in the long bones of teenagers, and BLs with a primary mammary localization have been seen in young lactating women. Some of them regressed when nursing was interrupted. Chronic inflammation may act in a similar way. EBV carrying body cavity lymphomas can be associated with pyothorax of 20 or more years’ duration. They carry HHV-8 as well. They present as diffuse large cell lymphomas, sometimes with an immunoblastic appearance. This is in line with numerous examples in the experimental literature, showing that chronic inflammation may act as a tumor promotor.

Hodgkin’s lymphoma (HL)

Almost half of the HL-cases in Western countries carry EBV-positive Hodgkin Reed-Sternberg (HRS) cells, that express EBERs, EBNA 1, LMP1 and presumably LMP2a, although this has been less extensively studied (Glaser et al., 1997; Levine et al., 1994; Ohshima et al., 1996; Lennette et al., 1995). The frequency of these presumably malignant cells is surprisingly low in the tumors (1–3%). Conceivably, the HRS cell orchestrates tissue derangement, by recruiting immune bystander cells such as non-neoplastic helper T lymphocytes, plasma cells, macrophages and eosinophilic granulocytes (Molin et al., 2001; Enblad et al., 1993; Weng et al., 2003). It is frequently surrounded by a rosette of CD4+ T-lymphocytes of both the Th1 and the Th2 type. EBV positive HLs show a shift towards Th1. The tumor is described as a “malignant inflammatory process.” It produces a large variety of cytokines (Dukers et al., 2000). The HRS cells carry non-functionally rearranged immunoglobulin heavy chain genes and contain somatic mutations in a high frequency, indicating post-germinal center derivation (Kuppers et al., 2002; Muschen et al., 2000; Spieker et al., 2000). They may have been frozen in a non-physiological state that prevents further differentiation or apoptosis.

Several findings suggest that EBV positive HRS originate from latently EBV infected B cells. Like BL and NPC cells, HL cells carry complete viral genomes in the form of multiple covalently closed episomal DNA. TR analysis revealed that viral genomes were clonal, suggesting that they have originated in a common proliferating precursor (Langerak et al., 2002). This argues against any role of virus replication in the establishment of the tumor cell.

Enhanced permissiveness and virus replication may still have a role as a risk factor, HD patients frequently have elevated antibody titres to EBV early antigen (EA) already at the presentation of disease and often years before. Significantly increased EBV genome load has been detected in the blood several years before the disease (Drouet et al., 1999). This suggests that the disease may be preceded by increased EBV reactivation and deregulation of the virus-host balance. Moreover,it has been shown that patients with acute infectious mononucleosis run an increased risk of developing HD (Amini et al., 2000; Axdorph et al., 1999).

In contrast to BL-cells and LCLs, it is difficult to grow HRS-cells in vitro. Only a dozen HL-/HRS-dervied cell lines have been established in vitro. They are EBV-negative, with only one exception. EBV-positive and negative HLs show no convincing differences in phenotype or clinical behavior. But while HD is less common in developing countries, it is much more frequently EBV-positive, (up to 90%). This is reminiscent of the difference between African and sporadic BLs (98% vs. 20% EBV positives).

Two studies have shown no difference in the prognosis of EBV positive and negative cases of HD. Morente et al. (1997) found however, that EBV-positivity was associated with improved overall survival and resulted in a higher complete therapeutic response, together with a significantly longer disease free interval. According to a fourth study (Murray et al., 1999) EBV positive tumors are easier to treat and treatment leads to longer disease free intervals.

EBV and nasopharyngeal carcinoma

Low differentiated or anaplastic NPC is the most regularly EBV associated tumor. It is the commonest malignant tumor among men in Southern China, the Guangzhou region in particular. The carcinoma cells carry multiple viral episomes, like BL cells. Terminal repeat (TR) analysis revealed that they have been derived from a single infectious event like the EBV positive BLs.

Some apparent paradoxes need to be reconciled. Given that the virus replicates lytically in epithelial cells, as particularly well shown in oral hairy leukoplakia (OHL, see below), why does it remain latent in NPC where it expresses only EBNA1, the EBERs and the LMPs (latency Ⅱ)? Is this due to the fact that NPC cells do not proceed to squamous differentiation? In OHL, the productive viral cycle is only switched on when the cells move upwards within the epithelium, to the level where they start engaging in squamous differentiation.

Early reports claiming that latent EBV could be detected in the basal layer of normal epithelia by in situ techniques have not been confirmed. Foci of lytic viral replication are only found in OHL, which is an EBV-induced lesion. It is found in immunodefectives and can be cursed by acylovir.

The robustly latent interaction between EBV and NPC is puzzling. It raises the question whether EBV inhibits the differentiation of NPC cells and, if so, whether this gives a clue to its role in the genesis of NPC.

It is not clear how and when the NPC-precursor cell becomes infected. In contrast to the EBV-infectability (and transformability) of B cells with EBV and the persistence of the virus in this compartment, epithelial cells are difficult to infect, unless a genetically engineered virus is used that carries a selectable marker like neomycin resistance.

Two experimental findings may offer possible clues. Comparing three different lines of EBV negative carcinoma cells with their EBV infected sublines, Nishikawa et al. (2003) found that the virus switched on the expression of a truncated basic hair keratin gene in all three lines. Conceivably, the truncated keratin may interfere with the production of full sized keratin and, thereby, differentiation.

But why would the keratin be truncated in the first place? This could stem from some of the multiple genetic changes found by PCR in NPC precursor lesions. Dolly Huang’s group has shown that some of the changes, particularly the frequent deletions affecting chr. 3p and 9p, occur prior to EBV infection (Lo & Huang, 2002). A possible scenario proceeds from an early genetic change that endows the precursor cell with the ability to produce a variant keratin, potentially capable of interfering with differentiation. This variant would be switched on by some viral product, expressed within the latency Ⅱ program. From the viewpoint of viral strategy, such a scenario would protect the virus from self-elimination by lytic infection and secure its persistence in latently infected, dividing, and undifferentiated cells.

Viral expression in carcinoma cells, cell behavior and host relationships in NPC

About two thirds of NPC tumors express LMP1 in vivo. In the non-expressors, the promoter region of the gene is hypermethylated (Hu et al., 1991). A comparison between non expressed LMP1 genes taken from NPC biopsies and the corresponding genes from LMP1 expressing tumors, showed that the former but not the latter could confer immunogenicity (rejection inducing capacity) on a non-immunogenic mouse mammary carcinoma, transplanted to syngeneic hosts (Hu et al., 2000). This suggested that the LMP1 expressors may have been sculpted by immunoselection in vivo that favored cells with genetic or epigenetic LMP1 inactivation.

LMP1 has been shown to convey increased agarose clonability and tumorigenicity on immortalized epithelial cells in vitro (Hu et al., 1993). Moreover, LMP1 expressing NPCs grew more expansively in immunodefective mice than non-expressors (Hu et al., 1995). Nevertheless, patients with LMP1 positive tumors showed better survival in a retrospective study than patients with LMP1 negative tumors, suggesting that the immunoselective sculpting of the LMP1 positives may still have left the tumors with a certain residual immunogenicity.

In addition to NPC, EBV genomes were also found in other solid tumors, notably gastric carcinomas, salivary gland tumors and a case of thymic carcinoma. Since these associations are not equally regular, they will not be included in this comparative discussion.

Immune surveillance and the oncogenic herpesviruses – the role of immunological “anticipation”

Viewed as a group, four of the potentially oncogenic herpesviruses, Marek’s disease herpesvirus (MDHV), H. saimiri (HVS), H. ateles (HVA) and EBV provide us with some important lessons about surveillance mechanisms that can protect against viral tumorigenesis, without reducing the spread of the virus. This results in a largely non-pathogenic “live and let live” situation.

MDHV is the only known DNA tumor virus that can cause tumors by epizootic infection. Unlike the three other viruses, MDHV is not ubiquitous in its natural host, the chicken. Infection of immunologically naive birds can therefore cause horizontally spreading epizootics. The frequency of the lymphoproliferative tumors and the level of their malignancy is influenced both by genetic and evironmental (e.g., stress) factors. One of the genetic resistance factors is linked to MHC. This is in line with the known role of the locus as an immune response regulator. It is reasonable to assume that a more general spread of the agent in chickens would have selected the natural host species for a similarly solid resistance that has been achieved by the other three viruses.

HVS infection is ubiquitous in the natural New World primate host, the squirrel monkey, and the same applies for HVA and its natural host, the spider monkey. Neither of the two viruses are known to be associated with any disease. But the number of examined animals and the observation periods are not comparable to what is known about EBV and the human host. The effect of immune defects is not known for the two simian viruses either.

In distantly related but HVS and HVA naive New World primates, such as marmosets, both HVS and HVA cause rapidly proliferating T-cell malignancy. The tumorigenic potential of both viruses is also consistent with their ability to immortalize human T-lymphocytes.

What makes the natural host solidly resistant to the tumorigenic effect of transforming viruses that can cause rapidly growing malignancies in related but immunologically naive species? Some years ago we performed an experiment with Friedrich Deinhardt’s group (Klein et al., 1973) that may throw a certain light on this question. We compared the primary antibody response of squirrel monkeys and marmosets to HVS inoculation soon after birth. Since all previously tested squirrel monkeys were found to carry HVS, fully mature fetuses were removed by Cesarian section from their mothers and were kept virus free by isolation, surrogate mothers and bottle feeding. After a few weeks they were inoculated with live HVS, in parallel with young marmosets. Both species responded with antiviral antibody production but while the squirrel monkeys reached peak antibody titers already 10–12 days after inoculation, the marmosets started responding only after 3 weeks. By that time the lymphoproliferative disease was already in progress, however.

Even though this study was restricted to humoral antibodies and gave no information about the more important cell mediated arm of the immune response, the time difference in the onset of the reaction indicated that the natural host of the virus has been selected for some kind of “immunological anticipation” of the impact of HSV that succeeds in infecting most and perhaps all members of the species under natural conditions.

EBV is also ubiquitous in its natural human host and causes only a self-limiting proliferative disease, mononucleosis, and only in about half of the primarily infected adolescents and adults. The other half and most children under the age of ten respond with “silent,” symptom-free seroconversion. Mononucleosis is characterized by the temporary proliferation of EBV transformed immunoblasts, followed by a rejection reaction, mediated by multiple immune effectors. In immunodefectives, whether of congenital (e.g., XLP) or iatrogenic (e.g., transplant recipient) or infectious (e.g., HIV) origin, immunoblastic proliferation may be fatal, as already discussed in the section on immunoblastomas. Moreover, and in further similarity to HVS and HVA, EBV can induce progressive lymphoproliferative malignancy in marmosets and owl monkeys, New World primates that do not have EBV-like viruses of their own. Old World primates that have their own EBV-like viruses carry cross reactive antibodies and are solidly resistant against EBV infection.

Neither BL nor NPC, nor any of the other malignancies where EBV is only found in a proportion of cases, are directly caused by EBV. Therefore, the relatively non-tumorigenic association of this ubiquitous virus with its natural human host is not very different from what HVS and HVA have achieved in their natural simian hosts. Our “immunological anticipation” of EBV is reflected by the surprisingly high frequencies of CD8+T-cells specific for latent and lytic EBV antigens in healthy virus carriers. As many as 5.5% circulating CD8+T cells in a virus carrier can be specific for a single EBV lytic protein epitope, as shown by tetramer staining (Tan et al., 1999). Lymphocytes carrying TCRs specific for latent proteins were found at somewhat lower, but still considerable (0.4–3.8%) frequencies. The preparedness of the immune system for the encounter with EBV transformed B-cells is also reflected by the fact that autologous T cells, admixed to EBV infected B-cells, undergo blast transformation and generate cytotoxicity at equally high levels as allogeneic, MHC-incompatible mixed lymphocyte cultures.

Surveillance of the human host against the development of EBV carrying neoplasia is thus virtually watertight. Occasional tumor development is due to an immunological or cytogenetic accident. Directly EBV-driven immunoblastomas can only arise in immunodefective hosts. Burkitt lymphoma is driven by the unrelated accident of the Ig/myc translocation, probably assisted by an anti-apoptotic effect of the minimal, non proliferation driving type Ⅰ viral expression program. In NPC, the role of EBV is even less clear. Our findings prompt the speculation that it may act by inhibiting squamous differentiation.

HHV-8 (KSHV)

This is the most puzzling virus of the DNA tumor virus family. It has hijacked more than a dozen cellular genes, many of which have potential tumor related functions. They include genes that influence the cell cycle, apoptosis, various other types of signaling and can create chromosomal imbalance. If a tumor biologist had been given the task of designing a tumor virus, he could not have done better. Surprisingly, however, the virus does not transform in vitro any target cell so far tested. The lack of an in vitro transformation system has hampered the functional analysis of tumorigenicity. Still, there are very good grounds to believe that the virus is responsible for three tumors, Kaposi sarcoma, multicentric Castleman’s disease (CD, a polyclonal B cell hyperplasia) and PEL, monoclonal body cavity lymphoma of the B cell series. Each of the three major proliferative syndromes associated with HHV-8 has a partially different virus expression program.

Only the viral ORF73-coded nuclear antigen, LANA1, is regularly expressed in all three HHV-8 related neoplasms. LANA1 binds to the histone H-1 which tethers the viral DNA to the chromosomes during mitosis, securing the maintainance of HHV-8 episomal DNA in infected cells and the delivery of the viral progeny to the daughter cells. LANA1 is a highly immunogenic protein, expressed predominantly by latent virus in KS cells and in PEL lines. LANA1 represses p53 transcription in a highly specific fashion (Friborg et al., 1999).

The viral cyclin D homologue, K cyclin is expressed from the same polycistronic transcript as LANA1. It can promote apoptosis and growth arrest (Verschueren et al., 2002). DNA synthesis, but not mitosis, continues, leading to multinucleation and polyploidy. Centrosome amplification leads to aneuploidy. In the absence of functional p53, aneuploid cells survive and expand. K cyclin expression in p53−/− and also in wild-type mouse embryonic fibroblasts induces massive centrosome amplification with multiple spindles and fusion bridges. In p53 knockout mice, viral K cyclin induces lymphomas.

Similarly to the small DNA tumor viruses, HHV-8 impairs both the p53 and the Rb pathway. But, unlike the small DNA tumor viruses, HHV-8 has developed multiple tactics to corrupt Rb functions. The viral cyclin K which is, like LANA1, constitutively transcribed in HHV-8 carrying cell lines, inactivates Rb and activates EF2 related transcription (Radkov et al., 2000). LANA 1 and Rb coexist as a complex in vivo. Similarly to adenovirus E1A, papillomavirus E7 and SV-40LT, LANA 1 targets the hypophosphorylated, active form of Rb. Moreover, LANA 1 can overcome the flat cell phenotype, induced by the Rb protein in SAOS2 cells. In cooperation with ras, LANA 1 transforms primary REF cells and renders them tumorigenic. While LANA 1 thus resembles SV40LT in being able to interfere with both Rb and p53 function, its ability to inactivate the two main tumor suppressor pathways is not as complete as the action of LT, because LANA 1 cannot transform by itself.

Double HHV8/EBV carrying PEL cells

The majority of the body cavity (PEL) derived lymphoma lines carry both viruses while some carry only HHV-8. EBV expresses its minimal (type I) program, characteristic for BL. The growth transformation associated, proliferation driving genes, characteristic for the type Ⅲ program of the LCLs, are not expressed, indicating that the double virus carrier lines are not driven by EBV. EBV may have a similar accessory function as it has in BL.

But are the PEL lines, whether single or double virus carriers, driven by HHV-8? This is a reasonable possibility, but it cannot be taken for granted. Body cavity lymphoma lines have numerous chromosomal changes. Some of them may be responsible for the proliferative activity. There is no single, common cytogenetic change like the Ig/myc translocations in BL. Multiple rearrangements may cover more specific changes, however, as in the cryptic Ig/myc translocations that often hide within the massively rearranged karyotypes of cell lines derived from human multiple myeloma, only detectable by the SKY technique (Gratama et al., 1988).

If HHV-8 occupies the driver’s seat in the body cavity lymhomas, RNAi or antisense techniques directed against either LANA1 or viral cyclin K should cause growth arrest or apoptosis. This experiment could go a long way in clarifying the role of the virus in PEL. In the absence of direct transformation systems, such evidence may be quite crucial.

Other HHV-8 genes that may be directly relevant for tumorigenesis include the pirated MIR1 and 2 genes, encoded by K3 and K5. They inhibit MHC class Ⅰ surface expression, through a unique mechanism not found in other viruses. They remove the MHC I proteins from the plasma membrane by enhanced endocytosis. V-FLIP, another pirated gene, is a powerful inhibitor of receptor mediated apoptosis. It is expressed constitutively on latent transcripts and it enhances the tumorigenicity of mouse lymphomas. In addition to the crippling of Rb and p53, and the virally encoded bcl-2, v-FLIP may provide additional protection against apoptosis.

Conclusions

EBV and HHV-8 associated malignancies show interesting parallels and contrasts. The main parallel is the universally occurring expression of the viral product required to maintain the viral episomes, EBNA1 in EBV, LANA1 in HHV-8. Both are nuclear proteins, but they act quite differently.

The most prominent and best known feature of EBV is its finely poised adaptation to the B cell compartment and its sophisticated exploitation of B cell biology – in different ways – for viral expansion, the control of that expansion, and for the latency of the persisting virus. It uses a B-cell surface receptor, CD21, as its receptor. It stimulates the infected B-cells to blast transformation, using or mimicking the normal immunoblastic transformation pathways, and it “immortalizes” its targets, driving them to continuous expansion in vivo and in vitro. The immune response of the host, directed against one of the immunogenic, growth transformation associated proteins (the choice depending on the MHC constitution of the host), rejects the expanded blasts, but not before they have substantially raised the viral load. Meanwhile, the virus secures its lifelong latency by downregulating its proliferation driving and its immunogenic proteins (the two categories overlap partly but not completely) and establishes permanent residence in long-lived, post-germinal center memory cells. Since successful bone marrow transplantation eradicates the resident virus population (Damania et al., 2000), all the persisting virus must reside in the hemopoetic compartment, and probably in B-cells.

We have no knowledge as yet of host cell gene expression pattern in latently HHV8 infected cells. It is therefore still an open issue whether the HHV8 tumorigenic process develops from a latent infection, from infection of “non-physiological” host cells or the abortion of a lytic infection. Several of the HHV8 proteins that have not yet been detected in tumors are interesting candidates in relation to tumor risks. Conceivably, the patterns of viral gene expression may differ in precursor cells of different phenotypes.

Interestingly, motifs of the two membrane proteins K1 and K15 mimic several features of LMP 2 a and LMP 1, which are frequently expressed in EBV-associated tumors. They contain potential NFkB- and ITAM-interacting motifs (Damania et al., 2000; Nicholas, 2003). In contrast to the smaller DNA tumor viruses, they may be less in need to control their long term persistence in the host cell and the fate of the host cell through long periods of time. On the other hand, that may be more dependent on modulators of the acute immune response and intracellular controls of proliferation and apoptosis, as part of their lytic virus replication.

Neither the proliferative, nor the resident phase of the viral life cycle requires crippling of the two main tumor suppressor pathways, Rb and p53. The situation is different in EBV carrying malignancies that are not driven to proliferate by EBV (like BL and NPC), but where the virus is present in an adjuvant capacity, probably as an accessory antiapoptotic device.

Most and perhaps all malignant tumors need to inactivate both the Rb and the p53 pathways. This can happen by a number of alternative mechanisms. In BL, the p53 pathway can be crippled by p53 mutation/deletion, ARF mutation or MDM2 amplification. The Rb pathway can be inactivated by Rb mutation/deletion, p16 inactivation or CDK4 amplification. There is no reason why the minimal (type I) EBV program expressed in BL cells should interfere with the Rb or the p53 pathway and there is no evidence that it does.

In HHV-8 associated tumors, the situation is quite different. In the absence of an in vitro transforming system, we cannot tell whether any of the viral genes (including the genes pirated from the host cell) can drive cell proliferation. We know, however, that two of the genes, LANA1, and viral cyclin K cripple the Rb and p53 pathways. This brings HHV-8 into the previously established DNA tumor virus fold.

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