The viruses associated with human cancer include Epstein Barr virus (EBV), human papilloma virus (HPV), human T-cell lymphotrophic virus (HTVL-1), Kaposi sarcoma virus (KSHV), hepatitis C virus (HCV), and hepatitis B virus (HBV). Our understanding of how these viruses operate has come a long way since Epstein Barr virus, the first human tumor virus, was identified from the cells of a patient with Burkitt's lymphoma in 1964. Those studies provide a framework for understanding how EBV perturbs cellular pathways, contributing to the development of cancer.

EBV, designated human herpesvirus 4 (HHV-4) in the herpesvirus family, infects more than 95% of the adult human population worldwide. Among known herpesviruses, EBV uniquely transforms human B-lymphocytes, establishing lifelong latent infections that sometimes reactivate. Although primary EBV infections in early childhood are usually subclinical, during adolescence they can cause mononucleosis, a self-limiting disease.

Additionally, because EBV can disrupt host-cell functions, it is implicated in a broad spectrum of human malignancies primarily of lymphoid and epithelial cell origin, including Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), AIDS-associated lymphoma, nasopharyngeal carcinoma (NPC), post-transplant lymphoma (PTL), and natural killer (NK)/T-cell lymphoma (Table 1). Moreover, individuals whose immune systems are compromised, such as AIDS patients or recipients of organ transplants, have a higher-than-normal probability of developing EBV-associated tumors. In addition to the immunological status of EBV-infected individuals, geography also matters. For instance, EBV-infected individuals in equatorial Africa are likely to develop BL, whereas NPC occurs at a relatively high frequency among Southern Asian populations.

In its linear form, the EBV genome is produced following virus replication, while the circular form arises within the nuclei of latently infected cells. The genome is an approximately 172-kb, double-stranded DNA molecule that encodes nearly 100 proteins. Many genes expressed during lytic phase have homologues in other human herpesviruses, but the genes expressed during latent phase appear to be unique to EBV. Several internal repeats are interspersed in the genome, which is bounded by several copies of direct terminal repeats.

EBV mainly infects epithelial cells of the oropharynx and resting B cells of the immune system. Infections of epithelial cells result in lytic replication and release of virions, whereas B-cell infections usually are latent. To infect B-cells, the EBV major viral envelope glycoprotein gp350 binds to the complement receptor CD21 (Fig. 1). The mechanism by which this virus enters epithelial cells, which lack CD21, is not understood. Like other herpesviruses, EBV has a distinct latent, or nonreplicative, and a lytic, or replicative, phase. The lytic phase of infection ensures replication, production of infectious virions, and transmission to other hosts. It is technically difficult to study lytic infection in vitro because no cell culture system supports such infections. However, researchers can use chemicals to induce latently infected EBV cell lines into a lytic phase.

EBV DNA replication begins at oriLyt, of which there are two copies in the viral genome. Altogether EBV encodes about 90 genes that are expressed in a temporally regulated manner during replication. By analogy to herpes simplex virus (HSV), these are classified as immediate early, early, and late genes. Transcription of immediate early genes begins immediately after EBV infects a cell, and is activated by viral proteins in the tegument of infectious virions. Their transcription does not require protein synthesis.

The EBV early genes, which are regulated by the immediate early genes, are involved in DNA replication and metabolism. Meanwhile, the late genes encode structural proteins that are involved in virion assembly, maturation, and egress. Viral genes expressed during lytic infection are involved not only in DNA replication and virion assembly but also in modulating the apoptotic pathway as well as inhibiting the activity of host interferon, which affects host immune responses. Various other proteins are also involved in lytic replication (Table 2).

EBV Affects the Host Cell Cycle

The eukaryotic cell cycle is highly regulated as it moves from G1 to S and G2 to mitosis (M), following a complex interplay of regulatory signals. DNA replicates during S phase, and then the newly duplicated chromosomes segregate during mitosis.

Members of the highly conserved family of protein kinases called cyclin-dependent kinases (CDK) orchestrate these steps. Specific cyclins bind and regulate the activity of the CDKs. While CDK levels remain constant throughoutthe cell cycle, cyclin levels oscillate via transcriptional regulation and protein degradation. Different cyclins are required at different phases of the cell cycle- for instance, cyclin D in G1, cyclin E and A in S phase, and cyclin B and A during mitosis.

In G1, active cyclin/CDK complex phosphorylates the retinoblastoma family of proteins, releasing the E2F family of transcriptional factors, activating transcription of genes responsible for cell cycle control, and initiating replication and DNA synthesis. In the absence of mitogenic signals, CDKs remain inactive, preventing aberrant proliferation. Proteins called CDK inhibitors (CKIs), including p15, p16, p21, p27 and p57, exert this negative control. The cell also protects the integrity of DNA replication, relying on the p53 tumor suppressor protein to repair damage. In response to damage, it either blocks the cell cycle to allow repair or induces apoptosis if the injury cannot be repaired. The p53 tumor suppressor enhances transcription of a specific CKI, p21, which in turn binds to and inhibits CDK2 to arrest the cell cycle.

EBV uses host cell components, commonly taking over pathways that control cell-cycle checkpoints and DNA repair. By dysregulating such key steps, EBV or other DNA tumor viruses can promote transformation. Some DNA tumor viruses encode unique oncoproteins- for example, large T antigen of SV40 and E6/E7 of human papilloma virus-that specifically target and abrogate the activity of the tumor suppressors Rb and p53.

In dysregulating the G1/S transition, EBV differs fundamentally from those and other small tumor viruses, which promote S phase transition and then use the cellular replication machinery to make their own DNA. Because B-lymphocyte proliferation is fundamental to EBV biology, the role of the EBV latent genes-specifically, EBNA2, EBNALP, LMP1, and EBNA3C-is the focus of much interest in the context of B-cell proliferation and transformation.


EBNA2, EBNALP, and LMP1 Can Disrupt the Cell Cycle

EBNA2 and EBNA-LP are the first latency proteins detected after EBV infects primary B-cells. EBNA2 is essential for transforming B-cells, while EBNA-LP may help to immortalize them by enhancing EBNA2-mediated transcriptional activation of the LMP-1 gene. Transfecting EBNA2 and EBNA-LP into resting B-cells can induce them into G1 phase.

EBNA2, a transcriptional activator, interacts with the basal transcription machinery but has no direct DNA-binding activity. Its ability to transactivate cellular and viral promoters is partly responsible for its ability to affect cell cycle progression. Although the proto-oncogene c-myc is a direct target for EBNA2, how EBNA2 activates c-myc is not fully understood. Because c-myc regulates expression of many genes involved in cell cycle signaling, activating c-myc likely links EBNA2 with the cell cycle. EBV also upregulates cyclin D2, which is a target of c-myc. Relatively little is known about EBNA-LP, which associates with Rb and p53 in vitro but does not interfere with their transcriptional activity.

Meanwhile, LMP1, an integral membrane protein, can transform established rodent fibroblasts and immortalize primary rodent fibroblasts. It also is a functional homologue of the tumor necrosis factor receptor family member and contributes substantially to the oncogenic potential of EBV by activating several signaling pathways, including nuclear factor κB (NF-κB), AP1, and JAK/STAT. LMP1 apparently upregulates the transcription of cyclin D1 via NF-κB signaling, accelerating G1/S phase and arresting the G2/M phase.


EBNA3C Acts as a Master Regulator

Our research on EBNA3C indicates it is key to dysregulating the cell cycle and absolutely required to transform B-cells in vitro. The protein itself is a large polypeptide of 992 amino acids with three putative nuclear localization signals, a leucine zipper domain, an acidic domain, and proline- and glutamine-rich domains.

EBNA3C, which interacts with both cellular and viral factors, is a transcriptional regulator, regulating chromatin by targeting acetylase, deacetylase, or other factors. EBNA3C binds to transcriptional repressor complexes that include histone-modifying deacetylase enzyme HDAC-1 and HDAC-2. Moreover, EBNA3C interacts with prothymosin α (ProTα) and the transcriptional coactivator p300, modulating histone acetylase transferase (HAT) activity. EBNA3C also interacts with the transcriptional corepressor C-terminal binding protein-1 (CtBP-1).

Further, EBNA3C is an oncoprotein that can cooperate with activated H-ras to transform rat embryo fibroblasts. Our studies show that EBNA3C directly suppresses the tumor suppressor proteins p53 and Rb.

In the case of Rb, EBNA3C becomes involved when the ubiquitin-proteasome pathway, which degrades proteins, is blocked. Under ordinary circumstances, the 26S proteasome degrades those proteins that ubiquitin ligases tagged with one or more ubiquitin molecules. We found that EBNA3C specifically recruits the ubiquitin ligase, Skp1/Cul1/F-box complex (SCF/Skp2), which adds ubiquitin to cell cycle regulators Rb and p27, marking them for subsequent degradation (Fig. 2). The interactions between SCF/Skp2 and Rb map to the amino terminus of EBNA3C, with residues 140-149 being absolutely essential.

The SCF/Skp2 complex is also important for the stability of p27, whose degradation activity is ordinarily associated with cyclin A complexes. Thus, we hypothesized that EBNA3C affects cyclin A activity to promote the G1/S transition. Indeed we found that there was enhanced cyclin A-associated kinase activity in EBNA3C-expressing cells. Moreover, EBNA3C promotes the G1/S transition and also disrupts the G2/M checkpoint by directly interacting with Chk2, an important player in the DNA damage repair pathway (Fig. 2). We find that EBNA3C can also stabilize the cellular oncoproteins c-Myc and Mdm2. Further, it either acts as a deubiquitinating enzyme (DUBs) or associates with DUBs.

EBNA3C also can suppress the function of p53 by stabilizing Mdm2 through deubiquitination. Mdm2 is an Ub ligase that negatively regulates the activity of p53 by facilitating its degradation through ubiquitination. EBNA3C was found to form a ternary complex with p53 and Mdm2 (Fig. 2). The binding site for Mdm2 was mapped to the amino-terminal domain of EBNA3C. Importantly, this binding is essential for Mdm2 stabilization. We are working on the mechanistic details of this interaction and how it provides a favorable environment for transformation and proliferation of EBV-infected cells. We also are continuing to study the signaling pathways through which EBNA3C transforms B-cells.


SUGGESTED READING

Kieff, E., and A. B. Rickinson. 2002. Epstein-Barr virus and its replication, p. 2511-2573. In D. Knipe and P. Howley (ed.), Fields virology, 4th ed., vol. 2.Lippincott Williams & Wilkins, Philadelphia.

Knight, J. S.,N. Sharma, and E. S. Robertson. 2005. Epstein-Barr virus latent antigen 3C can mediate the degradation of the retinoblastoma protein through an SCF cellular ubiquitin ligase. Proc. Natl. Acad. Sci. USA 102:18562-18566.

Knight, J. S., N. Sharma, and E. S. Robertson. 2005. SCFSkp2 complex targeted by Epstein-Barr virus essential nuclear antigen. Mol. Cell. Biol. 25:1749-1763.

Maruo, S., Y. Wu, S. Ishikawa, T. Kanda, D. Iwakiri, and K. Takada. 2006. Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells. Proc. Natl. Acad. Sci. USA 103:19500- 19505.

Middeldorp, J. M., and D. M. Pegtel. 2008. Multiple roles of LMP1 in Epstein-Barr virus induced immune escape. Semin. Cancer Biol. 18:388-396.

Robertson, Erle S. (ed.). 2005. Epstein-Barr virus. Caister Academic Press, Norwich, United Kingdom.

Robertson, Erle S. (ed.). 2010. Epstein-Barr virus: latency and transformation. Caister Academic Press, Norwich, United Kingdom.

O'Nions, J., and M. J. Allday. 2004. Deregulation of the cell cycle by the Epstein-Barr virus. Adv. Cancer Res. 92:119-186.

Saha, A., M. Murakami, P. Kumar, B. Bajaj, K. Sims, and E. S. Robertson. 2009. Epstein-Barr virus nuclear antigen 3C augments Mdm2-mediated p53 ubiquitination and degradation by deubiquitinating Mdm2. J Virol. 83:4652-4669.

Young, L. S., and A. B. Rickinson. 2004. Epstein-Barr virus: 40 years on. Nature Rev Cancer 4:757-768.