The Epstein-Barr virus (EBV) is associated with malignancies of lymphocytic and epithelial origin. EBV produces different infection states, cytolytic and latent, as well as cell immortalization, all of which are captured in cell lines, making them accessible to mechanistic studies. Currently, our research deals with viral latency, the ubiquitin-proteasome system in cell signaling, interferon regulatory factors (IRFs), invasion and metastasis, and antiviral drugs.
In EBV's cytolytic infection cycle we are focusing on two EBV gene products. One encodes EBV's sole protein kinase which phosphorylates other viral products, including the EBV DNA processivity factor EAD used in viral replication and viral maturation proteins involved in egress of viral nucleocapids from the nucleus. We are also studying a recently discovered EBV-encoded deubiquitinating enzyme BPLF1 and its role in the viral cytolytic cycle.
In latent infection we study how the cell cycle serves as a global regulator of viral latent gene expression through its effects on the major nuclear proteins, EBNA1 and EBNA2. We also study mechanisms of cell immortalization and oncogenesis through EBV's ability to stabilize and activate beta-catenin via the ubiquitin system. IRF7 was discovered in this laboratory, and how EBV is able to mount and evade immune responses through the ability of EBV LMP1 to induce and activate IRF7, now recognized as the master regulator of type I interferon responses, remains a principal focus. Finally we hold that EBV, in addition to being the etiologic agent for several malignancies, may also serve to promote tumor progression by the ability of its major oncoprotein, LMP1, to induce invasion, metastasis and angiogenic factors. Common to these areas is our emphasis on the role of tumor viruses in the ubiquitin-proteasomal system.
All trainees are encouraged to broaden their experience by also participating in the well known UNC-LCCC Postdoctoral Training Program, to assist in progressing toward their career goals. Fellows will also present their work in progress at one of the weekly meetings of the extended Virology Faculty at UNC.
We have proposed that the cell cycle is the master regulator of viral latent gene expression. Two distinct mechanisms are involved. EBNA-1, a nuclear factor that is required for replication of EBV episomes, is transcribed from the Q latency promoter in the most restricted form of EBV latency, Type 1. We have shown that QP is regulated by the reciprocal action of EBNA1 itself, which represses the promoter, and E2F, which overrides EBNA1 and activates the promoter during S-Phase. Thus there is essentially an "on / off" switch for transcription of EBNA1 mRNA that is placed under cell cycle control.
In the more complex Type 3 latency, at least nine viral gene products are expressed. All three of the promoters used for expression of these genes are transactivated by the product of one of them — EBNA2. We have shown that EBNA2 is hyperphosphorylated during mitosis. As a consequence, the activity of LMP1 (the principal EBV oncoprotein) is suppressed, and the level of LMP1 RNA is reduced. Since the promoters of all nine Type 3 latency genes are regulated by common mechanisms, implemented through the same viral and cellular proteins, we hypothesize that the expression of these latency genes may be suppressed in mitosis, as are many cellular genes.
This is the first evidence implicating the cell cycle in global control of latent viral gene expression.
Interferon Regulatory Factors
Studies of QP, which is a promoter used to transcribe EBNA1, led to the discovery of the 7th member of the interferon regulatory factor family (IRF7) and the first interferon stimulated response element (ISRE) identified in a mammalian viral promoter. IRF7 acts as a repressor of QP in EBV Type 3 latency, independent of the EBNA1 / E2F switch. LMP1 (which is expressed in Type 3 latency), induces IRF7 and activates it by phosphorylation, and more interestingly, by ubiquitination as discovered recently in our lab.
The increasing importance of IRF7 in host immune defenses is highlighted by recent discoveries that identify IRF7 as the master regulator of all type I interferon-dependent immune responses, the IRF7 signaling pathway involving Toll-like receptors (TLRs), and an IRF7 activation mechanism through ubiquitination that is independent of its major functional phosphorylation sites. Like TLR signaling, the ubiquitination pathway involved in LMP1 activation of IRF7 is also dependent on RIP (TNF receptor interacting protein) and TRAF6 (tumor necrosis factor receptor-associated factor 6). Moreover, we showed that the three C-terminal lysines sites on IRF7 are required for its ubiquitination-mediated activation, implying that ubiquitination on these sites may be a prelude to IRF7 phosphorylation.
In addition to being essential for induction of Type 1 interferons, IRF7 also induces expression of TAP2, STATs, and other ISGs which play important roles in cellular immunity. IRF7 thus appears to be a central regulator of EBV latency and cellular immunity. Finally, IRF7 appears to potentiate the oncogenic effects of LMP1, at least in part, by a regulatory circuit between the promoters for these two proteins, one cellular and one viral. Consistent with these findings, IRF7, which has oncogenic properties as detected in NIH 3T3 cell assays, may cooperate with LMP1 in EBV oncogenesis. The significance of these observations is underscored by the fact that IRF7 is overexpressed in EBV-positive CNS lymphomas.
The Ubiquitin-Proteasome System in Cell Signaling
Additionally, we have found that the β-catenin pathway is activated in Type 3 (but not Type 1) latency. We have discovered that whereas β-catenin is degraded by a proteasomal mechanism in Type 1 infection, the protein remains unexpectedly stable in Type 3 latently infected lymphocytes. The protein is also functional, as gauged by its ability to transactivate a TCF / LEF-responsive promoter, despite the integrity of the proteasome system. We have implicated the deubiquitinase (DUB) system in the stabilization of β-catenin and identified Type 3 viral proteins as candidates for inducing or activating one or more DUBs.
The β-catenin signaling pathway functions in many biologic contexts and may now be implicated in lymphomagenesis. This work discloses mechanisms whereby a tumor virus may activate this signaling pathway.
Invasion and Metastasis
We have established that EBV induces a constellation of invasion and metastasis factors. Nasopharyngeal carcinoma (NPC), which is latently infected with EBV, is an invasive tumor. We first discovered that one of the collagenases involved in tissue invasion and metastasis, matrix metalloproteinase-9 (MMP9), is induced by the EBV oncoprotein LMP1. MMP9 is upregulated in NPC and inhibited by salicylates which inhibit IκB kinase. LMP1 also induces COX2, VEGF, FGF2 and HIF1α. Thus, EBV not only itself transforms cells, but the virus can also program steps in the malignant process that are usually the cause of death, namely tumor invasion, angiogenesis and metastasis. LMP1 is the first viral oncoprotein shown to induce such factors, which are important in later stages in oncogenesis. Thus, as an alternative to an etiological relation, EBV may modify the phenotype of an established tumor.
Antiviral Drugs
Most of the studies on antiviral drugs that inhibit EBV replication have come from this laboratory. Current work focuses on maribavir, a potent drug that inhibits EBV (as well as cytomegalovirus) replication, but whose mechanism of action differs from that of established drugs. We have shown that the only EBV-encoded protein kinase (EBV PK) is responsible for phosphorylating the EBV DNA processivity factor EAD, which is an accessory to the viral polymerase. Maribavir inhibits this phosphorylation, but the effect of the drug on EBV PK is indirect.
EBV is directly responsible for fatal lymphoproliferative neoplasms in immunocompromised persons. Maribavir is the first non-nucleoside analog that could be potentially useful in interfering with the generation of new populations of infected lymphocytes during the polyclonal phase of lymphoproliferative diseases.
EBV is associated with several malignancies characterized by a latent infection state, which is not responsive to available antiviral drugs. HPMPC, a phosphonated nucleoside analog, offers promise for treatment for one of these cancers, NPC, in that the drug induces explosive apoptosis in NPC grown as xenografts in athymic mice. Findings with this and other drug family members may be adaptable for treatment of NPC in human beings.