Department of Medicine
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 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 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 b-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 EBNA-1 itself, which represses the promoter, and E2F, which overrides EBNA-1 and activates the promoter during S-Phase. Thus there is essentially an “on / off” switch for transcription of EBNA-1 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 — EBNA-2. We have shown that EBNA-2 is hyperphosphorylated during mitosis. As a consequence, the activity of LMP-1 (the principal EBV oncoprotein) is suppressed, and the level of LMP-1 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 phosphorylation. 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). More detailed mechanisms are under study and more important signaling molecules will be identified.
In addition to being essential for induction of Type 1 interferons, IRF7 also activates the TAP2 gene. 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 ß-catenin pathway is activated in Type 3 (but not Type 1) latency. We have discovered that whereas ß-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 ß-catenin and identified Type 3 viral proteins as candidates for inducing or activating one or more DUBs.
The ß-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 (MMP-9), is induced by the EBV oncoprotein LMP-1. MMP-9 is upregulated in NPC and inhibited by salicylates which inhibit I?B kinase. LMP-1 also induces COX-2, VEGF, FGF-2 and HIF1a. 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. LMP-1 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.
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, 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.
The most important recent discoveries from our laboratory are that:
- LMP1, the principal oncoprotein of EBV, activates IRF7 through a RIP- and TRAF6-dependent ubiquitination pathway.
- A tumor virus, EBV, can activate the ß-catenin signaling pathway in human lymphocytes.
- Activation of the ß-catenin pathway is through a novel mechanism, namely, the induction or activation of one or more deubiquitinating enzymes.
- IRF-7 has oncogenic properties that may potentiate those of LMP-1, the principal EBV oncoprotein, and it is overexpressed in CNS lymphomas.
- Among the invasion and metastasis factors that we have discovered are induced by EBV — MMP-9, COX-2, VEGF, FGF-2 — the most recent is HIF-1a. The EBV oncoprotein LMP-1 is again the first known tumor virus protein shown to induce HIF-1a, the sole transcription factor that mediates all known cellular responses to low oxygen tension.
Bentz GL, Whitehurst CB, Pagano JS (2011). Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) C-Terminal-Activating Region 3 Contributes to LMP1-Mediated Cellular Migration via Its Interaction with Ubc9. J Virol. 85(19):10144-53.
Bheda A, Yue W, Gullapalli A, Shackelford J, Pagano JS (2011). PU.1-dependent regulation of UCH L1 expression in B-lymphoma cells. Leuk Lymphoma. 52(7):1336-47.
Ning S, Pagano JS (2010). The A20 deubiquitinase activity negatively regulates LMP1 activation of IRF7. J Virol. 84(12):6130-8.
Bentz GL, Liu R, Hahn AM, Shackelford J, Pagano JS (2010). Epstein-Barr virus BRLF1 inhibits transcription of IRF3 and IRF7 and suppresses induction of interferon-beta. Virology. 402(1):121-8.
Wang FZ, Roy D, Gershburg E, Whitehurst CB, Dittmer DP, Pagano JS (2009). Maribavir inhibits epstein-barr virus transcription in addition to viral DNA replication. J Virol. 83(23):12108-17.
Bheda A, Shackelford J, Pagano JS (2009). Expression and functional studies of ubiquitin C-terminal hydrolase L1 regulated genes. PLoS One. 4(8):e6764.
Whitehurst CB, Ning S, Bentz GL, Dufour F, Gershburg E, Shackelford J, Langelier Y, Pagano JS (2009). The Epstein-Barr virus (EBV) deubiquitinating enzyme BPLF1 reduces EBV ribonucleotide reductase activity. J Virol. 83(9):4345-53.
Ning S, Campos AD, Darnay BG, Bentz GL, Pagano JS (2008). TRAF6 and the three C-terminal lysine sites on IRF7 are required for its ubiquitination-mediated activation by the tumor necrosis factor receptor family member latent membrane protein 1. Mol Cell Biol. 28(20):6536-46.
Gershburg E, Raffa S, Torrisi MR, Pagano JS (2007). Epstein-Barr virus-encoded protein kinase (BGLF4) is involved in production of infectious virus. J Virol. 81(10):5407-12.
Horikawa T, Yang J, Kondo S, Yoshizaki T, Joab I, Furukawa M, Pagano JS (2007). Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma.
Cancer Res. 67(5):1970-8.
Huye LE, Ning S, Kelliher M, Pagano JS (2007). Interferon regulatory factor 7 is activated by a viral oncoprotein through RIP-dependent ubiquitination. Mol Cell Biol. 27(8):2910-8.
Pagano JS (2007). Is Epstein-Barr virus transmitted sexually? J Infect Dis. 195(4):469-70
Kondo S, Yoshizaki T, Wakisaka N, Horikawa T, Murono S, Jang KL, Joab I, Furukawa M, Pagano JS (2007). MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling. J Virol. 81(4):1554-62.
Kondo S, Seo SY, Yoshizaki T, Wakisaka N, Furukawa M, Joab I, Jang KL, Pagano JS (2006). EBV latent membrane protein 1 up-regulates hypoxia-inducible factor 1alpha through Siah1-mediated down-regulation of prolyl hydroxylases 1 and 3 in nasopharyngeal epithelial cells. Cancer Res. 66(20):9870-7.
Yue W, Shackelford J, Pagano JS (2006). cdc2/cyclin B1-dependent phosphorylation of EBNA2 at Ser243 regulates its function in mitosis. J Virol. 80(4):2045-50.
Jang KL, Shackelford J, Seo SY, Pagano JS (2005). Up-regulation of beta-catenin by a viral oncogene correlates with inhibition of the seven in absentia homolog 1 in B lymphoma cells. Proc Natl Acad Sci U S A. 102(51):18431-6.
Ning S, Huye LE, Pagano JS (2005). Interferon regulatory factor 5 represses expression of the Epstein-Barr virus oncoprotein LMP1: braking of the IRF7/LMP1 regulatory circuit. J Virol. 79(18):11671-6.
Hahn AM, Huye LE, Ning S, Webster-Cyriaque J, Pagano JS (2005). Interferon regulatory factor 7 is negatively regulated by the Epstein-Barr virus immediate-early gene, BZLF-1. J Virol. 79(15):10040-52.
Yue W, Gershburg E, Pagano JS (2005). Hyperphosphorylation of EBNA2 by Epstein-Barr virus protein kinase suppresses transactivation of the LMP1 promoter. J Virol. 79(9):5880-5.
Wakisaka N, Yoshizaki T, Raab-Traub N, Pagano JS (2005). Ribonucleotide reductase inhibitors enhance cidofovir-induced apoptosis in EBV-positive nasopharyngeal carcinoma xenografts. Int J Cancer. 116(4):640-5.
Ning S, Huye LE, Pagano JS (2005). Regulation of the transcriptional activity of the IRF7 promoter by a pathway independent of interferon signaling. J Biol Chem. 280(13):12262-70.
1968-73 U.S.P.H.S. Research Career Development Award
1990 First Gertrude and Werner Henle Lectureship In Viral Oncology
1993-01 Member, Board of Directors, Burroughs Wellcome Fund
1996 The North Carolina Award in Science
1996 McLaughlin Visiting Professor, The University of Texas Medical Branch
1997 Norma Berryhill Distinguished Lecturer, University of North Carolina
1997 Harry Eagle Lecturer, Albert Einstein College of Medicine
1997-01 Member, Awards Assembly of the General Motors Cancer Research Foundation
1998- Institute of Medicine, Member
2002 Joseph and Ruth McCartney Hauck Lecturer, Mayo Clinic
2003 Lecturer, Japan Society for Head & Neck Cancer; 100th Anniversary, ENT Dept., Kanazawa Univ.
2004 Fellow, American Association for the Advancement of Science