Date of Award

5-1-2016

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Gershburg, Edward

Abstract

Epstein-Barr virus (EBV) is a human herpesvirus that infects more than 95% of the world’s population by adulthood. Like other herpesviruses, EBV has a biphasic infection cycle which includes latent and lytic programs. Upon primary infection, the virus establishes a lifelong latency in memory B lymphocytes characterized by a highly restricted gene expression and lack of virion production. Signals responsible for differentiation of mature B lymphocytes into plasma cells may activate viral lytic program in latently infected cells leading to a coordinated expression of majority of viral genes and production of progeny virions. Although most infections are asymptomatic, the primary clinical manifestation of EBV infection is infectious mononucleosis (IM), which accounts for 125,000 cases each year in the US. In addition, EBV is associated with several malignancies including Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma and gastric carcinoma, which account for approximately 200,000 new cases and 140,000 deaths each year worldwide. Cellular protein kinases have been reported to play a role in the viral life cycle, but their influence on different stages of EBV life cycle has not been studied extensively. Therefore, in the first part of my dissertation, I investigated the involvement of cellular protein kinases in the EBV lytic program. To this end, signaling pathways mediating Epstein-Barr virus (EBV) reactivation by antigen-bound B-cell receptor (BCR) were analyzed using a panel of 80 protein kinase inhibitors. Broad range protein kinase inhibitors Staurosporin, K252A, and PKC-412 significantly reduced the EBV genome copy numbers measured 48 hours after reactivation, perhaps due to their higher toxicity. In addition, selected inhibitors of the phosphatidylinositol-3-kinase (PI3K), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and nuclear factor B (NF-B) pathways, glycogen synthase kinase 3 (GSK-3), platelet-derived growth factor receptor-associated tyrosine kinase (PDGFRK), and epidermal growth factor receptor-associated tyrosine kinase (EGFRK) significantly reduced the EBV genome copy numbers as well. Of those, only U0126 and Erbstatin analog, which inhibit MAPK pathway and EGFRK respectively, did not inhibit viral reactivation assessed by expression of the EBV early protein, EA-D. None of the tested compounds, except for K252A, affected the activity of the EBV-encoded protein kinase in vitro. These results show that EBV reactivation induced by BCR signaling is mainly mediated through PI3K and PKC, whereas MAPK might be involved in later stages of viral replication. The goal of the second part of my dissertation was to comprehensively characterize IgG response to EBV infection in humans. Currently, EBV serology is used to confirm the EBV infection status of a patient, but there are no clinical markers available to date to diagnose EBV-associated cancers. Out of the 80 proteins that the virus expresses, EBV serology relies on detection of antibodies against only three antigens: BFRF3 (viral capsid antigen or VCA), BMRF1 (early antigen or EA), and BKRF1 (nuclear antigen or NA). Antibodies against two of these antigens, VCA and NA, are produced by almost all EBV carriers for their entire lifetime, and therefore, detection of these antibodies makes it impossible to differentiate between EBV carriers and EBV-associated cancer patients. To address this deficiency, the current project sought to determine if the human antibody response to EBV significantly differed between asymptomatic EBV carriers versus EBV-associated cancer patients. To this end, FLAG-tagged genes representing 90% of the EBV proteome were cloned into a mammalian expression vector, individually expressed in HEK293 cells, and immunoblotted with a monoclonal FLAG antibody (FLAG mAb), and each of the 55 human serum samples in the panel under study. My data confirmed the efficacy of the currently used markers (BFRF3, BKRF1), and several other previously studied potential markers (BNRF1, BdRF1, BLRF2, EBNA-3A, EBNA-3B, BHRF1, and BZLF1). I have also identified 5 novel immunodominant viral antigens (BPLF1, BKRF4, BRRF2, BLLF3, and BSLF2/BMLF1) that have never been described as major antigens of EBV. In addition, I have confirmed previous observations that the antibody response in sera from EBV-associated NPC patients differs significantly from that in sera from EBV seropositive individuals who did not develop an EBV-associated malignancy. I have identified multiple EBV antigens (BXLF1, BPLF1, BMRF1, BHRF1, BKRF4, BLLF3, BZLF1 and BSLF2/BMLF1) that were detected at higher frequency by sera from NPC patients. One of these antigens, BXLF1, was detected by 90% of sera from NPC patients, but only by 10% of the matched control sera, thus suggesting that this antigen may serve as a biomarker of NPC. These data advocate the feasibility of developing a serological assay that may be able to diagnose EBV-associated malignancies at early stages of the disease. Moreover, further studies are warranted to explore the prognostic value of such biomarkers, which could identify individuals prone to developing EBV-associated malignancies, allow their close monitoring, and early intervention.

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