Epstein-Barr virus (EBV) is etiologically associatedwith many malignant diseases, including nasopharyngeal and gastric cancer, and Burkitt's, Hodgkin's and non-Hodgkin's lymphoma. EBV remains atent in EBV-associated tumors and in normal B cells. Reactivation is required for the virus to spread between cells and among people. Reactivation from latency into the lytic cycle may be oncolytic. Lytic cycle products may also possess direct oncogenic effects. Our lab discovered and has intensively studied the EBV BZLF1 gene product, ZEBRA, which mediates the switch between latency and the lytic cycle. The global objective of work supported by CA12055 is to understandZEBRA's mechanism of action and regulation of its expression. In this application for an extensionof the MERIT award, we describe significant published and preliminary data of the past 4 years to accomplish this objective. We discoveredZEBRA point mutants which arrest the EBV lytic cycle at distinct phases of immediate-early, early and late gene expression. Each class of ZEBRA mutant elicits unique nuclear reorganization. We mapped the in vivo phosphorylation sites on ZEBRA and showed that phosphorylation of ZEBRA is required for its activity as a temporally regulated represser of viral late genes and for its activity in binding and activating the origin of lytic DMAreplication. We identified phosphoacceptor site mutants which specifically impaired ZEBRA's capacity to activate DMAreplication, but did not affect ZEBRA's ability to act as a transcriptional activator. We showed that induction of BZLF1 expression by histone deacetylase inhibitors and other inducing stimuli is mediated by newly synthesized cellular proteins. We found that a DMA methyltransferase inhibitor induces BZLF1 expression by a novel mechanism without detectably demethylating the promoter of this gene. We developed methods to separate lymphoma cells in which EBV is latent or lytic. Using this method we have shown that primary EBV infection is associated with an IgA response to early lytic antigens, such as ZEBRA. The proposed studies, utilizing tools of cell biology, biochemistry, molecular genetics, immunology and epidemiology, build on these seminal observations. The experiments address both basic questions of control of eukaryotic gene expression that are relevant to cancer and specific unresolved issues about molecular pathogenesis of an important human cancer virus.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Daschner, Phillip J
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Yale University
Schools of Medicine
New Haven
United States
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Park, Richard; Miller, George (2018) Epstein-Barr Virus-Induced Nodules on Viral Replication Compartments Contain RNA Processing Proteins and a Viral Long Noncoding RNA. J Virol 92:
Gorres, Kelly L; Daigle, Derek; Mohanram, Sudharshan et al. (2016) Valpromide Inhibits Lytic Cycle Reactivation of Epstein-Barr Virus. MBio 7:e00113
Wang'ondu, Ruth; Teal, Stuart; Park, Richard et al. (2015) DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA. PLoS One 10:e0126088
Gorres, Kelly L; Daigle, Derek; Mohanram, Sudharshan et al. (2014) Activation and repression of Epstein-Barr Virus and Kaposi's sarcoma-associated herpesvirus lytic cycles by short- and medium-chain fatty acids. J Virol 88:8028-44
Park, Richard; El-Guindy, Ayman; Heston, Lee et al. (2014) Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A)-binding protein are distinct processes mediated by two Epstein Barr virus proteins. PLoS One 9:e92593
McAllister, Shane C; Shedd, Duane; Mueller, Nancy E et al. (2014) Serum IgA to Epstein-Barr virus early antigen-diffuse identifies Hodgkin's lymphoma. J Med Virol 86:1621-8
El-Guindy, Ayman; Ghiassi-Nejad, Maryam; Golden, Sean et al. (2013) Essential role of Rta in lytic DNA replication of Epstein-Barr virus. J Virol 87:208-23
Yu, Kuan-Ping; Heston, Lee; Park, Richard et al. (2013) Latency of Epstein-Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA. Proc Natl Acad Sci U S A 110:8176-81
Daigle, Derek; Gradoville, Lyn; Tuck, David et al. (2011) Valproic acid antagonizes the capacity of other histone deacetylase inhibitors to activate the Epstein-barr virus lytic cycle. J Virol 85:5628-43
Park, Richard; Wang'ondu, Ruth; Heston, Lee et al. (2011) Efficient induction of nuclear aggresomes by specific single missense mutations in the DNA-binding domain of a viral AP-1 homolog. J Biol Chem 286:9748-62

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