Epstein-Barr virus (EBV) infection of oropharyngeal epithelial cells is associated with at least two types of disease: oral hairy leukoplakia (OHL), a tongue lesion caused by lytic EBV infection of differentiated epithelial cells, and nasopharyngeal carcinoma (NPC), a malignancy characterized by latent EBV infection of undifferentiated epithelial cells. AIDS patients have impaired control of lytic and latent EBV infection and increased incidence of OHL and NPC compared to normal hosts. The two EBV immediate-early proteins, BZLF1 (Z) and BRLF1 (R), promote the switch from latent to lytic EBV infection. Our recent studies have shown DNA methylation of lytic promoters enhances Z, but impedes R activation of lytic infection. Our exciting preliminary data reveal that the newly described epigenetic modification implicated in cytosine demethylation, 5-hydroxymethyl-cytosine (5-hmC), has a profound effect upon the ability of Z versus R to induce lytic EBV reactivation. Although epithelial cell differentiation has been linked to EBV lytic reactivation, studies of this relationship have been limited by the lack of an organotypic in vitro model that stably maintains EBV infection. We recently identified a telomerase immortalized normal oral keratinocyte (NOK) line that can be stably EBV infected, demonstrated that EBV+ NOKs undergo differentiation in raft cultures, and that lytic EBV reactivation occurs specifically in the more differentiated cells To date, EBV+ NOK is the only cell line shown to contain largely unmethylated EBV genomes, and the only EBV+ cell line known to reactivate following R, but not Z, expression. Thus, EBV+ NOKs are a unique tool for understanding the epigenetic mechanisms that link epithelial cell differentiation to the conversion of EBV latent to lytic infection and how EBV latent and lytic gene products influence the epigenetic state of infected oral epithelial cells.
In Specific Aim 1, we will characterize the epigenetic modifications of the host and viral genomes that occur during differentiation, and result in lytic reactivation and determine the role of BLIMP1 in reactivating EBV during NOK differentiation.
In Specific Aim 2, we will examine the effect of 5-hmC modification on lytic and latent EBV gene expression.
In Specific Aim 3, we will use the EBV+ NOK system to examine the effects of LMP1 and LMP2A on epithelial cell differentiation, viral replication, and the epigenetic state of the viral and cellular genomes.
In Specific Aim 4, we will characterize the extent of 5-hmC modification in NPC specimens and determine if the 5-hmC pathway is disrupted in NPC tumors. We hypothesize that a) methylation and 5-hmC modification of the EBV genome controls reactivation by Z versus R, b) epithelial differentiation regulates EBV reactivation at least partially via effects on viral genome methylation and 5-hmC modification, and c) EBV+ NPC tumors only occur in undifferentiated epithelial cells that promote viral latency at least partially via viral genome epigenetic modifications. The proposed studies should greatly enhance our understanding of how EBV normally replicates in differentiated epithelial cells yet achieves long term viral latency in undifferentiated epithelial tumor cells.

Public Health Relevance

Epstein-Barr virus (EBV) is a human herpesvirus that replicates in epithelial cells and establishes latency in B lymphocytes. Latent EBV infection is also characteristic of nasopharyngeal carcinoma (NPC), an aggressive epithelial tumor. In this application, we propose to use a new model of EBV epithelial infection to learn what determines whether EBV replicates or establishes latency. This knowledge may permit the development of new therapies for NPC.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE023939-03
Application #
8894306
Study Section
Special Emphasis Panel (ZDE1)
Program Officer
Rodriguez-Chavez, Isaac R
Project Start
2013-09-20
Project End
2016-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
3
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Djavadian, Reza; Hayes, Mitchell; Johannsen, Eric (2018) CAGE-seq analysis of Epstein-Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms. PLoS Pathog 14:e1007114
Nawandar, Dhananjay M; Ohashi, Makoto; Djavadian, Reza et al. (2017) Differentiation-Dependent LMP1 Expression Is Required for Efficient Lytic Epstein-Barr Virus Reactivation in Epithelial Cells. J Virol 91:
Wille, Coral K; Li, Yangguang; Rui, Lixin et al. (2017) Restricted TET2 Expression in Germinal Center Type B Cells Promotes Stringent Epstein-Barr Virus Latency. J Virol 91:
Wang, Anqi; Welch, Rene; Zhao, Bo et al. (2016) Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites. J Virol 90:2906-19
Djavadian, Reza; Chiu, Ya-Fang; Johannsen, Eric (2016) An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription. PLoS Pathog 12:e1005718
Makielski, Kathleen R; Lee, Denis; Lorenz, Laurel D et al. (2016) Human papillomavirus promotes Epstein-Barr virus maintenance and lytic reactivation in immortalized oral keratinocytes. Virology 495:52-62
Nawandar, Dhananjay M; Wang, Anqi; Makielski, Kathleen et al. (2015) Differentiation-Dependent KLF4 Expression Promotes Lytic Epstein-Barr Virus Infection in Epithelial Cells. PLoS Pathog 11:e1005195
Reusch, Jessica A; Nawandar, Dhananjay M; Wright, Kenneth L et al. (2015) Cellular differentiation regulator BLIMP1 induces Epstein-Barr virus lytic reactivation in epithelial and B cells by activating transcription from both the R and Z promoters. J Virol 89:1731-43
Wille, Coral K; Nawandar, Dhananjay M; Henning, Amanda N et al. (2015) 5-hydroxymethylation of the EBV genome regulates the latent to lytic switch. Proc Natl Acad Sci U S A 112:E7257-65
Ohashi, Makoto; Holthaus, Amy M; Calderwood, Michael A et al. (2015) The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth. PLoS Pathog 11:e1004822

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