The herpesvirus EBV is an extremely successful pathogen, which infects the majority of the population world- wide. Primary infection can be asymptomatic but is frequently associated with infectious mononucleosis, and there is a significant correlation between IM and the development of Hodgkins lymphoma. About 200, 000 malignancies per year are EBV-associated, and the majority of these are in epithelial cells. While the virus can infect both B and epithelial cells, resulting in tumors in both cell types, ver little is known about the role of epithelium in the lifecycle of EBV. EBV is transmitted to a nav host through saliva, and whether EBV replicates in epithelial cells to release virions into the ora cavity has long been debated. EBV has the characteristic glycoprotein profile of a virus generated in the epithelium, though little evidence of EBV infection of oral tissue in normal epithelium has been found until very recently. Surprisingly to many in the field, these cells harbored latent EBV (i.e., not productively replicating virus). Because latency is central to the life-long survival of EBV in the human host, it is very important to define latent infection in epithelium and determine the factors that favor latency, particularly given that EBV-associated tumors carry a latent infection. Although raft cultures clearly support productive replication, it s not clear whether or not they also support a latent infection. This application proposes to use organotypic raft cultures as a model for EBV infection of oral epithelium to determine whether a latently infected population of cells exists. To infect its host, a herpesvirus must bind to the hot cells, fuse with the cell membrane, and the viral genome must reach the nucleus, circularize and be maintained in the cell. If latency is not observed, investigation of these steps may provide clues as to why normal epithelium does not support latency while cancerous epithelium does. An understanding of the features of transformed cells that facilitate latent infection may suggest ways to block EBV latency in tumors. Because it now seems clear that the virus that initiates spread of infection originates in epithelial cells, it is important to understand how the virus traffics through the epithelium, either to establish a latent reservoir of infection or to propagat infection. The application poses some innovative hypotheses for the sites of a latent reservoir, which can be addressed experimentally by utilizing markers to identify cells infected by EBV, even it few viral genomes are expressed. The organotypic model will allow exploration of multiple steps in the virus life-cycle that cannot be addressed in monolayer culture or in vivo and should provide a greater understanding of the role of latent infection in epithelium and the factors that influence it, positively or negatively. In addition, the proposed experiments have the potential for a large impact on the development of antiviral strategies.
Epstein-Barr virus (EBV) is associated with ~200,000 malignancies per year, chiefly of epithelial origin. The proposed research is intended to fill the gaps in our knowledge regarding the lifecycle of EBV in epithelial cells. Because the oral mucosa is the site of entry and exit of the virus, epithelial cells play a major role in viral spred to uninfected individuals and thus, understanding their role in the viral life cycle will not only shed light on this important step but has the potential to impact all downstream events from EBV infection including malignancies.
