The development of oral hairy leukoplakia is a result of EBV infection in oral epithelial cells during HIV infection. Specific cofactors enhance the susceptibility of oral epithelial cells to infection (inadequate or inappropriate immune responses) and/or prolong survival of virus infected cells (coinfection with transforming viruses, inadequate immune responses). To provide insight into the events that determine how EBV infects epithelial cells and enhance our understanding of the pathogenesis of OHL. Procedures in the Proposal: To test the hypothesis a cell culture system will be established in which direct lytic infection by EBV or efficient induction of lytic infection following latent infection can be demonstrated in epithelial cells.
Specific aims are: 1) Analyze an immortalized, non-tumorigenic epithelial cell line that naturally expresses the virus receptor and can be stably infected upon selection with recombinant EBV. Explore how the receptor mediates attachment, internalization and signaling and determine which genes are expressed upon entry. Compare the ability of different virus strains to produce efficient infection and assess whether early infection has a lytic component. Determine whether early lytic replication can be activated by specific cytokines (as might be altered in HIV infection) and will study events that cause switching from the latent program established by recombinant virus selection in vitro to the lytic program that has been observed in vivo (inducers of differentiation, immediate early gene transfer, antagonists of oncogene expression). 2)Apply observations to the establishment of a normal epithelial cell culture system. Obtain normal oral keratinocytes of lateral tongue origin, keratinocytes from this site engineered to express HPV 16 E6 and/or E7 naturally arising preneoplastic and fully malignant from this site. These cells will be examined for receptor expression, infectibility, and for the type of EBV infection they are able to support in two types of conventional culture systems as well as in organotypic culture. The effects of exposure of cells to inflammatory mediators (as might be altered in HIV infection) will be assessed relative to receptor expression and the pattern of virus infection. 3)Study the biology of specific lytic viral genes in the epithelial line and oral keratinocytes in culture. Initial studies will focus on the immediate early transactivators. These genes will be introduced by retrovirus mediated gene transfer under the control of an inducible promotor and their capacity to alter the state of differentiation of the cell in the absence/ presence of EBV infection and together with specific inflammatory mediators will be assessed.
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