Subsets of human papillomaviruses (HPV) are associated with malignant and benign tumors of the anogenital region. Using a cell line derived from a biopsy of a cervical intraepithelial neoplasia type 1 (CIN 1), we recently demonstrated in an organotypic (raft) culture system the complete HPV replication cycle concomitant with the induction of a more complete differentiation program in vitro The use of our raft culture system with its ability to reproduce the complete HPV replication cycle allows for a complete description of HPV activity, transcriptional and translational, in human epithelial tissue actively propagating virus. Our working hypothesis is that HPV's replication cycle is intimately connected to the growth and terminal differentiation program of epithelial tissues. Changes that occur in epithelial cells as they undergo the process of differentiation directly control the viral replication cycle at the transcriptional and translational levels. To this end, we plan to define viral transcriptional and translational activity and correlate this activity to the temporal and spatial development of the natural host tissue, human squamous epithelium. In addition, the second aspect of our working hypothesis is that HPV infection and subsequent viral gene expression alters the differentiation program of the host tissue. In the case of the high risk HPV types, it is this interaction that results in the eventual progression of the neoplasia to malignancy. The first specific aim focuses on the patterns of HPV transcription in differentiating human epithelium during the complete viral replication cycle. The second specific aim focuses on HPV 31b protein expression patterns in differentiating human epithelium. A comparison of viral transcriptional and translational expression patterns will be made in (i) differentiating cervical epithelium that has been induced by 12-O- tetradecanoyl phorbol 13-acetate (TPA) to propagate HPV virions, (ii) differentiating cervical epithelium not treated with TPA but latently infected with HPV, and (iii) monolayer cultures of cervical cells. Using in situ technologies, we will map the spatial patterns of the HPV 31b gene expression in the context of the three-dimensional architecture in stratified and differentiated human epithelium. The use of the raft cultures will allow us to define the patterns of HPV gene expression during the temporal development of the host tissue. The expression of terminal epithelial differentiation markers including keratin 10, filaggrin, and involucrin will be examined and compared with HPV gene expression. The studies proposed in this application will enable us to test our working hypothesis that HPV gene expression and replication is contingent upon changes in the cellular environment of the epithelial cell as it stratifies and differentiates.
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