Human papillomavirus type 6 (HPV 6) causes the most prevalent viral sexually transmitted disease, condyloma acuminata or genital warts. On rare occasions it is associated with lower genital tract squamous cell carcinomas. The overall objective of this laboratory is to elucidate the mechanism of pathogenicity of HPV 6. In particular, in this proposal, we are concentrating on the regulation of the papillomavirus replication cycle by addressing the mechanism of regulation of viral gene expression during the productive viral life cycle in keratinocytes. The expression of the viral genome is tightly linked to the state of differentiation of the keratinocyte with little gene expression detectable in undifferentiated basal cells and minimally differentiated spinous cells and readily detectable gene expression in the more differentiated cell layers.
Three specific aims are proposed to test the hypothesi that changes in interactions with elements in the 5' half of the long control region (LCR), within the 3' untranslated region of the late mRNA (L1 3' UTR), contribute to low level expression in undifferentiated cells and high level gene expression in differentiated cells.
In specific aim 1 experiments in undifferentiated cells will be continued to analyze the DNA:protein interaction detected by gel shift and functional assays of mutants will be conducted to further delineate the silencer element within the 66-mer region, which negatively regulates the downstream E6 promoter. In subsequent experiments the activity of the silencer during differentiation will be determined by a) conducting functional analyses on keratinocytes induced to differentiate, and b) isolating the silencing protein, SIL, and characterizing it in undifferentiated and differentiated cells.
In specific aim 2 the mechanism by which the L1 3' UTR negatively regulates late gene expression will be determined. Whether the 66-mer is necessary and sufficient to perform this function, whether negative regulation of initiation of transcription completely explains the effect on late gene expression or whether other sequences and/or the presence of the sequence in the viral transcript is necessary will be determined. Whether there are changes in this effect during differentiation will be established.
In specific aim 3, the sequence of the 3' UTR will be determined for 12 L1-positive and 12 L1-negative biopsies from immunocompetent, non-pregnant women to determine whether L1 positivity falls into a limited number of groups suggesting the 3' UTR plays a role, or whether L1 positivity is scattered among all of the groups suggesting that other factors play a role. If L1 positivity remains limited to a few groups, the analysis will be extended to a similar set of samples from pregnant or immunocompromised patients since the frequency of L1 positivity is higher in these biopsies.
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