All ETDs from UAB

Advisory Committee Chair

Louise T Chow

Advisory Committee Members

Susan Ruppert

David Schneider

John A Thompson

Document Type

Dissertation

Date of Award

2012

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The large family of human papillomaviruses (HPVs) infects the cutaneous or mucosal epithelia causing benign hyper proliferative diseases. Infections by the high-risk (HR) HPV genotypes in the anogenital tracts can progress to high grade lesions and cancers in men and women. Because the viral productive program requires squamous differentiation of epithelia, HPV encodes oncoproteins that recondition the differentiated cells that have withdrawn from the cell cycle to support viral DNA amplification. The HR HPV E6 and E7 oncogenes are selectively expressed in cervical carcinomas and are necessary to maintain the malignant state. In vitro, constitutive expression of the HR HPV E6 and E7 proteins can immortalize primary human epithelial cells. The E7 oncoproteins of diverse HPV genotypes destabilize p130, a pocket protein related to pRB, resulting in S phase reentry by differentiated keratinocytes in which viral DNA amplifies during prolonged G2 phase. It is well established that the high-risk HPV E6 protein destabilizes p53 and many other host proteins. However, the critical E6 targets relevant to viral DNA amplification have not been identified because functionally significant E6 mutants cannot be stably maintained in transfected cells. Recently, our lab has developed just such a system which recapitulates a highly productive HPV-18 program in organotypic cultures of primary human keratinocytes. The goal of my research is to examine the role of the E6-p53 interaction in viral DNA amplification. We have developed an improved trans-complementation system and have constructed and tested an E6-null mutant and several E6 mis-sense mutants. Each was characterized in its ability to stabilize p53 and to support viral DNA amplification. Our results suggest high levels of p53 and viral DNA amplification were detected in separate cell populations. Trans-complementation results showed that over-expression of E6 may inhibit viral DNA amplification. We also identified several novel spliced early and late viral RNAs from wt and mutant-containing raft cultures.

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