Date of Award

Winter 12-2001

Document Type

Thesis

Degree Name

Honors College Theses

Department

Microbiology, Molecular Biology and Biochemistry

Second Advisor

Dr. Joyce W. O'Rourke

Third Advisor

Dr. Beverly Wade

Abstract

There are over seventy genotypes of Human Papillomaviruses (HPVs), some cause benign warts and others are risk factors for the development of carcinomas of the respiratory and genital tract. Research has proven that the high risk HPV 16 E5 protein in the presence of epidermal growth (EGF) acts as a survival factor resulting in elevated levels of activated epidermal growth factor receptor (EGFR) and activated levels of ERK 1/2 and Akt, two signaling molecules downstream of EGFR, in UVP irradiated human foreskin keratinocytes (HFKs). This leads to the protection of the cells from UVB induced apoptosis. The purpose of this experiment is to determine if the anti-apoptotic activity of the E5 protein is unique to the development of cancer or if the E5 protein is critical to the completion of the life cycle of both the low and high-risk viruses. The hypothesis being tested is that the anti-apoptotic activity of the E5 protein is fundamental to the completion of the life cycle of low and high-risk HPVs. If this is correct, then the low-risk HPV 6 E5 protein will block apoptosis. Therefore, to test this hypothesis HFKs were infected with the retroviruses encoding HPV 16E5, HPV 6 E5a, or the parent virus, LXSN. Then the cells were selected with G418, an antibiotic, exposed to UVB light, fixed in ethanol and incubated with propidium Iodine (PI) and Rnase A. Fluorescence Activated Cell Sorter (FACS) analysis was performed. The presence of cells in the subG1 peak would indicate apoptosis. In addition, to determine the potential effect of low risk E5 on survival pathways, cells were lysed, and Western blots were performed on the cell lysates to determine whether the EGFR had been activated.

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