The aim of this proposal is to understand at the molecular level the pathogenic mechanics by which human papillomavirus type 16 (HPV-16) transforms epithelial cells. Both HPV16 and 18 are associated with the majority of cases of premalignant and malignant disease of the cervix, while types 6 and 11 are associated with benign disease. When the whole genome of HPV16 was transfected into primary human foreskin keratinocytes it caused the inhibition of normal differentiation when examined in the collagen raft system. the morphology and abnormal cells observed was similar to immortalize and inhibit differentiation, but not as efficiently as the full length genome. HPV6 does not immortalize primary keratinocytes cultures. It is proposed to elucidate which combination of HPV16 genes are important for the immortalization and inhibition of differentiation and characterize their mechanisms of action. 1. We propose to examine which combinations of the three transcripts from the E6 and E7 region are important for immortalization and inhibition of differentiation and which other genes may cooperate. In addition, using an inducible promoter to express either E6 or E7 we hope to confirm that the continued presence of both is necessary for maintenance of the phenotype. 2. Identify important functional regions of the genes involved by site directed mutagenesis. 3. Investigate the effects of site directed mutagenesis at the protein level to see if function or stability is affected. 4. Determine the effects of the over expression of HPV6 genes on keratinocyte immortalization and differentiation. Also, investigate if combinations of HPV6 and 16 genes can immortalize cells. 5. Elucidate the transcription patterns of HPV6 and 16 containing keratinocytes by production of cDNAs. 6. Since papillomaviruses have overlapping reading frames, elucidate which cDNAs code for which proteins and assess the functional role of each cDNA. 7. Examine the effects of estrogens and progestagens in HPV6 and 16 transfected epithelial cells and examine the expression of their receptors. 8. Attempt to elucidate the mechanisms by which HPV16 inhibits epithelial cell differentiation. We will examine: a. the binding of the HPV16 E7 gene with the retinoblastoma gene product and, b. the expression of the retinoic acid receptor.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI030798-05
Application #
2065910
Study Section
Experimental Virology Study Section (EVR)
Project Start
1990-07-01
Project End
1995-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
McDade, Simon S; Patel, Daksha; McCance, Dennis J (2011) p63 maintains keratinocyte proliferative capacity through regulation of Skp2-p130 levels. J Cell Sci 124:1635-43
Menges, C W; McCance, D J (2008) Constitutive activation of the Raf-MAPK pathway causes negative feedback inhibition of Ras-PI3K-AKT and cellular arrest through the EphA2 receptor. Oncogene 27:2934-40
Thrash, Barry R; Menges, Craig W; Pierce, Robert H et al. (2006) AKT1 provides an essential survival signal required for differentiation and stratification of primary human keratinocytes. J Biol Chem 281:12155-62
Guess, Jennifer C; McCance, Dennis J (2005) Decreased migration of Langerhans precursor-like cells in response to human keratinocytes expressing human papillomavirus type 16 E6/E7 is related to reduced macrophage inflammatory protein-3alpha production. J Virol 79:14852-62
Westbrook, Thomas F; Nguyen, Don X; Thrash, Barry R et al. (2002) E7 abolishes raf-induced arrest via mislocalization of p21(Cip1). Mol Cell Biol 22:7041-52
Briggs, M W; Adam, J L; McCance, D J (2001) The human papillomavirus type 16 E5 protein alters vacuolar H(+)-ATPase function and stability in Saccharomyces cerevisiae. Virology 280:169-75
Adam, J L; Briggs, M W; McCance, D J (2000) A mutagenic analysis of the E5 protein of human papillomavirus type 16 reveals that E5 binding to the vacuolar H+-ATPase is not sufficient for biological activity, using mammalian and yeast expression systems. Virology 272:315-25
Stoppler, M C; Straight, S W; Tsao, G et al. (1996) The E5 gene of HPV-16 enhances keratinocyte immortalization by full-length DNA. Virology 223:251-4
Antinore, M J; Birrer, M J; Patel, D et al. (1996) The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors. EMBO J 15:1950-60
Sun, Y N; Lu, J Z; McCance, D J (1996) Mapping of HPV-11 E1 binding site and determination of other important cis elements for replication of the origin. Virology 216:219-22

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