Human papillomaviruses (HPVs) cause the most prevalent sexually transmitted diseases of viral etiology. Infections are either subclinical or manifested as benign papillomas and condylomata. Infects by the high-risk HPVs, notably types 18 and 16, cna progress to dysplasia and cancers. Because HPVs propagate only in human squamous epithelia undergoing terminal differentiation, we and others have adapted the technique of growing organotypic (raft) cultures of primary human keratinocytes (PHKs) to investigate HPV gene functions in vitro. We have shown that HPV-18 E7 gene expressed from HPV-18 enhancer-E6 promoter (URR) is differentiation-dependent and reactivates host DNA replication in differentiated PHKs in raft cultures. These results demonstrate that the function of the E7 protein, which binds to and inactivates pRB to release the E2F:DP transcription factors, is to facilitate viral DNA replication. We also found that ERR-E7 simultaneously induces, in differentiated PHKs, cyclin E and the universal cyclin-dependent kinase inhibitory p21cip1 protein. Induction of p21cip1 is mediated by post-transcriptional mechanisms. The induction of these two host proteins on the one hand and host and viral DNA synthesis on the other takes place in a mutually exclusive manner in differentiated PHKs in raft cultures and in benign papillomas, accounting for the heterogeneity of viral activities in patient specimens. Furthermore, certain E7 mutations activate a representative E2F-responsive host replication gene, the p180 subunit of the DNA polymerase alpha in differentiated PHKs but are unable to induce PCNA (a DNA polymerase delta co-factor) and therefore cellular DNA synthesis. These results suggest that the release of E2F:DP factors from pRB is necessary but not sufficient to activate all the DNA replication genes. This application is to investigate in depth the mechanisms in differentiated keratinocytes by which: (1) E7 activates host DNA replication such as pol-alpha and PCNA, and ultimately host DNA replication by using a panel of E7 mutations; (2) E7 activates the PCNA gene, with special attention to possible roles of cis elements in the first intron, the YY1 binding site which spans the RNA initiation sites, as well as additional regulatory elements in the promoter region; and (3) E7 induces cyclin E and p21cip1 proteins, with an emphasis on whether unscheduled cellular DNA synthesis in inhibited by the cyclin E or by the p21cip1 protein. The biochemical properties of E7 such as binding to tumor suppressors pRB, P107, and transcription factors TBP and YY1, and phosphorylation by casein kinase II will be determined and correlated with the biological consequences in just described. These studies will shed light on the pathways involved in controlling the cellular DNA replication machinery as well as HPV reproduction.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA036200-17
Application #
6632923
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Wong, May
Project Start
1984-08-01
Project End
2005-05-31
Budget Start
2003-08-26
Budget End
2005-05-31
Support Year
17
Fiscal Year
2003
Total Cost
$397,051
Indirect Cost
Name
University of Alabama Birmingham
Department
Biochemistry
Type
Schools of Medicine
DUNS #
063690705
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Bosch, F Xavier; Broker, Thomas R; Forman, David et al. (2013) Comprehensive control of human papillomavirus infections and related diseases. Vaccine 31 Suppl 5:F1-31
Bosch, F Xavier; Broker, Thomas R; Forman, David et al. (2013) Comprehensive control of human papillomavirus infections and related diseases. Vaccine 31 Suppl 8:I1-31
Chow, Louise T; Broker, Thomas R (2013) Human papillomavirus infections: warts or cancer? Cold Spring Harb Perspect Biol 5:
Bosch, F Xavier; Broker, Thomas R; Forman, David et al. (2013) Comprehensive control of human papillomavirus infections and related diseases. Vaccine 31 Suppl 6:G1-31
Chow, Louise T; Broker, Thomas R; Steinberg, Bettie M (2010) The natural history of human papillomavirus infections of the mucosal epithelia. APMIS 118:422-49
Banerjee, N Sanjib; Chow, Louise T; Broker, Thomas R (2005) Retrovirus-mediated gene transfer to analyze HPV gene regulation and protein functions in organotypic ""raft"" cultures. Methods Mol Med 119:187-202
Van Tine, Brian A; Broker, Thomas R; Chow, Louise T (2005) Simultaneous in situ detection of RNA, DNA, and protein using tyramide-coupled immunofluorescence. Methods Mol Biol 292:215-30
Van Tine, Brian A; Dao, Luan D; Wu, Shwu-Yuan et al. (2004) Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning. Proc Natl Acad Sci U S A 101:4030-5
Wiatrak, Brian J; Wiatrak, Deborah W; Broker, Thomas R et al. (2004) Recurrent respiratory papillomatosis: a longitudinal study comparing severity associated with human papilloma viral types 6 and 11 and other risk factors in a large pediatric population. Laryngoscope 114:1-23

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