The long-term objective of this proposal is to investigate the mechanism that regulate the productive life cycle of oncogenic human papillomaviruses (HPV). Papillomaviruses are small DNA viruses which induce a variety of proliferative lesions in most mammals including humans. Of the 71 types of human papillomaviruses that have been identified, a subset are associated at a high frequency with anogenital cancers and these are referred to as the high risk or oncogenic types (HPV 16, 18, 31, 33, and 51). Viral production occurs in low grade lesions which are only slightly altered in their pattern of differentiation from that seen in normal cells. The production of viral particles, genome amplification, capsid protein synthesis, and virion assembly is dependent upon differentiation and is restricted to suprabasal cells. In carcinomas, viral DNA is usually found integrated into host chromosome and no viral production is seen. Several years ago, we first used an in vitro system (raft cultures) for the stratification and differentiation of keratinocytes at the air-liquid interface to duplicate the life cycle of human papillomaviruses in tissue culture. When the CIN 612 cell line, which was derived from a patient biopsy and found to stably maintain episomal copies of HPV 31, was grown in raft cultures, the amplification of viral DNA was detected in suprabasal cells. Furthermore, upon the addition of phorbol esters to the growth media, the induction of late gene expression and virion production was observed. This system was then used to characterize the patterns of differentiation-dependent viral gene expression. Recently, we have extended these studies and synthesized HPV 31 in raft cultures from transfected cloned DNA templates. Using mutants in E1 which fail to establish episomal genomes, a requirement of extrachromosomal templates in the induction of the late gene expression was demonstrated. These methods provide the opportunity for a detailed genetic analysis of HPV functions. In this application, we propose to extend our studies on viral pathogenesis and perform a genetic analysis of the requirements for viral replication during the vegetative life cycle. The following specific aims are proposed: 1) Which cis sequences necessary for maintenance and amplification of HPV 31? 2) How do the known viral replication factors, E1 and E2, function in viral pathogenesis? 3) What signals activate the differentiation-dependent amplification of viral genomes?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA074202-04
Application #
6173189
Study Section
Virology Study Section (VR)
Program Officer
Read-Connole, Elizabeth Lee
Project Start
1997-08-01
Project End
2002-07-31
Budget Start
2000-08-01
Budget End
2001-07-31
Support Year
4
Fiscal Year
2000
Total Cost
$236,867
Indirect Cost
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Hong, Shiyuan; Laimins, Laimonis A (2017) Manipulation of the innate immune response by human papillomaviruses. Virus Res 231:34-40
Langsfeld, Erika; Laimins, Laimonis A (2016) Human papillomaviruses: research priorities for the next decade. Trends Cancer 2:234-240
Lee, Choongho; Wooldridge, Tonia R; Laimins, Laimonis A (2007) Analysis of the roles of E6 binding to E6TP1 and nuclear localization in the human papillomavirus type 31 life cycle. Virology 358:201-10
Moody, Cary A; Fradet-Turcotte, Amelie; Archambault, Jacques et al. (2007) Human papillomaviruses activate caspases upon epithelial differentiation to induce viral genome amplification. Proc Natl Acad Sci U S A 104:19541-6
Wang, Yi; Shupenko, Craig C; Melo, Luisa F et al. (2006) DNA repair protein involved in heart and blood development. Mol Cell Biol 26:9083-93
Hebner, Christy M; Wilson, Regina; Rader, Janet et al. (2006) Human papillomaviruses target the double-stranded RNA protein kinase pathway. J Gen Virol 87:3183-93
Wilson, Regina; Fehrmann, Frauke; Laimins, Laimonis A (2005) Role of the E1--E4 protein in the differentiation-dependent life cycle of human papillomavirus type 31. J Virol 79:6732-40
Oh, Stephen T; Longworth, Michelle S; Laimins, Laimonis A (2004) Roles of the E6 and E7 proteins in the life cycle of low-risk human papillomavirus type 11. J Virol 78:2620-6
Fehrmann, Frauke; Klumpp, David J; Laimins, Laimonis A (2003) Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation. J Virol 77:2819-31
Hubert, Walter G; Laimins, Laimonis A (2002) Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression. J Virol 76:2263-73

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