The oncogenic virus Bovine Papilloma Virus (BPV-1) replicates in synchrony with cellular DNA in transformed cells as a supercoiled plasmid, and as an autonomous replicon it provides an ideal system to study the replication of DNA in the cell cycle. We propose a program of experiments whose major goal is to understand the differences between regulated (latent) replication and vegetative replication. These experiments should add to our knowledge of how viral oncogenes usurp normal cell cycle controls. We propose to: 1. Clone and express from various vectors the viral genes which regulate and mediate this replication. 2. Continue our genetic analysis of the trans and cis requirements for latent replication. 3. Define when in the cell cycle the virus replicates and if the order of viral gene expression is critical to its apparent ability to replicate once per cell cycle. 4. Determine if the viral DNA encodes for a """"""""repressor of amplification."""""""" 5. Measure accurately the loss of plasmids as cells divide. Methods are described which will be very sensitive to mitotic instability. 6. Characterize the regulation of BPV-1 promoters both through genetic and in vitro transcription experiments. 7. Characterize the infectious """"""""BPV retroviruses"""""""" that we have made. These viruses will be useful in defining the genetic potential of various cell lines to support plasmid replication. 8. Raise polyclonal antibodies to BPV-1 early proteins.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA042414-04
Application #
3183695
Study Section
Virology Study Section (VR)
Project Start
1986-04-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
4
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of California Berkeley
Department
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Abbate, Eric A; Berger, James M; Botchan, Michael R (2004) The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2. Genes Dev 18:1981-96
Alexandrov, Alexander I; Botchan, Michael R; Cozzarelli, Nicholas R (2002) Characterization of simian virus 40 T-antigen double hexamers bound to a replication fork. The active form of the helicase. J Biol Chem 277:44886-97
Voitenleitner, Christian; Botchan, Michael (2002) E1 protein of bovine papillomavirus type 1 interferes with E2 protein-mediated tethering of the viral DNA to mitotic chromosomes. J Virol 76:3440-51
Harris, S F; Botchan, M R (1999) Crystal structure of the human papillomavirus type 18 E2 activation domain. Science 284:1673-7
Fouts, E T; Yu, X; Egelman, E H et al. (1999) Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J Biol Chem 274:4447-58
Lehman, C W; Botchan, M R (1998) Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc Natl Acad Sci U S A 95:4338-43
Lim, D A; Gossen, M; Lehman, C W et al. (1998) Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control. J Virol 72:1931-40
Lehman, C W; King, D S; Botchan, M R (1997) A papillomavirus E2 phosphorylation mutant exhibits normal transient replication and transcription but is defective in transformation and plasmid retention. J Virol 71:3652-65
Ferguson, M K; Botchan, M R (1996) Genetic analysis of the activation domain of bovine papillomavirus protein E2: its role in transcription and replication. J Virol 70:4193-9
Mendoza, R; Gandhi, L; Botchan, M R (1995) E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats. J Virol 69:3789-98

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