This proposal concerns the gene products of polyoma virus required for viral growth and neoplastic transformation. Our goal is to understand how the large T and middle T antigens act in both processes. This includes their interactions with elements of other cellular pathways which are, regulatory for cell growth. We emphasize phosphorylation, because phosphorylation appears central to their function. Our approach is both biochemical and genetic. Large T antigen, which is important for DNA replication and RNA transcription, has a complicated pattern of phosphorylation that mutant analysis relates to function. We will continue identification of the phosphorylation sites. As each site is identified, it will be subjected to oligonucleotide mutagenesis. We will express and study the properties of the C-terminal domain of the protein. We will probe the architecture of the large T molecule. Labeling experiments will be carried out to determine the key contact points between the N- and C-terminal domains, between large T in oligomers and between large T and DNA. Second-site revertants will be sought as a genetic way to determine important interactions. For the biochemical studies, we will express important mutants and the C-terminal domains using baculovirus vectors. For middle T we will complete the analysis and mutagenesis of the serine/threonine phosphorylation sites. Phosphatidylinositol-3-kinase is the target of middle T most closely linked with transformation. It has wide general significance because of its association with other oncogenes and growth factors. Our analysis of this enzyme will continue. Besides working out the details of its association with middle T, we will assess whether this enzyme is sufficient to regulate cell growth. Because a mutation in an NPXY sequence that has been associated with localization to coated pits renders middle T non-transforming, experiments on the endosomal targeting of middle T will be carried out. The effects of middle T on internalization and endosomal trafficking will also be tested. The interactions of polyoma T antigens with other pathways which are regulatory for cell growth win also be tested. It appears that a functioning ras pathway is required for middle T transformation, so elements of that pathway will be examined. Wild type p53 is a negative regulator of cell growth inactivated by SV40 transformation; we will determine whether polyoma transformation of cells is affected by p53 and whether p53 is in turn affected by polyoma virus.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA034722-13
Application #
2088739
Study Section
Experimental Virology Study Section (EVR)
Project Start
1983-04-03
Project End
1996-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
13
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02111
Pores Fernando, A T; Andrabi, S; Cizmecioglu, O et al. (2015) Polyoma small T antigen triggers cell death via mitotic catastrophe. Oncogene 34:2483-92
Utermark, Tamara; Schmit, Fabienne; Lee, Sang Hyun et al. (2014) The phosphatidylinositol 3-kinase (PI3K) isoform dependence of tumor formation is determined by the genetic mode of PI3K pathway activation rather than by tissue type. J Virol 88:10673-9
Hwang, Justin H; Pores Fernando, Arun T; Faure, Nathalie et al. (2014) Polyomavirus small T antigen interacts with yes-associated protein to regulate cell survival and differentiation. J Virol 88:12055-64
Harrison, Celia; Jiang, Tao; Banerjee, Pubali et al. (2013) Polyomavirus large T antigen binds symmetrical repeats at the viral origin in an asymmetrical manner. J Virol 87:13751-9
Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole et al. (2013) Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA. PLoS Pathog 9:e1003725
Andrabi, Shaida; Hwang, Justin H; Choe, Jennifer Kean et al. (2011) Comparisons between murine polyomavirus and Simian virus 40 show significant differences in small T antigen function. J Virol 85:10649-58
Utermark, Tamara; Schaffhausen, Brian S; Roberts, Thomas M et al. (2007) The p110alpha isoform of phosphatidylinositol 3-kinase is essential for polyomavirus middle T antigen-mediated transformation. J Virol 81:7069-76
Andrabi, Shaida; Gjoerup, Ole V; Kean, Jennifer A et al. (2007) Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner. Proc Natl Acad Sci U S A 104:19011-6
Gjoerup, Ole V; Wu, Jiaping; Chandler-Militello, Devin et al. (2007) Surveillance mechanism linking Bub1 loss to the p53 pathway. Proc Natl Acad Sci U S A 104:8334-9
Whalen, Kerry A; de Jesus, Rowena; Kean, Jennifer A et al. (2005) Genetic analysis of the polyomavirus DnaJ domain. J Virol 79:9982-90

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