The large-T antigen of SV40 and the middle-T antigen of polyoma virus play a central role in the transformation of established cell lines by their respective viruses, but so far we have a very poor understanding of the molecular basis of these activities. In this study we propose to extend our use of site-directed and site-specific mutagenesis and utilize our extensive collection of mutants to create further mutations within defined regions of SV40 large-T and polyoma virus middle-T. The sites we will mutate include an SV40 origin DNA binding domain and a putative non-specific DNA binding domain on large-T antigen, as well as defined regions within the amino-terminal half of middle-T which may be involved in pp60csrc binding. We also plan to produce fragments of large-T and chimaeric proteins including parts of large-T or middle-T fused to pyruvate kinase and parts of murine polyoma virus middle-T fused to equivalent sequences from small-t SV40 and middle-T of hamster papovavirus. The biochemical and biological properties of the mutated, fragmented and chimaeric proteins will be analyzed. In this way we hope (1) to define the precise limits of the SV40 origin DNA binding domain on SV40 large-T and identify the equivalent region on polyoma virus large-T; (2) to investigate whether the isolated domain independently retains DNA binding and other biochemical activities; (3) to determine whether SV40 large-T has a second domain involved in DNA binding, particularly to cellular DNA; (4) to establish whether the ability to bind to cellular DNA mediated by either site is essential to transformation by SV40; (5) to identify regions within the amino-terminal half of middle-T that are involved in pp60csrc binding; (6) to establish which regions of middle-T are dispensible for transformation; and (7) to accummulate further data on the molecular basis of polyoma virus induced transformation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA043186-05
Application #
2091106
Study Section
Experimental Virology Study Section (EVR)
Project Start
1986-07-01
Project End
1991-06-30
Budget Start
1990-09-21
Budget End
1991-06-30
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Integrated Genetics, Inc.
Department
Type
DUNS #
City
Framingham
State
MA
Country
United States
Zip Code
01701
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Cheng, S H; Espino, P C; Marshall, J et al. (1991) Structural elements that regulate pp59c-fyn catalytic activity, transforming potential, and ability to associate with polyomavirus middle-T antigen. J Virol 65:170-9
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Cheng, S H; Harvey, R; Espino, P C et al. (1988) Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus. EMBO J 7:3845-55
Anderson, R W; Tevethia, M J; Kalderon, D et al. (1988) Fine mapping two distinct antigenic sites on simian virus 40 (SV40) T antigen reactive with SV40-specific cytotoxic T-cell clones by using SV40 deletion mutants. J Virol 62:285-96
Cheng, S H; Piwnica-Worms, H; Harvey, R W et al. (1988) The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus. Mol Cell Biol 8:1736-47

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