The long-range goal of this laboratory is to understand the functional interactions between a tumor virus and the host cell. The approach has been to define the biological functions expressed by the SV40 transforming protein, large tumor antigen (T-ag), in infected and transformed cells. Our efforts during the previous funding period focused on characterizing the plasma membrane-associated form of SV40 T-ag and analyzing a viral mutant that encoded a cytoplasmic T-ag (cT-ag) defective for nuclear transport. We showed that surface T-ag is associated with host protein p53 in the membrane of transformed cells, that the complex is rapidly turned over in the membrane, that the conformation in the membrane exposes both amino and carboxy termini of T-ag on the exterior of the cell, and that the expression of surface T-ag correlates with cell growth. Studies of the nuclear-transport-defective viral mutant succeeded in identifying the single amino acid change responsible for blocking nuclear transport of T-ag. Finally, we found that T-ag is modified by glycosylation. These efforts culminated in a perspective that forms the basis of the current proposal, namely that the surface-associated form of SV40 T-ag is functionally important. Proposed objectives are logical extensions of our previous studies and are timely in the context of current thinking regarding possible molecular mechanisms in carcinogenesis. The following specific aims are proposed. (1) To categorize into complementation groups the transformation-related functions expressed by T-ag. The approach will include co-transfection/transformation assays using the mutant cT-ag-encoding plasmid and known cloned oncogenes in primary and immortalized host cells. (2) To further examine the observed relationship between surface T-ag expression and cell growth. (3) To test the hypothesis that surface-associated T-ag may be functioning in a growth factor pathway by mimicking an """"""""activated"""""""" growth factor receptor. Internalization of surface T-ag and processing by lysosomal proteases will be sought, and the possible requirement of complex formation with cellular protein p53 to generate an """"""""active"""""""" T-ag conformation examined. (4) To further characterize the glycosylation modification of T-ag, especially with respect to mapping the glycosylation sites on the polypeptide. T-ag may be a good model for O-glycoproteins in general. Finally, (5) To study the intracellular trafficking of T-ag. Special emphasis will be placed on determining the sites of T-ag synthesis and O-glycosylation in the cell, as well as investigating the sorting that separates T-ag molecules destined for nuclear and surface localizations. These studies should yeild important insights into the biological functioning of a prototype viral transforming protein and broaden our understanding of molecular mechanisms involved in cellular transformation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA022555-09
Application #
3165844
Study Section
Experimental Virology Study Section (EVR)
Project Start
1978-01-01
Project End
1988-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
9
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
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Lednicky, J A; Butel, J S (1997) Tissue culture adaptation of natural isolates of simian virus 40: changes occur in viral regulatory region but not in carboxy-terminal domain of large T-antigen. J Gen Virol 78 ( Pt 7):1697-705
Lednicky, J A; Butel, J S (1997) A coupled PCR and restriction digest method for the detection and analysis of the SV40 regulatory region in infected-cell lysates and clinical samples. J Virol Methods 64:1-9
Lednicky, J A; Stewart, A R; Jenkins 3rd, J J et al. (1997) SV40 DNA in human osteosarcomas shows sequence variation among T-antigen genes. Int J Cancer 72:791-800
Stewart, A R; Lednicky, J A; Benzick, U S et al. (1996) Identification of a variable region at the carboxy terminus of SV40 large T-antigen. Virology 221:355-61
Lednicky, J A; Garcea, R L; Bergsagel, D J et al. (1995) Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors. Virology 212:710-7
Lednicky, J A; Wong, C; Butel, J S (1995) Artificial modification of the viral regulatory region improves tissue culture growth of SV40 strain 776. Virus Res 35:143-53
Sawai, E T; Rasmussen, G; Butel, J S (1994) Construction of SV40 deletion mutants and delimitation of the binding domain for heat shock protein to the amino terminus of large T-antigen. Virus Res 31:367-78
Fromm, L; Shawlot, W; Gunning, K et al. (1994) The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice. Mol Cell Biol 14:6743-54
Sawai, E T; Butel, J S (1992) Epitope mapping and conformational analysis of SV40 T-antigen deletion mutants. Virology 189:782-5

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