Simian virus 40T antigen (TAg), which is the viral transforming protein, forms a stable, high molecular weight complex with the host tumor antigen (p53). The host p53 is found elevated in cells transformed not only by SV40, but also by a variety of DNA and RNA tumor viruses, chemical carcinogens, ionizing radiation and also in both animal and human tumors. The formation of the TAg p53 complex is inhibited in TAg mutants also affected in transformation, suggesting that the complex may play a role in transformation by the virus. We propose (1) to dissect the multiple sequential steps in the formation of the complex using subfraction-specific monoclonal antibodies and ATP analogs. We will devise a blocking assay for the ATP analog-binding, and sequence the analog-binding sites. We will study dissociation of the complex by iodoacetate and vanadium. (2) We will determine whether RNAs tightly associated with both TAg and p53 are covalently associated and characterize both the bonds and the RNAs. The sites of attachment of the RNAs will be sequenced. The possible involvement of the RNA-binding in ATPase activity will be determined. (3) We will determine the subcellular localization of the events in Tag p53 complex formation using (a) electron microscopy, specific monoclonal antibodies, SV40 mutants affecting localization, and the TAg-localization perturbant and anti-tumor drug taxol and (b) kinetic studies of the formation of the complex using cell fractionation. (c) We will identify various polypeptides recently found to co-purify or cross-react with TAg or p53.
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