Adenovirus 1p+ locus codes for a 19kd (175R) T antigen which plays an important role in cell transformation and tumorigenesis. We propose to carry out an in depth investigation to elucidate the structure and function of this protein. A library of Ad2 mutants with single amino acid substitutions as well as second-site revertants of selected viral mutants will be constructed by local mutagenesis. These mutants and revertants will be used to correlate the structure and functional domains of this protein with its involvement in cell transformation, tumorigenicity, and transformation associated characteristics. The transformation and tumorigenicity of the mutant DNAs will be studied using replication defective murine retrovirus vectors or by use of transformed cells in newborn hamsters. Ad2 mutants with possible compensating mutations mapping outside the 175R coding region will also be isolated in order to identify other viral genes that may interact with the 175R protein. Temperature sensitive mutants will be used to find out whether the 175R protein is involved in the initiation and/or maintenance of cell transformation. Mutants with specific alterations in the hydrophobic domains which are possibly involved in membrane association will be constructed in order to elucidate the structural requirements for membrane association and the relationship between membrane association and function. Genetic evidence indicates that the Ad12 163R protein may control tumorigenicity. We will investigate whether the Ad12 protein could contribute to differential tumorigenicity. We will investigate whether the Ad12 protein could contribute to differential tumorigenicity (i.e. the highly oncogenic nature of Ad12) using DNA constructs expressing Ad2 Ela region and Ad12 163R region employing murine retrovirus vectors. It appears that the 175R protein is required for CA++ mobilization in transformed cells. We will investigate whether the 175R protein could mediate the release of Ca++ from intracellular stores (endoplasmic reticulum) by an inositol triphosphate sensitive mechanism. Since 175R T antigen is also localized on the nuclear envelope, we will investigate whether this protein could play a role in the nucleocytoplasmic transport of RNA via enhanced nucleoside triphophatase activity.
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