Construction of adenovirus vectors for the efficient transfer and overexpression of polyomavirus middle tumor antigen in mammalian cells

To overproduce biologically active polyomavirus (Py) middle tumor (T) antigen in human 293 cells and to efficiently transfer the middle T gene into a variety of other mammalian cells, two helper-independent recombinant adenoviruses (Ad) were constructed. A hybrid transcription unit encoding Py middle T antigen was substituted in either orientation for most of the E1 region of the Ad5 genome. Permissive 293 cells infected with each recombinant virus produced elevated levels of Py-specific transcripts and middle T protein. The functionality of the recombinant virus-encoded middle T was evidenced by its ability to associate with and serve as a substrate for human pp60$ sp{{ rm c}-src}$ and to transform Rat-1 cells. Ad recombinants of similar structure expressing Py small T antigen alone and all three Py T antigens together were also constructed and characterized.The recombinant viruses expressing middle T antigen were modified by inclusion of the simian virus 40 3$ sp prime$ RNA-processing signals and/or the Py enhancer to improve their ability to act as expression and gene transfer vectors. The resulting Ad recombinants, constituting a matched set, synthesized middle T protein at different levels in human 293 cells ranging up to 75 times the level observed in a Py lytic infection of mouse 3T6 cells. The amount of stable middle T-pp60$ sp{{ rm c}-src}$ complex was proportional to the total amount of middle T expressed up to a certain level and did not further increase in conditions of extreme middle T overexpression. The presence of the Py enhancer in modified recombinant viruses augmented transient and stable middle T expression in selected cell lines which do not express the E1 functions.Two immunologically distinct middle T subpopulations, representing 30% and 70% of the total middle T present in the cell, were purified by immunoaffinity chromatography from recombinant adenovirus-infected 293 cells. While the smaller middle T subpopulation displayed associated tyrosine kinase activity, the larger one did not. Cellular proteins copurified differentially with each middle T subpopulation. The 92K, 85K, 61K, 36K proteins and pp60$ sp{{ rm c}-src}$ were exclusively found with the 30% subpopulation. The 70-72K doublet and 37K proteins associated with the middle T of both subpopulations.Ranges of transformation frequencies were observed after infection of a variety of murine and human cell strains and lines with recombinant viruses expressing Py middle T antigen alone or all three Py T antigens. Despite the resistance of human cells to transformation, a human mammary epithelial cell line (184A1N4) was successfully transformed. Independent transformed cell lines were isolated and properties indicative of oncogenic transformation were extensively analyzed, highlighting the critical role of the middle pp60$ sp{{ rm c}-src}$ association in middle T-mediated transformation. Transformation of 184A1N4 cells could provide an attractive model for the study of human breast cancer