The Polyoma virus 100K large T-antigen is not required for the maintenance of transformation

Abstract

Thirteen Rat-1 cell lines that were transformed in vitro by polyoma virus (PY) were examined for the following: the state of the viral sequences present (by blot hybridization ( E. Southern, 1975, J. Mol. Biol. 98, 503–515); the T-antigen species synthesized; the ability to display a transformed phenotype in vitro; and the ability to induce tumors in vivo. Eleven of the lines contained free viral genomes in addition to integrated viral sequences. Two lines (82-Rat and 53-Rat) contained no free viral DNA and only a single insert of integrated viral DNA. The alignment of the integrated viral sequences in these two lines was deduced by restriction enzyme analysis and blot hybridization as previously described by Botchan et al. ( M. Botchan, W. Topp, and J. Sambrook. 1976. Cell 9, 269–287). The viral sequences were found to be integrated into different regions of the rat cell DNA via linkages at different points on the viral genome. The integrated viral sequences in both lines were present in partial head-to-tail duplications. Neither line contained a complete early region. Discontinuities, either as a result of deletion or fusion to host sequences, affected the region which codes for the C-terminal portion of the 100K large T-antigen and which includes the position where the TS-A mutants have been mapped ( L. K. Miller and M. Fried, 1976, J. Virol. 18, 824–832). In both lines at least one copy of the region coding for both the 55K middle and 22K small T-antigen species was apparently unaltered. As expected from the maps of the integrated viral sequences neither line synthesized a full-size 100K large T-antigen but the 55K middle and 22K small T-antigens were both produced. Upon fusion to permissive mouse cells virus could be rescued readily from PY transformed lines containing a 100K large T-antigen and free viral genomes. However, with the 53-Rat line virus was rescued in reduced yield and with 82-Rat no rescue was detected. The 82-Rat and 53-Rat lines could not be distinguished from the other PY lines studied in their transformed phenotype in vitro or their ability to form tumors in vivo. Thus we find that the presence of the 100K large T-antigen (A gene product) is not essential for the maintenance of transformation in the 82-Rat and 53-Rat cell lines. Together with our previous studies in which we found that a Rat-1 cell line containing hr-t viral genomes in both integrated and free forms displayed a normal phenotype and synthesized a functional 100K large T-antigen but not the 55K middle nor 22K small T-antigen species ( L. Lania, M. Griffiths, B. Cooke, Y. Ito, and M. Fried, 1979, Cell. 18, 793–802) our data strongly suggest that it is the 55K middle and/or 22K small T-antigen species that are required for the maintenance of PY mediated transformation. The role of the 100K large T-antigen (A gene product) in the initiation of transformation and the excision of PY viral sequences is discussed.