Polyoma viral middle T-antigen is required for transformation

Abstract

To determine whether small or middle T-antigen (or both) of polyoma virus is required for transformation, we constructed mutants of recombinant plasmids which bear the viral oncogene and measured the capacity of these mutants to transform rat cells in culture. Insertion and deletion mutations in sequences encoding small and middle T-antigens (79.7, 81.3, and 82.9 map units) rendered the DNA incapable of causing transformation by the focus assay. Similar mutations in sequences that encoded middle but not small T-antigen (89.7, 92.1, and 96.5 map units) generally abolished the transforming activity of the DNA. However, two mutants (pPdll-4 and pPdl2-7) that carried deletions at 92.1 map units retained the capacity to transform cells; pPdll-4 did so at frequencies equal to those of the parental plasmid, whereas pPdl2-7 transformed at 10% the frequency of its antecedent. From these studies we conclude that small T-antigen alone is insufficient to cause transformation and that middle T-antigen is required for transformation, either in combination with small T-antigen or by itself.

Polyoma middle-sized T antigen can be phosphorylated on tyrosine at multiple sites in vitro.

“…Given the results of replacing tyrosine 315 and the observation that tyrosine 315 is not the sole phosphorylation site, there must be another explanation for the decreased kinase activity of dl-23 MT antigen. Possibly the kinase might bind to this highly acidic part of MT antigen, although there are other deletion mutations in this region whose MT antigens have high levels of associated protein kinase activity (Schaffhausen and Benjamin, 1981;Mes and Hassell, 1982;Nilsson et al, 1983). Furthermore, the hr-t mutant NG-59 MT antigen (Eckhart et al, 1979;Schaffhaussen and Benjamin, 1979), bearing a single amino acid substitution and insertion at position 179, and a series of NH2-terminal deletion mutants also lack protein kinase activity (D. Templeton, un-published observations), potentially implicating both these regions in recognition by the kinase as well.…”

“…Many of the tyrosine residues in the COOH-terminal region of the MT antigen can be removed by deletion without affecting transformation. For example, tyrosine 258 is missing in dl-8, tyrosines 289 and 297 in d-45, tyrosine 322 in dl-1013 (Magnusson et al, 1981) and tyrosine 315 in dl-1-4 (Mes and Hassell, 1982). Conversion of tyrosine 315 to phenylalanine may reduce but does not abolish the ability of MT antigen to transform (Oostra et al, 1983;G.Carmichael, personal communication).…”

Comparison of the DNA sequence of the Crawford small-plaque variant of polyomavirus with those of polyomaviruses A2 and strain 3

“…contains most of the postulated amino acid changes in middle T-antigen as well as two large T-antigen changes. Some segments of this region can be deleted from the polyoma genome without greatly affecting viral DNA replication or cellular transformation, functions ascribed to large T-antigen and middle T-antigen, respectively (2,18,31,32). However, at least one sequence including four consecutive glutamic acid residues and a tyrosine residue is crucial for transformation (18,35).…”

Properties of cells transformed by the middle T-antigen-coding region of polyomavirus

“…The properties of the series of LT-cells presented in this analysis add to the collection of data which demonstrate that transformation by polyomavirus of established rat fibroblasts can be maintained in the absence of large T antigen. These conclusions, based upon the correlation between the induction of a transformed phenotype and the presence of middle T-antigen DNA sequences or protein, were reached in experiments involving either infections with virus (4, 13, 15) or transfections with middle T-antigen cDNA plasmids (14,20,21). The latter group of experiments has the advantage of providing a means to study the separate properties of the polyomavirus early functions.…”