Detection and characterization of multiple forms of simian virus 40 large T antigen

Subclasses of simian virus 40 large T antigen in simian virus 40-transformed and -infected cells separated by zone velocity sedimentation in sucrose density gradients have been characterized. Three forms of large T antigen were distinguished: a 5 to 6S form, a 14 to 16S form, and a 23 to 25S form. These forms appeared to differ biochemically and biologically. Differential labeling experiments suggested that the 5 to 6S form was less highly phosphorylated than the faster-sedimenting forms. The 23 to 25S form which was complexed with one or more host phosphoproteins, as reported recently (D. P. Lane and L. V. Crawford Nature [London] 268:261-263, 1979; F. McCormick and E. Harlow, J. Virol. 34: 213-224, 1980), was prominent in extracts of transformed cells, but was also detected in productively infected cells. Pulse-chase experiments suggested that the 5 to 6S large T antigen is a precursor of the more stable, faster-sedimenting forms of T antigen. Monkey cells infected with a tsA mutant of simian virus 40 at 41 degrees C contained only 5 to 6S large T antigen, implying that this form is not active in the initiation of simian virus 40 DNA replication. In pulse-chase, shift-down experiments, DNA replication resumed, and the 5 to 6S large T antigen which had accumulated at 41 degrees C was partially converted at 33 degrees C to a fast-sedimenting form. However, shift-up experiments demonstrated that the fast-sedimenting large T antigen, once formed, remained stable at 41 degrees C, although it was unable to function in initiation. These experiments suggest that different biological functions of large T antigen may be carried out by different subclasses of this protein.