THE INHIBITION OF CULTURED MYOBLAST DIFFERENTIATION BY THE SIMIAN-VIRUS-40 LARGE T-ANTIGEN OCCURS AFTER MYOGENIN EXPRESSION AND RB UP-REGULATION AND IS NOT EXERTED BY TRANSFORMATION-COMPETENT CYTOPLASMIC MUTANTS

We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-term inal end, in a region that affects binding to p53, or in the nuclear t ransport signal, were also employed to determine whether the interfere nce was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiat ion of mouse C2 myoblasts, but this arrest occurred only after the syn thesis of myogenin, an initial step in biochemical differentiation. Ne ither the synthesis nor some basic activities of MyoD appeared to be a ffected by cvt SVLT. In these transformants, mitogen depletion elicite d an increase in the Rb level comparable to that in normal C2 cells; w t SVLT, however, promoted the phosphorylation of a large part of the i nduced Rb. Mutations affecting nuclear transport were far more critica l for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expressi on was;fully compatible,vith the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transformin g competence. The remaining SVLT mutants presented different degrees o f ability to inhibit differentiation (as shown by the expression of ti ssue-specific markers in transformants). The inhibiting mutants, inclu ding the Rb binding site mutant, were able to promote a higher state o f Rb phosphorylation than that observed in either normal cells or cyto plasmic-SVLT transformants.