THE REPLICATION FUNCTIONS OF POLYOMAVIRUS LARGE TUMOR-ANTIGEN ARE REGULATED BY PHOSPHORYLATION

Polyomavirus (Py) large T antigen (T Ag) contains two clusters of phos phorylation sites within the amino-terminal half of the protein. To ch aracterize possible regulatory effects of phosphorylation on viral DNA replication, Py T Ag was treated with calf intestinal alkaline phosph atase (CIAP). Incubation of the protein with a range of phosphatase co ncentrations caused progressive loss of phosphate without affecting it s stability. Treatment with smaller quantities of CIAP stimulated the ability of the viral protein to mediate replication of constructs cont aining the viral replication origin, while higher concentrations of CI AP caused a marked diminution of this replication function. Several bi ochemical activities of Py T Ag were examined after CIAP treatment. Py T Ag DNA unwinding and nonspecific DNA binding were only slightly aff ected by dephosphorylation. However, as determined by DNase I footprin ting experiments, treatment with smaller amounts of CIAP stimulated sp ecific binding to the Py replication origin by Py T Ag, while treatmen t with larger amounts of CIAP caused marked inhibition of origin-speci fic binding by the viral protein. Phosphotryptic maps of Py T Ag befor e or after treatment with CIAP revealed changes in individual phosphop eptides that were uniquely associated with either the stimulation or t he inhibition of replication. Our data therefore suggest that Py T Ag is regulated by both repressing and activating phosphates.