GENETIC-ANALYSIS OF POLYOMAVIRUS LARGE-T NUCLEAR-LOCALIZATION - NUCLEAR-LOCALIZATION IS REQUIRED FOR PRODUCTIVE ASSOCIATION WITH PRB FAMILYMEMBERS

Polyomavirus large T antigen (LT) is a multifunctional nuclear protein , LT has two nuclear localization signals (NLSs), one spanning residue s 189 to 195 (NLS1) and another spanning residues 280 to 286 (NLS2), S ite-directed mutagenesis showed that each signal contains at least two critical residues. The possibility of connections between NLSs and ad jacent phosphorylations has attracted much attention, Cytoplasmic LT ( CyT) mutants were underphosphorylated, particularly at sites adjacent to NLS2, However, since a nuclear LT bearing an inactivated NLS2 was p hosphorylated normally at adjacent sites, the signal was not directly required for phosphorylation, Conversely, LT could be translocated to the nucleus via NLS2 even when the adjacent phosphorylation sites were deleted, CyT was examined to probe the importance of LT localization, CyT was unable to perform LT functions related to interactions with r etinoblastoma susceptibility gene (pRb) family members, Hence, CyT was unable to immortalize primary cells or to transactivate an E2F-respon sive promoter, Consistent with these findings, CyT, though capable of binding pRb in vitro, did not cause relocalization of pRb in cells, As says of transactivation of the simian virus 40 late promoter and of th e human c-fos promoter showed that defects of CyT were not limited to functions dependent on pRb interactions.