Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Abstract Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126–132 that is recognized by the importin α/β1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction ∼50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102–110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110Rb). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110Rb implicate p110Rb binding as being responsible for the reduced nuclear accumulation, with the Ser106 phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110Rb and dependence thereof on negative charge at Ser106. The involvement of p110Rb in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110Rb, and how this may relate to transformation.