Nuclear translocation of IFN-gamma is an intrinsic requirement for its biologic activity and can be driven by a heterologous nuclear localization sequence

We have previously identified a nuclear localization sequence (NLS) in inte rferon-gamma (IFN-gamma). This NLS functions intracellularly by forming a c omplex with its transcription factor Stat1 alpha and the nuclear importer o f Stat1 alpha, the importin-alpha analog NPI-1. The stability of this compl ex and the subsequent nuclear translocation of the complexed Stat1 alpha ar e dependent on the integrity of this NLS, showing that Stat1 alpha nuclear import is mediated by the IFN-gamma NLS. In this study, to directly evaluat e the intrinsic requirement of nuclear IFN-gamma toward its biologic activi ties, we engineered a chimeric in which the IFN-gamma NLS has been substitu ted by a heterologous NLS, namely, the prototypical NLS of the SV40 large T antigen, which would drive nuclear translocation of IFN-gamma in a sequenc e-nonspecific manner. The chimeric, IFN-gamma -SV, was equally active in an tiviral and antiproliferative assays as the wild-type IFN-gamma. Interestin gly, IFN-gamma -SV was also translocated to the nucleus and was also recove red intracellularly as a complex with the Stat1 alpha importer NPI-1, like wild-type IFN-gamma. Comparison with an NLS deletion mutant showed that del etion or changes within the NLS motif of IFN-gamma were inconsequential to the high-affinity extracellular binding to the IFN-gamma receptor complex, yet the presence of an NLS was critical to the expression of the biologic a ctivities of IFN-gamma and its NPI-1 complexation ability. Our data conclus ively demonstrate that nuclear translocation of IFN-gamma is an intrinsic r equirement for the full expression of the biologic activities of IFN-gamma and strengthen the conclusion that nuclear chaperoning of Stat1 alpha is th e primary role of IFN-gamma nuclear translocation. This type of ligand impr inting by sequestering of activated Stat may contribute to the specificity of Stat nuclear transcription.