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 interferon-gamma (IFN-gamma). This NLS functions intracellularly by forming a complex with its transcription factor Stat1alpha and the nuclear importer of Stat1alpha, the importin-alpha analog NPI-1. The stability of this complex and the subsequent nuclear translocation of the complexed Stat1alpha are dependent on the integrity of this NLS, showing that Stat1alpha nuclear import is mediated by the IFN-gamma NLS. In this study, to directly evaluate the intrinsic requirement of nuclear IFN-gamma toward its biologic activities, we engineered a chimeric in which the IFN-gamma NLS has been substituted by a heterologous NLS, namely, the prototypical NLS of the SV40 large T antigen, which would drive nuclear translocation of IFN-gamma in a sequence-nonspecific manner. The chimeric, IFN-gamma-SV, was equally active in antiviral and antiproliferative assays as the wild-type IFN-gamma. Interestingly, IFN-gamma-SV was also translocated to the nucleus and was also recovered intracellularly as a complex with the Stat1alpha importer NPI-1, like wild-type IFN-gamma. Comparison with an NLS deletion mutant showed that deletion 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 activities of IFN-gamma and its NPI-1 complexation ability. Our data conclusively demonstrate that nuclear translocation of IFN-gamma is an intrinsic requirement for the full expression of the biologic activities of IFN-gamma and strengthen the conclusion that nuclear chaperoning of Stat1alpha is the primary role of IFN-gamma nuclear translocation. This type of ligand imprinting by sequestering of activated Stat may contribute to the specificity of Stat nuclear transcription.