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.

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The STAT transcription factors are mediators of signal transduction of a variety of factors, including interferons (IFNs), interleukins, growth factors, and peptide hormones. Subsequent to activation, STATs are translocated to the nucleus apparently through the well-described importin/Ran system, where they activate target genes. Molecules utilizing this nuclear import system require specific nuclear localization sequences (NLSs). Paradoxically, such NLSs are not identifiable on STATs, thus raising the question of how they are imported into the nucleus. Of considerable interest is the observation that ligands and/or receptors that signal through STATs contain putative NLSs and, where examined, either ligand or receptor undergoes nuclear translocation. We hypothesize that ligands and/or their receptors serve as vehicles for the nuclear translocation of STATs, and that they may be directly involved in signal transduction. Using IFNgamma as a model system, we provide a possible mechanism for how this direct role is fulfilled. A functional NLS has been identified in a C-terminal domain of IFNgamma. This domain and the NLS contained within are crucial for the biological properties of IFNgamma in that a peptide encompassing this domain is sufficient to induce an antiviral state. Further, this domain binds specifically to a membrane-proximal region internal cytoplasmic domain of the alpha subunit of the receptor complex in a region that is directly involved in the recruitment and activation of the JAK/STAT pathway. We suggest that this novel mode of receptor recognition and activation may be a driving force for nuclear translocation of molecules like STATs that are associated with the ligand-receptor complex.

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