apoptosis ͉ viral infection

IFN (2, 3). The crosslinking of 125I-IFNa to several human cell lines indicates that the IFNa/P receptor is a multiprotein complex, consisting of 95-, 115-, and 135-kDa subunits (3-5).Type I IFNs induce the transcription of specific early genes, the IFN-stimulated genes (ISGs), through the activation of the Jakl and Tyk2 protein-tyrosine kinases (PTKs) (6,7). PTK activation mediates the IFN-induced rapid tyrosine phosphorylation of the multiprotein latent cytosolic transcription factor ISGF3, which consequently translocates to the nucleus, where it interacts with conserved promoter elements in ISGs (8,9). This model of PTK activation resulting in the tyrosine phosphorylation of specific transcription factors and gene activation has been described for several cytokines (6,10,11) and serves as a paradigm for cytokine signaling. In contrast, the early events triggered by IFNa receptor occupancy and the functions of the various subunits are poorly characterized.A cDNA coding for one subunit (IFNaRl) of the human

Based on the reports of the activation of the transcription factor known as STAT3 (for signal transducers and activators of transcription) or APRF (for acute phase response factor) by various cytokines, we investigated the possible role of STAT3 in type I interferon (IFN) receptor signaling. We show that STAT3 undergoes IFNalpha-dependent tyrosine phosphorylation and IFNalpha treatment induces protein-DNA complexes that contain STAT3. In addition, STAT3 associates with the IFNAR-1 chain of the type I receptor in a tyrosine phosphorylation-dependent manner upon IFNalpha addition. The binding of STAT3 to the IFNAR-1 chain occurs through a direct interaction between the SH2 domain-containing portion of STAT3 and the tyrosine-phosphorylated IFNAR-1 chain. Furthermore, tyrosine-phosphorylated STAT3 bound to the IFNAR-1 chain also undergoes a secondary modification involving serine phosphorylation. This phosphorylation event is apparently mediated by protein kinase C, since it was blocked by low concentrations of the protein kinase inhibitor H-7. The biological relevance of IFN activation of STAT3 is further illustrated by the finding that STAT3 is not activated by IFN in a cell line resistant to the antiviral and antiproliferative actions of IFN alpha but in which other components of the JAK-STAT pathway are activated by IFNalpha.

The IFNAR chain of the type I interferon (IFN)

Type I interferons (IFNs), 1 consisting of ␣ and ␤ subtypes, are a multifunctional cytokine family capable of inhibiting cell proliferation and viral replication, in addition to modulating cellular immune functions (1). Type I IFNs compete with one another for binding to a common multisubunit receptor present on the surface of target cells. The type I IFN receptor consists of at least two distinct subunits IFNAR1 and IFNAR2 (2-5).The IFNAR1 chain appears to be involved primarily in signal transduction (2, 6, 7), while the IFNAR2 chain plays a role both in ligand binding and signal transduction (3-5). The IFNAR2 chain was originally identified as a Ϸ50-kDa protein (IFNAR2.1) (3), having a truncated cytoplasmic domain due to alternative mRNA splicing (8). A 100-kDa form of the IFNAR2 chain (IFNAR2.2) has been cloned which reconstitutes biological activity in both murine (4) and human cells (5). In most human cells IFNAR2.1 is expressed at low levels relative to the full-length IFNAR2.2 chain (9). These findings call into question the biological function of IFNAR2.1.We have examined the biological consequences of expression of the human IFNAR1 and IFNAR2.1 chains on the sensitivity of murine cells to several type I IFNs (IFN␣2, IFN␣8, and IFN␤1b). Murine L929 cells are completely unresponsive to human IFN (6, 7). We previously established that the IFNAR1 subunit acts as a species-specific signal transduction component of the human type I IFN receptor complex, but it does not directly bind IFN (6,7).In the present study, we demonstrate that IFNAR1 expression in L929 cells confers sensitivity to the antiviral, antiproliferative, and ISRE-dependent gel shift binding activities of IFN␣8 and to a lesser extent IFN␤1b. However, expression of IFNAR1 in L929 cells does not confer sensitivity to the biological activities of IFN␣2. IFNAR2.1 expression in murine cells does not confer sensitivity to any IFN subtype examined. Interestingly, cells expressing both receptor chains exhibited a markedly reduced sensitivity to the antiviral, antiproliferative, and ISRE-dependent gel shift binding activities of IFN␣8 and IFN␤1b, when compared with cells expressing only IFNAR1. These results suggest that IFNAR2.1 acts in a dominant negative manner for the induction of biological activity by several type I IFNs. MATERIALS AND METHODSCells and IFNs-Isolation of mouse L929 cells expressing IFNAR1, IFNAR2.1, or both IFNAR1 and IFNAR2.1 together has been described previously (10). IFNAR1 and IFNAR2.1 transfectants were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum and geneticin (1.5 mg/ml) or hygromycin (0.3 mg/ml), respectively. Stable transfectants expressing both receptor chains were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum, geneticin (1.5 mg/ml), and hygromycin (0.3 mg/ml).Recombinant IFN␣2 (4 ϫ 10 7 IU/mg), IFN␣8 (2 ϫ 10 7 IU/mg), and IFN␤1b (4 ϫ 10 7 IU/mg) was kindly provided by P. Trotta, ScheringPlough (Kenilworth, NJ), H. Hochkoppel, Ciba-Ge...