Dimerization of the Interferon Type I Receptor IFNaR2–2 Is Sufficient for Induction of Interferon Effector Genes but Not for Full Antiviral Activity

Abstract

We constructed chimeric receptors wherein the extracellular domain of the erythropoietin receptor (EpoR) was fused to the transmembrane and intracellular domains of the interferon (IFN) type I receptor subunits, IFNaR1 or IFNaR2-2. Transfection into 2fTGH and Tyk2-deficient 11,1 cells showed that EpoR/IFNaR2-2 alone was able to transduce a signal upon stimulation with erythropoietin (Epo), as judged by induction of the interferon type I-inducible 6-16 promoter. In contrast, protection against infection with encephalomyocarditis virus or vesicular stomatitis virus was reduced or absent, respectively. To further investigate the role of IFNaR1 in the induction of an antiviral state, we analyzed the Epo-versus IFN␣-induced transcription of a set of genes, involved in antiviral protection. Up to 24 h after stimulation with Epo or IFN␣, comparable transcription of the p56, dsRNA-dependent protein kinase, 2-5A synthetase, and MxA genes was seen. However, at later time points, only in the case of Epo induction, a sharp decrease of mRNA levels was observed. Western blotting analysis of dsRNA-dependent protein kinase showed a similar pattern at the protein level. Taken together, our results imply a role for IFNaR1 in the induction of sustained mRNA and protein levels that are likely required for optimal antiviral activity.It is generally accepted that activation of a cell by a cytokine is initiated by ligand-induced clustering of receptor subunits, which can occur as di-, tri-, or higher order oligomers, involving identical or related subunits. With the exception of the heptamembrane-spanning receptors, members from the hematopoietin/IFN, 1 tumor necrosis factor/nerve growth factor, and tyrosine kinase receptor families are all activated by this mechanism of cytokine-driven multimerization. Ligand binding to the first receptor component induces the association with additional receptor subunits eventually resulting in an increase of affinity for the ligand (1, 2). Alternatively, preformed receptor complexes may also exist on the cell membrane (3). Interferons belong to the class I cytokine family and are divided into type I interferons (at least 14 IFN␣ subtypes, IFN␤, IFN, and the bovine embryonic IFN) that have antiviral, cytostatic, and hematopoietic activities on many cell types, and the type II interferon or IFN␥ that is also involved in many immune functions. Both types of interferons bind to distinct receptors that belong to the class I cytokine receptors. The interferon type I receptor (IFNaR) is composed of two subunits, IFNaR1 and IFNaR2-2. As a result of alternative splicing, subtypes of the latter subunit do also exist: a cytoplasmic truncated transmembrane form (IFNaR2-1) and a soluble form (IFNaR2-3) (4 -8).A large body of evidence has shown that the class I cytokine receptors make use of associated kinases (JAKs) to start intracellular tyrosine phosphorylation, resulting in the activation of the so-called Stat proteins. This JAK/Stat pathway is essential for transcription of many of the cytokine-inducible ge...

Down-modulation of Type 1 Interferon Responses by Receptor Cross-competition for a Shared Jak Kinase

“…It is well known that the effectiveness of the various proteins involved in the multiple antiviral pathways of interferon action differs from virus to virus (39). Our findings imply that the IFN-based antiviral protection against VSV in human fibrosarcoma cells is more dependent on Ifnar1-mediated signaling than is the case for EMCV, which is in accordance with our previous observations (16). Down-modulation on IFN-␤-induced signals by Tyk2 sequestration was absent or extremely weak.…”

“…It was shown that Epo stimulation of the Epo-Ifnar2 chimera expressed in 2fTGH cells leads to efficient induction of IFN-sensitive genes but was extremely inefficient in antiviral protection. This result highlighted the indispensable role of Ifnar1 in the induction of an antiviral state (16). Here, we show that cells expressing the EpoR/Ifnar1 chimera do not exhibit an Epo-induced antiviral response.…”

“…In our previous study, the specific contribution of the intracellular domains of Ifnar1 and Ifnar2 was analyzed using Epodriven dimerization of chimeric receptors (16). It was shown that Epo stimulation of the Epo-Ifnar2 chimera expressed in 2fTGH cells leads to efficient induction of IFN-sensitive genes but was extremely inefficient in antiviral protection.…”

“…For the construction of the pSV-SPORT EpoR/Ifnar receptor chimeras, we refer to Ref. 16. The BS/IL-12R␤1 plasmid and the pEF-BOS expression vector containing the IL-12R␤2 cDNA (17) were gifts of U. Gubler.…”

“…Single neo R colonies were screened for EpoR/ Ifnar1 expression by RT-PCR (16 Immunoprecipitation and Western Blot Analysis-Cells were treated for 15 min (for detection of Jak kinases) or 30 min (for STATs) with increasing doses of Epo, IFN-␣2, or IFN-␤ (ranging from 1 pM to 1 nM) and lysed in a buffer containing 50 mmol/liter Tris-HCl, pH 8, 200 mmol/liter NaCl, 2 mmol/liter EDTA, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.05% SDS, 2 mmol/liter Pefablock (Merck KgaA, Darmstadt, Germany), 2 mmol/liter sodium orthovanadate, 30 g/ml aprotinin, and 30 g/ml leupeptin. Clarified lysates were immunoprecipitated with R5-7 Tyk2 antiserum (20) or with Jak1 M7 antiserum (a gift of A. Ziemiecki) and analyzed by immunoblotting with the anti-phosphotyrosine 4G10 mAB (Upstate Biotechnology, Inc.), the anti-Tyk2 T10 -2 mAb (21), or the anti-Jak1 M7.…”