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Study of the mechanisms by which interferon (IFN) treatment of cells induces resistance to virus infections has been complicated by the multiple biochemical changes induced. Over 20 proteins are increased by IFN, including the double-stranded (ds) RNA-activated protein kinase, (2'-5') oligo A synthetase, surface proteins such as the major histocompatibility complex (MHC) proteins, and various proteins with unknown functions. The availability of cloned complementary DNAs for several IFN-induced proteins now allows us to probe their roles in IFN action. For instance, the murine Mx protein has been shown to confer resistance, to influenza virus. We studied chinese hamster ovary (CHO) cell clones expressing high constitutive levels of (2'-5') A synthetase as a result of transfection with the cDNA encoding the enzyme form which has a relative molecular mass (Mr) of 40K. Elevated enzyme correlates directly with resistance to infection by a picornavirus such as Mengo, but does not make the cells resistant to vesicular stomatitis virus (VSV).

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Communicated by A.M.MichelsonRecently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69-or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(l).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69and 100-kd 2-5A synthetases were also identified by electrophoretic transfer inmmunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.

Rehovot 76100, Israel chains have been identified: IFNAR1 (Uze et al., 1990) 1 Corresponding author and IFNAR2-2 (or IFNAR2-c, Domanski et al., 1995;Lutfalla et al., 1995), which is a long form of IFN-α/βR The intracytoplasmic domain (IC) of cytokine receptors (Novick et al., 1994). Transduction of the signal generated provides docking sites for proteins which mediate by IFN-α,β,ω involves protein tyrosine kinases of the signal transduction. Thus, in interferon-α,β receptors Janus kinases (Jak) family and transcription factors of the (IFNAR1 and 2), the IC region binds protein-tyrosine Stat family (Velazquez et al., 1992;Darnell et al., 1994). and -serine/threonine kinases which phosphorylate the Proteins of the Jak-Stat pathways have been shown to receptor and the associated Stat transcription factors.bind to the intracytoplasmic (IC) domains of the IFNAR1 A two-hybrid screening was carried out to identify and 2 receptor chains. Jak1 is constitutively associated additional proteins which could interact with the IC with IFNAR2 (Novick et al., 1994), whereas tyk2 is bound domain of the IFNAR1 chain of the IFN-α,β receptor.to the IC domain of IFNAR1 (Abramovich et al., 1994b; Several positive clones representing a protein sequence Colaminici et al., 1994). IFNAR1 can also bind Stat2 (Abramovich et al., 1994b), and the docking site for latent designated IR1B4 were recovered from a human cDNA Stat2 was identified as a peptide containing phosphotyrolibrary. IR1B4 was identified as the human homolog sine Y466, adjacent to the tyk2 binding site (amino acids of PRMT1, a protein-arginine methyltransferase from 479-511) in the 100 amino acid long IFNAR1-IC region rat cells. Flag-IR1B4 fusion proteins bind to the isolated (Yan et al., 1996). IC was observed . Protein-tyrosine antibodies from untreated human cells. IR1B4/PRMT1 phosphatases PTP1C and D reversibly associate with is involved in IFN action since U266 cells rendered IFNAR1 upon IFN addition (David et al., 1995a). In deficient in this methyltransferase by antisense oligonuaddition, two serine/threonine protein kinases, the 48 kDa cleotides become more resistant to growth inhibition ERK2 MAP kinase (David et al., 1995b) and the cAMPby IFN. Methylation of proteins by enzymes which can activated protein kinase A (PKA; David et al., 1996) attach to the IC domains of receptors may be a signaling bind to the isolated membrane-proximal 50 residues of mechanism complementing protein phosphorylation.IFNAR1-IC. Therefore, the IC domains of type I IFN Among substrates methylated by PRMT1 are RNAreceptors serve as docking sites for multiple proteins binding heterogeneous nuclear ribonucleoproteins involved in phosphorylation and dephosphorylation. Both (hnRNPs) which are involved in mRNA processing, IFNAR1 and IFNAR2 become tyrosine phosphorylated in splicing and transport into the cytoplasm.response to IFN treatment, and binding of a tyrosineKeywords: interferon/methyltransferase/protein phosphorylated protein (βPTyr) to IFNAR1 is seen spemethylation/receptor/signaling cif...