ISGF-3 is an interferon-dependent positiveacting transcription factor that is cytoplasmically activated, possibly through direct interaction with the interferon receptor. The factor has been purified, Its component proteins have been separated, and its peptide sequences have been obtained. From the sequences, degenerate oligonucleotide probes were constructed to screen for cDNA clones. Sequencing of the selected clones shows that the 91-and 84-kDa components represent two forms of a previously unknown (to our knowledge) protein. Several antibodies raised against these proteins prove that they indeed do encode protein components of ISGF-3. This work provides reagents to explore the modification of this cytoplasmically activated transcription factor.The attachment of interferon a (IFN-a) to its specific cell surface receptor activates the transcription of a limited set of genes, termed the IFN-stimulated genes (1-3). Agents that affect second messenger levels do not activate transcription of these genes, leading us to propose that protein-protein interactions in the cytoplasm beginning at the IFN receptor might act directly in transmitting the signal generated by receptor occupation to the nucleus (4). To test this hypothesis, we began our experiments in the nucleus at the activated genes. We identified a DNA element, the IFN stimulation response element (ISRE), and a cognate transcription factor, ISGF-3, whose activation paralleled the transcriptional activation pattern of the IFN-stimulation genes in cells treated with . The observations that the proteins in ISGF-3 preexist in untreated cells, are promptly activated in an IFN-a-dependent fashion in the cell cytoplasm, and are subsequently translocated to the nucleus thus suggest that these proteins are a possible specific link between an occupied receptor and a limited set of genes (4,6).Partial purification of ISGF-3 followed by recovery of the purified proteins from a specific DNA-protein complex revealed that the complete complex was made up of four proteins (7). A 48-kDa protein termed ISGF-3y, because pretreatment of HeLa cells with IFN-y increased its presence, binds DNA weakly on its own (7-9). In combination with the IFN-a-activated proteins, termed collectively the ISGF-3a proteins, the ISGF-3y forms a complex that binds the ISRE with a 50-fold higher affinity (8). proteins comprise a set of polypeptides of 113, 91, and 84 kDa. All of the ISGF-3 components initially reside in the cell cytoplasm (6,10). However, after only about 5 min of IFN-a treatment, the active complex is found in the cell nucleus (6).To understand the mechanism of cytoplasmic activation of the ISGF-3a proteins, their transport to the nucleus, and their interaction with ISGF-3'y, we have purified the factor in sufficient quantity to obtain peptide sequence from each protein. Degenerate oligodeoxynucleotides that encode the peptides were constructed and used in a combination of cDNA library screening and PCR amplification of cDNA products copied from mRNA to identify cDNA clo...

Mutants in complementation group U3, completely defective in the response of all genes tested to interferons (IFNs) alpha and gamma, do not express the 91 and 84 kDa polypeptide components of interferon‐stimulated gene factor 3 (ISGF3), a transcription factor known to play a primary role in the IFN‐alpha response pathway. The 91 and 84 kDa polypeptides are products of a single gene. They result from differential splicing and differ only in a 38 amino acid extension at the C‐terminus of the 91 kDa polypeptide. Complementation of U3 mutants with cDNA constructs expressing the 91 kDa product at levels comparable to those observed in induced wild‐type cells completely restored the response to both IFN‐alpha and ‐gamma and the ability to form ISGF3. Complementation with the 84 kDa component similarly restored the ability to form ISGF3 and, albeit to a lower level, the IFN‐alpha response of all genes tested so far. It failed, however, to restore the IFN‐gamma response of any gene analysed. The precise nature of the DNA motifs and combination of factors required for the transcriptional response of all genes inducible by IFN‐alpha and ‐gamma remains to be established. The results presented here, however, emphasize the apparent general requirement of the 91 kDa polypeptide in the primary transcriptional response to both types of IFN.

(6). That cDNA sequence shows some similarity to the ISGF-2 (IRF-1) DNA (7, 8) and encodes a protein differentfrom the three larger ISGF-3 proteins. We have now cloned and sequenced a cDNA encoding the 113-kDa proteint and antiserum against this protein also reacts with the ISGF-3 DNA complex. Comparison of the sequences of the 113-kDa and the 91/84-kDa proteins reveals that they are encoded by closely related but distinct genes. Inspection of the 113-kDa and 91/ 84-kDa sequences reveals heptad leucine repeats in a potentially helical region (9, 10) that could account for complex formation after IFN-a treatment. In addition, one short conserved region ofthe two genes encodes a sequence similar to the Src homology 2 (SH2) domains noted in various tyrosine kinases and substrates for these enzymes (11). We speculate that the genes encoding the 113-and 91/84-proteins are members of a gene family whose products serve directly to receive information that a specific receptor has bound its ligand and subsequently transduce this information to the nucleus.MATERIALS AND METHODS The purification of ISGF-3 for microsequencing (12) was described earlier (1, 5). Sequences obtained by microsequencing ofthe ISGF-3 113-kDa component are underlined in Fig. 1. Based on peptide E, we designed a degenerate oligonucleotide, AAYACIGARCCIATGGARATYATT, which was used to screen a cDNA library basically as described (13). Briefly, the degenerate oligonucleotides were labeled by polynucleotide kinase with [y-32P]ATP and hybridizations were carried out overnight at 400C in 6x SSTE (0.9 M NaCI/60 mM Tris'HCl, pH 7.9/6 mM EDTA)/0.1% SDS/2 mM Na4P207/6 mM KH2PO4/10 x Denhardt's solution (13) in the presence of salmon sperm DNA (100 ,g/ml).The nitrocellulose filters then were washed four times (10 min per wash) with the same hybridization conditions but without labeled probe and salmon sperm DNA. Autoradiography was carried out at -80oC with an intensifying screen for 48 h. A PCR product (14) was also obtained by using oligonucleotides designed according to peptides D and E. The sequence ofthis PCR product was identical to a region in clone fli. The full-length cDNA clones ofthe 113-and 91-kDa proteins were transcribed in vitro and transcribed RNAs were translated in vitro with rabbit reticulate lysate (Promega; conditions as described in the Promega protocol).

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The early events that occur after treatment of the highly interferon a (IFN-a)-sensitive human lymphoblastoid Daudi cell line with human leukocyte IFN-a have been examined. IFN-a treatment ofDaudi cells results in a rapid and transient increase in the cellular content of diacylglycerol, which occurs in the absence of inositol phospholipid turnover, or an increase in intracellular calcium concentration. Furthermore, IFN-a treatment results in a selective, time-dependent activation of the Ca2+-independent E isoform of protein kinase C (PKC), while the a isoform is unaffected by IFN-a treatment. In contrast, IFN-a treatment of an IFN-resistant subclone of Daudi cells had no effect on the diacylglycerol content of cells and on the activation of PKC-e. The selective PKC inhibitor staurosporine blocked the transcriptional activation of IFN-a-stimulated genes, the cytoplasmic accumulation of mRNAs for these genes, and the induction of antiviral activity by IFN-a against vesicular stomatitis virus in IFN-sensitive cells. These observations suggest that transmembrane signaling of IFN-a involves diacylglycerol production and activation of PKC-E in Daudi cells.-,411, and -y) and Ca2+-independent (PKC-8, -E, -s, and -ti) enzymes. Furthermore, PKC-E has been shown to have Ca2+-independent phorbol ester binding activities and exhibits substrate specificity distinct from other characterized PKC isoforms (10, 11).We have investigated the early events that occur upon treatment of human Daudi lymphoblastoid cells with IFN-a in order to identify the biochemical pathways of transmembrane signaling. IFN-a treatment of these cell lines results in cessation of cell growth, protection against viral infection, and the rapid transcriptional activation of ISGs (12, 13). We report that the outstanding features of the signal transduction pathway of IFN-a in Daudi cells are the generation of DAG in the absence of inositol phospholipid turnover or Ca2+ elevation and the activation of the calcium-independent E isoform of PKC. Furthermore, staurosporine, a potent inhibitor of PKC activity blocks the transcriptional activation of ISGs, the cytoplasmic accumulation of ISG mRNA, and the induction of antiviral activity against vesicular stomatitis virus (VSV) in Daudi cells by IFN-a.