©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcriptional Induction by Double-stranded RNA Is Mediated by Interferon-stimulated Response Elements without Activation of Interferon-stimulated Gene Factor 3 (*)

(Received for publication, May 15, 1995; and in revised form, June 8, 1995)

Sudip K. Bandyopadhyay (1) George T. Leonard , Jr. (1) (2) Tanya Bandyopadhyay (1) George R. Stark (1) Ganes C. Sen (1) (2)(§)

From the  (1)Department of Molecular Biology, Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the (2)Department of Biochemistry, Case Western Reserve University Medical School, Cleveland, Ohio 44106

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Many genes induced by type I interferons (IFNs) are also induced by double-stranded (ds) RNA. In this study, we investigated the mechanism of this induction process. Using cell lines from which the type I IFN genes have been deleted, we established that induction by dsRNA of the IFN-inducible 561 gene is direct and not mediated by the intermediate synthesis of IFN. Unlike 561 mRNA, the IFN-inducible 6-16 mRNA was induced poorly by dsRNA. Transfection studies demonstrated that the sequence difference between the core IFN-stimulated response elements (ISREs) of these two genes is not responsible for their differential inducibility by dsRNA. A point mutation in the 561 ISRE that abolished its response to IFN-alpha also made it unresponsive to dsRNA, thus demonstrating that the ISRE is the relevant cis-acting element for dsRNA signaling. The roles of different known ISRE-binding protein and tyrosine kinases in transducing the signal elicited by dsRNA were evaluated in genetically altered cell lines. dsRNA failed to induce 561 mRNA in cells expressing an antisense RNA for interferon regulatory factor 1, whereas it was induced strongly in cells expressing the corresponding sense mRNA. 561 mRNA was also induced strongly by dsRNA, but not by IFN-alpha, in mutant cell lines that do not express functional tyrosine kinases Tyk2 or JAK1 or ISRE binding protein, p48, or STAT2, all of which are required for IFN-alpha signaling. However, in cells devoid of functional STAT1, which is also required for IFN-alpha signaling, the induction of 561 mRNA by dsRNA was very low. Expression of transfected STAT1alpha protein, but not of STAT1beta protein, in these cells greatly enhanced the dsRNA inducibility of the 561 gene. These studies indicate that the major ISRE-mediated signaling pathway used by dsRNA requires interferon regulatory factor 1 and STAT1alpha. This pathway, however, does not require the other known cytoplasmic components used for IFN-alpha signaling.


INTRODUCTION

Double-stranded (ds) RNA, (^1)which is often produced in a cell during virus replication, is a strong inducer of transcription of specific cellular genes(1, 2, 3) . The mechanism of this induction process and the identity of the cellular transcription factors that take part in it remain ill defined. Genes encoding interferon (IFN)-beta and IFN-alpha are among the most extensively studied dsRNA-induced genes(4) . They contain multiple positive and negative cis-acting elements which are required for induction. Moreover, many of these elements can bind to several protein factors which have different effects on transcription. As a result, the induction process is quite complex and activation of several trans-acting factors by dsRNA is required for the assembly of an active transcription complex. Of these factors, NFkB is a crucial member whose participation is necessary but not sufficient to activate transcription of the human IFN-beta gene(4, 5) . How dsRNA activates the other necessary factors remains to be understood.

Another class of dsRNA-inducible genes is also induced by type I IFNs which act through the IFN-stimulated response element (ISRE) present in these genes(6, 7, 8, 9, 10, 11) . All IFN-inducible genes are, however, not equally induced by dsRNA. We have been studying the mechanism of induction of the 561 gene in human cells. The transcriptional regulatory region of this gene is not complex. It contains ISRE but no kB site or site for binding of any other known transcription factors. Thus, this simply regulated gene is highly suited for studying the mechanism of activation of ISRE-containing genes by dsRNA. We have previously shown that 561 mRNA is strongly induced by dsRNA or by virus infections that produce dsRNA(10, 11) . We have also shown that this induction process is direct and does not require ongoing protein synthesis. It, however, is blocked by 2-aminopurine, an inhibitor of RNA-activated protein kinase(11) . Our studies provided persuasive, but indirect, evidence to indicate that the induction process is not mediated by the synthesis of IFNs as intermediates. In this paper, we present more definitive evidence in support of the above characteristics of 561 mRNA induction by dsRNA.

Since ISREs, which receive the transcriptional signal generated by IFNs, also appear to be involved in mediating the signal elicited by dsRNA, we were interested to determine the extent of overlap between the two signaling pathways. The major signaling pathway used by IFN-alpha to signal through ISREs is the JAK-STAT pathway(12, 13, 14, 15, 16, 17, 18) . Binding of IFN-alpha to its receptor on the cell surface leads to tyrosine phosphorylation of two protein tyrosine kinases, Tyk2 and JAK1, followed by tyrosine phosphorylation of the STAT1 and STAT2 proteins. Tyrosine phosphorylation of the STAT proteins results in their nuclear translocation and association with the p48 protein. The trimeric complex containing STAT1, STAT2, and p48 is IFN-stimulated gene factor 3 (ISGF3), which binds to ISREs and stimulates transcription. ISRE containing genes fail to respond to IFN-alpha in mutant cell lines defective in any component of the JAK-STAT pathway.

Another ISRE-binding protein is IRF-1(19, 20) . IRF-1 has a DNA sequence recognition specificity partially overlapping but distinct from the binding specificity of ISGF3. IRF-1 can bind to the positive regulatory domain I element of the IFN-beta gene and is thought to be involved in its transcriptional induction by dsRNA(19, 21) . It is not clear whether IRF-1 is involved in ISRE-mediated signal transduction by IFN-alpha. The most compelling indication for such an involvement comes from studies with cell lines missing functional IRF-1(22) . In these cells, induction of several genes by IFN-alpha was partially impaired. Whether this was due to a direct or indirect effect of IRF-1 remains to be determined.

Here we report that IRF-1 is essential for transduction of the dsRNA-elicited signal to the ISREs. In contrast, Tyk2, JAK-1, p48, and STAT2, all components of the JAK-STAT pathway, are not absolutely required for this signaling process. 561 mRNA could be induced, albeit weakly, by dsRNA in cells missing STAT1 which suggests that although this component is required for the major pathway of signaling, a STAT1-independent minor pathway may exist.


MATERIALS AND METHODS

Cell culture materials and G418 were obtained from Life Technologies, Inc. The source of pure IFN-alpha has been described previously(23) . Cycloheximide and poly(I)-poly(C) (dsRNA) were from Boehringer Mannheim, 2-aminopurine was from Sigma, and luciferase assay reagents kit were from Promega. All radioactive chemicals were from DuPont.

Cell Culture and Treatment

M007 glioma cells were obtained from Rufus S. Day (University of Alberta, Edmonton, Canada), and GRE and SAN glioma cells were obtained from Paul Kornblith (Albert Einstein College of Medicine, Bronx, NY) through Rufus Day(24) . Glioma cells, 2fTGH, and mutant cell lines derived from 2fTGH were cultured in Dulbecco's modified Eagle's medium plus 10% heat-activated fetal bovine serum supplemented with glutamine, penicillin, and streptomycin. Human fibroblast lines, transfected with sense IRF-1 (S1), transfected with antisense IRF-1 (AS11), and transfected with empty vector (C1), were obtained from Jan Vilcek (New York University Medical Center, New York) and maintained as described by Reis et al.(21) . U3A cells expressing STAT1alpha, STAT1beta, or a mutant STAT1alpha were obtained from James E. Darnell, Jr. (Rockefeller University, New York). IFN-alpha (500 Units/ml), cycloheximide (50 µg/ml), 2-aminopurine (10 mM), and dsRNA (100 µg/ml) in medium containing 0.5% serum were used to treat cells for 4 h, unless otherwise indicated.

RNA Isolation and Northern Blot Analysis

Total RNA was prepared from 75-85% confluent cells after the indicated treatments by the method of Chomczynski and Sacchi(25) . RNA samples were electrophoresed in 1% agarose-formaldehyde gel and then transferred to either GeneScreen (DuPont NEN) or Nytran (Schleicher and Schuell) membranes. cDNA probes were prepared by random priming using a Boehringer Mannheim kit to an efficiency of >2 10^8 cpm/µg of DNA. Hybridization and washing were performed according to standard procedure. Blots used for 561 mRNA level estimations were reprobed with actin cDNA for normalization purpose. The levels of actin mRNA as estimated by PhosphorImager analysis of the blots were within 20% of one another.

Reporter Gene Constructs

The 561 CAT and 6-16 CAT constructs have been described before(26) . The 561 luciferase reporter gene contains -134 to +1 nucleotides of the 561 gene. For mutating the 561 ISRE to 6-16 ISRE or to null ISRE, polymerase chain reactions were done using the 561 CAT plasmid and appropriate primers. The reporter luciferase genes were produced using by transferring the 561 promoters to pGL2-Basic (Promega).

Transient Transfection

Cells in 100-mm plates were transfected with 10 µg of DNA using the modified calcium phosphate procedure(27) . Cells were shocked with 16% Me(2)SO for 90 s, 16 h after transfection. After another hour, cells were trypsinized and pooled from several plates which had been transfected with the same plasmid. Equal number of cells were replated on several plates which were used for treatment with IFN-alpha or dsRNA. This protocol ensured that any variation in transfection efficiency from plate to plate would not influence the results. Three hours after replating the cells, they were treated with the indicated concentrations of IFN or dsRNA for 4 h in 2 ml of medium containing 0.5% serum. Eight ml of medium containing 10% serum was then added to the plates. Cells were harvested for preparing extracts 16 h later. Equal amounts of cell extract protein were used for CAT and luciferase assays. CAT assays were done as described before(17, 26) . Acetylated and unacetylated forms of chloramphenicol were quantitated after chromatography using a PhosphorImager (Molecular Dynamics). Luciferase activity was measured according to the protocol provided by Promega. Light emission was measured by a luminometer (model ML2250, Dynatech).

RNase Protection Assay

RNase protection analysis (27) was performed using probes yielding protected fragments of 260 and 130 base pairs for 561 and -actin(15) , respectively. Probes were generated using T7 RNA polymerase for 561, and SP6 RNA polymerase for -actin, and hybridized with 15 µg of total RNA with 100,000 cpm of each probe at 45 °C overnight. RNase A (20 µg/ml) together with RNase T1 (1 µl/ml, Life Technologies, Inc.) were used for digestion at 30 °C for 1 h. After phenol extraction and ethanol precipitation, the samples were separated on 5% urea-acrylamide gels which were then dried and autoradiographed.


RESULTS

Induction of 561 mRNA by dsRNA in Cells Missing Type I IFN Genes

561 mRNA is strongly induced by type I IFNs (IFN-alpha and IFN-beta) and by dsRNA or virus infection(10, 11, 28, 29) . We provided indirect evidence that the observed induction was directly mediated by dsRNA without an involvement of IFN-alpha or IFN-beta which are also known to be induced by dsRNA. To conclusively establish this fact, we have now examined dsRNA induction of 561 mRNA in three human glioblastoma lines, GRE, M007, and SAN, all of which have been shown to be missing the region of chromosome 9 that encompasses the IFN-alpha and IFN-beta genes(24) . Since both alleles of all type I IFN genes are absent, these cells cannot synthesize any type I IFN. As shown in Fig. 1, dsRNA could efficiently induce 561 mRNA in all three cell lines, thus demonstrating that IFNs are not involved as intermediates.


Figure 1: Induction of IFN-inducible 561 mRNA by dsRNA in IFN-minus cell lines. Glioma cell lines GRE, M007, and SAN were either untreated or treated with 100 µg/ml dsRNA in 0.5% serum containing medium for 4 h. Twenty µg of total RNA was used for each sample in Northern blot with labeled 561 DNA as probe.



Characteristics of 561 mRNA Induction

The kinetics of 561 mRNA induction in GRE cells is shown in Fig. 2. No 561 mRNA was detectable in untreated cells or in cells treated with dsRNA for 0.5 h or 1 h. The maximum level of induction was at 4 h after which the level started to decline. This experiment showed that, like induction by IFN-alpha, induction of 561 mRNA is also rapid but transient.


Figure 2: Kinetics of induction of IFN-inducible 561 mRNA by dsRNA in GRE cells. Cells were treated with 100 µg/ml dsRNA for the indicated time. Twenty µg of total RNA was used for each sample in the Northern blot, which was probed with labeled 561 DNA. The relevant portion of the autoradiogram is shown. The same blot was deprobed and hybridized with actin cDNA for normalization. The actin mRNA levels in each lane were within 20% of one another.



Effects of inhibitors on the induction process were examined in the experiment shown in Fig. 3. Cycloheximide, an inhibitor of protein synthesis, did not inhibit 561 induction by dsRNA in any of the three lines. This result indicates that ongoing protein synthesis is not required for the induction and that pre-existing proteins are sufficient for this process. Cycloheximide alone did not induce 561 mRNA (data not shown) but more mRNA accumulated in cells treated with dsRNA and cycloheximide than in cells treated with dsRNA alone (Fig. 3). This was probably due to stabilization of the labile 561 mRNA in the presence of cycloheximide. We have previously shown that 2-aminopurine, a known inhibitor of RNA-activated protein kinase, blocks 561 mRNA induction by dsRNA in HeLaM cells(11) . Similar results were obtained with the glioma cell lines, in which 2-aminopurine almost completely blocked 561 mRNA induction by dsRNA (Fig. 3).


Figure 3: Effects of cycloheximide and 2-aminopurine on the induction of 561 mRNA by dsRNA. GRE, M007, and SAN cells were untreated or treated as indicated for 4 h. Cycloheximide (CHX) and 2-aminopurine (2-AP) were added to the cells 15 min prior to the dsRNA treatment. 561 mRNA levels were determined as described in the legend to Fig. 2.



Differential Induction of 6-16 mRNA

6-16 is another gene whose transcription is augmented by IFN-alpha and IFN-beta in many cell lines(30) . As expected, IFN-alpha strongly induced 6-16 mRNA synthesis in the glioma cell lines as well (Fig. 4). However, in contrast to 561 mRNA, induction of 6-16 mRNA by dsRNA was very poor in these cells. The differential induction of the 561 and 6-16 resident genes was also observed with transfected reporter genes driven by the regulatory regions of the two genes (Fig. 5). GRE cells were transiently transfected with 561 CAT or 6-16 CAT. IFN-alpha and dsRNA were almost equally efficient in inducing CAT activity in cells transfected with 561 CAT. In cells transfected with 6-16 CAT, however, only IFN-alpha, but not dsRNA, induced CAT activity. The induction of 6-16 CAT by IFN-alpha was actually 4-fold more efficient than that of 561 CAT although the former was not induced at all by dsRNA. It should be noted that the 6-16 CAT gene contains two identical ISREs, whereas the 561 CAT contains only one. These results demonstrate that the 6-16 regulatory region present in the reporter gene contains the element(s) responsible for the observed differential induction by IFN-alpha and dsRNA of the native 6-16 gene.


Figure 4: Induction of 6-16 mRNA by dsRNA. GRE, M007, and SAN cells were either untreated or treated with dsRNA or IFN-alpha for 4 h. The same blot as used for Fig. 3was deprobed and hybridized again with labeled a 6-16 cDNA probe, washed, and autoradiographed.




Figure 5: Induction of 561 CAT and 6-16 CAT expression in GRE cells. 561 CAT and 6-16 CAT constructs were transiently transfected into GRE cells. Cells were either untreated or treated with dsRNA or IFN-alpha for 16 h as indicated. CAT activity was measured in extracts with equal amount of protein as described under ``Materials and Methods.''



Role of ISRE in dsRNA-mediated Signal Transduction

Transcriptional signals generated by IFN-alpha and IFN-beta are known to be received by the ISREs present in IFN-inducible genes. To examine the role of ISREs in the induction of the same genes by dsRNA, we used a newly constructed 561 luciferase reporter gene. This reporter gene, when transfected to GRE cells, was equally induced by IFN-alpha and dsRNA (Fig. 6). There is a difference of only one base between the two core ISRE sequences present in the 6-16 gene and the core ISRE sequence of the 561 genes. When the ISRE sequence of the 561 luciferase gene was mutated to the 6-16 ISRE sequence, the reporter gene remained equally inducible by the two inducers (Fig. 6, lanes 4-6), thus suggesting that the one base difference in the ISRE core sequences of the two genes is not responsible for their differential induction by dsRNA.


Figure 6: Effects of ISRE mutations on gene induction by dsRNA. 561 luciferase (lanes 1-3), 561 to 6-16 ISRE luciferase (lanes 4-6), and 561 to null ISRE luciferase (lanes 7-9) constructs were transiently transfected into GRE cells. ISRE core sequences of different constructs are shown in the figure with the open letter indicating the mutated nucleotide. The cells were treated as indicated and luciferase activities were measured as described under ``Materials and Methods.''



Whether the ISRE sequence is involved in the induction of 561 luciferase by dsRNA at all was addressed in the next experiment (Fig. 6, lanes 7-9). A specific point mutation, known to destroy the binding of IRF-1 and ISGF3 to ISREs, was introduced into the ISRE of 561 luciferase. As expected, the mutated gene could not respond to IFN-alpha. It was also not induced by dsRNA, thus indicating that the ISRE sequence is required for response to this agent as well.

Role of IRF-1 in the Induction Process

Once it had been established that the dsRNA-elicited signal is mediated by ISREs, the possible role of known ISRE-binding transcription factors was evaluated. One such protein, IRF-1, is known to bind to ISREs and to the positive regulatory domain I element of the IFN-beta gene. We tested the possible role of IRF-1 in 561 mRNA induction by dsRNA in human fibroblast lines stably transfected with vectors expressing sense or antisense IRF-1 RNAs(21) . The two transfected lines as well as the mock transfected line were treated with dsRNA in the presence of cycloheximide so that direct induction of 561 mRNA could be measured. The cellular levels of 561 mRNA before and after induction were measured using a sensitive and quantitative RNase protection assay. Actin mRNA levels were measured by the same assay concurrently to serve as an internal control. As shown in Fig. 7, IFN-alpha induced 561 mRNA in all three lines. Induction by dsRNA was more efficient in the S1 line, which overexpresses IRF-1 mRNA, than in the C1 line, which was mock transfected. More importantly, the induction by dsRNA was completely abolished in cells (AS11) expressing the IRF-1 antisense RNA. These results suggest that IRF-1 may transmit the dsRNA-elicited signal to the ISRE of the 561 gene for inducing its transcription.


Figure 7: 561 mRNA induction by dsRNA in cell lines expressing sense or antisense IRF-1 RNA. Cultures of C1 (expressing only vector), S1 (expressing sense IRF-1), and AS11 (expressing antisense IRF-1) cells were treated in the presence of cycloheximide for 4 h with 100 µg/ml dsRNA (lanes 2, 5, and 8) or with 500 units/ml IFN-alpha (lanes 3, 6, and 9) or with only medium (lanes 1, 4, and 7). The levels of mRNA 561 were measured by RNase protection assay using 15 µg of total RNA for each sample. Lane 10 is the antisense 561 probe digested with RNase and lane 11 is the probe without RNase digestion. An antisense actin probe was used in the same experiment for estimating the levels of actin mRNA (data not shown). The amounts of actin mRNA in each sample were within 20% of one another.



Possible Involvement of ISGF3 in Transmitting the dsRNA-elicited Signal

ISGF3 is the primary trans-acting factor which transmits the signal generated by IFN-alpha to the ISRE present in IFN-inducible genes. Since the ISRE is also required for the induction of the 561 gene by dsRNA, we wondered whether ISGF3 or any of its components is involved in this process. To address this issue, we used mutant human cell lines that are missing different components of the ISGF3 complex or the tyrosine kinases which are required for its activation by IFN-alpha. Five mutant lines and the corresponding 2fTGH wild-type cells were treated with either dsRNA or IFN-alpha and the induction of 561 mRNA was measured by Northern analysis (Fig. 8). As expected, IFN-alpha could not induce the 561 mRNA in any of the mutants but did induce the mRNA efficiently in the wild-type cells. In contrast, dsRNA could induce 561 RNA in all the mutants, although to varying degrees.


Figure 8: 561 mRNA induction by dsRNA in IFN-alpha unresponsive mutant cell lines. Cellular levels of 561 mRNA were measured by Northern blot analysis of 20 µg of total RNA from 2fTGH and its mutant cell lines. Treatments were for 4 h as indicated in the figure.



Since the extent of induction of 561 mRNA by dsRNA was different in different mutant lines, we quantitated the levels of this mRNA by using an RNase protection assay. Actin mRNA levels were also measured using the same total RNAs and the same assay. 561 mRNA levels were normalized using the actin mRNA values and the normalized level of 561 mRNA in dsRNA-treated 2fTGH cells was assigned a value of 100 units. All untreated cells contained undetectable (less than 1 unit) levels of 561 mRNA (Table 1). IFN-alpha treatment of 2fTGH cells, but not of mutant cells, increased the level of 561 mRNA to 164 units. In U1A cells, lacking Tyk2, and U4A cells, lacking Jak1, dsRNA induced 51 units of 561 mRNA. In U2A cells, lacking p48, 96 units of 561 mRNA was induced by dsRNA and in U6A cells, lacking STAT2, the corresponding level was 29 units. These results demonstrated that although their absence affects the level of induction quantitatively, Tyk2, Jak1, p48, and STAT2 are not required for 561 mRNA induction by dsRNA. In contrast, the induction level in U3A cells, which lack STAT1, was quite low (11 units). Expression of one of the two STAT1 proteins, STAT1alpha (p91), by complementing U3A cells with the corresponding expression vector, greatly enhanced the dsRNA inducibility of 561 mRNA. However, the same was not true for the STAT1beta (p84) protein; 561 mRNA was poorly induced in cells expressing STAT1beta but lacking STAT1alpha. Thus, among the known cytoplasmic components of the IFN-alpha signaling pathway, STAT1alpha appears to be the most important factor in the ISRE-mediated signaling pathway used by dsRNA. A mutant STAT1alpha, in which residue 701 has been changed from tyrosine to phenylalanine, cannot function in the signaling pathways used by IFN-alpha or IFN- (31) . This mutant also failed to complement the U3A cells for their ability to transmit the signal generated by dsRNA (Table 1).




DISCUSSION

Our previous studies strongly indicated that the induction of 561 mRNA transcription in human cells in response to exogenous dsRNA or virus infection is a direct process(10, 11) . However, since type I IFNs are also induced by the same inducers and 561 is an IFN-inducible gene, it was necessary to rigorously rule out the possible involvement of IFNs as the proximal inducer. Here, we provide genetic evidence in favor of the direct induction mechanism. dsRNA could induce 561 mRNA efficiently in two types of mutant cell lines: one that cannot produce IFNs in response to dsRNA because the IFN genes are missing and the second that cannot respond to type I IFN because components of the IFN-signal transduction pathway are defective. Thus, it is highly likely that dsRNA activates pre-existing cellular proteins, leading to enhanced transcription of the 561 gene. The rapid kinetics of induction and its insensitivity to cycloheximide also support the above conclusion. Similar conclusions have been made by other investigators, using cell lines missing IFN genes, about the mechanism of induction of other IFN-inducible genes by dsRNA(6, 32) . It is curious to note that, like its induction by IFN-alpha, induction of 561 mRNA by dsRNA was also transient.

Our experiments demonstrated that all type I IFN-sensitive genes are not equally induced by dsRNA. In the three IFN-minus mutant lines, dsRNA failed to induce the 6-16 mRNA appreciably, although it was very well induced by IFN-alpha. The testing of dsRNA inducibility of an IFN-sensitive gene in these cell lines is more meaningful than in normal cell lines, since intermediate synthesis of IFN does not complicate the picture. For this reason, much of the information in the literature regarding dsRNA inducibility of other IFN-sensitive genes needs to be re-examined in IFN-minus or IFN-insensitive mutant lines. Our experiments show that the observed difference in dsRNA inducibility is not due to the single nucleotide difference in the two core ISREs. It remains possible that the observed difference is not due to a difference in the ISRE sequences, but due to the presence of a negative element in the 6-16 regulatory region.

The same point mutation in the 561 ISRE that abolishes its response to IFN-alpha also abolishes its response to dsRNA, thus establishing a crucial role for the ISRE in the latter response. This mutation is known to eliminate binding of ISGF3 and IRF-1 to the ISRE(18) . Thus, these two factors are attractive candidates for mediating the response to dsRNA. Our results with the IFN-alpha unresponsive 2fTGH-derived mutant cell lines clearly demonstrated that ISGF3 does not mediate the action of dsRNA. Furthermore, these experiments established that the p48 and STAT2 components of ISGF3 and the Tyk2 and JAK1 tyrosine kinases are not absolutely required for the ISGF3-independent pathway of transducing the dsRNA signal. However, there were noticeable quantitative differences in the extent of 561 mRNA induction in different mutant cell lines missing these proteins. The biochemical basis of these differences remains to be determined. The requirement of the STAT1alpha protein, on the other hand, was more pronounced. The signal was markedly reduced, although still detectable, in cells missing STAT1alpha. Our results suggest that the STAT1alpha protein may be an ancillary protein for an ISRE-binding factor or its absence may indirectly affect the function or the synthesis of a protein which is directly involved in the signal transduction pathway used by dsRNA. Alternatively, parallel pathways, one of which uses STAT1alpha and another which does not, may exist. It appears that the function of STAT1alpha in dsRNA signaling cannot be replaced by STAT1beta as is the case for IFN-, but not IFN-alpha, signaling pathway.

In contrast to the lack of a role of ISGF3, our studies clearly indicate an important role for IRF-1 in ISRE-mediated signal transduction by dsRNA. This signaling pathway is distinct from ISRE-mediated IFN-alpha/beta signaling and kB-mediated signaling by dsRNA or virus infection. There is somewhat conflicting information in the literature about the role of IRF-1 in the latter two pathways. In cells or mice devoid of functional IRF-1, type I IFN could be efficiently induced by dsRNA or virus infection and IFN-alpha could induce IFN-stimulated genes normally(22, 33) . However, in another study, overexpression of IRF-1 in a human cell line enhanced IFN-beta mRNA induction by dsRNA and the expression of IRF-1 antisense RNA reduced the level of its induction, thus demonstrating the need of IRF-1 in IFN-beta mRNA induction(21) . Induction of some genes by IFN-beta was also partly affected by the absence of IRF-1. Results presented here with the antisense IRF-1 RNA-expressing cells clearly demonstrated that ISRE-mediated signaling by dsRNA also requires IRF-1.

Although we provide here genetic evidence for the involvement of IRF-1 and possibly p91 in ISRE-mediated signal transduction, confirming biochemical evidence is yet to come. Unlike the report by Daly and Reich(6) , we could not detect any dsRNA-activated factor which binds to ISREs under conditions in which dsRNA activation of NFkB was readily detected by electrophoretic mobility shift assays (data not shown). The observation that the Y701F mutant of STAT1alpha cannot support dsRNA signaling suggests that tyrosine phosphorylation of STAT1alpha is required for this process. However, we failed to detect tyrosine phosphorylation of STAT1alpha in response to dsRNA although such phosphorylation was apparent in response to IFN-alpha or IFN- treatment (data not shown). This observation indicates that STAT1alpha may not be directly involved in the signaling process since the only known mechanism of its activation is by tyrosine phosphorylation(12) . However, the formal possibility remains that STAT1alpha can be activated by other alternative means. Our failure to detect a specific dsRNA-stimulated IRF-1-ISRE complex by mobility shift assays could be due to technical difficulty. The induced complex may have a mobility very similar to that of the constitutive complexes. These complexes have indeed been shown to be highly heterogenous, containing different proteins but having similar mobilities(34) . On the other hand, it is also quite possible that the dsRNA-elicited signal does not change the DNA-binding ability of the relevant factor (such as IRF-1) but changes its function. This will be similar to the case for cAMP-induced transcriptional regulation of genes, which contain the cis-acting cyclic AMP-response element to which the transcription factor CRE-binding protein binds(35) .


FOOTNOTES

*
This study was supported by National Institutes of Health Grants CA-62220 and CA-68782. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology, The Cleveland Clinic Foundation, 9500 Euclid Ave., NC20, Cleveland, OH 44195. Tel.: 216-444-0636; Fax: 216-444-0512.

(^1)
The abbreviations used are: ds, double-stranded; IFN, interferon; ISRE, interferon-stimulated response element; JAK, Janus kinase; STAT, signal transducer and activator of transcription; ISGF, interferon-stimulated gene factor; IRF, interferon regulatory factor; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We are grateful to Mathias Müller and Ian M. Kerr for sharing unpublished data and 2fTGH and its mutant cell lines, R. S. Day III for glioma cells, Jan Vilcek for C1, S1, and AS11 cells, James E. Darnell, Jr. and Ke Shuai for STAT1alpha, STAT1-beta, and mutant STAT1alpha complemented U3A cells. We also thank Laura Tripepi for secretarial assistance.


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