Synergism between Multiple Virus-induced Factor-binding Elements Involved in the Differential Expression of Interferon A Genes*

(Received for publication, January 14, 1997, and in revised form, May 30, 1997)

José Bragança , Pierre Génin , Marie-Thérése Bandu , Nicole Darracq , Madeleine Vignal , Céline Cassé , Janine Doly and Ahmet Civas Dagger

From the Laboratoire de Régulation de l'Expression des Gènes Eucaryotes, CNRS, UPR 37, UFR Biomédicale des Saints-Pères, Université René Descartes, Paris V, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides -57 and -46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055-5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G-57 right-arrow C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A-78 right-arrow G and the G-57 right-arrow C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.


INTRODUCTION

Type I interferons (IFN-A1 and IFN-B) belong to the group of cytokines that constitute an early response to viral infection in eukaryotic cells. Whereas IFN-B is encoded by a single gene, which is expressed essentially in fibroblastic cells, IFN-A is represented by a large family of structurally related genes, which are predominantly expressed in lymphoid human cells or murine fibroblasts and macrophages (2-6). The IFN-A genes characterized so far have been shown to be coordinately induced in virus-infected cells. However, differences in the expression of individual IFN-A mRNAs are observed in both human and mouse cells, which reflect the transcriptional activity of the corresponding promoter regions in a particular cell type (2, 7-9).

Virus-induced expression of IFN-A and IFN-B genes is mediated by regulatory sequences located within 200 base pairs upstream of the transcription start site of their promoters (10-14). IFN-B gene expression is regulated by multiple factors that interact with positive regulatory domains (PRDs) located within virus-responsive element B (VRE-B): interferon regulatory factor-1 (IRF-1), which binds to both the PRDI and PRDIII domains, also known as IRF elements (15-18); the nuclear factor NF-kappa B, which recognizes the PRDII domain (19-21); and the ATF-2/c-Jun heterodimer, which binds to the PRDIV domain and is required for maximal virus inducibility in mouse L929 and human HeLa cells (22). Binding of both the NF-kappa B and ATF-2/c-Jun complexes is facilitated by HMGI(Y), members of the high mobility group of proteins that bend the DNA and allow VRE-B-binding factors to assemble in a higher order nucleoprotein complex called an "enhanceosome," thus promoting the virus-induced transcription of the IFN-B gene (23, 24). Postinductional repression and maintenance of a poised state of repression seem to be mediated by IRF-2 (and its truncated form), which recognize the IRF elements and prevent the binding of IRF-1. Other factors have been implicated in the repression of transcription by their interaction with the IRF elements or the negative regulatory domains surrounding VRE-B (18, 25-29).

Factors specifically involved in regulation of the IFN-A genes are less well defined. The virus-responsive element of the human IFN-A1 gene (VRE-A1) and the inducible element of the murine IFN-A4 gene have been shown to contain a PRDI-like site, thus involving IRF-1 in the induced expression of the IFN-A genes (30-32). A comparative study of the IFN-A4 and IFN-A6 gene promoters displaying virus inducibility has led to the detection, by EMSA, of a binding activity, AF1. It has been proposed that AF1-forming proteins cooperate with IRF-1 in the virus-induced transcription of IFN-A genes (4, 33, 34). Another factor possibly involved in the specific regulation of IFN-A gene regulation is the TG protein, which binds to the hexameric GAAATG repeats that confer virus inducibility by an IRF-1-independent pathway. The so-called "TG sequence" is adjacent to the PRDI-like motif in the human VRE-A1 and is conserved in most human or murine IFN-A (30, 35). However, the AF1-related and TG-binding proteins have yet to be characterized.

Interestingly, the deletion of the gene coding for IRF-1 did not affect virus-inducible expression of the type I IFN genes, thus calling into question its role (36, 37). Another member of the IRF family, IFN-stimulated gene factor 3gamma (ISGF3gamma ) has been suggested to participate in the regulation of the IFN-B gene promoter both alone and as a heteromeric complex (ISGF3) formed with Stat1alpha and Stat2 (from the signal transducer and activator of transcription family) (38, 39, 40, 41). ISGF3 has been shown to be induced secondarily by the virus and involved in the amplification of type I IFN gene expression triggered initially by virus infection of cells. However, the primary transcription factor, directly activated by the virus and mediating the PRDI-dependent initiation of transcription of the type I IFN genes, remains to be identified.

We have previously shown that the differential expression of IFN-A4 and IFN-A11 genes, in NDV-induced L929 cells, was due in part to the negative effect of the A-78 right-arrow G substitution that distinguishes the inducible element of IFN-A4 (IE-A4) from the equivalent region of the IFN-A11 gene promoter and also to the presence of negatively acting sequences located upstream of the inducible element of the IFN-A11 promoter (42). By comparative analysis of the -109 to -64 fragments of the IFN-A4 and IFN-A11 gene promoters, which include their inducible elements, we identified a virus-induced binding activity that is stimulated within 1 h of virion contact with cells (1). This virus-induced factor (VIF) recognizes specifically the PRDI-like domain shared by both the inducible elements and the TG-like domain of IE-A4.

In the present study, we report that VIF also binds a second PRDI-like motif located between the IE-A4 and the TATA box of the IFN-A4 promoter. We show that this motif contributes to the high level of NDV-induced transcription of the IFN-A4 gene by cooperating with the IE-A4 subdomains and that the low levels of IFN-A11 gene expression are caused essentially by the absence of both the TG-like and second PRDI-like domain in this promoter. Our data lead us to propose a model for the differential regulation of the IFN-A gene family members and to delineate the involvement of VIF in this regulation.


EXPERIMENTAL PROCEDURES

Plasmid Construction

The Bi series of plasmids pIF4 and pIF11 carrying, respectively, the -211 to +19 and the -198 to +19 promoter regions of the IFN-A4 and IFN-A11 genes and the J series deleted up to position -119 were derived from the plasmids pIF4T and pIF11T previously described (42) by the subcloning of polymerase chain reaction-amplified fragments. The 3'-primers were p17 for pIF4Bi or pIF4J and p18 for pIF11Bi or pIF11J, with p3 and p4 as the 5'-primers for the Bi and J constructs, respectively. These fragments, digested by PstI and BamHI, were subcloned into the plasmid pBLCAT3 (44). The constructs carrying a point mutation at position -78, pIF11[V4]Bi and pIF4[V11]Bi, were obtained similarly from the corresponding T series (42). The IFN-A11/A4 hybrid plasmid pIFH3Bi was derived from subcloning of the fragment amplified with the primers p3 and p18 with the plasmid pIFH66T (42). The pIFH1Bi and pIFH2Bi constructs were obtained in two steps by a procedure previously described (43); the fragments amplified in the first round using, respectively, pIF11T (or pIFH66T) as the template and p3 and p10 (or p3 and p9) as primers, were used as the 5'-primer in a second polymerase chain reaction using either p17 and the hybrid plasmid pIFH66T or p18 and the plasmid pIF11T as the 3'-primer and template, respectively. The construct pIFH4Bi was obtained similarly from pIF11T, using primers p3 and p5 in the first step, and the amplified fragment was used with primer p18 and plasmid pIF11T in the second polymerase chain reaction. To construct the plasmids pIF4T-3S and pIF4T-1S or plasmids pIF4T-4S and pIF4T-2S, either plasmid pIF4T or plasmid pIF4[V11]T was used as a template in each round. p17 was the primer for the second step, whereas either p6 or p7, respectively, was used as a 3'-mutagenic primer with p1 as the 5'-primer in the first round. Similarly, to construct pIF11T-3S and pIF11T-1S, pIF11T was used as template in both steps with p18 in the second and p5 or p8 as the mutagenic primer used with p2, whereas pIF11[V4]T was used as template to obtain the plasmids pIF11T-4S and pIF11T-2S. In each case, the final products were purified, digested by PstI and BamHI, and inserted between these sites in the pBLCAT3 polylinker. The promoter constructs carrying internal deletions were obtained by the ligation of two separately amplified fragments, one digested by PstI, the other by BamHI, to PstI-BamHI-digested pBLCAT3. Thus, pIF4Delta Bi and pIF11Delta Bi were constructed by ligation of two fragments amplified with p3/p21 and p19/p17 or with p3/p21 and p20/p18, respectively, in the presence of pIF4Bi or pIF11Bi as template. The pV4, p[B]2 and p[C4]2 constructs were previously described (1). The constructions depicted in Fig. 6A were obtained by insertion of synthetic fragments of the IFN-A4 and IFN-A11 promoters at the HindIII and BamHI sites upstream of the herpes simplex virus thymidine kinase promoter fused to the CAT gene (pBLCAT2 modified by G. Schütz). The correct sequences of the constructions presented were confirmed by DNA sequence analysis. The sequence information for polymerase chain reaction mutagenesis is available upon request.


Fig. 6. Correlation between the VIF binding and differential virus inducibility. A, HSV-tk constructs harboring different subdomains of the inducible element of IFN-A4 and IFN-A11 as described under "Experimental Procedures." The construct pdC4 contains two copies of the TG-like domain of IFN-A4 separated by an adenine residue. B, the -98 to -74 fragments of both the IFN-A4 and the IFN-A11 promoters, namely BC4 (lanes 1 and 2) and BC11 (lanes 3 and 4); the C4 or C11 dimers separated by an adenine residue, namely dC4 (lanes 5 and 6); and dC11 (lanes 7 and 8) were used as probes with nuclear extracts prepared from L929 uninduced (-) or induced for 4 h with NDV (+).
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Transfection and CAT Assays

Mouse L929 cells (ATCC CCL 1) were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% horse serum. HeLa S3 cells (ATCC CCL 2.2) were maintained as monolayers in the same medium supplemented with 10% newborn calf serum. Cells were transfected by the calcium phosphate method (45) and induced with NDV (La Jolla strain, New Jersey) or mock-induced as described previously (1). Measurements of CAT activity were performed on cytoplasmic extracts obtained 24 h after induction (46). The protein concentration was determined with the Coomassie G250 procedure as described previously (1). The results are derived from the average CAT values obtained in at least three independent transfection experiments, each performed in triplicate with two or three different clones.

Preparation of Nuclear Extracts

L929 cells and HeLa cells were grown in suspension, respectively, in Dulbecco's modified Eagle's medium and suspension-modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum. Virus induction for 4 h was performed by keeping the cells in contact with NDV for 1 h in serum-free medium containing 50 µg/ml of cycloheximide and by maintaining the cells for a further 3 h in the presence of 50 µg/ml cycloheximide and 2% fetal calf serum. IFN induction was carried out with recombinant mouse IFN-alpha 11 (103 units/ml) for 2 h. After washing and centrifugation, the cells were harvested immediately, and the nuclear extracts were prepared according to Dignam et al. (47).

Gel Mobility Shift Assays

The oligonucleotides BC4 and BC11 correspond to the -98 to -74 region of the IFN-A4 and IFN-A11 promoters, respectively. In the dC4 and dC11 oligomers, the module B is replaced by C4 in BC4 or by C11 in BC11. The B2 oligonucleotide corresponds to the dimer of the -98 to -87 GAAAGTGAAAAG sequence of the IFN-A4 and IFN-A11 promoters. The C42 and D42 represent, respectively, two copies of the -85 to -74 GAATTGGAAAGC and -57 to -46 GAAAGGAGAAAC sequences of IFN-A4, whereas C112 and D112 refer, respectively, to the dimers of their mutated forms GAATTGGGAAGC and CAAAGGAGAAAC present in the IFN-A11 promoter (the substituted residues are underlined).

ISRE-ISG15 (gatcCTCGGGAAAGGGAAACCGAAACTGAAGCC) and GIRE-Fcgamma R (ctagATTTCCCAGAAAAGGAACATGATGAATctag) are, respectively, the binding sites for ISGF3 and Stat1 dimers (48, 49). Preparation of the probe was performed using single-stranded oligonucleotides 5'-labeled with [gamma -32P]ATP (Amersham, UK; 3000 Ci/mmol) and annealed to a slight excess of the unlabeled complementary strand. ISRE was labeled by using the Klenow fragment of DNA polymerase I. Binding reactions and electrophoresis were performed as described previously (1). For competition assays, the molar excess of unlabeled competitor was mixed with the probe prior to the addition of the nuclear extract, and the bound DNA was quantified using a PhosphorImager (Molecular Dynamics; 445-SI). For experiments with antibodies, the binding reaction was preincubated for 1 h on ice with the appropriate antisera before the addition of the labeled probe. Anti-ISGF3gamma (sc-496 X) and anti-Stat1alpha /beta (sc-464 X) antibodies were obtained from Santa Cruz Biotechnology, Inc., and the anti-phosphotyrosine 4G10 antibodies were from Euromedex, France.


RESULTS

Identification of a Virus-responsive Domain between the Inducible Element and the TATA Box in the IFN-A4 Promoter

We have shown that the difference in NDV inducibility of two members of the IFN-A gene family, namely IFN-A4 and IFN-A11, is in part due to a A-78 right-arrow G substitution and to the presence of negatively acting sequences located between -244 and -199 in the IFN-A11 gene promoter (42, 50). Transient transfection experiments were therefore carried out to identify the additional regulatory element(s) involved in the differential NDV-induced response conferred by these two promoters.

A comparison of the results obtained with constructions carrying different 5'-deleted fragments of these promoters showed that the region between -198 and -119 did not affect the NDV inducibility of the IFN-A11 promoter, whereas, as described previously by Raj et al. (31), the deletion of the corresponding region exerted a slight positive effect on the virus-induced transcription of IFN-A4 (Fig. 1). However, the virus inducibility of IFN-A4 remained consistently higher than IFN-A11, even when the promoters were devoid of any modulatory region upstream of their inducible element (the Bi or J series of constructions). The creation of a TG-like domain in IFN-A11 by the G-78 right-arrow A mutation increased its inducibility, but not to the expression levels conferred by IFN-A4 (Fig. 2A). The disruption of the TG-like domain in IFN-A4 decreased the induced transcription levels without reducing them to those of IFN-A11. Thus, the G-78 right-arrow A substitution alone is not sufficient to explain the difference between the virus inducibility of these promoters. Transfection of the IFN-A11/A4 hybrid promoters showed that the substitution of the nucleotides (TAC)A11, located from -59 to -57, by (CCG)A4 significantly increased the virus inducibility (pIFH4Bi compared with pIF11Bi), suggesting the presence of an additional virus-responsive sequence located between the inducible element and the TATA box in the IFN-A4 promoter. Alternatively, a negatively acting sequence could be present in the corresponding region of IFN-A11. To investigate these hypotheses, the residues -59 to -57 were deleted in the Bi series of constructs or interchanged between the two promoters extending up to nucleotides -470 to -457. The (CCG)A4 deletion significantly decreased the virus inducibility of IFN-A4, whereas a (TAC)A11 deletion in IFN-A11 did not have any effect (Fig. 2B). This suggests that the CCG deletion disrupts a virus-inducible element in the IFN-A4 promoter. Furthermore, the substitution of (CCG)A4 by (TAC)A11 decreased the virus inducibility of the native IFN-A4 promoter fragment 3-fold, whereas the reciprocal substitution increased the IFN-A11 inducibility more than 6-fold (Fig. 3). These data confirmed the presence of a virus-inducible enhancer in the IFN-A4 promoter. The more pronounced effect of (TAC)A11 conversion in IFN-A11 in the T series compared with the Bi series may be due to the deletion of the negatively acting sequences located between nucleotides -244 and -199 in this promoter because the inducibility of pIF11Bi is already the 2-3-fold higher.


Fig. 1. Comparison of the virus-induced transcriptional activities conferred by the IFN-A4 and IFN-A11 proximal promoters. Constructions containing the -470/-457 (T series), -211/-198 (Bi), or -119 (J) deletions of IFN-A4 and IFN-A11 inserted upstream of the CAT reporter gene are schematically represented in the left with open and shaded boxes corresponding to IFN-A4 and IFN-A11, respectively. The hatched box corresponds to the TATA box. The inducible element located between -109 and -75 in IFN-A4 and the corresponding region of IFN-A11 are boxed, and the -78 A right-arrow G substitution is indicated by a filled circle. L929 cells were transiently transfected as described under "Experimental Procedures" with 2 µg of the indicated reporter plasmids. Cells induced by NDV (solid bars) or mock-induced (open bars) 48 h after transfection were harvested 24 h later to determine the CAT activities. Bar values represent the average of CAT activities expressed in bacterial CAT units. The S.D. values obtained in at least three independent transfection experiments are indicated. Relative transcription is expressed as the percentage of NDV-induced CAT activity obtained with pIF4T, and inducibility is expressed by the induction ratio of the NDV-induced to mock-induced CAT activities.
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Fig. 2. Identification of an additional virus-responsive domain in the IFN-A4 proximal promoter. A, the symbols are as described in Fig. 1. pIF4[V11]Bi differs from pIF4Bi by a A-78 right-arrow G substitution indicated by a filled circle, whereas pIF[11V4]Bi contains the reciprocal G-78 right-arrow A mutation in the IFN-A11 promoter. The hybrid promoter constructs pIFH1Bi, pIFH2Bi, and pIFH3Bi carry, respectively, the -24 to +19, -44 to -24, or -44 to +19 regions of IFN-A4 in the IFN-A11 promoter. The TAC residues (-59 to -57) are substituted by CCG in pIFH4Bi. Putative regulatory elements in the -64 to -55 regions are indicated. Relative transcription is expressed as the percentage of NDV-induced CAT activity obtained with pIF4Bi, and the inducibility is expressed by the induction ratio of the NDV-induced to mock-induced CAT activities. B, deletion of the nucleotide -59 to -57 residues in pIF4Delta Bi and pIF11Delta Bi is represented by a space between the inducible element and the TATA box.
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Fig. 3. Cooperativity between the proximal PRDI-like domain (-57 to -46) and the inducible element IE-A4. The A-78 and -59 to -57 (CCG)A4 nucleotides of IFN-A4 (open boxes) and the G-78 and -59 to -57 (TAC)A11 nucleotides of the IFN-A11 promoter (shaded boxes) are represented by open and filled circles, respectively. Construction of plasmids and their transfection into L929 and HeLa S3 cells are detailed under "Experimental Procedures."
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The Identified Virus-responsive Domain Is a Second PRDI-like Motif Acting in Synergy with IE-A4

Since we were able to show the -78 A/G substitution had an effect on virus inducibility, we measured the cumulative effect of the -78 and -59 to -57 substitutions in both promoters. The substitution of (CCG)A4 by (TAC)A11 together with the A-78 right-arrow G mutation caused a dramatic decrease in the IFN-A4 inducibility, reducing the transcription rate to the level conferred by the IFN-A11 promoter (Fig. 3). The values obtained, compared with those conferred by the IFN-A4 constructs carrying only the A-78 right-arrow G or the (TAC)A11 substitutions, clearly indicated a synergistic effect between the newly identified virus-responsive enhancer and the inducible element IE-A4. Consistently, reciprocal substitutions in the IFN-A11 promoter augment the virus-induced transcription. In this case, the cooperative effect of the enhancer with the inducible element was hindered, probably due again to the 2-3-fold inhibitory effect of the negatively acting sequences located in the -244 to -199] region of the IFN-A11 promoter.

Sequence analysis of the IFN-A4 and IFN-A11 promoters revealed that in the -70 to -40 region, the -59 to -57 substitutions may affect potential binding sites for the CCAAT/enhancer-binding protein beta , NFkappa B, and IRF family of transcription factors. Although CCAAT/enhancer-binding protein beta  has been involved in the regulation of several cytokine genes in response to stimulation by lipopolysaccharide (51, 52), we did not find any evidence concerning its potential role in IFN regulation. NFkappa B, which is involved in the virus-induced expression of the IFN-B gene, does not seem to participate in IFN-A gene regulation (53, 54). We therefore focused our attention on the potential effect of the PRDI-like motif located between nucleotides -57 and -46. in IFN-A4, which is disrupted by the G-57 right-arrow C substitution in IFN-A11.

Comparison of the results presented in Fig. 3 indicates that the negative effect of the G-57 right-arrow C substitution alone on the virus-induced transcription of IFN-A4 was comparable with the inhibition observed with the (TAC)A11 substitutions. The A-78 right-arrow G and G-57 right-arrow C mutations were sufficient to reduce the transcription levels of a highly inducible 500-base pair IFN-A4 promoter to those of a very weakly induced IFN-A11 promoter (pIF4T-1S, pIF4T-3S, and pIF4T-2S). Interestingly, the C-57 right-arrow G substitution alone or in combination with the G-78 right-arrow A substitution (pIF11T-1S and pIF11T-2S) exerted a reverse effect on the IFN-A11 promoter, increasing considerably its virus-induced transcription. Taken together, these results indicate that the proximal PRDI-like site participates in the regulation of the IFN-A4 promoter by cooperating with the inducible element to enhance its virus-induced transcription and that the disruption of both the TG-like and the proximal PRDI-like motifs by the A-78 right-arrow G and G-57 right-arrow C mutations, respectively, contributes to the low level of IFN-A11 gene expression.

A similar series of transfection experiments performed in the human HeLa cells indicated that the -470 to +19 fragment of IFN-A4 was highly inducible after virus infection in comparison with the equivalent IFN-A11 promoter (Fig. 3). Moreover, the single mutation of either the -78 A/G or the -57 G/C and the simultaneous substitution at both positions had the same effects on NDV-induced transcription as those observed in L929 cells. However, no difference could be observed in the values between these mutated constructs and the native IFN-A4 or IFN-A11 promoter fragments due to the constitutive expression levels, which increased concurrently with the virus-induced transcription levels of the constructs. However, the same regulatory elements, i.e. the TG-like domain of IE-A4 and the proximal PRDI-like domain, apparently ensured maximal NDV-induced transcription of the IFN-A4 gene in HeLa cells, suggesting that human cell lines also contain factors able to interact with these domains.

We therefore compared the EMSA patterns obtained with nuclear extracts prepared from NDV-infected or mock-induced L929 and HeLa cells using as DNA probes different oligonucleotides corresponding to dimers of the subdomains of the inducible element IE-A4 that have been shown previously to confer virus inducibility to the HSV-tk promoter in L929 cells (1). The results presented in Fig. 4 indicate that two copies of the PRDI-like and TG-like motifs of the inducible element IE-A4, denoted B2 and C42, respectively, were also virus-responsive in HeLa cells and that the inducibility may be correlated with a binding activity stimulated by the virus infection, which we have denoted VIC. The fact that this complex was the only one detected with the inducible TG-like motif (Fig. 4, lanes 5-8) strongly suggested that VIF, the factor forming the complex VIC, was responsible for the virus inducibility conferred by these motifs in both the HeLa and L929 cell lines. IRF-2 activity was only detected with the PRDI, essentially in mock-induced nuclear extracts, yet with a reduced intensity in HeLa cells (Fig. 4, lanes 1 and 3). An explanation for this decrease could be the presence of lower amounts of this repressive factor in HeLa cells, which may also account for the higher constitutive expression levels observed in the transfection experiments.


Fig. 4. Correlation between the virus inducibility of two copies of the PRDI-like and the TG-like domains of IE-A4 and the detection of VIC in L929 and HeLa S3 cell lines. The PRDI-like (-98 to -87) and the TG-like (-85 to -74) domains of IFN-A4, respectively referred to as B2 (solid boxes) and C42 (open boxes), were inserted in tandem upstream of the HSV-tk promoter in pBLCAT2 (see "Experimental Procedures") or used as probes in EMSA. The NDV inducibility of each construction is indicated at the top. The solid and open bars represent the NDV-induced and mock-induced CAT activities, respectively. The binding activities detected with B2 (lanes 1-4) and C42 (lanes 5-8) were obtained with nuclear extracts prepared from L929 cells (lanes 1, 2, 5, and 6) and HeLa S3 cells (lanes 3, 4, 7, and 8), mock-induced (-) or induced for 4 h with NDV (+).
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The Virus-induced Factor VIF and IRF-2 Recognize the Dimers of the Proximal PRDI-like Domain of the IFN-A4 Promoter

Since the G-57 right-arrow C substitution decreased NDV inducibility of the IFN-A4 promoter by disrupting the proximal PRDI-like domain, we performed EMSA with two copies of the -57 to -46 GAAAGGAGAAAC sequence of the IFN-A4 promoter corresponding to this domain and with its mutated form CAAAGGAGAAAC present in IFN-A11, denoted as D42 and D112, respectively. The patterns obtained with D42 were highly similar to those with B2: three IRF-2-containing complexes (all supershifting with anti-IRF-2 antibodies; data not shown) and a complex that displayed identical mobility and very similar competition patterns to VIC (Fig. 5). IRF-2-containing complexes were easily removed by the PRDI-related oligomers used as competitors but not by the TG-like domain of IFN-A4 (Fig. 5, lanes 8-13), the latter having been shown previously to be recognized with very low affinity by IRF-1 and IRF-2 (1). The complex corresponding to VIC could be competed out by a 50-fold molar excess of B2 or C42 and by a 100-fold molar excess of D42, but not by the D112 (Fig. 5, lanes 2-13). These data indicate that the binding of VIF is affected by the G-57 right-arrow C substitution and suggest that this factor is also involved in the NDV-induced transcription mediated by the D element of IFN-A4.


Fig. 5. Detection of VIC with the dimer of the proximal PRDI-like domain of the IFN-A4 promoter. Nuclear extracts prepared from L929 cells induced for 4 h with NDV were incubated with D42 (lanes 1-13) and D112 probes (lane 14) as described under "Experimental Procedures." Different unlabeled competitors indicated above each lane were added at 50- (lanes 2, 5, 8, and 11), 100- (lanes 3, 6, 9, and 12), and 200-fold molar excess (lanes 4, 7, 10, and 13).
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VIF Binding Activity Correlates with the Virus Inducibility

Of the heterologous HSV-tk constructs, pBC4 (carrying the PRDI-like and TG-like domains, which correspond to the core sequence of the inducible element IE-A4) and pBC11 (containing the -98 to -74 fragment of the IFN-A11 promoter), only pBC4 displayed virus inducibility (Fig. 6A). This difference in response suggests that the A-78 right-arrow G substitution present in the BC11 fragment disrupts the sequence required for the binding of the factor involved in the virus-induced activation of transcription. On the other hand, the similarities between the inducibility ratios of pBC4 and pdC4 suggested that the PRDI-like and the TG-like domains of IFN-A4 should both be recognized with similar affinity by this factor. EMSA performed with the BC4, BC11, and dC4 probes in the presence of virus- or mock-induced L929 nuclear extracts revealed that VIF was detected only when the DNA fragment was virus-inducible, the unresponsive combinations displaying no VIF binding activity (Fig. 6B). These results were confirmed by competition experiments that also showed that VIF binding affinity for BC4 was 4-fold higher than for BC11 (data not shown). Thus, the virus responsiveness of the PRDI-like and TG-like domains of the inducible element IE-A4 is correlated with the binding of VIF to these motifs.

VIF Is Different from ISGF3

Since the B and D motifs behaved also as binding sites for IRF-2, we cannot exclude the possibility that other members of the IRF family, particularly IRF-1 or ISGF3gamma , also recognize these motifs to mediate the virus-induced transcription. In fact, the interaction of ISGF3 with the virus-responsive element of the human IFN-A1 gene promoter via ISGF3gamma was recently reported, suggesting the requirement of this secondarily NDV-induced factor in the amplification of IFN-A gene expression (41). Since we showed that VIF was different from IRF-1 and IRF-2 (1), we wondered whether any relationship existed between VIF and ISGF3. Competition experiments indicated that the ISRE motif of the ISG15 gene promoter (48), a ISGF3 binding site, inhibited the formation of VIC with the same efficiency as PRDI and TG4 dimers (at a 25-fold molar excess for a 60-80% inhibition), whereas a mutated form of ISRE that is not recognized by ISGF3 did not affect the VIF binding, suggesting a similarity in the composition of VIF and ISGF3 (Fig. 7, lanes 8 and 9). We therefore attempted using B2 and C42 as specific competitors to identify VIC among the ISRE complexes detected in NDV-induced nuclear extracts. Strikingly, other than the IRF-2-containing complexes inhibited only by ISRE and B2, no complex was competed out by ISRE, B2, or C42 with the same efficiency (data not shown). Our inability to detect the VIF binding activity with ISRE may be due to the heteromeric structure of this factor, which could have at least one subunit unrecognized by the probe. When used as a competitor, ISRE could then titrate the remaining subunit(s) shared by VIF and ISGF3. The ISRE core sequence contains a binding site for the IRF family members and particularly for ISGF3gamma ; the component titrated by competition could therefore correspond to this subunit. However, antibodies raised against ISGF3gamma did not prevent the formation of VIC (Fig. 7, lane 4), whereas they efficiently inhibited ISGF3 (data not shown), indicating that VIC did not contain ISGF3gamma . Moreover, the unreactivity of VIF with anti-phosphotyrosine antibodies, which recognized the activated ISGF3 in control experiments, suggested also that VIF was different from ISGF3 (Fig. 7, lane 6). The slight inhibition of VIF binding activity observed in the presence of anti-Stat1alpha /beta antibodies (Fig. 7, lane 5) suggested, however, that Stat1alpha or Stat1beta may participate in the formation of VIF. Stat1 has been shown to homodimerize for binding to different IFNgamma activation site (GAS) motifs and to heterodimerize with Stat3 for recognizing various natural palindromic Stat-binding elements of cytokine-responsive genes or with ISGF3gamma and Stat2 for binding to the ISRE sequences (55). The contribution of Stat1 homodimers in the formation of VIF is unlikely, since they do not bind to the ISRE-ISG15 sequence (56), which efficiently competed out VIF, and since the GIRE-Fcgamma R sequence containing the GAS motif, which is recognized by Stat1 homodimers (49, 56), did not inhibit the VIF binding activity (data not shown). These data strongly suggest that VIF is different from ISGF3, although they may share the Stat1 subunit.


Fig. 7. VIF is different from ISGF3. EMSAs were performed using B2 as a probe with nuclear extracts prepared from L929 cells induced 4 h with NDV (lanes 1-9). Extracts were preincubated with polyclonal antibodies (1:10 dilution) raised against the 244-257 peptide of the human IRF-1 protein (lane 2), the 244-258 peptide of the human IRF-2 protein (lane 3), and the 374-393 peptide of the human ISGF3gamma (p48) (lane 4). Monoclonal antibodies raised against the 613-739 peptide of human Stat1alpha /beta (p91/p84) (lane 5) and anti-phosphotyrosine (lane 6) were also used. The controls were performed in the absence (lane 1) or in the presence of preimmunized serum (lane 7). A 200-fold molar excess of the ISRE motif of the ISG15 promoter and a mutant form of ISRE were used as unlabeled competitors (lanes 8 and 9).
[View Larger Version of this Image (51K GIF file)]


DISCUSSION

The results reported in this study, together with previously published data (4, 30), allow insight into the modular architecture of the IFN-A gene promoters. Thus, we suggest that the virus-responsive element of IFN-A4 may be considered to be composed of four enhancer motifs exhibiting different properties: the A motif, represented by the -103 to -93 GTAAAGAAAGT sequence, which is not virus-inducible even in multiple copies and requires a juxtaposition with the B domain to respond to virus induction (34); the B and C motifs, corresponding, respectively, to the -98 to -87 GAAAGTGAAAAG and -85 to -74 GAATTGGAAAGC sequences, which are virus-responsive once multimerized or in combination with each other (see Ref. 1 and this study); and finally the D motif, represented by the -57 to -46 GAAAGGAGAAAC sequence, which is identified in this report and here shown to cooperate with the B and C domains to confer maximal NDV inducibility to the IFN-A4 promoter in L929 cells (Fig. 8).


Fig. 8. Model for differential virus-induced activation of transcription of murine IFN-A genes. At the top, mRNA levels of four murine IFN-A genes detected in L929 cells induced by NDV (solid bars) or noninduced (open bars) are represented relative to the levels of expression of murine IFN-B mRNA (2, 8, 9). IFN-A4, IFN-A2, and IFN-A11 promoter sequences are indicated below. The numbering is relative to the transcription start of the promoter, the TATA box being indicated in each case. Nucleotides distinguishing these sequences are represented by larger characters. The A-78 right-arrow G and the G-57 right-arrow C mutations disrupting, respectively, the C and D motifs are indicated by arrows. Localization of the binding sites for potential transactivators, namely alpha F1 sequence (the A motif), PRDI-like domain (the B and D motifs, represented by PRDI*) and TG-like domain (the C motif, represented by TG*) used throughout and the factors binding to these domains are indicated. The combination of these domains in the promoters of the IFN-A genes defines the virus-responsive elements that are indicated below each sequence.
[View Larger Version of this Image (24K GIF file)]

In the L929 cell line, determination of the relative NDV-induced expression levels between individual murine IFN-A mRNAs has revealed that IFN-A4 is the predominant species over IFN-A2, while IFN-A6 and IFN-A11 are very weakly expressed (8, 9, 54). The difference in expression between the IFN-A4 and IFN-A6 genes has been attributed to substitutions disrupting the A and B motifs in the IFN-A6 promoter (31). However, this finding alone is not sufficient to explain the lower expression levels of IFN-A2 and IFN-A11, since they show perfect homology with IFN-A4 with respect to these motifs in their promoters. In fact, in the -109 to -75 region, which corresponds to the previously delimited inducible element IE-A4, the IFN-A2 and IFN-A11 promoters are distinguishable from IFN-A4 by the G-80 right-arrow A and A-78 right-arrow G substitutions in IFN-A2, and only by the A-78 right-arrow G substitution in IFN-A11. By transient transfection experiments performed with the IFN-A4 and IFN-A11 promoters extending up to -470 nucleotides from the transcription initiation site, as well as with hybrid IFN-A4/A11 promoter constructs, we showed that the difference between the virus responsiveness of the IFN-A4 and the IFN-A11 promoters was due essentially to the A-78 right-arrow G and G-57 right-arrow C substitutions that disrupt, respectively, the C and D motifs in IFN-A11. In fact, abrogation of either the C or D domain affected only partially the virus-induced transcription of IFN-A4. Interestingly, a similar organization exists naturally in IFN-A2 where the C motif is disrupted by the A-78 right-arrow G substitution, but the D motif is intact. In NDV-induced L929 cells, the lower levels of IFN-A2 expression, in comparison with IFN-A4, are thus most likely due to attenuated cooperation among the A, B, and D domains instead of maximal synergy levels conferred by the four motifs. Differential virus-induced expression of IFN-A genes seems then to be directly dependent on the number of cooperating enhancer motifs within their promoter.

The similarity of the results obtained in transfection experiments in HeLa cells with different IFN-A4 and IFN-A11 constructs and the detection of VIF binding activity in the nuclear extracts obtained from the NDV-induced HeLa S3 cells suggest that the model proposed in this study may also be valid for human cell lines. The results obtained here with the transiently expressed IFN-A4 or IFN-A11 promoter constructs correlate well with the endogenous expression patterns of the corresponding IFN-A genes in L929 cells. In contrast, the expression of endogenous human IFN-A genes, as well as the CAT activity of the SV-CAT reporter constructs containing the human IFN-A1 promoter, were undetectable upon NDV or Sendai virus induction of HeLa cells (7, 57, 58). This discrepancy may be due to the presence of a negative regulatory sequence located in the human IFN-A1 promoter between the two highly conserved motifs equivalent to C and D domains of murine IFN-A4. This negatively acting region has been suggested to be the binding site for a repressor that affects human IFN-A gene expression, especially in restrictive cell types such as HeLa cells (58). Within their proximal -120 to +19 promoter fragment, the murine and human IFN-A genes exhibit sequence divergence; the putative repressor may therefore not affect the inducibility of murine IFN-A promoters in these cell lines.

The minimal virus-inducible combination, BC4, is the binding site for the VIF that was previously detected with two copies of the B and C4 modules (1). In contrast, VIF is not detected with the BC11 combination, which carries the A-78 right-arrow G substitution in the C domain and fails to respond to virus induction. VIF also recognizes the dimerized D motif of IFN-A4, a motif that is shown to participate in virus-induced transcription. Conversely, VIF does not recognize the equivalent region of IFN-A11 disrupted by the G-57 right-arrow C substitution. The D motif also recognized by IRF-2 may be involved, as is the B motif, in IRF-2-mediated repression of transcription. However, the B and D motifs described as PRDI-like domains (or IRF elements) and the C motif denoted as the TG-like domain should be considered as VIF-binding sites. Thus, we suggest that VIF is involved in virus-induced activation of IFN-A gene transcription by recognizing the B, C, and D motifs in the IFN-A4 promoter, whereas it binds only to the B and D motifs of IFN-A2 and the B motif of IFN-A11 (Fig. 8).

The fact that VIF recognizes the IRF elements and is competed out by the ISRE motif, which contains an IRF-binding site in its inner core sequence, suggests that this factor may contain a DNA-binding subunit related to the IRF family. However, EMSA performed in the presence of antibodies raised against different members of the IRF family indicated that VIF did not correspond to or contain IRF-1 and IRF-2 and that ISGF3gamma is not involved in VIF formation. Nevertheless, the possibility still cannot be excluded that VIF contains a DNA binding subunit related to other recently cloned members of the IRF family (59-63), although to date there is no evidence to suggest their involvement in virus-induced type I IFN gene expression. These factors were shown, in fact, to recognize the ISRE motif and to exert repressive effects on IFN-stimulated gene transcription. Recently, binding activities stimulated by double-stranded RNA (DRAFs) characterized in HeLa cells treated with poly(rI·rC) or infected with a mutant of adenovirus lacking the e1a oncogene were suggested to participate directly in the virus-activated transcription of some IFN-stimulated genes (64, 65). A relationship between VIF and ISRE-binding DRAF activities seems unlikely, since VIF is not detected by the ISRE probe and the DRAFs were shown not to recognize the IRF elements. In addition, the absence of reactivity between VIF and anti-phosphotyrosine antibodies indicated, as did anti-ISGF3gamma antibodies, that VIF was different from ISGF3. Furthermore, we have shown that in NDV-induced L929 cells, VIF, unlike ISGF3, appears in the nuclei within 1 h of contact between the cells and the virions, without requiring de novo protein synthesis, and that VIF activation precedes the secondary stimulation of ISGF3 (1, 40, 66). We therefore suggest that VIF, which we have shown to be different from IRF-1 and ISGF3, corresponds to the primary transcription factor directly activated by NDV and mediates initiation of the IFN-A gene transcription. The role of IRF-1 and ISGF3 may be to maintain transcription initially activated by VIF via interaction with the B and D domains.


FOOTNOTES

*   This work was supported by the Center National de la Recherche Scientifique, the Université René Descartes Paris V, and grants from the Association pour la Recherche sur le Cancer (Contrat 1042) and the Ligue Nationale Française contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 33-1-42-86-22-84; Fax: 33-1-42-60-55-37; E-mail: civas{at}citi2.fr.
1   The abbreviations used are: IFN, interferon; PRD, positive regulatory domain; PRDI-PRDIV, positive regulatory domains I-IV, respectively; VRE, virus-responsive element; IRF, interferon regulatory factor; EMSA, electrophoretic mobility shift assay; ISG, IFN-stimulated gene; ISGF, ISG factor; NDV, Newcastle disease virus; VIF, virus-induced factor; IE, inducible element; CAT, chloramphenicol acetyltransferase; HSV-tk, herpes simplex virus thymidine kinase; GAS, IFNgamma activation site; DRAF, double-stranded RNA-activated factor; ISRE, interferon-stimulated response element.

ACKNOWLEDGEMENTS

We especially thank Moshe Yaniv for critical reading of the manuscript. We are also indebted to Nobumasa Watanabe and Tadatsugu Taniguchi for providing the mouse anti-IRF-1 and anti-IRF-2 antisera, John Hiscott for the human anti-IRF-1 and anti-IRF-2 antibodies, and Keiko Ozato for the human ISGF3gamma antibodies.


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