Type I interferon gene expression: Differential expression of IFN-A genes induced by viruses and double-stranded RNA

Biochimie (1998) 80, 673-687 © Socidt6 fran~:aise de biochimie et bMogie moldculaire / Elsevier, Paris

Type I interferon gene expression: Differential expression of IFN-A genes induced by viruses and douMe-stranded RNA Jos6 B r a g a n q a , A h m e t C i v a s * UPR 37-CNRS, Laboratoire de R~gulation de l'Expression des Gbnes Eucar3'otes, UFR Biomddicale des Saints-Pbres, Universitd Paris V, 45, rue des Saints-Pbres, 75270 Paris cedex 06, France

(Received 29 April 1998; accepted 9 June 1998) A b s t r a c t - The family of interferon regulatory transcription factors (IRF) participates in the virus-induced and dsRNA-stimulated transcriptional regulation of either type I 1FN genes or a definite set of genes which can also be activated by IFN. In this review, we place emphasis on the role of IRF-3 that associates with the coactivators CBP and/or p300, together or not with IRF-7. These complexes bind to the PRDI, PRDl-like domains or to a number of ISRE sequences located in the promoter of these virus-inducible genes. We also discuss the involvement of the IRF-3-related complexes in the differential regulation of IFN-A genes. © Socirt6 franqaise de biochimie et biologie moirculaire / Elsevier, Paris IFN-A genes I IFN-B I |RF family I transcription factors I gene regulation

Viral infection of eukaryotic cells causes early and transient expression of various cytokine and chemokine genes that constitutes a crucial step in the stimulation of cellular defense mechanisms. In this stimulation process, transcriptional activation of interferon (IFN) genes and the simultaneous or subsequent activation of other genes stimulated by virus and/or by IFN lead to the synthesis of a specific set of proteins that mainly participate in the cell growth inhibition and ill the establishment of an antiviral state in different cell types. These cellular functions are controlled by signal transduction pathways mediating the activation of the [FN system including type I IFN (IFNct subtypes and IFNI'5) secreted by virus-infected cells, and type II IFN (the immune IFNy) secreted mainly by Th-! lymphocytes and natural killer cells [ 11. The IFN system also includes the proteins encoded by IFN-stimulated genes (ISG), activated by type I and/or type II IFN [2-91. Little is known about the virus-activated signal transduction pathways required for the transcription of target genes. The early steps of virus entry in cells, such as the binding of virions on their cellular receptors and membrane fusion events are poorly understood and considered to have only a limited effect on the virus-induced signal transduction and activation of transcription, even if envelope glycoproteins involved in membrane fusion are also shown to induce IFN [10, 11]. In opposition, the next step of infection consisting on the release of the viral genome, nucleoproteins and envelope glycoproteins in the cyto-

plasm of infected cells is considered to be more important. For instance, the double-stranded RNA (dsRNA) contained in the viral genome or often produced during virus replication is suggested to be the main inducer signal of IFN and ISG gene transcription I12, 13]. If transduction pathways triggered by different viruses remain to be elucidated, transcriptional activation mechanisms of target genes, particularly those concerning the IFN-B gene, are better defined. Thus, virus induction of the IFN-B gene promoter was shown to be mediated by several IRF (interferon regulatory factors) family members binding to the DNA motifs called positive regulatory domains PRDi and PRDIII, and by NF-KB and ATF-2/c-Jun complexes interacting with the PRDI! and PRDIV domains, respectively [14]. Together, these regulatory domains constitute the virus responsive element of IFN-B (VRE-B) which shares, within the PRDIII-PRDI region, sequence homology with the VRE elements of different IFN-A gone promoters and the IFN-stimulated response elements (ISRE) of type: I IFN-stimulated genes. Recent data indicate that virus infection or treatment of cells with dsRNA not only induces type I IFN, but also directly activates the transcription of some ISGs [[ 5-19], suggesting that there exist similar pathways between these transactivation mechanisms. Furthermore, the ISGF3 complex, initially described as the primary transcriptional factor involved in ISRE-mediated activation of ISGs, is also shown to be secondarily induced by viruses and to be involved in the amplification of type I IFN gene expression [20, 21 !.

* Correspondence and reprints

In this review, we will attempt to summarize recent progress in the field of virus-stimulated or dsRNA-

1. Introduction

674 induced activation of either type I IFN gene and ISG transcription and we will discuss the roles attributed to different IRF family members on the regulation of IFN-A genes.

2. The role of positive regulatory domains (PRDs) in virus.induced transcription of IFN.B gene promoter The DNA sequences required for virus induction of IFN-B gene promoter have been localized to the first 120 bp immediately upstream of the initiation site of transcription. This region acting as a virus-inducible transcriptional enhancer (also referred to as VRE-B) contains four positive regulatory domains involved in the activation of transcription, initiated without requiring newly synthesized proteins. These domains also participate in the post-inductional repression mediated by the inducible repressors synthesized during virus infection (for review see [14, 22, 231). The PRDI and the PRDIII domains, formed by the juxtaposition of hexameric GAAANN motifs and presenting a high degree of sequence similarity are considered as binding sites for at least some members of the IRF family of transcription factors (figure i). None of these domains in itself is sufficient for virus induction, whereas, in multiple copies each confers virus inducibility to heteroiogous promoters. The PRDII domain corresponding to the core sequence GGGAAATTCC, binding site for the NF-nB/Rel family of transcription factors, also plays a critical role in the virus-induced transcription of IFN-B gene promoter. The PRDIV domain, shown to be required for maximal virus induction in various cell lines (human HeLa or murine L929 cells), contains the ATGTAAAT motif presenting similarity with the consensus binding sites for the ATF/CREB family of transcription factors. As in the case of PRD! and PRDIII, only multimers of PRDII or PRDIV can exhibit the properties of a virus-responsive enhancer: Besides their virusresponsiveness, multiple copies of PRDI or PRDIII also confer IFN type I and II inducibility, whereas multimers of PRDII are inducible by LPS, PMA or TNFa and those of PRDIV are cAMP-responsive. The native IFN-B promoter containing a combination of these domains, each in a single copy, does not respond to any of these external stimuli, but is highly inducible by virus or by dsRNA, The specificity and the potency of virus inducibility were suggested to be due to a combination of cooperative interactions between PRD-binding transcription factors and between the resulting multicomponent complex and the basal transcription apparatus 1141. Evidence that transcriptional activation requires the assembly of a higher order transcription enhancer complex (enhanceosome) was brought out by the studies of Thanos and Maniatis 1241. They have shown that this complex consists of at least three distinct transcription factors IRF-I, NF-~:B, ATF2/c-Jun and the architectural protein HMGI(Y) and

Bragan~:a and Civas that both the in vitro assembly and in vivo transcriptional activity of the enhanceosome require a precise helical relationship between individual PRD binding sites. The importance of the positioning of the HMGI(Y) binding to four A-T rich sequences (two in PRDIV, one in PRDII and the last one between PRDII and the TATA box) and the relevance of PRD binding sites on the DNA helix were demonstrated by the analysis of helical phasing mutations in the IFN-B promoter [25]. These experiments indicated that the high level of synergistic activation exerted by all of the PRD-binding factors decreases significantly when a half-helical turn of 6 bp is introduced between PRDI and PRDII, whereas insertion of full-helical turn (10 pb), which reestablishes the relative positions of PRD binding sites on the face of the DNA helix, almost fully restores the activation of the IFN-B promoter. By circular permutation and phasing analysis, the authors have shown that the PRDII and PRDIV sites have distinct intrinsic DNA curvatures which are partially counteracted by HMGI(Y), leading to the recruitment of PRD-binding transcription factors by protein-protein interactions [261. Proteininduced bending orients the bZIP domain of ATF2/c-Jun bound to PRDtV toward the Rel homology domain of NF-~cB bound to PRD II. The DNA curvature is reversed by these interactions and this effect is further enhanced by HMGI(Y), revealing a critical and complex role for HMGI(Y) in the assembly and function of the enhanceosome. According to the authors, these d~!a suggest that the DNA bending at PRDII and PRDIV m"~ht be required to bring NF-~:B and ATF2/c-Jun into contact with the PRDIII-PRDI binding factors necessary for virus induction. Recently, it has been shown that the cAMP-responsive element-binding protein (CBP), a transcriptional coactivatot" which potentiates the activity o1" several groups of transcription factors127-321, including STATla and STAT2 (signal transducers and activators of transcription), is also involved in the viral activation of IFN-B ex~:~ression [331. Actually, cotransfection of a CBP expression vector along with the IFN-B promoter in either mock- or Sendai virus-induced COS cells caused a 3- to 4-fold increase in the virus-induced levels, suggesting that the transcriptional synergy observed in the context of the enhanceosome requires recruitment of the CBP/p300 coactivator to the enhanceosome 1331. Consistently, transfection of a CBP derivative containing only the enhanceosome-interacting domain of CBP decreased the levels of virus induction (four-fold), indicating that this fragment of CBP competes with the endogenous CBP protein for recruitment into the enhanceosome. In addition, the expression of EYA, an inhibitor of CBP, virtually abolished virus induction which was restored by coexpression of CBP. These results suggest that CBP recruitment is necessary but not sufficient for synergistic activation of transcription by the enhanceosome. The transcriptional synergism requires a specific region in the p65 subunit of

Differential expression of [FN-A genes

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NF-~zB, located next to the activation domain of p65 and interacting with the activation domains of p65, IRF- I and ATF2/c-Jun [33 I. Therefore, the activation domain of p65 is suggested to direct distinct functions in the context of enhanceosome: that is, a direct involvement in the process of transcriptional activation by contacting the components of the basal apparatus such as TFIID/A/B and an indirect involvement by recruiting CBP via the synergism dornain. Furthermore, enhanceosomes bearing the activation domain of p65 placed on IRF-I or the activation domain of IRF-I placed in p65 are weaker activators of transcription when compared to the wild type enhanceosome, either with or without overexpressed CBP. The authors propose that in the context of the enhanceosome, transactivators bound to the enhancer are assembled in a functional unit that juxtaposes their activation domains to form a novel activating surface that interacts optimally with CBP and that the activation domains of the activators are not fully interchangeable. Deletion, substitution, or rearrangement of any one of the activation domains of these factors in the context of the enhanceosome was shown to decrease both recruitment of CBP and transcriptional synergy.

3. The importance of PRDI-like domains in the regulation of different IFN-A gene promoters The PRDl-like domains, also referred to as IRFelements, are also present in the virus responsive element (VRE-A) of IFN-A gene promoters suggesting that IRF family of transcription factors also plays a role in the transcriptional regulation of these genes. IFN-A genes are represented by a multigenic family of intronless genes clustered in a 400 kb region containing also the IFN-B gene on the human chromosome 9 and on chromosome 4 in the mouse [34, 35]. They have been shown to oe coordinately induced in virus-infected human and mouse cells and to exhibit differences in the expression of their individual mRNAs. The differential expression is shown to be dependent on the transcriptional activity of the corresponding IFN-A gene promoter in a particular cell type. Thus. human IFN-A1, IFN-A2 and IFN-A4 genes are highly expressed in Sendai virusinfected PBMC and lymphoblastoid Namalwa cells. whereas the expression of IFN-A5, IFN-A7, IFN-A8 and IFN-AI4 mRNA levels are 5- to 20-fold lower in the same

676 ceils[361. Similarly, in NDV-induced L929 cells, the murine IFN-A4 expression is predominant in comparison to IFN-A2, IFN-A5 and IFN-A6, whereas IFN-A1 and IFN-AII are very weakly expressed 137-391. However, the VRE of each IFN-A promoter which would account for the level of its virus-induced expression and the factors specifically involved in this regulation are less well defined. The virus responsive element of human IFN-AI gene promoter (VRE-Ai) and the murine IFN-A4 inducible element (IE-A4) gene promoter have also been shown to contain a PRDl-like site, thus involving IRF-I in the induced expression of the IFN-A genes, since IRF-i was shown to be PRDl-specific [40-421. A comparative study of the murine IFN-A4 and IFN-A6 gene promoters displaying differential virus-inducibility led to the detection by EMSA of a binding activity called AFI/B. It was proposed that AFl/B-forming proteins cooperate with IRF-I in the virus-induced transcription of IFN-A genes 143-451. Another factor suggested to be specifically involved in IFN-A gene regulation is the TG-protein which binds to the hexameric GAAATG repeats shown to confer virus-inducibility by a IRF- 1-independent pathway. The so-called 'TG-sequence' is adjacent to the PRDl-like motif in the human VRE-AI (figure 1) and is conserved in most human and murine IFN-A gene promoters [42, 461. However, the AFl-binding proteins and the TG-protein have not yet been characterized. As already mentioned, in L929 cells, NDV infection induces transcription of high levels of IFN-A4 mRNAs, whereas in the same conditions the ,r~urine IFN-AI 1 gene is very weakly expressed, despite the striking homology existing between both promoter regions. Actually, the region of tile IFN-A I I gene promoter corresponding to the IE-A4 element, located between the nucleotides (-109 to =75), differs only by a A/G substitution at position -78, and the low levels of IFN-Ail expression after NDV induction is in part due to this substitution, which disrupts a virus-responsive enhancer module, also referred to as the TG-like domain 1471 (figure 2). Further comparative analysis of IFN-A4 and IFN-AI 1 or IFN-A4/All hybrid promoters in transient transfection experiments led to the characterization of another positive regulatory domain located between the IE-A4 and the TATA box within the IFN-A4 gene promoter 1481, This virus-responsive enhancer domain which turned out to be another PRDl-like domain is disrupted by the -57G---)C substitution in the IFN-AII promoter (figure2), thus affecting its virus induced expression. This proximal PRDl-like site participates in the regulation of the IFN-A4 promoter by cooperating with the inducible element to enhance its virus-induced transcription and disruption of both the TG-like and the proximal PRDl-like motifs by the -78A--,G and - 5 7 G ~ C mutations respectively, largely contributes to explain the low level of IFN-All gene expression. These results, together with data provided by

Braganqa and Civas other groups (140--441 and references cited therein) gave an insight into the modular organization of the IFN-A gene promoters. Thus, the virus-responsive element of IFN-A4 is shown to be composed of lbur modules (A to D) (figure 2) exhibiting different enhancing properties: the AFI domain represented by the (-103 to -93) GTAAAGAAAGT sequence, which is not virus-inducible even in multiple copies and requires a juxtaposition with the PRDl-like domain to respond to virus induction [44]; the PRDI-like and the TG-like domains, 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; and finally the proximal PRDl-like domain represented by the (-57 to --46) GAAAGGAGAAAC sequence which cooperates with the other domains to confer maximal NDV-inducibility to the IFN-A4 promoter in L929 cells. In this cell line, the differential expression of IFN-A4 and IFN-A6 genes was attributed to substitutions disrupting essentially the (A) and (B) modules in the IFN-A6 promoter[41]. This finding is not sufficient to explain the lower expression levels of IFN-A2 and IFN-AII which show perfect homology with IFN-A4 within these motifs in their promoter. The difference between the virusresponsiveness of IFN-A4 and IFN-AI1 promoters was essentially due to the disruption of the (C) ~md (D) modules in IFN-AI1. Mutational analyses indicated that abrogation of either the (C) or (D) domain alone affected only partially the virus-induced transcription of IFN-A4. Interestingly, in the IFN-A2 promoter the (C) module is disrupted, but the (D) module is intact, therefore the lower levels of IFN-A2 expression ill comparison to IFN-A4 are most likely due to attenuated cooperation between the (A), (El) and (D) instead of maximal synergy levels conferred by four modules. Thus the dil'ferenti:d virus-induced expression o1' IFN-A genes seems then rather to be directly dependent on the number of cooperating enhancer motifs within their promoter. Electrophoretic mobility shil't assys (EMSA) carried out with the virus responsive elements of the IFN-A4 and IFN-AII gene promoters have allowed the identification of a virus-induced binding activity containing a factor which recognizes specifically either the (B) module shared by both virus responsive elements or the (C) and (D) modules of IFN-A4. In contrast, this virus-induced factor, VIF, is not detected with the virus-unresponsive (C) and (D) modules of IFN-A! 1 promoter carrying the -78A---)G and the -57G---)C substitution, respectively 147, 481. The fact that VIF recognizes the IRF-elements and is competed out by the ISRE motif, which contains a IRF-binding site in its inner core sequence, suggests that VIF 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 does not correspond to IRF-I, IRF-2 or

Differential expression of IFN-A genes

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ISGF3, and that ISGF3y is not involved in VIF formation. Nevertheless, the authors have not excluded that VIF may contain a DNA-binding subunit related to other recently cloned members of the IRF family [21, 49-55]. In NDVinduced L929 cells, VIE unlike ISGF3, appears in nuclei within 1 h of contact between cells and virions, without requiring de novo protein synthesis and its activation preceded the secondary stimulation of ISGF3 [20, 47, 56]. Experiments on time course analysis of induction have

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also suggested that VIF preexists in the cytoplasm and is translocated into the nucleus upon viral induction. On the other hand, laser cross-linking experiments performed on the VIF complex suggested an heteromeric structure for this factor. The stimulation of the binding activity of this factor was then suggested to require the assembly of different oligomerization partners preexisting in latent forms in the cytoplasm before induction. Taken together, these results have indicated that VIF participates in the

678 virus-induced activation of IFN-A gene transcription and can correspond to the primary transcription factor directly activated by NDV. 4. Effect of IRF-I and I ~ - 2 on virus-induced and IFN-induced gene transcription The PRDI and PRDIII domains of the IFN-B promoter, the PRDl-like sequences found in IFN-A gene promoters and the ISRE elements found in most IFN-stimulated gene promoters contain several copies of AANNGA motif or the permutated GAAANN form of this motif [57]. Multimerization of certain types of these hexanucleotides generates sequences that can be distinguished by their virusor IFN-inducibility when inserted upstream of a minimal promoter [421. Among the members of the growing IRF family of transcription factors which recognize the VRE or the ISRE elements containing different subsets of the GAAANN motif, IRF-1 was the first one to be described as an activator involved in the transcriptional activation of either type I IFN gene or type I IFN-stimulated gene promoters (see for review [581). Screening of a ~,gtll library of cDNAs prepared from mouse L929 cell-derived mRNAs with a DNA probe containing GAAAGT repeats led to the isolation of IRFI 1591. The search for cDNA clones that cross-hybridized with the IRF! eDNA in a similarly prepared library permitted the cloning of IRF2 1601. These factors were shown to manifest very similar or identical binding properties for the recognition sequences consisting of the motif G(A)AAASYGAAASY (S: G/C, Y: C/T) 161 I. However, unlike IRFI, IRF2 does not stimulate transcription and manifests repressive at.tivity on the repeated GAAAGT sequence that functions either as a virus-inducible enhancer element or as a silencing element to juxtaposed viral enhancers in induced and uninduced cells, respectively. Overexpression of IRF-1 in COS cells has been shown to increase endogenous type I IFN gene expression in the absence ot" viral induction, the detected antiviral activity (160 to 320 U/5 × 10 6 cells), being attributed to IFNa, 15 and to, NDV induction of COS cells did not lead to lFNct production, but caused a very high level (5000 U/5 x 10 6 cells) of IFNf5 I621. The difference observed between the IFN-B expression levels has been explained by the relieved control mechanisms or by delivered additional signals, following viral infection, The events causing the induction of IFN-A genes by IRF-1 overexpression, but not by NDV infection, have not been understood. Harada et al. reported that transfection of IRF-1 eDNA in undifferentiated PI9 embryonal carcinoma (EC) cells results mainly in the activation of endogenous IFN-A genes, IFN-B being induced at a very low level 1631. Following differentiation, induction of IFN-B gene has been shown to be more evident. The authors suggested that in undif-

Braganqa and Civas ferentiated cells, many if not all of the IFN-A gene loci may be more accessible to IRF-I action than IFN-B gene locus. The IFN induction was efficiently blocked by the coexpression of IRF-2 cDNA and the IRF-2 effect predominated after differentiation in P I9 cells, as it did in L929 cells. These results suggested a model based on the alternation of interaction between IRF-I and IRF-2 with the target genes that regulates the IFN system depending on the nature of the stimulus. According to the model, in undifferentiated cells where IRF-I and IRF-2 are not expressed, ectopic expression of IRF-I can lead to an efficient induction due to the absence of IRF-2. In differentiated cells (differentiated EC cells, L929 cells, etc.), IRF-2 is bound to the IFN gene promoters, and the low levels of IRF-I induced upon cytokine treatment (IFN, TNF or IL-1) cannot efficiently compete with IRF-2. In contrast, when cells are induced by a virus, the IRF- I level is increased (first signal), and it undergoes modifications that generate a form of IRF-! with higher DNA binding affinity (second signal), leading to the activation of the IFN system. Yet, the precise mechanism of this event, particularly the nature of the modified IRF-I form is not established. Subsequent cessation of inducing signals results in a reversal of IFN regulatory process through IRF-2 binding. The critical role attributed to IRF-1 in the transcriptional activation of the IFN-B gene and in the IFN-stimulated transcription of some ISGs has also been supported by the obtention of significantly higher IFN-B, 2'-5' oligoadenylate synthetase (2'-5' OAS) or class I HLA mRNA and protein levels in NDV- or poly(1).poly(C)induced stable transfectants of human skin fibroblasts GM-637 constitutively expressing IRF-I, than in control cells 1641. Overexpression of IRF-! led also to induction of the murine IFN-A4 and IFN-A6 promoters but only under conditions of transient and not of permanent transIbrmation 143 I. Other analyses have shown that IRF-I gene transcription, protein levels and DNA-binding activity were increased 3- to 5-fold by IFNct and 10- to 20-fold by IFNy, yet without transcription of the chromosomal IFN-B gene [651. In these experiments carried out in HeLa cells, in contrast to the clear correlation between the presence of ISGF3 and high transcription rates of ISGs, the presence of IRF- i did not correlate with transcription of either ISGs or IFN-B. IRF-I was then suggested to be part of a regulatory network activated by IFN treatment, as a modulator of transcription of genes whose primary activation depends on other factors. Alternatively, IRF-1 could be the primary activator of genes that are secondary participants in the cellular response to IFN [651. Furthermore, determination of uninduced or viral-induced expression of IFN-A and IFN-B transcripts in both murine embryonic stem (ES) cells and in the ES cells in which both IRF-I alleles were disrupted, showed that virusinduced levels of IFN-A mRNA were similar to the levels found in wild-type and IRF-I °~° cells and there was little

Differential expression of IFN-A genes induction on IFN-B mRNA on either cell type [66]. In 8-day differentiated cells the levels of virus-induced IFN-B mRNA, but not of IFN-A mRNA, were about 10-fold higher than in undifferentiated cells and only slightly higher in wild-type than in IRF-1 °m cells. These data revealed that, although IRF-1 at high levels may elicit or augment induction of IFN genes unaer certain circumstances, it is not essential for IFN gene induction by virus. Lack of IRF-I had no effect on IFN-induced expression levels of the ISGs tested, however the authors noticed there was little or no constitutive expression of 2'-5' OAS in IRF- 1o/o ES, in contrast to wild-type cells, These results suggest the existence of two different mechanisms for induction of IFN-B gene promoter, since IFN-B transcripts can be induced in the absence of IRF-I, but they also can clearly be induced by overexpression of IRF-I, at least in certain cell lines.

5. Existence of IRF-I independent pathways in virus induced transcription Utilizing mice engineered to lack IRF-I or IRF-2 expression through homologous recombination in ES cells, Matsuyama et aa. have examined the role of IRFs in type I IFN induction in the presence of two inducers, poly(I).poly(C) and NDV [67]. They observed that the induction of type 1 IFN genes was markedly reduced in IRF-I deficient cells treated by poly(1).poly(C). In contrast, the level of induction remained essentially the same as that of wild-type upon NDV infection. In IRF-2deficient cells, peak induction of type I IFN mRNA was consistently enhanced in NDV-infected hornozygous mutant embryonic fibroblasts (EFs) and macrophages. In contrast, no difference was observed between IRF-2 wild-type and mutant EFs with induction by poly(1). poly(C), implying that the induction of type ! IFN with poly(l).poly(C) is not affected by the presence of the negative regulator IRF-2. Similar experiments carried on mice homozygous for disrupted IRF- ! genes showed that the levels of type I IFN activity in serum, and of IFN-A or IFN-B mRNAs in the various organs tested after induction with virus or poly(1). poly(C) are similar in IRF-1 °/° and in wild-type mice 11681. This study has also confirmed that IRF-! knock-out mouse Efs responded normally to NDV induction in opposition to a diminished expression of IFN-A and IFN-B mRNA following poly(l).poly(C) treatment. The pretreatment of cells with type I IFN proteins before induction increased type I IFN gene expression either in normal or mutant EFs, bringing them to about the same level. It has been reported that in some cell lines, the inducibility of the IFN-B gene by both poly(l).poly(C) and virus is dependent on priming by IFN 169, 701. The enhancement of transcription by priming with IFN requires protein synthesis since it is abolished by cycloheximide and exerts its

679 effects at the transcription level [711. This effect may be related to the activation of ISGF3 or to the increased concentrations of" its components, such as ISGF3y or STAT! (see the section on the autocrine amplification mediated by ISGF3). These results, taken together, clearly demonstrated the existence of IRF-l-dependent and IRF-l-independent pathways for type I IFN induction. Thus, the signal for type I IFN induction by poly(1).poly(C) is suggested to be primarily mediated by IRF-1, while additional signal cascades were suspected to be involved in NDV infection. The existence of an alternative pathway is also supported by the observation that type I IFN induction by poly(1). poly(C) in IRF-l-deficient cells is restored by priming with IFN~ [68]. The authors assumed that, since IFN~ alone could not induce type l IFN, the factors induced by IFN~ need to be modified through a signal elicited by the virus or poly(I).poly(C). They indicated that this pathway seems not to operate in some cell lines, such as GM-637, in which inhibition of IRF-1 expression correlates with inhibition of type I IFN expression. Reis et al. proposed that some factor X which is constitutively present at effective levels in organs of wild-type and IRF-1 °/° mice, as well as in wild-type EFs but not in IRF-i °m EFs, is essential for the induction of both IFN-A and IFN-B genes by poly(1).poly(C). The maintenance of effective levels of factor X in the EFs is dependent on IRF-!, but this factor can be induced by IFN in an IRF-I independent process in these cells. Reis et al. suggested also that induction by NDV utilizes, at least in part, a different pathway (designated Y), because induction of IFN genes by virus is not impailvd in IRF-I °m EFs [68]. They claim that it cannot be argued that full inducibility by NDV in IRF-i °m EFs is due to "self-priming' by amounts of IFN synthesized early after infection, since cycloheximide super-induces IFN type ! mRNA levels at the same extent in NDV-infccted wild-type and IRF-1 °/° cells. It should bc mentioned that the mechanism of superinduction is not entirely clear il 31. However; the knock-out studies invalidated the critical role attributed to IRF-! in the induction of type 1 IFN. As regard the expression of the ISGs, IRF-1 has been shown to be also dispensable for induction by type I IFN of at least some of the ISGs, whereas IFNy-induced expression of the inducible fon~n of the NO synthase (iNOS) is severely impaired in macrophages derived from IRF-1 °/° mice. These data suggested that IRF-1 might be necessary for the activation of some ISGs by type II IFN: this observation has been further confirmed 172-741.

6. The IFN-stimulated response elements (ISREs) contain PRDI-like domains IFN signal transduction pathways leading to the transcriptional activation of ISGs are better defined than virus-signaling pathways resulting in type I IFN gent

680 regulation [751. Schematically, either IFNa subtypes or IFNI3 bind to the same cell surface receptor and activate the protein tyrosine kinases Jakl and Tyk2, which phosphorylate STATIa/[~ and STAT2. These factors translocate into the nucleus and associate with ISGF3y to form the ISGF3 complex which interacts with the interferonstimulated response element (ISRE) of ISG promoters activated by type I IFN. The IFNy signaling is mediated by a different JakSTAT pathway. In this case, binding of type II IFN to its specific cell surface receptor activates Jakl and Jak2. This leads to fort tion of STATI homodimers (gamma activated factor, ~,F) which are capable of binding directly on the I F N - : nma activated site (GAS) of the ISG promoters. S'I~fl homodimers displaying GAS-binding affinity are also formed upon type I IFN stimulation (alpha activated factor, AAF), which explains, at least in part, the transcriptional activation of some ISGs by both types of IFN. This overlapping expression is suggested to be also modulated by the IFNy-stimulated formation td STATI homodimers which interact with ISGF3y and thus bind to ISRE motifs. The difference between the gene expression mediated by ISGF3 and by the complex formed by STATI homodimers and ISGF3? resides in the higher affinity of ISGF3 for the ISRE sequences and the capability of ISGF3 to form at low concentrations of STATI and ISGF3y 1761. Another difference comes from the phosphorylation kinetics of STATI depending on the cell type and the inducer, Thus, STATI phosphorylation is often much slower and more prolonged in response to IFNy than in response to IFNa It is suggested that the complex tbrmed by STAT! homodimers and ISGF3y is more likely to play an important role in prolonged transcriptional responses to IFNy, instead o1' all essential role when STATI is transiently phosphorylated. ISGF3 binds ISRE with much greater affinity than does the ISGF3y protein alone. However, ISGF3y was shown to bind the 9-nt core sequence of ISRE of the ISG 15 gene promoter, yet with a DNA-binding specificity distinct from those of IRF-1 and IRF-2 [77, 7 8 1 . The core ISRE (GGGAAACCGAAACTG) is in fact, closely related to the PRDI core sequence (AGAGAAGTGAAAGTG) from IFN-B promoter, but the latter is not sufficient to mediate transcriptional induction by IFN-~t or to be recognized by ISGF3 I781. Mutational analyses indicate that ISGF3y prefers a cytosine residue in the position underlined in the ISRE sequence, whereas IRF-1 which binds efficiently to PRDI is not affected by the C/G mutation at this position 1781. The authors have also shown that the ISGF3 complex requires a longer DNA sequence than is recognized by ISGF3y alone, contacting at least the guanines located upstream the core sequence that are not in contact with ISGF3y. Thus, ISGF3 binding, and therefore IFN-ct inducibility is suggested to be restricted to a subset of IRF-binding sites that contain these additional residues.

Braganc,a and Civas

7. Autocrine amplification of type I IFN gene expression mediated by ISGF3 The comparison of virus-induced type I IFN mRNA levels in different primary cells isolated from wild type mice and mice with targeted disruption of the ISGF3y gene indicated that ISGF3y plays a critical role in the induction of IFN-A and B genes and that the dependency of IFN gene expression on ISGF3~ varies among the cell types [20]. Thus, NDV induced IFN-A mRNA levels were dramatically reduced (about 40-fold) in ISGF3y°/° embryonic stem cells, and IFN-B mRNA was reduced about two-fold. In macrophages the mRNA levels were reduced for IFN-A and IFN-B about 100-tbld and 10-tbid respectively. The induction of TNF-Q mRNA by NDV infection was not affected in both cells. A similar defect in type l IFN expression was also observed in cells lacking STATI or type I IFN receptor, further demonstrating the role of IFN signaling in the induction of IFN genes 1201. EMSA performed using the virus-inducible element from the mouse IFN-A4 promoter, the major IFN-A subtype induced in EFs, revealed a NDV-inducible binding activity in wild-type but not in ISGF3? °/° or IFNR°/° cells. Moreover, cotransfection of a human ISGF3"~ expression plasmid with a reporter gene that consists of luciferase gene driven by a minimal promoter and the inducible element from the mouse IFN-A4 promoter showed that the activation of IFN-A promoter by NDV was significantly augmented by ISGF3y expression, indicating that ISGF3y can bind to and activate a IFN-A promoter as a component of ISGF3 or a ISGF3-1ike complex 1201. The authors have further shown that the priming with IFN also requires the presence of ISGF3y. These results argued for the existence of a two-stage process of type ! IFN gene induction, in the initial stage, viral infection is suggested to induce the synthesis of a small amount of IFN through the activation of a factor(s) distinct from ISGF3. In the second stage, ISGF3 formed by the binding of the produced IFN to the type I IFN receptor could activate the type I IFN genes either directly or indirectly. Since the degree of dependency of the IFN gene expression on ISGF3 is different between EFs and macrophages and since in splenocytes the induction of both IFN-A and IFN-B mRNA is not affected at all by the disruption of the ISGF3y gene, the authors have raised the possibility that one can imagine that there is a subset of IFN-A genes with high ISGF3y-dependency which are only expressed in EFs or macrophages. This does not seem to be the case, because they notice that induction of all the IFN-A subtypes detected in fibroblasts and macrophages is essentially not affected in splenocytes. According to the authors, these observations indicate that the ISGF3ydependency of IFN induction is dependent on the cell type rather than IFN-A gene subtype. The generation of a deletion mutant of ISGF3y, Ap48, which lacks a portion of the DNA-binding domain, but retains the domain for

Differential expression of IFN-A genes interaction with ISGF3~ crealed a dominam inhibilor for ISGF3 activation [21 ]. NDV induction of mutant 1,929 cells stably expressing high levels of this dominantnegative form of ISGF3y showed that induction of IFN-A genes was dramatically inhibited (more than 20-fold), whereas IFN-B gene was partially but significantly inhibited (3- to 4-fold). When poly(I).poly(C) was used as an inducer, A948 almost completely inhibited the induction of IFN-A and IFN-B gene expression. These results confirmed that the induction of ISGF3 is necessary for the efficient induction of type I IFN by virus or dsRNA. Moreover, the addition of neutralizing anti-IFN sera in the culture medium of NDV-infected L929 cells resulted in a significant decrease in the ISGF3 level, with a concomitant decrease in gene activation mediated by PRD I, indicating that the positive feedback activation by IFN is necessary for efficient induction of IFN genes in L929 cells. Thus, it appeared that the autocrine ISGF3 pathway is essential for the amplification of IFN type I gene expression initially triggered by virus or double-stranded RNA [21]. According to the authors, the putative factor mediating the initiation of transcriptional activation must be activated directly by virus infection or dsRNA. They consider that a virus-induced PRDI-binding activity observed in cell lines deficient in the IFN receptor (HEC-I) or IFN locus (U87MG, U iI8MG, Reh, K562, RS4:I !) might be a candidate for the primary activator. This binding activity, distinct fi'om iSGF3, was shown to exhibit similar gel shift mobility to DRAF1, an ISREbinding activity induced by dsRNA 117-19[. The fact that type I IFN treatment does not directly induce type 1 IFN gene expression is attributed by the authors to the PRDII and PRDIV binding factors required for full induction of IFN-B promoter and to the presence of other types of elements which have yet not been identified in the IFN-A gene promoters [211. According to the authors, virus inl'ection may trigger multiple activators, whereas IFN treatment primarily induces ISGF3, which alone is insufficient to activate IFN gene. They indicate that additional mechanisms should be involved in the initial transcription process since repeated PRDI motifs strongly induced by NDV are only weakly induced by IFN treatment. They mention the involvement of transcriptional co-activators which do not interact directly with the promoter elements that could not be detected with the conventional EMSA methods. Alternatively, they indicate that negative PRDI-binding activities which disappear following virus infection or dsRNA treatment may be a part of these mechanisms. In transient transfection assays using EFs derived from IRF-1 and ISGF3y double knock-out mice, mutations in the IRF-elements present within IFN promoters markedly reduced, but did not completely abrogate type I IFN induction by NDV, suggesting that other IRF family members play a role in type I IFN gene transcription 1791.

681 8o Role of [RF-3 ~ d ~RF-7 in the regulation of type | IFN system T:ze IRF family includes also IRF-3 and other factors such as IFN consensus sequence binding protein (ICSBP). IRF-4 and IRF-7 150, 51, 54, 55] (see 180] for a review). These apparently redundant regulators suggest a complex network of virus-induced and/or IFN-regulated genes. The characteristic feature of factors belonging to this family consists of the conservation of the DNA-binding domain (DBD), located in the N-terminal 115 amino acids carrying a cluster of tryptophan residues, which is also found in the DBD of Myb oncoproteins. The crystal structure of the IRFI-DBD region bound to the PRDI domain revealed that IRF-1 binds to this motif as a monomer by a helix-turn-helix motif that latches onto DNA through three of the five conserved tryptophans [81]. Contacts to bases within the major groove are shown to be localized onto a GAAA core sequence within the PRDI element. The authors suggest that the presence of two GAAA core sequences in the PRDIII domain bound by IRF-! and the 1SRE elements bound by ISGF3y may indicate that these elements accommodate two IRF molecules. The interactions within the major groove are mediated by arginine 82, cysteine 83, asparagine 86 and serine 87 residues of the recognition helix c~3. Arginine 82 is totally conserved within the IRF family, whereas cysteine 83 is substituted by serine in IRF-3. The importance of these amino acids is also revealed by the abrogation of DNA binding by a mutation of either residue to alanine. ICSBP is induced exclusively in cells of macrophagc and lymphoid lineages and has a similar roic to IRF-2 in antagonizing the effect of IRF- 1 either on the induction of IFN genes or IFN-inducible genes [50]. The IRF-4 gene expressed specifically in lymphoid tissues in both B- and T-cell lines encodes for a factor that when expressed in hunaan carcinoma cells exerts, like IRF-2 and ICSBP. repressive effects on IFN-et mediated activation of constructs carrying 2'-5' OAS or H-2L d promoters or two tandem repeats of GBP-ISRE fused to tk-luciferase 1541. IRF-2 and ICSBP repress also the IFN-a oi" IFN-y activation of a promoter carrying tandem repeats of ISGI5-1SRE, whereas IRF-4 had a repressive effect only on IFN-,/ response of this construct. The authors suggested that IFN-stimulated gene expression is regulated through differential repression by the multiple members of IRF family and that in some genes IRF-4 exerts different repressive effects according to which activator comes in. When co-expressed in NIH3T3 cells, IRF-4 and PU.I. a member of Ets family transcription factors, function as mutually dependent transactivators of the immunoglobulin light chain promoter. These results suggested a dual function of IRF-4, a transcriptional repressor when it binds directly to its target sequence as a monomer protein, an activator binding to composite response elements when an interaction partner is present ]541.

682 IRF-3 gene, identified by its primary sequence homology to the IRF family, encodes a 50 kDa protein, previously shown to bind specifically to the ISRE sequences of different ISGs [511. In L929 cells, transfection of a IRF-3 expression plasmid was shown to stimulate a reporter construct containing the ISGI5 promoter. In the same conditions, IRF,3 alone was unable to induce the mmine IFN,A4 promoter, but enhanced (- 3-fold) the NDVmediated induction of this promoter. Recently, IRF3-DBD was also shown to bind to the IRF-elements present in the virus,inducible element of murine IFN-A4 promoter and to the PRDIII domain of the human or murine IFN-B promoter, whereas it interacted weakly with the PRDI domain of IFN-B [82]. Cotransfection of IRF-3 and RelA (p65) expression plasmid in human 293 cells (an embryonic kidney endothelial cell line), activates the expression of a IFN-B-CAT reporter, without affecting the activity of a IFN-A4-CAT construct that does not contain the NF-KB binding site, indicating that IRF-3 alone does not stimulate the induction of IFN genes, but can cooperate with the p65 subunit of NF-~:B to stimulate the IFN-B promoter. Other ,ecent studies have shown that a dominant negative mutant of IRF-3 can inhibit virus-induced activation of chromosomal type I IFN genes and ISGs 1831. The gene activation by IRF-3 was correlated with a DNA-binding activity detected in whole cell extracts prepared from either L929 cells or stable transformants expressing hemagglutinin-tagged human IRF-3, induced for 12 h with NDV. This complex detected either with the PRDIIIPRDI sequence of the human IFN-B promoter or with the ISG ! 5-1SRE sequence is referred to as virus-activated IRF (VA-IRF). This binding activity was shown to react either with anti-human IRF-3 antibodies or antibodies raised against the co-activators CBP and p300 1831. These lindings suggested tt'.lt IRF-3 and CBP/p300 play an important role in the activation of virus-induced type ! IFN genes as well as in the virus-induced transcription of some IFN-responsive genes. In unstimulated cells, IRF-3 is present in its inactive form, restricted to the cytoplasm due to a continuous nuclear export mediated by a nuclear export signal (NES) located between amino acid residues 129 and 190. The latent cytoplasmic IRF-3 which exhibits few DNA,binding properties is phosphorylated on specific serine residues upon virus infection of cells, but not tbilowing IFN treatment. This modification allows IRF-3 to translocate into the nucleus and to interact with CBP/p300 and specific DNA-binding sites. The authors consider that the viral dsRNA generated in the cytoplasm may act as a direct stimulus for the specific phosphorylation of IRF-3, yet the transduction signals triggered by virus or dsRNA resulting in the phosphorylation of IRF-3 and the mechanism which prevents the export of the phosphorylated IRF-3 from the nucleus remain to be characterized. The association of CBP/p300 with IRF-3 is suggested to alter the conformation of its DNA-binding domain, then to induce specific DNA binding of VA-IRE

Braganqa and Civas However, the possibility of direct participation of CBP/p300 in DNA binding is not ruled out, since the association with CBP/p300 is indispensable for DNA binding of IRF-3. The authors suggest that CBP/p300 which has associated histone acetylase activity, may also have an active role in converting the chromatin from the inert to the activated conformation, which allows recruitment of different transcription factors to the enhanceosome to maximize gene activation. The post-translational modification of IRF-3 following virus infection (Sendai virus-infected 293 cells) was further shown to occur by protein phosphorylation at multiple serine and threonine residues located in the carboxy-terminus of IRF-3 between amino acids 395 and 407 [841. Point mutation of serine 396 and serine 398 eliminated virus-induced phosphorylation of IRF-3 protein and abrogates its binding to CBP. Substitution of the serine and threonine sites with the phosphomimetic aspartic acid generated a constitutively activated form of IRF-3 that functions as very strong activator of promoters containing PRDIII-PRDI or ISRE regulatory elements. Phosphorylation also appeared to represent a signal for virus-mediated degradation, since the virus-induced turnover of IRF-3 was prevented by mutation of IRF-3 serine-threonine cluster or by proteasome inhibitors. The composition of the dsRNA-activated factor 1 (DRAFI), a ISRE-binding activity detected in response to infection of HeLa $3 cells and HEC-IB cells deficient in autocrine IFN signaling by adenovirus, by NDV or by dsRNA has been recently characterized 117-191. Two of the components of DRAF1 have been identified as IRF-3 and the transcriptional co-activator CBP/p300. These results confirm~'d the association o1" IRF-3 with CBP upon viral infection ~,~ndclearly indicated that the direct virus induction oi" a set of ISGs is mediated by IRF-3. Finally, IRF-7 which is expressed predominantly, in spleen, thymus and peripheral blood ieukocytes, bind to ISRE sequences and repress transcriptional activation by both IFN and IRF-I 1551. This factor was recently shown to interact with IRF-3 to form a complex in uninfected cells, but either IRF-3 or IRF-7 is unable to bind to DNA or activate transcription whereas, in virus-infected cells, IRF-3 and IRF-7 interact with CBP and form a virusactivated factor, VAF that accumulates in the nucleus and binds to the promoters of virus-inducible genes [851. VAF binds with high affinity to the ISRE of IFN-stimulated and virus-inducible genes, but not to the ISRE of genes that are induced by IFN only. The authors have shown that recombinant IRF3 and IRF-7 or VAF bind very weakly to the PRDIII-PRDI region of the IFN-B promoter and that they are exclusively recruited to the endogenous IFN-B promoter as part of a protein complex that includes ATF-2/c-Jun and NF-~cB, in response to virus infection. By in vivo protein-DNA cross-linking analyses, they also showed that a DNA fragment corresponding to the ISG-56 gene promoter could be amplified from the DNA immu-

Differential expression of |FN-A genes noprecipitated from Sendal virus-infected cells with eithcr anti-IRF-3 or anti-lRF-7 antibodies. This promoter could not have been amplified I?om DNA immunoprecipitated with antibodies raised against IRF-I, NF-lcB subunits or ATF-2 and c-Jun, indicating that IRF-3 and IRF-7 associate with the ISG 56-|SRE fragment. In virus-infected cells, the IFN-B promoler has been amplified from all immunoprecipitates except those amplified from antiIRF-1. According to authors, this implies that not only is IRF-1 dispensable for virus-induction of the IFN-B gene, but it cannot substitute for IRF-3 and IRF-7 in the formation of a virus-activated multicornponent enhancer complex in vivo.

9. Concluding remarks on the virus-induced differential expression of IFN-A genes The studies summarized in the previous sections indicate that IRF-3 and IRF-7 participate in the virus-induced and dsRNA-stimulated transcriptional activation of either IFN-B gene or a definite set of genes which can also be activated by IFN (figure 3). In murine L929 cells and human 293 cells, overexpression of IRF-3 alone did not show significant effect on the murine IFN-A4 promoter construct. In NDV-induced cells, the virus-induced response of the same construct has been shown to be up-regulated by IRF-3 in L929 cells, and down-regulated in human 293 cells 151,821. The activation is explained by the association of IRF-3 with virus-modified cellular factors resulting in an increase of IRF-3 binding to the virus-responsive element of the IFN-A4 promoter, whereas the inhibition observed in 293 cells remains to be clarified. However, the fact that the IRF3-GST fusion protein recognizes the minimal inducible element (IE-A4) of the IFN-A4 promoter and that a trans-dominant negative lbrm o!" IRF-3 down-regulates the overall NDVinduced IFN-A mRNA levels indicates that this factor participates also in the activation of IFN-A genes, at least in L929 cells 1831. Now, the question i~, if IRF-3, or the complexes containing IRF-3 and CBP (VA-IRF or DRAF1), or the IRF-3/IRF-7/CBP complex (VAF) corresponds to the primary activator which triggers the initiation of virus-induced transcription of type I IFN genes and a definite subset of ISGs. In this context, the relationship between these complexes and the previously identified virus-induced factor VIF 1471 that recognize different subdomains of the virus-responsive element of the highly inducible murine IFN-A4 gene promoter remains to be determined. It is not yet known if the differential expression of IFN-A genes in a given cell type could be attributed to IRF-3, IRF-7 and/or to different multiprotein complexes formed by these factors, the relative affinity for their binding sites being affected by the nucleotide substitutions occuring in the individual virus-responsive element of

683 different IFN-A promoters. Since IFN-A gene expression is also dependent on the aulocrine amplification mediated by 1SGF3, the high expression levels of the IFN-A4 gene induced by virus may also be related to the affinity of ISGF3 for the corresponding promoter. Recent studies indicate that the induction of |FN-A genes is not completely abolished, but severely impaired in Statl knockout cells, and that IFN-A4 is the only subtype synthesized in these cells [861. IFN-A4 is also shown to be the unique subtype in wild-type cells treated with a protein synthesis inhibitor during NDV infection. Priming with exogenous IFN-ct partially restores the induction of IFN-A genes other than IFN-A4. These results suggest that a Statl- and protein synthesis-dependent pathway is required for induction of IFN-A genes other than IFN-A4 in response to NDV infection. Decoding the mechanisms of virus-activated type I IFN gene transcription can now give an insight into the understanding of different signals triggered by virus infection of host cells, interfering with numerous cell functions, including cell survival, proliferation, some forms of apoptosis and modulation of the immune response. These signals which can be dependent on the virus entry, such as the viral haemagglutinin-mediated, pHdependent membrane fusion of Influenza virus or the conformational change in the viral envelope glycoprotein induced by interactions of viruses (Rous sarcoma virus, HIV-I gp120/41) with their cellular receptor(s)[87-89]. Envelope-mediated signal transduction through chemokine receptor CCR5, shown to induce chemotaxis of T-cells, is suggested to contribute to the pathogenesis of HIV by chemo-attracting activated CD 4+ cells to sites of viral replication o1" to influence viral cytopathicity or apoptosis 1901. The signals triggered by virus during the intracellular transport of viral proteins and genomes to cndoplasmic reticulum and to the nucleus and those gcncratcd during viral replication and assembly of new progeny may also contribute to the transcriptional regulation of IFN genes and the genes encoding for other cytokines (TNFot, IL-6), chemokines or a definite group of ISGs. The identification of either other proteins and coactivators interacting with IRF-3 and IRF-7, or the kinases involved in the phosphorylation of these factors will form the new avenues leading to the discovery of strategies evolved by viruses to recognize and discriminate the target host cells.

Acknowledgments We thank Genevibve Vincent tbr reading the manuscript. This work was supported by the grants from the Association de Recherche contre le Cancer (ARC, contrat 1042), Ligue Nationale contre le Cancer and F6d6ration Nationale des Groupements des Entreprises Franqaises et la Lutte contre le Cancer (FEGEFLUC).

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References I1] De Maeyer E., De Maeyer-Guignard J., lnterferons and other regulatory eytokines, New York, John Wiley and Sons, 1988. 121 Cheng Y.S., Becker-Manley M.E, Chow T.P., Horan D.C., Affinity purification of an interferon-induced human guanylate-binding protein and its characterization, J, Biol. Chem, 260 (1985) 15834-15839, 131 StaeheliP., HailerO., Boll W., Lindenmann J., WeissmannC.. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx eDNA confers selective resistance to influenza virus, Cell 44 (1986) 147-158.

Figure3. A, B. Virus-induced activation of transcription of type I IFN genes. The virus entry (consisting on the interaction of viral envelope with cell membrane receptor, penetration of the viral genome, proteins and ribonucleoproteins into the cell) lead to the specific activation of pre-existing inactive transcription factors. A putative signal transduction pathway stimulated by virus or dsRNA via the NF-~B-inducing kinase (NIK) and IKB kinases (IKK-~t and IKK-~) is indicated [91-931. The virus-activated transcription factors initiate the transcription of type I IFN in the nucleus and lead to the production and secretion of small amounts of type I IFN. The primary role of IRF-3 and IRF-7 in transcription is described in the text. The binding of IFNs to their ceil-surface receptor activates ISGF3 and lead~ to the autocrine (or paracrine) amplification of type I gene transcription initially induced by virus. A. Differential expression of murine IFN-A genes. The expression of IFN-A genes is dependent on the number of positive regulatory modules contained in their promoters. The role of PRDi-like domains corresponding to the B, C and D modules in IRF3- or IRF7-mediated transcription remains 1o be established. B. Activation ot" IFN-B gene transcription. Transcriptional activation of IFN-B gene promoter requires the assembly of a higher order transcription enhancer complex formed by ATF2/c-Jun, NF-~:B, IRF-3 and IRF-7 and the architectural HMGI(Y) protein, termed enhanceosome, which cooperates with CBPIp300 coactivator.

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