Interferon Response Sequence Research Articles

Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus.

Activation of cellular interferon-stimulated genes (ISGs) after infection with herpes simplex virus type 1 (HSV-1) or human cytomegalovirus (HCMV) was investigated. The level of ISG54-specific RNA in human fetal lung (HFL) or human foreskin (BJ) fibroblasts increased substantially after infection with either virus in the presence of cycloheximide. HSV-1 particles lacking glycoprotein D or glycoprotein H failed to induce ISG54-specific RNA synthesis, demonstrating that entry of virus particles rather than binding of virions to the cell surface was required for the effect. A DNA-binding complex that recognized an interferon-responsive sequence motif was induced upon infection with HSV-1 or HCMV in the presence of cycloheximide, and the complex was shown to contain the cell proteins interferon response factor 3 (IRF-3) and CREB-binding protein. IRF-3 was modified after infection with HSV-1 or HCMV to a form of lower electrophoretic mobility, consistent with phosphorylation. De novo transcription of viral or cellular genes was not required for the activation of IRF-3, since the effect was not sensitive to inhibition by actinomycin D. Infection of HFL fibroblasts with HSV-1 under conditions in which viral replication proceeded normally resulted in severely reduced levels of the IRF-3-containing complex, defining the activation of IRF-3 as a target for viral interference with ISG induction. In BJ fibroblasts, however, significant activation of IRF-3 was detected even when the viral gene expression program progressed to later stages, demonstrating that the degree of inhibition of the response was dependent on host cell type. As a consequence of IRF-3 activation, endogenous interferon was released from BJ cells and was capable of triggering the appropriate signal transduction pathway in both infected and uninfected cells. Activation of ISG54-specific RNA synthesis was not detected after infection of human U-373MG glioblastoma cells, showing that the induction of the response by infection is cell type dependent.

Characterization of a gastric tumor cell line defective in MHC class I inducibility by both alpha- and gamma-interferon.

Alpha/beta and gamma type interferons (IFN), act through distinct cell surface receptors and induce transcription of an overlapping sets of genes. MHC class I genes are inducible by both type of interferons. We have analyzed a gastric tumor cell line, AGS, which was completely defective in MHC class I response to interferon-alpha and gamma. Northern blot analysis demonstrated that the lack of IFN response was related with the absence of up-regulation of specific HLA class I mRNA. Electrophoretic mobility shift assays in various tumor cell lines after IFN-alpha and IFN-gamma treatment showed differential binding of the transcriptional factors to MHC class I regulatory elements. Comparison of kappa-B binding activity showed that IFN-alpha and IFN-gamma induced opposite changes in NF-kappa B binding activity in AGS cells, indicating that the absence of MHC class I response in AGS appears to be independent of kappa-B activity. In contrast, there were remarkable differences in the level of transcriptional factor binding to an interferon-responsive sequence element (IRSE), between AGS and other interferon-responsive tumor cell lines. This result suggests that the low level of transcriptional factor binding to IRSE in AGS cells was responsible of the lack of induction of MHC class I antigens. In this context, overlapping factors in the signal transduction pathway of both type I and II interferons may be involved in the non-responsiveness of this gastric carcinoma tumor cell line.

The cattle major histocompatibility complex (MHC) class-I possesses HLA-like promoters

To study the genetic regulation of the cattle major histocompatibility complex (MHC) class-I, a cattle MHC class-I promoter DNA fragment was isolated and characterized for the first time. Semi-degenerate PCR was performed on cattle genomic DNA and the resulting product was isolated, subcloned and sequenced. Sequence comparison of the HLA-A, -B and -C promoters to the cloned product, designated BL3-6prmtr, revealed the cattle MHC class-I promoter to have close homology to human MHC class-I promoters. To address the ability of the cattle MHC class-I promoter to initiate transcription, BL3-6prmtr was subcloned into a luciferase reporter vector and transiently transfected into cattle and human B-lymphoblastoid cell lines. A strong transcription initiation ability of BL3-6prmtr was observed, including the ability of the enhancerA and interferon response sequence ( IRS) to upregulate transcription initiation.

The human interferon α-receptor protein confers differential responses to human interferon-β versus interferon-α subtypes in mouse and hamster cell transfectants

The human interferon α-receptor (IFNAR gene product or IFNαR protein) was expressed in hamster CHO cells and in mouse A9 cells. The response of the IFNαR cDNA transfectants to human IFNs was studied by measuring induction of (2′–5′) A synthetase (2′–5′ AS). In the murine cells, the IFNαR protein conferred response to the human IFN-α-8 (α-B) subtype, but not to huIFN-α-2 (α-A) or to huIFN-β. In murine huIFNαR cDNA transfectants, containing a hygromycin B resistance gene placed under the control of the 2′–5′ AS gene Interferon Response Sequence (IRS), survival and growth of the cells in the presence of hygromycin B was induced by huIFN-α-8 but not by huIFN-α-2, indicating that the effect of huIFNαR is transcriptional. In hamster CHO cells, the huIFNαR protein conferred a completely different pattern of response to human IFN subtypes. Thus, the CHO-IFNαR transfectants responded to huIFN-β by 2′–5′ AS induction as well as by activation of the ISGF3 and IRF-1 transcription factors. In contrast, the CHO-IFNαR cells showed no response to huIFN-α-8. The differential response conferred by the huIFNαR protein in the two types of rodent cells, indicates that IFN subtype recognition is influenced by another component contributed by the rodent host cell. The ability of human cells, and of human-mouse hybrid cells containing human chromosome 21, to respond to all IFN subtypes is likely to depend also on interactions of the IFNαR protein with additional receptor components.

The enhancer A/IRS region of a cattle MHC class I promoter.

In addition to the TATA and CAAT box sequences necessary for RNA polymerase II positioning, the major histocompatibility complex (MHC) class I promoter contains an upstream enhancer region (EnhancerA) that partially overlaps the Interferon Response Sequence (IRS; Kimura et al. 1986). This EnhancerA/IRS region is necessary for effecting class I transcription modulation through cytokine stimulation (Harms and Splitter 1992), viral infection (Maudsley and Pond 1991 ), and carcinogenesis (Blanchet et al. 1992). Genetic information about the cattle MHC class I, known as the Bo LA complex, has been limited to studies of cattle class I cDNA sequences; little is known of the genetic control of Bo LA expression. To better define genetic regulation of the cattle MHC class I, a Bo LA class I promoter EnhancerAdRS region DNA fragment was isolated, sequenced, and functionally tested in a luciferase reporter gene expression system. Genomic DNA, isolated from the B-lymphosarcoma cell line, BL3 (Harms and Splitter 1992), was amplified by PCR, using primer pairs designed from the HLA-A2 promoter sequence (Koller and Orr 1985). The PCR fragment was subcloned into the vector, pGem-3Z (Promega, Madison, WI), and dideoxy sequenced. The resultant Bo LA EnhancerA/IRS sequence was named, BoLAenh-9 (Fig. 1). Transcription initiation ability of BoLAenh-9 was assayed, using a luciferase reporter gene system. The cattle class I EnhancerA/IRS region DNA fragment, BoLAenh-9, was subcloned into the Bgl II site of the luciferase expression vector, pGL Promoter (Promega), and the resulting vector, pGL BoLAenh-9, along with negative and positive control vectors pGL Promoter (SV40 promoter, no enhancer) and pGL Control (SV40 promoter, SV40 enhancer), were transfected into Vero cells (ATCC CCL 81), using the liposome reagent, TransfectAM1NE (Gibco BRL, Gaithersburg, MD). Cell lysates were assayed for luciferase activity 24 h after transfection, using a Monolight 2010 luminometer (ALL, San Diego, CA) and the level of sample luminescence recorded as relative light units. Results (Fig. 2) indicate that BoLAenh-9 has a strong transcription initiation ability. The cattle MHC class I EnhancerA/IRS region clone, BoLAenh-9, was isolated by using PCR incorporating degenerate primers designed from HLA-A2. Sequence comparison of the EnhancerA/IRS in BoLAenh-9 with that of HLA-A2 was only 59% (data not shown). Nevertheless, transcription initiation ability was remarkably strong, using the SV40 promoter compared with SV40's native enhancer (Fig. 2). The cattle MHC class I enhancer, BoLAenh-9, will be useful in further research of class I genetic regulation of expression.

Interleukin-6 activates and regulates transcription factors of the interferon regulatory factor family in M1 cells.

Activation of (2'-5') A synthetase gene expression in interleukin-6 (IL-6)-treated myeloleukemic M1 cells correlates with protein binding to the interferon response sequence enhancer (IRS). A new interferon response sequence complex, F6, is induced by IL-6 independently of interferon and is identified here as comprising the interferon regulatory factor-1 (IRF-1) and IRF-2, by use of specific antibodies in DNA mobility shift assays with probes containing IRF binding sites. IRF-1 and IRF-2 have, respectively, positive and negative transcriptional effects on interferon-beta and interferon-inducible genes. In the IL-6-treated M1or cells, IRF-1 binding is activated early and maximally at 1 h, whereas the onset of IRF-2 binding is delayed. In a cell variant M1res, where (2'-5') A synthetase is no more induced, IRF-2 binding is constitutive, and IRF-1 binding is not seen before or after IL-6 treatment. In sensitive M1or cells, IL-6 rapidly induces IRF-1 mRNA, but in M1res cells, IRF-1 mRNA is constitutively high and not changed by IL-6. IRF-2 mRNA levels are also constitutive and not inducible by IL-6 even in M1or cells. The dissociation between induction of mRNAs and of protein binding observed suggests that the activity of the IRF proteins is regulated by IL-6. Transcripts of a third member of the IRF gene family, ICSBP, encoding a protein known to act as repressor, were found to be strongly down-regulated by IL-6. The rapid activation of IRF-1 and the modulation of the other transcription factors of this family may play a role in the early phase of IL-6 action on the M1 cells.

A role for the interferon response DNA sequence in directing transcription of the T18 d Tla gene

T18d of BALB/c mice is a member of the Tla category of class I genes of the major histocompatibility complex of the mouse and is highly restricted in expression. Deletion analysis implies that an element essential to T18d expression resides within the region -4 to +54. The homologous region of T3d, a Tla gene which normally is not expressed in BALB/c mice, also has promoter activity. Thus the expressibility of T18d vs T3d is unlikely to be due to sequence differences in this region. A DNA-binding protein, factor VI, was found to bind to the region -33 to +54. DNase I footprinting analysis indicated that the DNA fragment 5'-ACTATAGTTTCACTTTTT-3' (+3 to +20) was protected by factor VI. This region includes the interferon response sequence (IRS). Homologous DNA segments of other class I genes, Ld and Dd, competed for factor VI in DNA-protein binding assay with lower affinity as compared with T18d. In mutation analysis, the 3' portion of the IRS is more important than the 5' portion with respect to binding affinity of factor VI and to transcriptional activity in transfected cells. This result signifies a role of IRS in T18d transcription and suggests that the mechanism of T18d transcription might be unusual.

IMBALANCE OF MHC CLASS I EXPRESSION IN 3LL TUMOUR CELLS

Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack of beta 2m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low '37' phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Characterization of an IFN-α induced factor that binds to the interferon response sequence of the 202 gene in murine leukemia cells

Characterization of an IFN-α induced factor that binds to the interferon response sequence of the 202 gene in murine leukemia cells

Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon‐response sequence (IRS) and to the IRS‐containing fragment of the human c‐myc gene

The human c-myc proto-oncogene was recently found to contain a regulatory sequence similar to the consensus interferon-response sequence (IRS) of interferon-activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c-myc, lacking the main part of 5'-nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene. Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c-myc gene or the interferon-dependent 6-16 gene. Moreover, each of these factors 'cross-binds' to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c-myc and 6-16 genes were determined. In vitro transcription of the human full-length c-myc gene (i.e. the gene containing the complete 5'-noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed.

Identification of cis Sequences Controlling Efficient PositionIndependent Tissue-Specific Expression of Human Major Histocompatibility Complex Class I Genes in Transgenic Mice

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.

Interleukin-6 induces the (2′–5′) oligoadenylate synthetase gene in M1 cells through an effect on the interferon-responsive enhancer

Interleukin-6 (IL-6) activates (2'-5') A synthetase (2'-5' AS) gene expression in differentiating myeloleukemic M1 cells. Antibodies to type I interferon (IFN) inhibit 2'-5' AS induction but not differentiation. Analysis of the mechanism of 2'-5' AS induction shows that it does not result from increased IFN formation, but from a synergism between IL-6 and endogenously secreted IFN. IL-6 can activate expression of a CAT construct fused to the interferon response sequence (IRS) of the 2'-5' AS gene. In extracts of IL-6-treated M1 cells, changes in protein binding to IRS DNA can be demonstrated. One of the effects of IL-6 on M1 cells is, therefore, to induce DNA binding factors, some of which act on the same enhancer sequence as IFNs, resulting in a synergistic gene activation. M1 variants resistant to differentiation by IL-6 have lost the ability to induce the 2'-5' AS gene.

Human c-myc gene contains a regulatory site similar to consensus of interferon response sequence (IRS)

Expression of c-myc proto oncogene is regulated by multiple mechanisms. Here, we report that the consensus of the regulatory region of interferondependent genes, GGAAAN 1−3 GAAA, was found after computer search in the 5'-terminal flank of human c-myc gene in position (−76:−67). In vitro transcription of c-myc gene fragments showed that the consensus region competes with oligonucleotide GGGAAAATGAAACT for binding to specific protein(s). This oligonucleotide was shown to bind selectively the interferon-dependent positive transcription factor [1]. Transcription of c-myc fragments lacking 5'-terminal region up to positions −101 or +71 was initiated at two sites located in the first intron. These sites did not coincide with P 1 in vivo RNA cap-site. Binding of the protein factor(s) to the regulatory region of c-myc gene −76:−67 blocked the in vitro transcription initiated in the first intron.

Differential regulation of HLA class I genes by interferon

Allele-specific differences in the regulation of HLA class I genes by type I interferon (IFN) were observed after transfection of eight HLA-B, -A, or -C genes into mouse L cells. HLA-B7 and -Bw64 gene expression was significantly more inducible by type I IFN than the genes coding for HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3, and HLA-A2 antigens. Modification of the 5' end of HLA-B7 and HLA-B27 genes revealed the presence of enhancer sequences responding to IFN treatment in the 5' untranslated region of HLA-B7, but not of HLA-B27 and suggested further, independently acting enhancer elements downstream of the transcription initiation site. Comparison of 5' enhancer region sequences in correlation with type I IFN inducibility of the different HLA class I alleles indicated that the exchange of only two nucleotides in the interferon response sequence (IRS) or enhancer A region of HLA-B7 or -Bw64 could account for nonregulated promoters in all other HLA-A, -B, or -C alleles analyzed. Thus, type I IFN stimulation of HLA class I genes in mouse L cells appears to predominantly operate in most alleles by a mechanism targeted to enhancer sequences downstream of the gene's transcription initiation site.

Characterization of human IFN-? response using somatic cell hybrids of hematopoietic and nonhematopoietic origin

A panel of 27 rodent-human somatic cell hybrids composed of cells of hematopoietic (nonadherent cells) and nonhematopoietic origin (adherent cells) was used to identify the chromosomes involved in the biological response to human IFN-gamma (Hu-IFN-gamma). We found that the stimulation of class-I histocompatibility antigen expression correlates with the presence of human chromosomes 6 and 21 in adherent cell hybrids, while human chromosome 6 alone is sufficient in nonadherent hybrids. Scatchard analysis of the binding of radiolabeled Hu-IFN-gamma to nonadherent cell hybrids gave a Kd value similar to that found on human cell lines. Induction of a reporter gene placed under the transcriptional control of the interferon responsive sequence (IRS) in adherent cell hybrids requires both chromosomes 6 and 21. The antiviral protection by Hu-IFN-gamma in adherent cell hybrids was reached at physiological doses (2 units/ml) when human chromosomes 6 and 21 were present, while higher doses of Hu-IFN-gamma (5000 units/ml) were required for hybrids lacking chromosome 21. Thus, we demonstrate that differences exit in the response to Hu-IFN-gamma depending on the origin of the cell type.

A gamma-interferon-induced factor that binds the interferon response sequence of the MHC class I gene, H-2Kb.

Transcription of class I genes of the major histocompatibility complex (MHC) can be induced by interferons. Treatment of HeLa cells with interferon-gamma induces a DNA-binding factor, IBP-1, specific for a site within the interferon response sequence (IRS) of the H-2Kb promoter. The mol. wt of IBP-1, as estimated by photoactivated protein-DNA crosslinking analysis, is approximately 59 kd. Point-mutation of this binding site abolishes IBP-1 interaction and the ability of the MHC promoter to respond to interferon. Induction of this binding activity is rapid and closely parallels the previously reported time course of transcriptional activation of endogenous MHC class I genes. Treatment of cells with cycloheximide, a protein synthesis inhibitor, blocked the induction of the DNA-binding activity. An oligonucleotide derived from the virus- and double-stranded RNA-inducible promoter of the interferon-beta 1 gene is able to bind IBP-1. Sequences similar to the IBP-1 binding site are found upstream of many interferon-responsive genes.

Enhancer functions and in vitro protein binding of native and mutated interferon-responsive sequences.

Mutants of the Interferon responsive sequence (IRS) of the mouse and human (2'-5') A synthetase (moE-IRS and hE-IRS) were tested for their Interferon (IFN)-inducible enhancer functions and for protein binding in vitro. Two complexes R1 and R3, were formed specifically with the hE-IRS. R3 migrated much faster and was about ten times more abundant than R1. R1 and R3 are increased about 2-fold in IFN-treated HeLa extracts relatively to extracts from non-treated cells. R1 and R3 seem to involve the same DNA sequence in the probe since they react identically to competitors. Two proteins of 69 and 46 kDa form the IRS specific complexes as revealed by UV cross-linking. Identical DNA probes bearing either the hE-IRS or moE-IRS form complexes of different characteristics with nuclear proteins, suggesting that the two IRS variants are the targets of binding of different proteins or of different protein complexes.

Enhancer-like interferon responsive sequences of the human and murine (2′-5′) oligoadenylate synthetase gene promoters.

The human (2'-5') oligo(A) synthetase gene contains two independent cis-acting DNA elements, A and B, which act as transcriptional enhancers. Element A alone is not activated by IFN treatment. Element B alone confers IFN-inducibility to the herpes tk promoter. Two murine (2'-5') oligo(A) synthetase genes were isolated and their promoter sequences show high conservation of element A and B. A synthetic oligonucleotide, containing 16 bp of the human element B, or 14 bp of the homologue murine element B, was linked to a TK-CAT construct. These oligonucleotides were shown to be sufficient to activate the TK promoter in the presence of IFN. When multiple repeats of the interferon-responsive sequence (E-IRS) were cloned in 5' of the TK promoter, the activation ratio was increased. In vitro, specific binding of nuclear protein(s) is observed to the radiolabelled synthetic human E-IRS. This binding is competed by the addition of cold synthetic mouse E-IRS or fragments of genomic DNA containing the E-IRS.

A common positive trans-acting factor binds to enhancer sequences in the promoters of mouse H-2 and beta 2-microglobulin genes.

Using gel retardation and in vitro "footprinting", we have analyzed the interactions between nuclear proteins derived from various mouse cells and the enhancer and interferon response sequences of the H-2Kb gene. We have found that a protein factor binds a site in the enhancer sequence that partially overlaps the interferon response sequence. This factor also binds to a similar sequence lying in the opposite orientation in the promoter of the mouse beta 2-microglobulin gene, suggesting a common regulatory mechanism. Transfection competition experiments indicate that this factor acts as a positive element in the expression of H-2 and beta 2-microglobulin genes.