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PubMed2001

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity.

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.

Adenosine Triphosphatases↗
PubMed1997

The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR.

We demonstrate that the interferon-induced, double-stranded (ds) RNA-activated kinase, PKR, is able to bind to and phosphorylate the human immunodeficiency virus type 1 (HIV-1) trans-activating protein, Tat. Furthermore, Tat can inhibit the activation and activity of the kinase. Phosphorylation of Tat by PKR is dependent on the prior activation of PKR by dsRNA and occurs on serine and threonine residues adjacent to the basic region important for TAR RNA binding and Tat function. Activated PKR efficiently phosphorylates both the two-exon form of Tat (Tat-86) and the single exon form (Tat-72). Mutagenesis indicates that the interaction between PKR and Tat requires the RNA-binding region of Tat. Tat competes with eukaryotic initiation factor 2, a well-characterized substrate of PKR, for phosphorylation by activated PKR. Tat also inhibits the autophosphorylation of PKR by dsRNA. This biochemical evidence of an intimate relationship between Tat, an important regulator of HIV transcription, and PKR, a pleiotropic cellular regulator, may provide insights into HIV-1 pathogenesis and, more generally, virus/host interactions.

Amino Acid Sequence↗
PubMed1980

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells.

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.

2',5'-Oligoadenylate Synthetase↗
PubMed2005

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA.

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic-polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-gamma, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 microM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.

Antigens, CD↗
PubMed2005

Alteration of TGA factor activity in rice results in enhanced tolerance to Xanthomonas oryzae pv. oryzae.

In dicotyledonous plants broad-spectrum resistance to pathogens is established after the induction of the systemic acquired resistance (SAR) response. In Arabidopsis the NPR1 protein can regulate SAR by interacting with members of the TGA class of basic, leucine-zipper transcription factors to alter pathogenesis-related (PR) gene expression. Overexpression of (At)NPR1 in Arabidopsis enhances resistance to multiple pathogens. Similarly, overexpression of (At)NPR1 in rice enhances resistance to the bacterial pathogen, Xanthomonas oryzae pv. oryzae (Xoo). These results suggest that components of the (At)NPR1-mediated SAR defense response may be conserved between monocots and dicots. To determine whether or not rice TGA factors are involved in disease resistance responses, the effect of altering the function of rice TGA2.1 was analyzed in transgenic plants. Transgenic rice overexpressing an rTGA2.1 mutant, that can no longer bind DNA, and transgenic rice that have the endogenous rTGA2.1 silenced by dsRNA-mediated silencing were generated. Both types of transgenic rice displayed increased tolerance to Xoo, were dwarfed, and had altered accumulation of PR genes. The results presented in this study suggest that wild-type rTGA2.1 has primarily a negative role in rice basal defense responses to bacterial pathogens.

DNA-Binding Proteins↗
PubMed1984

Interferon-mediated protein kinase activity in different fractions of mouse L-929 cells.

The interferon (IFN)-mediated protein kinase activity in extracts from mouse L-929 cells is manifested by the phosphorylation of an endogenous 67 kD molecular weight (mw) protein in the presence of double-stranded (ds) RNA. This protein kinase activity can also be assayed after partial purification on poly(I) X poly(C)-Sepharose under phosphatase-free conditions. By the use of this latter technique, here we investigated the distribution of the protein kinase activity in different cellular compartments. Most of the protein kinase activity is found in the post-ribosomal supernatant (S100) fraction, while a small portion of it is associated with the ribosomal salt wash (RSW: 0.5 M KCl eluate of ribosomal pellet) and nuclear fractions. These results are in contrast to several observations in the literature in which the protein kinase activity is thought to be associated with the ribosomal pellet. This controversy results from the conditions used for assay of the protein kinase activity. In fact, when the kinase is assayed in crude extracts supplemented with dsRNA, very little kinase activity is detectable in the S100 fraction compared to the RSW fraction. The S100 fraction contains a high level of phosphatase(s) activity which interferes with the protein kinase assay and might account for the misinterpretation observed in the literature. Some recent results have implicated a correlation between the dsRNA-dependent protein kinase responsible for the phosphorylation of the 67 kD protein and a polyamine-dependent protein kinase which phosphorylates a similar molecular weight protein, subunit of ornithine decarboxylase (Orn Dcase). Here, we show that Orn Dcase does not bind to poly(I) X poly(C)-Sepharose and polyamines do not substitute the requirement of dsRNA for the phosphorylation of the 67 kD protein.

Animals↗
PubMed1997

Surprising specificity of PKR binding to delta agent genomic RNA.

A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity.

Base Sequence↗
PubMed2005

A20 is a negative regulator of IFN regulatory factor 3 signaling.

IFN regulatory factor 3 (IRF-3) is a critical transcription factor that regulates an establishment of innate immune status following detection of viral pathogens. Recent studies have revealed that two IkappaB kinase (IKK)-like kinases, NF-kappaB-activating kinase/Traf family member-associated NF-kappaB activator-binding kinase 1 and IKK-i/IKKepsilon, are responsible for activation of IRF-3, but the regulatory mechanism of the IRF-3 signaling pathway has not been fully understood. In this study, we report that IRF-3 activation is suppressed by A20, which was initially identified as an inhibitor of apoptosis and inducibly expressed by dsRNA. A20 physically interacts with NF-kappaB-activating kinase/Traf family member-associated NF-kappaB activator-binding kinase 1 and IKK-i/IKKepsilon, and inhibits dimerization of IRF-3 following engagement of TLR3 by dsRNA or Newcastle disease virus infection, leading to suppression of the IFN stimulation response element- and IFN-beta promoter-dependent transcription. Importantly, knocking down of A20 expression by RNA interference results in enhanced IRF-3-dependent transcription triggered by the stimulation of TLR3 or virus infection. Our study thus demonstrates that A20 is a candidate negative regulator of the signaling cascade to IRF-3 activation in the innate antiviral response.

Cell Line↗
PubMed1999

Inhibition of double-stranded RNA- and tumor necrosis factor alpha-mediated apoptosis by tetratricopeptide repeat protein and cochaperone P58(IPK).

P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.

3T3 Cells↗
PubMed2006

Analysis of the interaction between human TLR3 ectodomain and nucleic acids.

Toll-like receptor 3 (TLR3) recognizes dsRNA of viral origin and polyriboinosine-polyribocytidylic acid (poly (I:C)). TLR3 mediates the activation of IRF-3 and NF-kappaB and thereby the secretion of type I interferons and inflammatory cytokines. However, the mechanism of this activation is poorly understood. To study the molecular recognition events and biochemical interactions between TLR3 and dsRNA, human TLR3 ectodomain (ECD) fused with a signal peptide at the N-terminus and 6 x His-tag at the C-terminus (TLR3-ECD) was obtained by the baculovirus expression system. To examine the various nucleic acids binding to TLR3-ECD in vitro, a filter binding assay was carried out at pH 4.2-7.6. Interaction of TLR3-ECD with various nucleic acids (particularly dsRNA, in vitro transcripts of tRNA and HCV NS3 aptamer) required an acidic pH.

Binding Sites↗
PubMed1992

Identification of novel factors that bind to the PRD I region of the human beta-interferon promoter.

Treatment of cells with virus or synthetic double-stranded RNA (dsRNA) leads to the transient transcriptional activation of the beta-interferon gene. Genetic analysis has revealed that the 5' regulatory sequence responsible for this induction contains multiple positive and negative elements. One of these, Positive Regulatory Domain I (PRD I), has been shown to bind the positively-acting transcription factor IRF-1. In this study we show that this element is inducible under conditions where IRF-1 cannot be detected, suggesting that additional cellular factors are involved in the induction process. To investigate the existence of such factors we have analysed the range and properties of PRD I-binding activities present in HeLa cells. In addition to the repressor protein IRF-2, several novel factors can bind to PRD I in uninduced cells: two of these have properties consistent with a role in negative regulation; levels of two others increase upon priming, and may be alternative candidates for activators. Upon induction we also observe a novel factor whose appearance does not depend upon de novo protein synthesis, and which appears to be a truncated form of IRF-2. The potential involvement of these factors in regulating the beta-interferon gene is discussed.

Base Sequence↗
PubMed1999

Host cell proteins bind specifically to the capsid-cleaved 5' end of Leishmaniavirus RNA.

Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt), via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.

5' Untranslated Regions↗
PubMed2006

Structure-function analysis of rotavirus NSP2 octamer by using a novel complementation system.

Viral inclusion bodies, or viroplasms, that form in rotavirus-infected cells direct replication and packaging of the segmented double-stranded RNA (dsRNA) genome. NSP2, one of two rotavirus proteins needed for viroplasm assembly, possesses NTPase, RNA-binding, and helix-unwinding activities. NSP2 of the rotavirus group causing endemic infantile diarrhea (group A) was shown to self-assemble into large doughnut-shaped octamers with circumferential grooves and deep clefts containing nucleotide-binding histidine triad (HIT)-like motifs. Here, we demonstrate that NSP2 of group C rotavirus, a group that fails to reassort with group A viruses, retains the unique architecture of the group A octamer but differs in surface charge distribution. By using an NSP2-dependent complementation system, we show that the HIT-dependent NTPase activity of NSP2 is necessary for dsRNA synthesis, but not for viroplasm formation. The complementation system also showed that despite the retention of the octamer structure and the HIT-like fold, group C NSP2 failed to rescue replication and viroplasm formation in NSP2-deficient cells infected with group A rotavirus. The distinct differences in the surface charges on the Bristol and SA11 NSP2 octamers suggest that charge complementarity of the viroplasm-forming proteins guides the specificity of viroplasm formation and, possibly, reassortment restriction between rotavirus groups.

Amino Acid Sequence↗
PubMed2005

Expression and DNA binding activity of the recombinant interferon regulatory factor-1 (IRF-1) of mouse.

Interferon regulatory factor-1 (IRF-1) is a positive transcription factor for genes involved in immune response, cell growth regulation and apoptosis in mammalian cells. Many agents like virus, interferon (IFN), double-stranded RNA (dsRNA), proinflammatory cytokines, prolactin etc. induce IRF-1 at transcriptional level. IRF-1 transcriptionally activates many IRF-1-regulated genes during normal physiological and pathological conditions. We have expressed recombinant mouse IRF-1 (329 amino acids) as a GST(glutathione-S-transferase)-IRF-1 fusion protein from a 1029 bp IRF-1 cDNA in pGEX-2TK expression vector in Escherichia coli XL-1 blue cells. Recombinant GST-IRF-1 was highly expressed as a approximately 66 kDa soluble protein by IPTG-induction, and was biologically active in terms of its DNA binding activity with a 24 bp specific oligonucleotide, i.e. 32P(GAAAGT)4 but not with a similar but nonspecific oligonucleotide i.e. 32P(GAAA)6. GST-alone expressed from the vector did not bind 32P(GAAAGT)4. We observed multiple (1-4) GST-IRF-1-(GAAAGT)4 protein-DNA complexes which were competed out by 25x- to 100x-fold molar excess of (GAAAGT)4 showing that the complexes 1-4 were specific for IRF-1. Such GAAANN (N = any nucleotide) hexanucleotides occur in the promoters of many virus- and interferon-inducible mammalian genes. Multimeric GAAAGT/C sequences are inducible by virus, IFN, dsRNA and IRF-1. Specificity of DNA binding by IRF-1 lies in the 5th and 6th nucleotides in the GAAAGT sequence. Multiple IRF-1-DNA complexes should stimulate transcription by IRF-1.

Animals↗
PubMed1998

Rotavirus RNA replication requires a single-stranded 3' end for efficient minus-strand synthesis.

The segmented double-stranded (ds) RNA genome of the rotaviruses is replicated asymmetrically, with viral mRNA serving as the template for the synthesis of minus-strand RNA. Previous studies with cell-free replication systems have shown that the highly conserved termini of rotavirus gene 8 and 9 mRNAs contain cis-acting signals that promote the synthesis of dsRNA. Based on the location of the cis-acting signals and computer modeling of their secondary structure, the ends of the gene 8 or 9 mRNAs are proposed to interact in cis to form a modified panhandle structure that promotes the synthesis of dsRNA. In this structure, the last 11 to 12 nucleotides of the RNA, including the cis-acting signal that is essential for RNA replication, extend as a single-stranded tail from the panhandled region, and the 5' untranslated region folds to form a stem-loop motif. To understand the importance of the predicted secondary structure in minus-strand synthesis, mutations were introduced into viral RNAs which affected the 3' tail and the 5' stem-loop. Analysis of the RNAs with a cell-free replication system showed that, in contrast to mutations which altered the structure of the 5' stem-loop, mutations which caused complete or near-complete complementarity between the 5' end and the 3' tail significantly inhibited (>/=10-fold) minus-strand synthesis. Likewise, incubation of wild-type RNAs with oligonucleotides which were complementary to the 3' tail inhibited replication. Despite their replication-defective phenotype, mutant RNAs with complementary 5' and 3' termini were shown to competitively interfere with the replication of wild-type mRNA and to bind the viral RNA polymerase VP1 as efficiently as wild-type RNA. These results indicate that the single-strand nature of the 3' end of rotavirus mRNA is essential for efficient dsRNA synthesis and that the specific binding of the RNA polymerase to the mRNA template is required but not sufficient for the synthesis of minus-strand RNA.

Base Sequence↗
PubMed2003

Presence of potential allergy-related linear epitopes in novel proteins from conventional crops and the implication for the safety assessment of these crops with respect to the current testing of genetically modified crops.

Mitochondria of cytoplasmic male sterile crop plants contain novel, chimeric open reading frames. In addition, a number of crops carry endogenous double-stranded ribonucleic acid (dsRNA). In this study, the novel proteins encoded by these genetic components were screened for the presence of potential binding sites (epitopes) of allergy-associated IgE antibodies, as was previously done with transgenic proteins from genetically modified crops. The procedure entails the identification of stretches of at least six contiguous amino acids that are shared by novel proteins and known allergenic proteins. These stretches are further checked for potential linear IgE-binding epitopes. Of the 16 novel protein sequences screened in this study, nine contained stretches of six or seven amino acids that were also present in allergenic proteins. Four cases of similarity are of special interest, given the predicted antigenicity of the identical stretch within the allergenic and novel protein, the IgE-binding by a peptide containing an identical stretch reported in literature, or the multiple incidence of identical stretches of the same allergen within a novel protein. These selected stretches are present in novel proteins derived from oilseed rape and radish (ORF138), rice (dsRNA), and fava bean (dsRNA), and warrant further clinical testing. The frequency of positive outcomes and the sizes of the identical stretches were comparable to those previously found for transgenic proteins in genetically modified crops. It is discussed whether novel proteins from conventional crops should be subject to an assessment of potential allergenicity, a procedure which is currently mandatory for transgenic proteins from genetically modified crops.

Journal Article↗
PubMed1993

Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B.

We have studied the role of protein turnover in the induction of NF-kappa B DNA binding activity. Treatment of cells with tumour necrosis factor (TNF), double-stranded RNA (dsRNA), or phorbol esters is shown to be associated with an increase in the rate of p105 to p50 processing, and the loss of immunologically detectable MAD3/I kappa B alpha. Phosphate-labelling experiments indicate that these events are preceded by the phosphorylation of MAD3 and p105. The protease inhibitors TLCK (N alpha-p-Tosyl-L-Lysine Chloromethyl Ketone) and TPCK (N alpha-p-Tosyl-L-Phenylalanine Chloromethyl Ketone) inhibit both p105 to p50 processing and MAD3 degradation, and also cause a complete block to NF-kappa B activation. These data suggest a model for NF-kappa B activation in which phosphorylation destabilises the NF-kappa B/MAD3 complex but that, in vivo, this is insufficient to lead to activation in the absence of an obligatory mechanism that degrades MAD3.

Animals↗
PubMed1999

Functional interactions between conserved motifs of the hepatitis C virus RNA helicase protein NS3.

The hepatitis C virus NS3 gene encodes a RNA helicase with several sequence motifs conserved among the members of the DExH box protein family. The contributions of the sequence motifs to enzyme activity were assessed in this study by substitution of alanine for the Lys in the ATP binding motif GxGK (referred to as K1236A mutation), or for the Asp in the DExH motif (D1316A), or for the Arg in the middle of the QRxGRxGR motif known for RNA binding (R1490A). Histidine-tagged recombinant proteins of Mr 54,000 were expressed in Escherichia coli and purified by chromatography on nickel agarose. All three mutants were severely defective in ATPase and RNA helicase activities, but loss of the ATPase activity was not dependent on polynucleotide cofactors. With the exception of R1490A mutant, a stable complex was formed between dsRNA substrates and recombinant proteins, indicating that the arginine-rich motif is required for efficient RNA binding. Complex formation was not affected by omission of ATP or substitution by a non-hydrolyzable analog AMP-PCP, suggesting that neither binding nor hydrolysis of ATP is required for RNA binding. Moreover, the K1236A mutant which was defective in binding ATP exhibited an unusually strong affinity for RNA duplex. These results suggest that the conserved motifs cooperatively constitute a large functional domain rather than act as individual domains with strictly independent functions, and that alteration of one motif affects functions of other motifs in a mutually interactive fashion.

Adenosine Triphosphatases↗