Synthetic dsRNA Research Articles

Suppression of nitric oxide (NO)-dependent behavior by double-stranded RNA-mediated silencing of a neuronal NO synthase gene.

We have used double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) to disrupt neuronal nitric oxide (NO) synthase (nNOS) gene function in the snail Lymnaea stagnalis and have detected a specific behavioral phenotype. The injection of whole animals with synthetic dsRNA molecules targeted to the nNOS-encoding mRNA reduces feeding behavior in vivo and fictive feeding in vitro and interferes with NO synthesis by the CNS. By showing that synthetic dsRNA targeted to the nNOS mRNA causes a significant and long-lasting reduction in the levels of Lym-nNOS mRNA, we verify that specific RNAi has occurred. Importantly, our results establish that the expression of nNOS gene is essential for normal feeding behavior. They also show that dsRNA can be used in the investigation of functional gene expression in the context of whole animal behavior, regardless of the availability of targeted mutation technologies.

Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development.

Th cell polarization toward Th1 or Th2 cells is strongly driven by exogenous cytokines, in particular IL-12 or IL-4, if present during activation by Ag-presenting dendritic cells (DC). However, additional Th cell polarizing mechanisms are induced by the ligation of cell surface molecules on DC and naive Th cells. In the present study, the role of LFA-1/ICAM-1 ligation in human Th cell polarization was investigated. Triggering of LFA-1 on anti-CD3/CD28 stimulated naive Th cells with immobilized Fc-ICAM-1, in the absence of DC and exogenous cytokines, induced a marked shift toward Th1 cell development, accompanied by a dose-dependent decrease in GATA-3 expression and a dose-dependent increase in T-bet expression. Th1 polarization by LFA-1 ligation could be demonstrated only under low cytokine conditions, as it was largely overruled by IL-12 or IL-4. This IL-12-independent Th1-driving mechanism appears to be operated by certain subsets of effector DC. Maturation of DC by poly(I:C), a synthetic dsRNA, used as an in vitro model for viral infections, leads to the generation of Th1-driving effector DC (DC1), which express elevated levels of ICAM-1 but produce only low levels of IL-12p70. Blocking the ICAM-1/LFA-1 interaction in cocultures of these DC with naive Th cells attenuated their Th1-driving capacity. The molecular mechanism by which LFA-1 signaling supports Th1 differentiation is blocked by specific inhibitors of extracellular signal-regulated kinase phosphorylation. The present data indicate the existence of an IL-12-independent, extracellular signal-regulated kinase-mediated mechanism, through which high ICAM-1-expressing DC1 can drive Th1 polarization. This mechanism may be operational during viral infections.

Short 5′-phosphorylated double-stranded RNAs induce RNA interference in Drosophila

Double-stranded (ds) RNA causes the specific degradation of homologous RNAs in a process called “RNA interference (RNAi)” [1–4]; this process is called “posttranscriptional gene silencing (PTGS)” in plants [5–7]. Both classes of gene silencing have been reviewed extensively [8–13]. The duplex RNA becomes processed by Dicer [14] or another RNase III-like enzyme to short dsRNA fragments of about 21–23 nucleotides (nt) [15], which are incorporated in the RNA-induced silencing complex (RISC) [16] that directs target-specific RNA degradation [17, 18]. Here, we show that different synthetic dsRNA cassettes, consisting of two 5′-phosphorylated RNA strands of 22 nt each, can initiate RNAi in Drosophila embryos. The cassettes were active at similar quantities required to initiate RNAi by conventional dsRNA. Their sequence specificity was confirmed using synthetic dsRNA cassettes for two different genes, Notch and hedgehog; each time, only the relevant embryonic phenotype was observed. Introduction of point mutations had only a moderate effect on the silencing potential, indicating that the silencing machinery does not require perfect sequence identity. 5′-phosphorylated synthetic RNA was more active than its hydroxylated form. Substitution of either RNA strand by DNA strongly reduced activity. Synthetic cassettes of siRNA will provide a new tool to induce mutant phenotypes of genes with unknown function.

Mechanisms of β-Cell Death in Response to Double-Stranded (ds) RNA and Interferon-γ: dsRNA-Dependent Protein Kinase Apoptosis and Nitric Oxide-Dependent Necrosis

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate beta-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs beta-cell function and induces beta-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates beta-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-gamma stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-gamma on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and N(G)-monomethyl-L-arginine, attenuate poly IC + IFN-gamma-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. N(G)-monomethyl-L-arginine fails to prevent poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-gamma-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-gamma is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-gamma-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway.

Pancreatic β-Cell Damage Mediated by β-Cell Production of Interleukin-1: A NOVEL MECHANISM FOR VIRUS-INDUCED DIABETES

Viral infection is one environmental factor that may initiate beta-cell damage during the development of autoimmune diabetes. Formed during viral replication, double-stranded RNA (dsRNA) activates the antiviral response in infected cells. In combination, synthetic dsRNA (polyinosinic-polycytidylic acid, poly(I-C)) and interferon (IFN)-gamma stimulate inducible nitric-oxide synthase (iNOS) expression, inhibit insulin secretion, and induce islet degeneration. Interleukin-1 (IL-1) appears to mediate dsRNA + IFN-gamma-induced islet damage in a nitric oxide-dependent manner, as the interleukin-1 receptor antagonist protein prevents dsRNA + IFN-gamma-induced iNOS expression, inhibition of insulin secretion, and islet degeneration. IL-1beta is synthesized as an inactive precursor protein that requires cleavage by the IL-1beta-converting enzyme (ICE) for activation. dsRNA and IFN-gamma stimulate IL-1beta expression and ICE activation in primary beta-cells, respectively. Selective ICE inhibition attenuates dsRNA + IFN-gamma-induced iNOS expression by primary beta-cells. In addition, poly(I-C) + IFN-gamma-induced iNOS expression and nitric oxide production by human islets are prevented by interleukin-1 receptor antagonist protein, indicating that human islets respond to dsRNA and IFN-gamma in a manner similar to rat islets. These studies provide biochemical evidence for a novel mechanism by which viral infection may initiate beta-cell damage during the development of autoimmune diabetes. The viral replicative intermediate dsRNA stimulates beta-cell production of pro-IL-1beta, and following cleavage to its mature form by IFN-gamma-activated ICE, IL-1 then initiates beta-cell damage in a nitric oxide-dependent fashion.

The plasmid pcDNA3 differentially induces production of interferon-α and interleukin-6 in cultures of porcine leukocytes

An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides. Here we describe the interferon-α (IFN-α) and interleukin-6 (IL-6) inducing properties of a commonly used eukaryotic expression vector, pcDNA3, in porcine leukocytes. The magnitude of the cytokine response was compared to that induced by the synthetic ds RNA analogue poly(I):poly(C), inactivated preparations of Aujeszky’s disease virus (ADV) and the Gram-negative bacteria Actinobacillus pleuropneumoniae. The plasmid, as well as poly(I):poly(C), required lipofectin to induce IFN-α production whereas both preparations induced IL-6 irrespective of preincubation with lipofectin. However, the nucleic acid-induced levels of IL-6 were low compared to those induced by A. pleuropneumoniae. The IFN-α response elicited by pcDNA3 in the presence of lipofectin was as high as, or higher than that induced by ADV. Interestingly, also A. pleuropneumoniae induced a substantial production of IFN-α when preincubated with lipofectin. Plasmid expression was not necessary for induction of IFN-α. Furthermore, the IFN-α inducing capacity of pcDNA3 was not reduced when the two predicted immunostimulatory sequences 5′AACGTT3′ were deleted. Nor did the ability of the plasmid to induce IFN-α production decrease when the ampicillin resistance (ampR) gene was replaced with the kanamycin resistance (kanR) gene. However, methylation of all cytidines in CpG dinucleotides of pcDNA3 abolished the IFN-α inducing capacity. These in vitro results indicate an immunomodulatory role of bacterial DNA also in the pig. Unmethylated CpG dinucleotides are crucial for induction of IFN-α by the plasmid, but other CpG motifs than those within the 5′AACGTT3′ sequences of the ampR gene contribute to this induction in porcine cells.

Chimeric double-stranded RNA-specific adenosine deaminase ADAR1 proteins reveal functional selectivity of double-stranded RNA-binding domains from ADAR1 and protein kinase PKR.

The RNA-specific adenosine deaminase (ADAR1) and the RNA-dependent protein kinase (PKR) are both interferon-inducible double-stranded (ds) RNA-binding proteins. ADAR1, an RNA editing enzyme that converts adenosine to inosine, possesses three copies of a dsRNA-binding motif (dsRBM). PKR, a regulator of translation, has two copies of the highly conserved dsRBM motif. To assess the functional selectivity of the dsRBM motifs in ADAR1, we constructed and characterized chimeric proteins in which the dsRBMs of ADAR1 were substituted with those of PKR. Recombinant PKR-ADAR1 chimeras retained significant RNA adenosine deaminase activity measured with a synthetic dsRNA substrate when the spacer region between the RNA-binding and catalytic domains of the deaminase was exactly preserved. However, with natural substrates, substitution of the first two dsRBMs of ADAR1 with those from PKR dramatically reduced site-selective editing activity at the R/G and (+)60 sites of the glutamate receptor B subunit pre-RNA and completely abolished editing of the serotonin 2C receptor (5-HT(2C)R) pre-RNA at the A site. Chimeric deaminases possessing only the two dsRBMs from PKR were incapable of editing either glutamate receptor B subunit or 5-HT(2C)R natural sites but edited synthetic dsRNA. Finally, RNA antagonists of PKR significantly inhibited the activity of chimeric PKR-ADAR1 proteins relative to wild-type ADAR1, further demonstrating the functional selectivity of the dsRBM motifs.

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

DADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated.

Induction of Mx protein by interferon and double-stranded RNA in salmonid cells

Mx protein is one of several antiviral proteins that are induced by the type I interferons (IFN), IFN α and β, in mammals. In this work induction of a 76kDa Mx protein by double-stranded RNA (dsRNA) or type I IFN-like activity in Atlantic salmon macrophages, Atlantic salmon fibroblast cells (AS cells) and in Chinook salmon embryo cells (CHSE-214) is reported. Type I IFN-like activity was produced by the stimulation of Atlantic salmon macrophages with the synthetic dsRNA polyinosinic polycytidylic acid (poly I:C). A correlation appeared to exist between Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) induced by IFN in CHSE-214 cells. Several observations in the present work suggest that, as in mammals, the induction of Mx protein by dsRNA in fish cells primarily occurs via induction of type I IFN. First, type I IFN-like activity but not poly I:C, induced Mx protein expression in CHSE-214 cells. These cells apparently lack the ability to produce IFN in response to poly I:C. Second, the putative IFN induced maximal Mx protein expression 48h earlier than poly I:C in AS cells. Third, the peak expression of Mx protein in macrophages induced by poly I:C occurred after 48h whereas peak in IFN-like activity was observed by 24h after addition of poly I:C. The present work supports the notion of using Mx protein as a molecular marker for the production of putative type I IFN in fish.

Viral Double-Stranded RNA, Cytokines, and the Flu

The symptoms of the flu, such as fever, drowsiness, and malaise, are the sole means by which this common clinical syndrome is defined. The syndrome is usually the first clinical manifestation of both acute bacterial and viral infections. In the case of acute bacterial infections, several proinflammatory cytokines induced by bacterial products have been implicated as the causative agents of the flu syndrome. Viruses induce similar cytokines to bacteria, plus substantial amounts of interferon-alpha (IFN-alpha), although the direct association of these cytokines with the viral flu syndrome is less clear. Furthermore, the viral inducer(s) of cytokines has not been defined. The best candidate cytokine inducer associated with a majority of viral infections is virus-associated double-stranded RNA (dsRNA). This review examines the essential physical properties of toxic dsRNA, the cytokines induced by it, its viral and cellular sources, evidence for its presence in infected cells, its quantities in normal and infected cells, its cytotoxic mechanisms, and its cell-penetration properties. Toxic effects of viruses and dsRNA are compared. Energetics and extraction artifact issues are also discussed. Whereas most research on dsRNA toxicity has employed synthetic dsRNA, studies with virus-associated dsRNA are featured when available. Finally, a model for how viral dsRNA might initiate systemic disease is presented.

Interferon-gamma improves muscle flap microcirculation in double-strand RNA-induced inflammation.

Endothelial cell (EC) activation and subsequent expression of leukocyte adhesion molecules are initial events in multiple pathological processes. Viral double-strand ribonucleic acid (dsRNA) induces EC adhesion protein expression and leukocyte adhesion in vitro. Interferon-gamma (IFN-gamma) has been demonstrated to modulate the expression of certain adhesion proteins. The purpose of this study was to measure the inflammatory response to a viral mimetic--a synthetic dsRNA, polyinosinic-polycytidylic acid (poly-I:C)-on the microcirculation of a muscle flap in a rat model and to determine whether IFN-gamma attenuated the response. Two-stage surgery to create a cremaster muscle end-organ tube flap was performed on 18 male Sprague-Dawley rats in three groups. After intra-arterial injection into the abdominal aorta, the reagents (phosphate-buffered saline-bovine serum albumin [PBS-BSA] in groups I and II, and IFN-gamma in group III) were kept for 1 hour in this end-organ system. During the second stage at 16 hours, after injection into the penile vein (PBS-BSA in group I, poly-I:C in groups II and III), the flap was prepared for intravital microscopic measurement. The following parameters were measured: red blood cell velocity; vessel diameter; number of functional capillaries; and number of rolling, sticking, and transmigrating neutrophils and lymphocytes. Wilcoxon's rank sum test was used for statistical comparison. Poly-I:C caused a 70% increase in the main artery diameter and a 7% increase in velocity. But as a consequence of dynamic activation of leukocyte interaction, a 30% drop in functional capillary perfusion was observed. Injury to the entire vascular endothelium was confirmed by a 160% increase in transmigrating leukocytes. Treatment with IFN-gamma inhibited the poly-I:C-induced inflammation, as shown by 88%, 63%, and 85% decreases in rolling, sticking, and transmigrating leukocytes respectively, and by a 28% increase in capillary perfusion. Treating the system with IFN-gamma in advance, inhibited poly-I:C-induced inflammation, shown by marked decreases in rolling, adhering, and transmigrating leukocytes, and a notable increase in perfused capillaries. These observations reflect an inhibitory effect of IFN-gamma on leukocyte adhesion molecule expression in vascular endothelium in response to dsRNA in a muscle flap at the microcirculatory level.

Polyriboinosinic Polyribocytidylic Acid (Poly(I:C)) Induces Stable Maturation of Functionally Active Human Dendritic Cells

For vaccination strategies and adoptive immunotherapy purposes, immature dendritic cells (DC) can be generated from adherent monocytes using GM-CSF and IL-4. Presently, the only clinically applicable method to induce stable maturation of DC is the use of supernatants of activated monocytes (monocyte-conditioned medium (MCM)). MCM contains an undefined mixture of cytokines and is difficult to standardize. Here we report that stable maturation of DC can be simply induced by the addition of polyriboinosinic polyribocytidylic acid (poly(I:C)), a synthetic dsRNA clinically applied as an immunomodulator. Poly(I:C)-treated DC show a mature phenotype with high expression levels of HLA-DR, CD86, and the DC maturation marker CD83. This mature phenotype is retained for 48 h after cytokine withdrawal. In contrast to untreated DC, poly(I:C)-treated DC down-regulate pinocytosis, produce high levels of IL-12 and low levels of IL-10, induce strong T cell proliferation in a primary allo MLR, and effectively present peptide Ags to HLA class I-restricted CTL. In conclusion, we present a simple methodology for the preparation of clinically applicable mature DC.

Serotonin-2C Receptor Pre-mRNA Editing in Rat Brain andin Vitro by Splice Site Variants of the Interferon-inducible Double-stranded RNA-specific Adenosine Deaminase ADAR1

The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat serotonin-2C receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5-HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 revealed a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre-mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.

Activation of the Interferon-Inducible (2′-5′) Oligoadenylate Synthetase by the Epstein–Barr Virus RNA, EBER-1

The 2′-5′ oligoadenylate synthetases and the protein kinase PKR are both interferon-induced, double-stranded RNA-dependent proteins that play important roles in the antiviral effects of the interferons and in cellular growth control. Both enzymes are activated by natural or synthetic dsRNAs and by single-stranded RNAs that possess extensive secondary structure. This report describes the effects of the small Epstein–Barr virus-encoded RNA EBER-1 on the regulation of 2–5(A) synthetase activity. We demonstrate that EBER-1 RNA binds to and activates the human 40-kDa 2–5(A) synthetase in a dose-dependent manner. The efficiency of EBER-1 as an activator of 2–5(A) synthetase is approximately 25% of that of the synthetic double-stranded RNA poly(I)/poly(C), and poly(I)/poly(C) further stimulates enzyme activity even in the presence of a high concentration of EBER-1. Conversely, EBER-1 neither stimulates nor inhibits 2–5(A) synthetase that has been activated by a high concentration of poly(I)/poly(C). Competitive binding assays suggest that the relative affinity of the enzyme for poly(I)/poly(C) is considerably higher than that for EBER-1. Our data indicate that EBER-1, like VAI RNA of adenovirus, TAR RNA of HIV-1, and Rex-RE RNA of HTLV-1, is able to activate the 2–5(A) synthetases. The significance of why several viruses may activate the 2–5(A) synthetase/RNase L-mediated RNA degradation pathway is discussed.

Synthetic influenza viral double-stranded RNA induces an acute-phase response in rabbits

Numerous studies have characterized the physiological effects of synthetic, high-molecular-weight, homopolymeric, double-stranded RNA (dsRNA), particularly polyriboinosinic.polyribocytidylic acid [Carter and De Clercq (1974): Science 186:1172-1178], but limited information exists regarding the physiological effects of dsRNA of viral composition and size. In this report, we determined sleep and fever responses of rabbits to intracerebroventricular injection of different doses of synthetic viral dsRNA (either 108 base pairs or 661 base pairs) derived from the N-terminal sequence of gene segment 3 of the A/PR/8/34-H1N1 (PR8) influenza virus. Both the108-mer and the 661-mer dsRNAs increased nonrapid eye movement sleep, suppressed rapid eye movement sleep, and induced fever. The 661-mer dsRNA had more potent somnogenic and pyrogenic effects than the 108-mer dsRNA on the basis of weight. Neither single-stranded RNA from the corresponding sequences had significant effects on sleep or brain temperature. These results demonstrate for the first time that low-molecular-weight, viral dsRNA has the stability in vivo that is required to induce the fever and sleep changes found in natural viral infections, and the hypothesis is supported that virus-associated dsRNA may be responsible for initiating the acute-phase response during viral infections.

The RNase L inhibitor (RLI) is induced by double-stranded RNA.

The (2-5A)-RNase L pathway is an important component of interferon (IFN) action. Its central role in the antiviral effect of IFN against Picornaviridae has been clearly demonstrated. We have characterized and cloned a new component of this pathway, the RNase L inhibitor (RLI). RLI is a cellular protein whose mRNA is not regulated by IFN but is induced by viruses, such as encephalomyocarditis virus (EMCV). RLI inhibits RNase L during the time course of EMCV infection, and overexpression of RLI in HeLa cells partially reverses the antiviral action of IFN against EMCV. The replicative complexes of several viruses consist of double-stranded RNA structures. These dsRNAs could activate gene transcription as demonstrated for IFNs and could be responsible for RLI induction. We describe the increased expression of RLI mRNA and RLI protein induced by synthetic dsRNAs, such as poly(I):poly(C). This induction gives rise to an inhibition of the 2-5A-binding activity of RNase L. The inhibition of RNase L activity is transcient, probably due to the rapid turnover of RLI protein.

Double-Stranded RNA-Specific Adenosine Deaminase: Nucleic Acid Binding Properties

The RNA-specific adenosine deaminase (ADAR1, herein referred to as ADAR) is an interferon-inducible RNA-editing enzyme. ADAR catalyzes the C-6 deamination of adenosine in double-stranded (ds) structures present in viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates. ADAR possesses three functionally distinct copies of the highly conserved double-stranded RNA binding R motif (RI, RII, RIII) implicated in the recognition of dsRNA structures within the substrate RNAs. ADAR is also a Z-DNA-binding protein. Two Z-DNA binding motifs (Zα and Zβ) present in ADAR correspond to repeated regions homologous to the N-terminal region of the vaccinia virus E3L protein. Here we describe assay methods for measurement of ADAR enzymatic activity, dsRNA binding activity, and Z-DNA binding activity.

Loss ability to produce IFN-alpha in response to HIV-1 as monocytes differentiate into macrophages. Induction through a mechanism independent of double-stranded RNA.

Abstract IFN-alpha is an antiviral cytokine detected in plasma of HIV-1-infected patients during acute viremia and during late-stage disease. Monocytes produced IFN-alpha in response to HIV-1:1) IFN-alpha was produced predominantly by adherent cells; 2) depleting CD14+ cells nearly abolished HIV-1-induced IFN-alpha production; and 3) intracytoplasmic IFN-alpha was detected in CD14+ cells. During cell culture, monocytes differentiated into macrophages and lost their ability to produce IFN-alpha when challenged with HIV-1. These cells remained capable of producing IFN-alpha in response to other stimuli such as poly(I:C), a synthetic dsRNA. Thus, we examined two negative-stranded RNA viruses that have dsRNA intermediates, Newcastle disease virus and Sendai virus, and a DNA virus, herpes simplex virus type I (HSV-1). Macrophages lost their ability to produce IFN-alpha in response to HSV-1, but not to Sendai virus or to Newcastle disease virus. Thus, HIV-1 and other viruses were capable of inducing IFN-alpha through a mechanism that was independent of dsRNA. In conclusion, these data suggest that there are dsRNA-dependent and -independent mechanisms for the induction of IFN-alpha production, and that as monocytes differentiate into macrophages, they selectively lose their ability to produce IFN-alpha through the dsRNA-independent mechanism.

Differential up-regulation of HLA class I molecules on neuronal and glial cell lines by virus infection correlates with differential induction of IFN-beta.

Adult neurons normally lack the expression of MHC class I molecules, which has implications on virus clearance from the central nervous system. The author previously demonstrated that HLA class I up-regulation in measles virus (MV)-infected glial cells is primarily mediated by IFN-beta. In contrast, this study demonstrates that MV-infection of the neuronal cell lines IMR-32 and CHP-126 fails to up-regulate HLA class I expression, which was associated with an inability of MV to induce IFN-beta in the neuronal cell lines. However, treatment with IFN-beta on coculture of the IMR-32 neuronal cell line with MV-infected glioma cells resulted in the up-regulation of HLA class I on the former, which could be neutralized by anti-IFN-beta Ab. The inability of MV to up-regulate HLA class I expression on the neuronal cell line IMR-32 was not virus specific because similar findings were observed with mumps virus or stimulation with the synthetic dsRNA polyinosinic polycytidylic acid (PIPC). Induction of IFN-beta gene expression by virus requires binding of NF-kappa B to the positive regulatory domain II element of the IFN-beta promoter. Our studies indicate that MV, TNF-alpha, or PIPC induces NF-kappa B (p50 and p65 subunits) binding to positive regulatory domain II in the glioma cell line. In contrast, such activity was induced by TNF-alpha but not MV or PIPC in the neuronal cell line IMR-32. This indicated that HLA class I expression is differentially regulated in glial and neuronal cell lines in response to MV, which correlates with differential binding of NF-kappa B to the IFN-beta promoter and induction of IFN-beta gene expression.