scholarly journals Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase.

1995 ◽  
Vol 15 (10) ◽  
pp. 5376-5388 ◽  
Author(s):  
J B Patterson ◽  
C E Samuel
Keyword(s):  
Adenosine Deaminase ◽  
Fusion Proteins ◽  
Immunoblot Analysis ◽  
Binding Activity ◽  
Human Cells ◽  
Cell Fractionation ◽  
Double Stranded Rna ◽  
Nuclear Extracts ◽  
Dsrna Binding ◽  
Ifn Treatment

A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.

scholarly journals A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus

2019 ◽  
Vol 48 (1) ◽  
pp. 304-315 ◽  
Author(s):  
Guifang Chen ◽  
Li-Chung Ma ◽  
Shanshan Wang ◽  
Ryan L Woltz ◽  
Emily M Grasso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Binding Activity ◽  
Restriction Factor ◽  
Amino Acid Residues ◽  
Ns1 Protein ◽  
Double Stranded Rna ◽  
Host Restriction ◽  
Host Restriction Factor ◽  
Dsrna Binding

Abstract Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

scholarly journals The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification.

1994 ◽  
Vol 14 (8) ◽  
pp. 5425-5432 ◽  
Author(s):  
L Saccomanno ◽  
B L Bass
Keyword(s):  
Adenosine Deaminase ◽  
Rna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Meiotic Maturation ◽  
Protection Factor ◽  
Independent Manner ◽  
Double Stranded Rna ◽  
Dsrna Binding

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

scholarly journals Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3.

1988 ◽  
Vol 8 (1) ◽  
pp. 273-283 ◽  
Author(s):  
L A Schiff ◽  
M L Nibert ◽  
M S Co ◽  
E G Brown ◽  
B N Fields
Keyword(s):  
Transcription Factor ◽  
Capsid Protein ◽  
Binding Activity ◽  
Zinc Binding ◽  
Outer Capsid Protein ◽  
Double Stranded Rna ◽  
Transcription Factor Iiia ◽  
Terminal Fragment ◽  
Dsrna Binding ◽  
Outer Capsid

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

scholarly journals Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

1995 ◽  
Vol 15 (3) ◽  
pp. 1389-1397 ◽  
Author(s):  
M A O'Connell ◽  
S Krause ◽  
M Higuchi ◽  
J J Hsuan ◽  
N F Totty ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Amino Acid Level ◽  
Brain Slices ◽  
Nuclear Targeting ◽  
Binding Motifs ◽  
Double Stranded Rna ◽  
Dsrna Binding ◽  
Calf Thymus ◽  
Adenosine Deaminase Activity ◽  
Targeting Signal

Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain.

scholarly journals Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

2010 ◽  
Vol 84 (6) ◽  
pp. 3004-3015 ◽  
Author(s):  
Kathleen C. Prins ◽  
Sebastien Delpeut ◽  
Daisy W. Leung ◽  
Olivier Reynard ◽  
Valentina A. Volchkova ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Ebola Virus ◽  
Structural Features ◽  
Binding Activity ◽  
Mutant Virus ◽  
Wild Type ◽  
Minimal Growth ◽  
Double Stranded Rna ◽  
Dsrna Binding

ABSTRACT Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-α/β responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-α/β production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

scholarly journals Mechanism of Interferon Action: Double-Stranded RNA-Specific Adenosine Deaminase from Human Cells Is Inducible by Alpha and Gamma Interferons

Virology ◽  
1995 ◽  
Vol 210 (2) ◽  
pp. 508-511 ◽  
Author(s):  
John B. Patterson ◽  
Daniel C. Thomis ◽  
Sherrie L. Hans ◽  
Charles E. Samuel
Keyword(s):  
Adenosine Deaminase ◽  
Human Cells ◽  
Double Stranded Rna

scholarly journals Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

2006 ◽  
Vol 80 (11) ◽  
pp. 5168-5178 ◽  
Author(s):  
Washington B. Cárdenas ◽  
Yueh-Ming Loo ◽  
Michael Gale ◽  
Amy L. Hartman ◽  
Christopher R. Kimberlin ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Binding Activity ◽  
Wild Type ◽  
Antagonist Activity ◽  
Double Stranded Rna ◽  
Beta Interferon ◽  
Double Stranded Dna ◽  
Dsrna Binding ◽  
Alpha Beta

ABSTRACT The Ebola virus (EBOV) VP35 protein blocks the virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor critical for the induction of alpha/beta interferon (IFN-α/β) expression. However, the mechanism(s) by which this blockage occurs remains incompletely defined. We now provide evidence that VP35 possesses double-stranded RNA (dsRNA)-binding activity. Specifically, VP35 bound to poly(rI) · poly(rC)-coated Sepharose beads but not control beads. In contrast, two VP35 point mutants, R312A and K309A, were found to be greatly impaired in their dsRNA-binding activity. Competition assays showed that VP35 interacted specifically with poly(rI) · poly(rC), poly(rA) · poly(rU), or in vitro-transcribed dsRNAs derived from EBOV sequences, and not with single-stranded RNAs (ssRNAs) or double-stranded DNA. We then screened wild-type and mutant VP35s for their ability to target different components of the signaling pathways that activate IRF-3. These experiments indicate that VP35 blocks activation of IRF-3 induced by overexpression of RIG-I, a cellular helicase recently implicated in the activation of IRF-3 by either virus or dsRNA. Interestingly, the VP35 mutants impaired for dsRNA binding have a decreased but measurable IFN antagonist activity in these assays. Additionally, wild-type and dsRNA-binding-mutant VP35s were found to have equivalent abilities to inhibit activation of the IFN-β promoter induced by overexpression of IPS-1, a recently identified signaling molecule downstream of RIG-I, or by overexpression of the IRF-3 kinases IKKε and TBK-1. These data support the hypothesis that dsRNA binding may contribute to VP35 IFN antagonist function. However, additional mechanisms of inhibition, at a point proximal to the IRF-3 kinases, most likely also exist.

scholarly journals The Properties of a tRNA-Specific Adenosine Deaminase from Drosophila melanogaster Support an Evolutionary Link between Pre-mRNA Editing and tRNA Modification

2000 ◽  
Vol 20 (3) ◽  
pp. 825-833 ◽  
Author(s):  
Liam P. Keegan ◽  
André P. Gerber ◽  
Jim Brindle ◽  
Ronny Leemans ◽  
Angela Gallo ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Adenosine Deaminase ◽  
Mrna Editing ◽  
Double Stranded Rna ◽  
Adenosine Deaminases ◽  
Binding Domains ◽  
Dsrna Binding ◽  
The Central Nervous System ◽  
Chromosome Ii ◽  
Deaminase Domain

ABSTRACT Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned fromSaccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastorisand purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system.

Distinct binding sites for zinc and double-stranded RNA in the reovirus outer capsid protein sigma 3

1988 ◽  
Vol 8 (1) ◽  
pp. 273-283
Author(s):  
L A Schiff ◽  
M L Nibert ◽  
M S Co ◽  
E G Brown ◽  
B N Fields
Keyword(s):  
Transcription Factor ◽  
Capsid Protein ◽  
Binding Activity ◽  
Zinc Binding ◽  
Outer Capsid Protein ◽  
Double Stranded Rna ◽  
Transcription Factor Iiia ◽  
Terminal Fragment ◽  
Dsrna Binding ◽  
Outer Capsid

By atomic absorption analysis, we determined that the reovirus outer capsid protein sigma 3, which binds double-stranded RNA (dsRNA), is a zinc metalloprotein. Using Northwestern blots and a novel zinc blotting technique, we localized the zinc- and dsRNA-binding activities of sigma 3 to distinct V8 protease-generated fragments. Zinc-binding activity was contained within an amino-terminal fragment that contained a transcription factor IIIA-like zinc-binding sequence, and dsRNA-binding activity was associated with a carboxy-terminal fragment. By these techniques, new zinc- and dsRNA-binding activities were also detected in reovirus core proteins. A sequence similarity was observed between the catalytic site of the picornavirus proteases and the transcription factor IIIA-like zinc-binding site within sigma 3. We suggest that the zinc- and dsRNA-binding activities of sigma 3 may be important for its proposed regulatory effects on viral and host cell transcription and translation.

The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification

1994 ◽  
Vol 14 (8) ◽  
pp. 5425-5432
Author(s):  
L Saccomanno ◽  
B L Bass
Keyword(s):  
Adenosine Deaminase ◽  
Rna Binding ◽  
Binding Protein ◽  
Protein S ◽  
Meiotic Maturation ◽  
Protection Factor ◽  
Independent Manner ◽  
Double Stranded Rna ◽  
Dsrna Binding

Here we describe studies of double-stranded RNA (dsRNA) adenosine deaminase in Xenopus laevis, in particular during meiotic maturation, the period during which a stage VI oocyte matures to an egg. We show that dsRNA adenosine deaminase is in the nuclei of stage VI oocytes. Most importantly, we demonstrate that the cytoplasm of stage VI oocytes contains a factor that protects microinjected dsRNA from deamination when dsRNA adenosine deaminase is released from the nucleus during meiotic maturation. Our data suggest that the protection factor is a cytoplasmic dsRNA-binding protein or proteins that bind to dsRNA in a sequence-independent manner to occlude dsRNA from binding to dsRNA adenosine deaminase. The cytoplasmic double-stranded RNA-binding protein(s) does not bind to other nucleic acids and can be titrated at high concentrations of dsRNA. These studies raise the question of whether all dsRNA-binding proteins share endogenous substrates and also suggest potential means of regulating dsRNA adenosine deaminase in vivo.

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