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PubMed2004

The exonuclease ISG20 is directly induced by synthetic dsRNA via NF-kappaB and IRF1 activation.

Many interferon (IFN)-stimulated genes are also induced by double-stranded RNA (dsRNA), a component closely associated with the IFN system in the context of virus-host interactions. Recently, we demonstrated that the IFN-induced 3' --> 5' exonuclease ISG20 possesses antiviral activities against RNA viruses. Here we show that ISG20 induction by synthetic dsRNA (pIpC) is stronger and faster than its induction by IFN. Two families of transcription factors are implicated in the transcriptional activation of ISG20 by dsRNA. Initially, the NF-kappaB factors p50 and p65 bind and activate the kappaB element of the Isg20 promoter. This is followed by IRF1 binding to the ISRE. As pIpC often induces protein movements in the cells, we questioned whether it could influence ISG20 localization. Interestingly and contrary to IFN, dsRNA induces a nuclear matrix enrichment of the ISG20 protein. dsRNA induction of ISG20 via NF-kappaB and its antiviral activity led us to suggest that ISG20 could participate in the cellular response to virus infection.

Binding Sites↗
PubMed1994

Interferon-stimulated response element and NF kappa B sites cooperate to regulate double-stranded RNA-induced transcription of the IP-10 gene.

To understand the mechanisms involved in dsRNA-induced gene expression, we analyzed the poly(I/C)-induced transcription of the IFN-inducible chemokine gene IP-10 using the GRE cell line in which type I IFN genes have been deleted. Accumulation of IP-10 mRNA in GRE cells was more strongly stimulated by treatment with dsRNA than by IFN-alpha or IFN-gamma and was independent of protein synthesis. This same pattern of response was produced when GRE cells were transiently transfected with a plasmid containing 243 bases of sequence from the promoter of the murine IP-10 gene linked to the chloramphenicol acetyltransferase reporter gene. Deletion- and site-specific mutagenesis of the 243 base pair fragment indicated that an ISRE located between residues -204 and -228 was a primary target site for the action of dsRNA on this promoter. This was confirmed by results showing that two copies of this ISRE tandemly arrayed in front of the thymidine kinase promoter were able to mediate reporter gene transcription in dsRNA-stimulated cells. At least one of the two NF kappa B binding sites present in the 243 base pair IP-10 promoter is also necessary for response to dsRNA; mutation of both sites eliminates promoter activity. Thus the ISRE and one NF kappa B site cooperate to produce transcriptional response to dsRNA.

Base Sequence↗
PubMed1988

Viroid-induced phosphorylation of a host protein related to a dsRNA-dependent protein kinase.

Viroids are very small, unencapsidated RNAs that replicate and induce severe disease in plants without encoding for any proteins. The mechanisms by which the viroid RNA regulates these events and interacts with host factors are unknown. An Mr 68,000 host-encoded protein has been identified that is differentially phosphorylated in extracts from viroid-infected and mock-inoculated tissues. This phosphoprotein is immunologically related to a double-stranded (ds) RNA-dependent protein kinase from virus-infected, interferon-treated human cells. Further, nucleotide photoaffinity labeling indicates that the protein has an ATP binding site. This protein is similar to dsRNA-dependent protein kinases implicated in mammalian systems in the regulation of protein synthesis and virus replication.

Molecular Weight↗
PubMed1989

Double-stranded RNA activates binding of NF-kappa B to an inducible element in the human beta-interferon promoter.

The human beta-interferon promoter contains at least two positive acting domains (PRD I and PRD II). PRD I has been previously shown to stimulate basal transcription and to respond to induction by double-stranded RNA (dsRNA). Here we show that PRD II functions independently as a constitutive element that also responds to induction. A cellular factor that specifically binds to PRD II has been identified, and the levels of this factor increase markedly in extracts from cells treated with dsRNA. The inducible factor has a binding specificity that is indistinguishable from the transcription factor NF-kappa B. As has been shown for NF-kappa B, the PRD II-specific factor can be activated in uninduced extracts by treatment with detergent, suggesting that the inactive state is due to association with an inhibitory factor. Induction by dsRNA therefore provides a novel means for the post-translational activation of NF-kappa B. Potential binding sites for NF-kappa B are present in the 5' flanking regions of a number of genes involved in the immune response, several of which are inducible by dsRNA. These findings demonstrate a role for NF-kappa B in the physiological activation of genes in non-lymphoid cells.

Animals↗
PubMed2000

Gill-associated virus of Penaeus monodon prawns: an invertebrate virus with ORF1a and ORF1b genes related to arteri- and coronaviruses.

A 20089 nucleotide (nt) sequence was determined for the 5' end of the (+)-ssRNA genome of gill-associated virus (GAV), a yellow head-like virus infecting Penaeus monodon prawns. Clones were generated from a approximately 22 kb dsRNA purified from lymphoid organ total RNA of GAV-infected prawns. The region contains a single gene comprising two long overlapping open reading frames, ORF1a and ORF1b, of 4060 and 2646 amino acids, respectively. The ORFs are structurally related to the ORF1a and ORF1ab polyproteins of coronaviruses and arteriviruses. The 99 nt overlap between ORF1a and ORF1b contains a putative AAAUUUU 'slippery' sequence associated with -1 ribosomal frameshifting. A 131 nt stem-loop with the potential to form a complex pseudoknot resides 3 nt downstream of this sequence. Although different to the G/UUUAAAC frameshift sites and 'H-type' pseudoknots of nidoviruses, in vitro transcription/translation analysis demonstrated that the GAV element also facilitates read-through of the ORF1a/1b junction. As in coronaviruses, GAV ORF1a encodes a 3C-like cysteine protease domain located between two hydrophobic regions. However, its sequence suggests some structural relationship to the chymotrypsin-like serine proteases of arteriviruses. ORF1b encodes homologues of the 'SDD' polymerase, which among (+)-RNA viruses is unique to nidoviruses, as well as metal-ion-binding and helicase domains. The presence of a dsRNA replicative intermediate and ORF1a and ORF1ab polyproteins translated by a-1 frameshift suggests that GAV represents the first invertebrate member of the Order NIDOVIRALES:

Amino Acid Sequence↗
PubMed2002

Novel role for calcium-independent phospholipase A(2) in the macrophage antiviral response of inducible nitric-oxide synthase expression.

The double-stranded (ds) RNA-dependent protein kinase (PKR) is a primary regulator of antiviral responses; however, the ability of dsRNA to activate nuclear factor-kappa B (NF-kappa B) and dsRNA + interferon gamma (IFN-gamma) to stimulate inducible nitric-oxide synthase (iNOS) expression by macrophages isolated from PKR(-/-) mice suggests that signaling pathways in addition to PKR participate in antiviral activities. We have identified a novel phospholipid-signaling cascade that mediates macrophage activation by dsRNA and viral infection. Bromoenol lactone (BEL), a selective inhibitor of the calcium-independent phospholipase A(2) (iPLA(2)), prevents dsRNA- and virus-induced iNOS expression by RAW 264.7 cells and mouse macrophages. BEL does not modulate dsRNA-induced interleukin 1 expression, nor does it affect dsRNA-induced NF-kappa B activation. Protein kinase A (PKA) and the cAMP response element binding protein (CREB) are downstream targets of iPLA(2), because selective PKA inhibition prevents dsRNA-induced iNOS expression, and the inhibitory actions of BEL on dsRNA-induced iNOS expression are overcome by the direct activation of PKA. In addition, BEL inhibits dsRNA-induced CREB phosphorylation and CRE reporter activation. PKR does not participate in iPLA(2) activation or iNOS expression, because dsRNA stimulates iPLA(2) activity and dsRNA + IFN-gamma induces iNOS expression and nitric oxide production to similar levels by macrophages isolated from PKR(+/+) and PKR(-/-) mice. These findings support a PKR-independent signaling role for iPLA(2) in the antiviral response of macrophages.

Animals↗
PubMed1997

Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze the unwinding of double-stranded RNA substrates.

RNA-dependent ATPase and helicase activities have been identified associated with the purified VP6 protein of bluetongue virus, a member of the Orbivirus genus of double-stranded RNA (dsRNA; Reoviridae family) viruses. In addition, the protein has an ATP binding activity. RNA unwinding of duplexes occurred with both 3' and 5' overhang templates, as well as with blunt-ended dsRNA, an activity not previously identified in other viral helicases. Although little sequence similarity to other helicases was detected, certain similarities to motifs commonly attributed to such proteins were identified.

Adenosine Triphosphatases↗
PubMed1987

The double-stranded RNA-dependent protein kinase is also activated by heparin.

The double-stranded(ds)-RNA dependent protein kinase from human cells is a Mr 68,000 protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by dsRNA, the p68 kinase becomes autophosphorylated. The phosphorylated p68 kinase then can catalyze the phosphorylation of exogenous substrates, such as eIF2 and histone. The second phosphorylation step can take place in the absence of dsRNA. Here we show that, besides dsRNA other polyanions, especially heparin, can also activate the p68 kinase for the autophosphorylation reaction. Heparin activation of the p68 kinase is reversible since it can be prevented by addition of antithrombin III, heparin-binding protein. However, when antithrombin III is added after autophosphorylation of the p68 kinase then phosphorylation of histone is not affected. The p68 kinase binds to heparin-Sepharose. Further evidence that the p68 kinase can be activated by heparin was provided by photoaffinity labeling with 8-azido-[alpha-32P]ATP. This ATP analog can bind to the p68 kinase only in the presence of heparin or dsRNA. Thus suggesting that the activation of the p68 kinase triggers a conformational modification allowing the binding of ATP. Basic proteins, histone and protamine, prevent the activation process induced by heparin. This is probably due to binding of these basic proteins to heparin and thus sequestering the activator of the protein kinase.

Anions↗
PubMed2004

Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

Active Transport, Cell Nucleus↗
PubMed1994

Identification of the nucleic acid binding domain of the rotavirus VP2 protein.

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.

Animals↗
PubMed1997

Effects of varying lengths of double-stranded RNA on binding and activation of 2'-5'-oligoadenylate synthetase.

We investigated the effects of varying the length of the double-stranded (ds) RNA cofactor on activation of 2'-5'-oligoadenylate [2-5 (A)] synthetase. dsRNA of 40, 55, 67, 85, and 110 bp lengths were generated by in vitro transcription of complementary strands and annealing and trimming of the single-stranded overhangs. The dsRNA molecules were radiolabeled by polynucleotide kinase and purified by gel electrophoresis. Their abilities to bind to recombinant mouse 9-2 2-5 (A) synthetase, as monitored by electrophoretic mobility shift assays, were comparable. When used at a saturating concentration of 25 microg/ml, all dsRNA molecules were equally effective in activating the enzyme. At a subsaturating concentration, however, longer RNAs were better activators. At 100 nM concentration, there was a linear relationship between the length of the dsRNA and its ability to activate 2-5 (A) synthetase.

2',5'-Oligoadenylate Synthetase↗
PubMed1992

Atomic force microscopy imaging of double stranded DNA and RNA.

A procedure for imaging long DNA and double stranded RNA (dsRNA) molecules using Atomic Force Microscopy (AFM) is described. Stable binding of double stranded DNA molecules to the flat mica surface is achieved by chemical modification of freshly cleaved mica under mild conditions with 3-aminopropyltriethoxy silane. We have obtained striking images of intact lambda DNA, Hind III restriction fragments of lambda DNA and dsRNA from reovirus. These images are stable under repeated scanning and measured contour lengths are accurate to within a few percent. This procedure leads to strong DNA attachment, allowing imaging under water. The widths of the DNA images lie in the range of 20 to 80nm for data obtained in air with commercially available probes. The work demonstrates that AFM is now a routine tool for simple measurements such as a length distribution. Improvement of substrate and sample preparation methods are needed to achieve yet higher resolution.

Aluminum Silicates↗
PubMed1997

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase.

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zalpha) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5' to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zalpha has allowed identification of motifs common to this class of nucleic acid binding domain.

Adenosine Deaminase↗
PubMed1996

Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription.

Base Sequence↗
PubMed2001

The left-handed double helical nucleic acids.

The conversion of right-handed dsDNA and dsRNA to the left-handed Z-conformation involves a reorganization of the nucleotides relative to each other. This conversion can be facilitated by the tight binding of a Z-conformation-specific protein domain from the editing enzyme dsRNA adenosine deaminase. This may influence the modification of both pre-mRNAs as well as some replicating RNA viruses.

Adenosine Deaminase↗
PubMed2002

RNA-binding activity of the rotavirus phosphoprotein NSP5 includes affinity for double-stranded RNA.

Phosphoprotein NSP5 is a component of replication intermediates that catalyze the synthesis of the segmented double-stranded RNA (dsRNA) rotavirus genome. To study the role of the protein in viral replication, His-tagged NSP5 was expressed in bacteria and purified by affinity chromatography. In vitro phosphorylation assays showed that NSP5 alone contains minimal autokinase activity but undergoes hyperphosphorylation when combined with the NTPase and helix-destabilizing protein NSP2. Hence, NSP2 mediates the hyperphosphorylation of NSP5 in the absence of other viral or cellular proteins. RNA-binding assays demonstrated that NSP5 has unique nonspecific RNA-binding activity, recognizing single-stranded RNA and dsRNA with similar affinities. The possible functions of the RNA-binding activities of NSP5 are to cooperate with NSP2 in the destabilization of RNA secondary structures and in the packaging of RNA and/or to prevent the interferon-induced dsRNA-dependent activation of the protein kinase PKR.

Acid Anhydride Hydrolases↗
PubMed2002

Regulation of PKR and IRF-1 during hepatitis C virus RNA replication.

The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.

DNA-Binding Proteins↗
PubMed2001

The Hsp90 chaperone complex is both a facilitator and a repressor of the dsRNA-dependent kinase PKR.

PKR, a member of the eukaryotic initiation-factor 2alpha (eIF-2alpha) kinase family, mediates the host antiviral response and is implicated in tumor suppression and apoptosis. Here we show that PKR is regulated by the heat shock protein 90 (Hsp90) molecular chaperone complex. Mammalian PKR expressed in budding yeast depends on several components of the Hsp90 complex for accumulation and activity. In mammalian cells, inhibition of Hsp90 function with geldanamycin (GA) during de novo synthesis of PKR also interferes with its accumulation and activity. Hsp90 and its co-chaperone p23 bind to PKR through its N-terminal double-stranded (ds) RNA binding region as well as through its kinase domain. Both dsRNA and GA induce the rapid dissociation of Hsp90 and p23 from mature PKR, activate PKR both in vivo and in vitro and within minutes trigger the phosphorylation of the PKR substrate eIF-2alpha. A short-term exposure of cells to the Hsp90 inhibitors GA or radicicol not only derepresses PKR, but also activates the Raf-MAPK pathway. This suggests that the Hsp90 complex may more generally assist the regulatory domains of kinases and other Hsp90 substrates.

Dimerization↗