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PubMed1998

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis.

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.

Amino Acid Sequence↗
PubMed2004

RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae.

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (dsbetagal; note that "ds" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsbetagal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsbetagal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

Alphavirus↗
PubMed2001

Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines.

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression, referred to as RNA interference (RNAi). In invertebrates, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-nt-long short interfering (si) duplex RNAs, acting as effectors of RNAi. siRNAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are impeded by its nonspecific effects, mediated by dsRNA-dependent protein kinase PKR and RNase L. Here, we report that the RNAi response can be induced effectively by long dsRNA in nondifferentiated mouse cells grown in culture. Transfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results in a sequence-specific decrease in the level of proteins expressed from either exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediated by siRNAs, which are generated by cleavage of dsRNA by the RNaseIII-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into approximately 23-nt fragments and that Dicer localizes to the cytoplasm of EC and HeLa cells.

Animals↗
PubMed1991

Cyclic AMP mediates the direct antiproliferative action of mismatched double-stranded RNA.

Previous experiments have demonstrated that double-stranded RNAs (dsRNAs) can exert an antiproliferative effect on human tumor cells, independent of interferon (IFN) induction. However, the mechanism by which dsRNAs inhibit tumor growth has not been elucidated. As a first step in determining the molecular events responsible for growth arrest, we have explored the role of signal transduction through the cAMP system in the antiproliferative effect of the mismatched dsRNA, r(I)n.r(C12,U)n (Ampligen). These studies utilized the human glioma cell line A1235, which does not produce detectable levels of IFN-alpha, -beta, or -gamma in response to mismatched dsRNA treatment. Treatment of A1235 cells with mismatched dsRNA in combination with either 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), which inhibits cAMP-dependent protein kinase and protein kinase C, or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), which preferentially inhibits the cAMP-dependent protein kinase, yielded an antagonism of the mismatched dsRNA-induced antiproliferative effect. Measurement of adenylate cyclase activation showed a dose-dependent increase in activity at antiproliferative mismatched dsRNA concentrations, but not at lower, nonantiproliferative doses. This increase in activity was rapid, seen as early as 30 sec after initiation of treatment, and it was sustained at peak levels for 1-2 hr. Analysis of the intracellular cAMP concentration gave similar kinetics of induction. Exposure of cells to the stable cAMP analogue dibutyryl cAMP yielded dose-dependent inhibition of cell growth. The cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also inhibited proliferation. In contrast, neither H-7 nor HA1004 had an effect on growth inhibition induced by human natural IFN-alpha treatment. In addition, antiproliferative doses of IFN-alpha did not increase cAMP concentrations. These results indicate that the cAMP system is utilized by mismatched dsRNA as an early signal transduction mechanism for growth control. Furthermore, the antiproliferative effects induced by mismatched dsRNA and IFN can occur by different mechanisms of action.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Amino Acid Sequence↗
PubMed2005

Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA+ interferon-gamma-induced apoptosis in primary pancreatic beta-cells.

Viral infections and local production of cytokines probably contribute to the pathogenesis of Type 1 diabetes. The viral replicative intermediate double-stranded RNA (dsRNA, tested in the form of polyinosinic-polycytidylic acid, PIC), in combination with the cytokine interferon-gamma (IFN-gamma), triggers beta-cell apoptosis. We have previously observed by microarray analysis that PIC induces expression of several mRNAs encoding for genes downstream of Toll-like receptor 3 (TLR3) signaling pathway. In this report, we show that exposure of beta-cells to dsRNA in combination with IFN-alpha, -beta, or -gamma significantly increases apoptosis. Moreover, dsRNA induces TLR3 mRNA expression and activates NF-kappaB and the IFN-beta promoter in a TRIF-dependent manner. dsRNA also induces an early (1 h) and sustained increase in IFN-beta mRNA expression, and blocking IFN-beta with a specific antibody partially prevents PIC plus IFN-gamma-induced beta-cell death. On the other hand, dsRNA plus IFN-gamma does not induce apoptosis in INS-1E cells, and expression of TLR3 and type I IFNs mRNAs is not detected in these cells. Of note, disruption of the STAT-1 signaling pathway protects beta-cells against dsRNA plus IFN-gamma-induced beta-cell apoptosis. This study suggests that dsRNA plus IFN-gamma triggers beta-cell apoptosis by two complementary pathways, namely TLR3-TRIF-NF-kappaB and STAT-1.

Animals↗
PubMed2000

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.

Animals↗
PubMed2002

The effectiveness of double-stranded short inhibitory RNAs (siRNAs) may depend on the method of transfection.

RNA interference (RNAi) is a recently described powerful experimental tool that can cause sequence-specific gene silencing, thereby facilitating functional analysis of gene function. Consequently, we became interested in using RNAi to determine the function of aberrantly expressed ErbB3 in the KAS-6/1 human myeloma cell line. Despite the wealth of information available on the use of RNAi, dsRNA target design, and the transfection of dsRNA in vitro, little information is available for transfecting dsRNA into nonadherent cells from any species. In the present study, we report that gene silencing of ErbB3 was not observed in myeloma cells when dsRNA targeting ErbB3 was introduced using conventional transfection agents and protocols that have proved successful for several adherent cell lines. Silencing of ErbB3, however, was observed in T47D cells, an adherent breast carcinoma cell line, using the same transfection methods, indicating that our target sequence was functional for gene silencing of ErbB3. Interestingly, ErbB3 was silenced in myeloma cells when the dsRNA target was introduced by electroporation. Thus, our studies illustrate the striking dependence of dsRNA-mediated gene silencing in some cells on the methods of dsRNA transfection.

Electroporation↗
PubMed2002

Ribonuclease activity and RNA binding of recombinant human Dicer.

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.

Animals↗
PubMed2002

The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity.

The p53-induced mouse wig-1 gene encodes a Cys2His2-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56-75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST-wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1-dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.

3T3 Cells↗
PubMed2001

A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola.

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.

Amino Acid Sequence↗
PubMed2004

RNA interference evidence that growth differentiation factor-9 mediates oocyte regulation of cumulus expansion in mice.

In mouse ovaries, growth differentiation factor-9 (GDF9) is an oocyte-derived growth factor that plays an essential role during early follicular development. However, the role of GDF9 during later stages of follicular development is uncertain. In the present study, a long double-stranded (ds) RNA interference approach was used to investigate the possible role of GDF9 in mediating oocyte regulation of cumulus expansion. Fully grown mouse oocytes injected with Gdf9 dsRNA, Bmp15 dsRNA, or injection buffer were cultured for 24 h and processed for measurement of Gdf9 and Bmp15 mRNA levels using real-time reverse transcription-polymerase chain reaction (RT-PCR) and for measurement of GDF9 protein levels using Western blot analysis and immunofluorescence. Injection with Gdf9 dsRNA knocked down Gdf9, but not Bmp15, mRNA expression in oocytes, and vice versa. Furthermore, GDF9 protein levels were reduced in the Gdf9 dsRNA-injected oocytes. To investigate the role of GDF9 in cumulus expansion, two endpoints were used to evaluate cumulus expansion: Has2 and Ptgs2 mRNA levels were measured in cumulus cells using real-time RT-PCR, and assessment of cumulus expansion was undertaken morphologically. After 24 h of culture in the presence of 0.5 IU/ml of FSH, cumulus shells cocultured with buffer- and Bmp15 dsRNA-injected oocytes exhibited a high degree of expansion, whereas cumulus shells cocultured with Gdf9 dsRNA-injected oocytes exhibited only limited expansion. Supporting this observation, Has2 and Ptgs2 mRNA levels after 8 h of coculture were lower in cumulus cells cocultured with Gdf9 dsRNA-injected oocytes than in those cocultured with buffer-injected oocytes. The present results strongly support the concept that GDF9 is a key mediator of oocyte-enabled cumulus expansion in mice.

Animals↗
PubMed1989

The characterization of equine encephalosis virus and the development of genomic probes.

Equine encephalosis virus (EEV) is an orbivirus associated with a peracute illness of horses in southern Africa. The virus has now been partially purified for the first time and characterized on a molecular level. The virion is composed of 10 dsRNA segments and a protein capsid consisting of at least seven structural proteins that vary in Mr from 36,000 to 120,000. Partial clones of six of the dsRNA segments of EEV serotype Cascara were obtained and analysed for possible use as serotype-specific or group-specific probes in the detection of EEV dsRNA. Cloned fragments of genome segments 3, 8 and 10 were found to show high conservation of these segments, hybridizing to dsRNA from the six EEV serotypes under conditions that indicated more than 90% sequence homology. The genome segment 2-specific probe did not hybridize with dsRNA from any of the other EEV serotypes, suggesting that this segment encodes the serotype-specific antigen of EEV. Cross-hybridization of probes from genome segments 3 and 5 with dsRNA from bluetongue virus (BTV), epizootic haemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) indicated that EEV is more closely related to BTV and EHDV than to AHSV. Both probes can be used to distinguish between EEV and AHSV dsRNA.

African Horse Sickness↗
PubMed1995

Activation of the double-stranded-RNA-activated protein kinase and induction of vascular cell adhesion molecule-1 by poly (I).poly (C) in endothelial cells.

Double-stranded RNA (dsRNA) induces the vascular cell adhesion molecule VCAM-1 to high levels of expression in human umbilical vein endothelial (HUVE) cells. Although VCAM-1 is also induced by the cytokine interleukin 1 beta (IL-1 beta), activation of the dsRNA-activated protein kinase (PKR) occurs only in response to incubation with dsRNA but not with IL-1 beta. Incubation of HUVE cells with the synthetic dsRNA, poly (I).poly (C), activates PKR with increased autophosphorylation, increased phosphorylation of the translation factor eIF2 alpha, and increased activation of the transcription factor NF-kappa B. Promoter analysis in HUVE cells using a VCAM-1 promoter linked to CAT reporter gene demonstrates that poly (I).poly (C) responsiveness resides in the minimal VCAM-1 promoter that contains two NF-kappa B sites, and deletion of the NF-kappa B sites eliminates basal and poly (I).poly (C)-induced CAT activity, supporting the importance of NF-kappa B in the poly (I).poly (C)-mediated induction of VCAM-1. In vitro studies using purified reagents demonstrate that PKR is capable of phosphorylating I kappa B alpha (the inhibitory subunit of NF-kappa B) in a dsRNA-dependent manner. This suggests that phosphorylation of I kappa B alpha by PKR could be an initial step in the activation of NF-kappa B by dsRNA. NF-kappa B is also activated by IL-1 beta in HUVE cells, but this activation occurs without increased PKR autophosphorylation or eIF2 alpha phosphorylation. Poly (I).poly (C) induces VCAM-1 mRNA levels that are dramatically higher and sustained longer than levels induced by IL-1 beta. Although phosphorylation of eIF2 alpha interferes with protein translation, sufficient VCAM-1 mRNA translation occurs in response to poly (I).poly (C) to yield VCAM-1 protein levels that are similar to levels that are induced by IL-1 beta. This suggests that the higher, sustained VCAM-1 mRNA levels that occur in response to incubation with poly (I).poly (C) compensate for the partial translational block resulting from increased eIF2 alpha phosphorylation. These studies indicate that transcriptional and translational regulatory events that occur in response to activation of PKR by dsRNA are important in the regulation of VCAM-1 gene expression in HUVE cells.

Cell Adhesion↗
PubMed2005

Double-stranded RNA stimulates chemokine expression in microglia through vacuolar pH-dependent activation of intracellular signaling pathways.

During neurotropic virus infection, microglia act as a source of chemokines, thereby regulating the recruitment of peripheral leukocytes and the multicellular immune response within the CNS. Herein, we present a comprehensive study on the chemokine production by microglia in response to double-stranded RNA (dsRNA), a conserved molecular pattern of virus infection. Transcriptional analyses of chemokine genes revealed that dsRNA strongly induces the expression of CXC chemokine ligand 10 (CXCL10) and CC chemokine ligand 5 (CCL5) in microglia. We also observed that the dsRNA stimulation triggered the activation of signaling pathways mediated by nuclear factor kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK). The microglial CXCL10 response to dsRNA was induced via NF-kappaB, p38, and JNK pathways, whereas the dsRNA-induced CCL5 production was dependent on JNK, but not on the other signal-transducing molecules tested. In addition, the acidic environment of intracellular vesicles was required for the activation of cellular signaling in response to dsRNA. Taken together, these results suggest that the recognition of dsRNA structure selectively induces the CXCL10 and CCL5 responses in microglia through vacuolar pH-dependent activation of NF-kappaB and MAPK signaling pathways.

Acids↗
PubMed1977

Biochemical and physiological studies of the yeast virus-like particle.

A study was made of the virus-like particle (VLP) of Saccharomyces cerevisiae S7. This strain contains elevated amounts of P1 double-stranded ribonucleic acid (dsRNA) but no P2 dsRNA. The amount of dsRNA contained in cells grown on a fermentable carbon source (glucose) was compared with that in cells grown on a nonfermentable carbon source (ethanol). It was found that ethanol-grown cells contain higher levels of dsRNA than glucose-grown cells. In the former, the amount of dsRNA increased during the logarithmic phase of growth, whereas in the latter it increased during the transition from the logarithmic to the stationary phase. A method was devised to isolate VLPs from these cells by using CsCl gradients, and the yield was assessed by monitoring the recovery of dsRNA. Three proteins were found to be tightly associated with these particles. They have molecular weights of 75,000, 53,000, and 37,000. Together they account for almost all of the coding capacity of the P1 dsRNA that the VLP contains.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Double-stranded RNA binding by a heterodimeric complex of murine cytomegalovirus m142 and m143 proteins.

In response to viral infection, cells activate a variety of antiviral responses, including several that are triggered by double-stranded (ds) RNA. Among these are the protein kinase R and oligoadenylate synthetase/RNase L pathways, both of which result in the shutoff of protein synthesis. Many viruses, including human cytomegalovirus, encode dsRNA-binding proteins that prevent the activation of these pathways and thereby enable continued protein synthesis and viral replication. We have extended these analyses to another member of the beta subfamily of herpesviruses, murine cytomegalovirus (MCMV), and now report that products of the m142 and m143 genes together bind dsRNA. Coimmunoprecipitation experiments demonstrate that these two proteins interact in infected cells, consistent with their previously reported colocalization. Jointly, but not individually, the proteins rescue replication of a vaccinia virus mutant with a deletion of the dsRNA-binding protein gene E3L (VVDeltaE3L). Like the human cytomegalovirus dsRNA-binding protein genes TRS1 and IRS1, m142 and m143 are members of the US22 gene family. We also found that two other members of the MCMV US22 family, M23 and M24, encode dsRNA-binding proteins, but they do not rescue VVDeltaE3L replication. These results reveal that MCMV, like many other viruses, encodes dsRNA-binding proteins, at least two of which can inhibit dsRNA-activated antiviral pathways. However, unlike other well-studied examples, the MCMV proteins appear to act in a heterodimeric complex.

Dimerization↗
PubMed2006

Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon.

Expression of alpha/beta interferon (IFN-alpha/beta) in virus-infected vertebrate cells is a key event in the establishment of a sustained antiviral response, which is triggered by double-stranded RNA (dsRNA) produced during viral replication. These antiviral cytokines initiate the expression of cellular proteins with activities that limit the replication and spread of the invading viruses. Within this response, the dsRNA-dependent protein kinase R (PKR) that is expressed at constitutive levels and upregulated by IFN-alpha/beta acts as an important antiviral effector that can block the cellular translational machinery. We previously demonstrated that efficient replication of influenza B virus depends on the viral dsRNA-binding NS1 protein that inhibits the transcriptional activation of IFN-alpha/beta genes. Here we tested the postulate that the viral NS1 protein counteracts antiviral responses through sequestering intracellular dsRNA by analyzing a collection of recombinant influenza B viruses. As expected, viruses expressing dsRNA-binding-defective NS1 proteins were strongly attenuated for replication in IFN-competent hosts. Interestingly, these virus mutants failed to prevent activation of PKR but could effectively limit IFN induction. Conversely, a mutant virus expressing the N-terminal dsRNA-binding domain of NS1 prevented PKR activation, but not IFN induction, suggesting an important role for the NS1 C-terminal part in silencing the activation route of IFN-alpha/beta genes. Thus, our findings indicate an unexpected mechanistic dichotomy of the influenza B virus NS1 protein in the suppression of antiviral responses, which involves at least one activity that is largely separable from dsRNA binding.

Cell Line↗