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PubMed1993

Differential kinetics of polypeptide expression and different biological activities in the human fibroblast response to dsRNA or interferon treatment.

Using two-dimensional electrophoresis on total and nuclear extracts of human fibroblasts, we compared polypeptide patterns of cells treated with interferon-beta (IFN-beta), IFN-gamma, or with dsRNA in the presence of anti-IFN antibodies. The analysis of whole-cell extracts revealed that, after a 6-h treatment, the three agents induce the synthesis of a common set of proteins in addition to others that are specifically induced either by IFNs or by dsRNA. After a 15-h treatment, this common set of proteins was only induced by IFNs. Furthermore, at this time, IFNs also regulated proteins whose synthesis was specifically induced or repressed by poly(I).poly(C) in the 6-h treated cells. These results indicate that poly(I).poly(C) regulates protein expression more rapidly and more transiently than IFNs. The analysis of nuclear extracts showed similar differential kinetics of protein expression. However, a greater number of polypeptides was found to have their synthesis specifically induced by dsRNA. Moreover, poly(I).poly(C) was found to be mitogenic in these cells and did not induce a significant resistance to vesicular stomatitis virus (VSV). This study provides evidence for an overlap in the expression of proteins by dsRNA and IFNs, although these compounds do not share the same biological activities.

2',5'-Oligoadenylate Synthetase↗
PubMed1998

Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.

Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.

Amino Acid Sequence↗
PubMed1998

Activation of the dsRNA-dependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling.

The dsRNA-dependent protein kinase (PKR) is considered to play a key role in interferon-mediated host defense against viral infection and conceivably malignant transformation. To investigate further the mechanisms of PKR-induced growth inhibition, we have developed tetracycline-inducible murine cell lines that express wild-type PKR or a catalytically inactive PKR variant, PKRdelta6. Following induction, the growth of the wild-type PKR-expressing cells was similar to that of cells transfected with vector alone, while cells expressing PKRdelta6 became malignantly transformed. Significantly, treatment with dsRNA caused the wild-type PKR-overexpressing cells to undergo programed cell death while, conversely, cells expressing PKRdelta6 were completely resistant. Our studies demonstrated that activation of PKR induces the expression of members of the tumor necrosis factor receptor (TNFR) family, including Fas (CD95/Apo-1) and pro-apopotic Bax. In contrast, transcripts representing Fas, TNFR-1, FADD (Fas-associated death domain), FLICE, Bad and Bax were ablated in cells expressing PKRdelta6. The involvement of the death receptors in PKR-induced apoptosis was underscored by demonstrating that murine fibroblasts lacking FADD were almost completely resistant to dsRNA-mediated cell death. Thus, PKR, a key cellular target for viral repression, is a receptor/inducer for the induction of pro-apoptotic genes by dsRNA and probably functions in interferon-mediated host defense to trigger cell death in response to virus infection and perhaps tumorigenesis.

3T3 Cells↗
PubMed2004

Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes.

Double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation in most eukaryotic organisms via a conserved pathway known as RNA interference (RNAi). Post-transcriptional gene silencing by RNAi is also connected with transcriptional silencing of cognate sequences. In plants, this transcriptional silencing is associated with sequence-specific DNA methylation. To address whether this mechanism operates in mammalian cells, we used bisulfite sequencing to analyze DNA in mouse oocytes constitutively expressing long dsRNA against the Mos gene. Our data show that long dsRNA induces efficient Mos mRNA knockdown but not CpG and non-CpG DNA methylation of the endogenous Mos sequence in oocytes and early embryos. These data demonstrate that dsRNA does not directly induce DNA methylation in the trans form of this sequence in these mammalian cells.

Animals↗
PubMed2006

Plasmodium falciparum DNA helicase 60. dsRNA- and antibody-mediated inhibition of malaria parasite growth and downregulation of its enzyme activities by DNA-interacting compounds.

Helicases are ubiquitous enzymes that play important roles in all types of DNA transaction in the cells. Recently we have reported the characterization of the first DEAD-box helicase [Plasmodium falciparum DNA helicase 60 (PfDH60)] from Plasmodium falciparum and have shown that it is a unique, dual bipolar helicase expressed in a stage-specific manner. In this study, we show the further characterization of PfDH60. For analyzing the significance of this enzyme in parasite growth, we studied the effect of dsRNA and specific antibodies on growth of the parasite. The studies indicate that the parasite cultures treated with PfDH60 dsRNA exhibited approximately 50% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA. It was interesting to note that purified immunoglobulins against PfDH60 induced approximately 62% inhibition of in vitro growth of P. falciparum and that this inhibitory effect was associated with morphologic damage to the parasite. DNA-interacting compounds inhibit DNA helicase and ssDNA-dependent ATPase activities of PfDH60. Of various compounds tested, only actinomycin, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the enzyme activities of PfDH60, with apparent IC50 values for helicase inhibition of 0.8, 0.3, 2.0, 1.2 and 1.5 microm, respectively. It may be proposed that these compounds form a complex with DNA and specifically inhibit helicases due to obstruction in the translocation of the enzyme. These compounds also inhibited parasite growth in culture. This is the first study to show inhibition of growth of the parasite by the dsRNA of a helicase, and most probably this is due to interference with cognate mRNA expression.

Adenosine Triphosphatases↗
PubMed1992

Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase.

The double-stranded RNA-dependent protein kinase (dsRNA-PK) is thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs). Studies examining the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Autophosphorylation and consequent activation of dsRNA-PK in vitro and in vivo result in phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2) and inhibition of protein synthesis. Expression of a functionally defective mutant of human dsRNA-PK in NIH 3T3 cells resulted in malignant transformation, suggesting that dsRNA-PK may function as a suppressor of cell proliferation and tumorigenesis.

Amino Acid Sequence↗
PubMed2004

Long dsRNA and silent genes strike back:RNAi in mouse oocytes and early embryos.

RNA interference (RNAi) refers to the selective degradation of mRNA induced by double-stranded RNA (dsRNA), first discovered in Caenorhabditis elegans. Homology-dependent silencing phenomena related to RNAi have been observed in many species from all eukaryotic kingdoms. RNAi and related mechanisms share several conserved components. The hallmark of these phenomena is the presence of short dsRNA molecules (21-25 bp long), termed short interfering RNA (siRNA), which are generated from dsRNA by the activity of Dicer, a specific type III RNAse. These molecules serve as a template for the recognition and cleavage of the cognate mRNA. As it is beyond the scope of a single review to cover all aspects of RNAi, this review will focus on certain steps of the pathway relevant to mammals and on the use of long dsRNA to specifically silence genes in mammalian cells permissive to this technique, such as oocytes and early embryos.

Animals↗
PubMed2003

Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections.

BACKGROUND: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour. RESULTS: We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation. CONCLUSION: Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

Complex Mixtures↗
PubMed1995

The large genome segment of dsRNA bacteriophage phi6 is the key regulator in the in vitro minus and plus strand synthesis.

Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription.

Bacteriophage phi 6↗
PubMedDate not supplied

Herpes simplex virus infection in primary neuronal cultures and antiviral activity of dsRNA.

Central neurons in culture represent a limitless substratum for research in neurobiology and experimental neurology. Primary cultures of NIH mouse neurons have shown that about 83% of total cells in the cultures are neuron clumps, detected by their reaction with the neuron specific-enolase (NSE) marker. Herpes Simplex Virus type 1 (HSV-1) can grow efficiently in these cultures, as it does in nonneuronal cultures usually used for antiviral drugs testing. For that reason, the primary neuronal cultures were used for testing antiviral activity against HSV-1, after an overnight treatment with different concentrations of dsRNA from phi 6 bacteriophage. The dsRNA started to be toxic for the cells at concentrations of 4 micrograms/ml, but it was found that 1 microgram/ml of this dsRNA protected all the neuronal cultures from HSV-1 infection. The dsRNA value for effective dose (ED50) was 0.27 microgram/ml.

Animals↗
PubMed1997

Serine palmitoyltransferase (scs1/lcb2) mutants have elevated copy number of the L-A dsRNA virus.

The microsomal fraction isolated form serine palmitoyltransferase (lcb2/scs1) mutants is enriched in a 90 kDa protein. The protein was identified as the major coat (Gag) protein of the L-A dsRNA virus particles by partial sequencing and by its interaction with anti-Gag antibodies. The total amount of Gag in whole-cell lysates of scs1/lcb2 mutant cells is greater than in wild-type lysates indicating that the enrichment of the protein in the microsomal fraction of scs1/lcb2 mutant cells may result from increased copy number of the L-A dsRNA virus. This is supported by the findings that the mutants also have increased levels of L-A dsRNA. Altered sphingolipid synthesis in the scs1 mutant cells appears to increase the copy number of the L-A viral particles.

Acyltransferases↗
PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. II. Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex.

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations. The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical. The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell. Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses. Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure. The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Bacteriophage phi 6↗
PubMed1995

Genomic organization and chromosomal location of the human dsRNA adenosine deaminase gene: the enzyme for glutamate-activated ion channel RNA editing.

The structure of the human gene encoding the double-stranded RNA (dsRNA) adenosine deaminase (DRADA) was characterized. This nuclear localized enzyme is involved in the RNA editing required for the expression of certain subtypes of glutamate-gated ion channel subunits. The DRADA gene span 30 kb pairs and harbors 15 exons. The transcription of the DRADA gene driven by the putative promoter region, which contains no typical TATA or CCAAT box-like sequences, is initiated at multiple sites, 164 to 216 nucleotides upstream of the translation initiation codon. The three dsRNA binding motifs (DRBM), 70 amino acid residues long, are each encoded by two exons plus an intervening sequence that interrupts the motif at the identical amino acid position. This finding is consistent with the notion that the dsRNA binding domains may be composed of two separate functional subdomains. Fluorescent in situ hybridization localized the DRADA gene on the long arm chromosome 1, region q21. The gene structure and sequence information reported in this study will facilitate the investigation of involvement of DRADA in hereditary diseases that may be the result of malfunction of glutamate-gated ion channels.

Adenosine Deaminase↗
PubMed1995

Sequence of the M28 dsRNA: preprotoxin is processed to an alpha/beta heterodimeric protein toxin.

The killer and immunity phenotypes of K28 killer strains of Saccharomyces cerevisiae are determined by the 1.75-kb M28 dsRNA virus. In the plus strand, M28p, the K28 preprotoxin gene, comprises bases 13-1047 and is followed, after an additional 85 bases, by a 63-bp poly(A) sequence and a 553-base 3'-sequence. This 3'-sequence contains two potential stem-loop structures predicted to bind the L-A encoded cap-pol protein, initiating encapsidation; high-level expression results in curing of M1 dsRNA. Expression of M28p confers the complete K28 killer and immunity phenotype on a cell lacking M28 dsRNA. K28 toxin is a disulfide-bonded heterodimer of alpha (10.5 kDa) and beta (11 kDa) components whose N-termini correspond to M28p residues 50-61 and 246-257, respectively. alpha is preceded by a potentially redundant pair of secretion signal peptides; deletion of the first reduces toxin secretion by 75%. While M28p bears no sequence similarity to M1p, the K1 preprotoxin, the predicted patterns of processing by glycosylation and cleavage are remarkably similar. The beta N- and C-termini are probably processed by Kex2p and Kex1p, respectively; the mechanism of cleavage at the less typical sites bounding the alpha component is under investigation. While a kex2 delta mutation prevents toxin secretion, secreted toxin retains 20% activity in a kex1 delta mutant. Neither mutation affects immunity.

Amino Acid Sequence↗
PubMed1998

Characterization of the thermosensitive ts453 reovirus mutant: increased dsRNA binding of sigma 3 protein correlates with interferon resistance.

The mutation harbored by the reovirus ts453 thermosensitive mutant has been assigned to the S4 gene encoding the major outer capsid protein sigma 3. Previous gene sequencing has identified a nonconservative amino acid substitution located near the zinc finger of sigma 3 protein in the mutant. Coexpression in COS cells of the sigma 3 protein presenting this amino acid substitution (N16K), together with the other major capsid protein mu 1, has also revealed an altered interaction between the two proteins; this altered interaction prevents the sigma 3-dependent cleavage of mu 1 to mu 1C. This could explain the lack of outer capsid assembly observed during ts453 virus infection at nonpermissive temperature. In the present study, we pursued the characterization of this mutant sigma 3 protein. Although the N16K mutation is located close to the zinc finger region, it did not affect the ability of the protein to bind zinc. In contrast, this mutation, as well as mutations within the zinc finger motif itself, can increase the binding of the protein to double-stranded RNA (dsRNA). It also appears that the N16K mutant protein is more efficiently transported to the nucleus than the wild-type protein, an observation consistent with the postulated role of dsRNA binding in sigma 3 nuclear presence. The lack of association with mu 1, and/or the increased dsRNA-binding activity of sigma 3, could be responsible for a partial resistance of the ts453 virus to interferon treatment and this could have important consequences in the context of protein synthesis regulation during natural reovirus infection.

Animals↗
PubMed2006

Complete nucleotide sequences of three dsRNA segments from Raphanus sativus-root cv. Yidianhong [corrected] with leaf yellow edge symptoms.

The two minor dsRNA bands, previously detected in symptomatic leaves of Raphanus sativus-root cv. Yidianhong [corrected] were subjected to further analysis. cDNA cloning and sequencing revealed that the smaller of the two dsRNA bands is actually a doublet consisting of two co-migrating dsRNA segments and the resulting three segments were designated as RasR 3, RasR 4, and RasR 5. RasR 3 was 1717 bp in length and potentially encoded a protein of about 55.3 kDa, containing all of the six conserved motifs shared by the RNA dependent RNA polymerases of members of the family Partitiviridae. RasR 4 and RasR 5, which co-migrated in the 5% polyacrylamide gel, were 1521 and 1485 bp in length and each encoded a putative protein of unknown function. Their molecular masses, as calculated from the deduced amino acid, were 38.2 and 38.8 kDa, respectively. The 5' UTRs of all three segments shared regions of high sequence similarities, but were distinct from those of the RasR 1 and RasR 2. Taken together, these results along with those described in the previous report, suggest that more than one partitivirus was co-infecting radish leaves.

5' Untranslated Regions↗
PubMed2026

Molecular characterization of a novel partitivirus harboring an additional third dsRNA segment from Trichoderma harzianum.

We report the complete genome sequence of a novel partitivirus identified from Trichoderma harzianum NFCF092 strain, designated Trichoderma harzianum partitivirus 4 (ThPV4). Unlike canonical members of the family Partitiviridae, which possess a bipartite genome consisting of two double-stranded RNA (dsRNA) segments encoding an RNA-dependent RNA polymerase (RdRP) and a capsid protein (CP), ThPV4 harbors a third dsRNA segment encoding a protein of unknown function. The complete genome consists of dsRNA1 (1,950 bp; encoding the RdRP), dsRNA2 (1,772 bp; encoding the CP), and dsRNA3 (1,629 bp; encoding a protein with unknown function). Sequence analysis shows that each segment possesses a single open reading frame (ORF). The deduced amino acid sequence of the RdRP shows the highest similarity (90.5% identity) to that of Trichoderma gamsii alphapartitivirus 1. Phylogenetic analyses based on the RdRP indicate that ThPV4 clusters within the genus Alphapartitivirus of the family Partitiviridae. To our knowledge. ThPV4 is the first member of the genus Alphapartitivirus identified from T. harzianum to possess an additional, conserved third dsRNA segment.

Phylogeny↗
PubMed2005

Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana.

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four Arabidopsis Dicer-like proteins (DCL1-4), Arabidopsis HYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein-protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

Arabidopsis↗