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PubMed2003

Identification of parental origin of cognate dsRNA genome segment(s) of rotavirus reassortants by constant denaturant gel electrophoresis.

Rotaviruses are the single most important etiologic agents of severe diarrhea in infants and young children worldwide. They possess a triple capsid morphology and a genome of 11 segments of double-stranded (ds) RNA. During the course of the development of various live, attenuated reassortant rotavirus vaccines, we often experienced difficulty in identifying the parental origin of certain genome segment(s) of a reassortant vaccine candidate. Various assays have been utilized for determination of the parental origin of reassortant virus genes, including polyacrylamide gel electrophoresis (PAGE), DNA and/or RNA hybridization assays and gene sequence analysis. The traditional PAGE is simple and easy to perform, however, it is common to find that certain cognate dsRNA segment(s) cannot be differentiated by this assay due to a high degree of sequence homology among different rotavirus strains. Constant denaturant gel electrophoresis (CDGE) is one of several methods that have been used to screen DNA fragments for small sequence changes or point mutations. By using the CDGE, we were successful in partially denaturing rotavirus dsRNA thereby changing the physical properties of the genome segment(s) in the gel and thus differentiating the cognate genome segment(s) of rotavirus reassortants. The CDGE provides a simple and reliable assay system for identification of parental gene origins of a rotavirus reassortant.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

Inhibition of mRNA binding to ribosomes by localized activation of dsRNA-dependent protein kinase.

The initiation of protein synthesis can be regulated in mammalian cells by protein kinases which phosphorylate the alpha subunit of initiation factor eIF-2. This phosphorylation results in a block in the recycling of eIF-2 and in the inhibition of messenger RNA binding to 80S initiation complexes. After eIF-2 alpha is phosphorylated, the mRNA becomes associated with 48S complexes consisting of a 40S ribosomal subunit, eIF-2 (alpha P), GDP and Met-tRNAf. One of the eIF-2 alpha kinases is activated by low concentrations of double-stranded RNA (dsRNA). This kinase (PKds) is present at a basal level in all mammalian cells investigated and its synthesis is induced in cells treated with interferon. The PKds may be involved in the inhibition of translation of viral mRNA in interferon-treated cells infected with RNA viruses, as it is activated by viral replicative complexes. It is not known, however, if the activated PKds preferentially inhibits the translation of viral mRNA when cellular protein synthesis proceeds at a normal rate in infected cells. We now report that mRNA covalently linked to dsRNA is preferentially inhibited from binding to 80S complexes by a localized activation of PKds. This suggests that in interferon-treated cells the binding of some nascent viral mRNAs to functional initiation complexes may be preferentially inhibited by a similar mechanism.

Enzyme Activation↗
PubMed2000

Intrahepatic mRNA expression of interferon-inducible antiviral genes in liver diseases: dsRNA-dependent protein kinase overexpression and RNase L inhibitor suppression in chronic hepatitis C.

As a part of the defense mechanism of the host to viral infection, interferons induce the transcription of several genes. These interferon-inducible genes contribute to the eradication of the viruses. Whereas some studies suggested the participation of a dsRNA-dependent protein kinase in the host reaction to hepatitis C virus infection, the involvement of other interferon-inducible genes has not been evaluated. Furthermore, there has been no analysis on the expression profile of multiple interferon-inducible genes. The aim of this study was to clarify the hepatic mRNA expression profile of interferon-inducible genes with a special concern to chronic hepatitis C. A total of 76 liver biopsy samples (28 with chronic hepatitis C, 10 with chronic hepatitis B, 9 with alcoholic liver disease, 14 with autoimmune hepatitis, 10 with primary biliary cirrhosis, and 5 of normal liver) were enrolled. The expression of the following genes was quantified by competitive reverse transcription-polymerase chain reaction and was compared according to the etiology; dsRNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (2,5-AS), latent cellular endoribonuclease (RNase L), RNase L inhibitor, and MxA. As a result, PKR mRNA was significantly overexpressed in the liver of chronic hepatitis C compared with those of other etiologies (P =.0178), and it correlated significantly with serum alanine transaminase values (r =.51, P =.0054). Also, the expression of the RNase L inhibitor showed a significant reduction in chronic hepatitis C (P =.0184). The expressions of 2,5-AS, RNase L, and MxA were not different significantly irrespective to the etiology. In conclusion, hepatic overexpression of PKR and reduced expression of RNase L inhibitor seem to contribute to the anti-HCV mechanism characteristically.

ATP-Binding Cassette Transporters↗
PubMed1992

MAK10, a glucose-repressible gene necessary for replication of a dsRNA virus of Saccharomyces cerevisiae, has T cell receptor alpha-subunit motifs.

The MAK10 gene is necessary for the propagation of the L-A dsRNA virus of the yeast Saccharomyces cerevisiae. We have isolated MAK10 from selected phage lambda genomic DNA clones that map near MAK10. This gene encodes a 733-amino acid protein with several regions of similarity to T cell receptor alpha-subunit V (variable) regions. We show that MAK10 is essential for optimal growth on nonfermentable carbon sources independent of its effect on L-A. Although loss of L-A by mak10-1 mutants is partially suppressed by loss of the mitochondrial genome, no such suppression of a mak10::URA3 mutation was observed. Using MAK10-lacZ fusions we show that MAK10 is expressed at a very low level and that it is glucose repressed. The highest levels of expression were seen in tup1 and cyc8 mutants, known to be defective in glucose repression. These results suggest that the mitochondrial genome and L-A dsRNA compete for the MAK10 protein.

Acetyltransferases↗
PubMed1986

The stiffness of dsRNA: hydrodynamic studies on fluorescence-labelled RNA segments of bovine rotavirus.

The sedimentation coefficients of dsRNA segments of bovine rotavirus were determined in the analytical ultracentrifuge. The eleven segments were separated by preparative gel electrophoresis, and isolated by elution from gel pieces. The RNA was labelled by the intercalating fluorescent dye ethidium bromide at a ratio bound dye per base pair between 0.003 to 0.018. The analytical ultracentrifuge was equipped with a fluorescence recording optics. Sedimentation coefficients could be determined with amounts of RNA as little as 8 ng. All sedimentation coefficients were extrapolated to zero-concentration, zero-dye binding, and zero-impurities from the preparative gel electrophoresis. The hydrodynamic model of flexible cylinders was applied for the interpretation of the sedimentation coefficients. All dsRNA segments of rotavirus (663-3409 base pairs) and the dsRNA5 of cucumber mosaic virus (335 base pairs) fit the model of a "worm-like" or flexible cylinder with a persistence length of 1125 A and a hydrated diameter of 30 A. The results are compared with data from the literature on the persistence lengths of the B- and Z-forms of dsDNA and of viroids.

Animals↗
PubMed1992

Atomic force microscopy of reovirus dsRNA: a routine technique for length measurements.

Atomic force microscopy (AFM) was used to image reovirus double stranded RNA (dsRNA) deposited from diluted buffer solution onto a chemically treated mica surface. This procedure allows AFM images of dsRNA molecules to be obtained with a quality close to that obtained with conventional electron microscopy. The length of the molecules were measured directly on a computer display using the digitally acquired images. The lengths of the molecules varied between 0.2 and 1.8 microns. Statistical analysis showed a multimodal distribution with clear maxima at 0.4, 0.65 and 1.05 microns. These data are in a good agreement with those obtained by electron microscopy and gel electrophoresis.

Microscopy, Scanning Tunneling↗
PubMed1997

Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme.

During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.

Adenosine Triphosphate↗
PubMed1986

5S RNA and tRNA-like molecules are associated with killer virus dsRNA of yeast.

A population of oligonucleotides co-purified with the dsRNA genomic segments of the killer virus of Saccharomyces cerevisiae during electrophoresis through agarose gels. These smaller RNA molecules must be separated from the viral genome in order to determine the structure of the dsRNA molecules. Sequence analysis of these isolated oligonucleotides showed that the population contained tRNA-like molecules, as well as 5S RNA, which are presumably encoded by the host cell genome.

Base Sequence↗
PubMed2000

A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin.

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Abscisic Acid↗
PubMed2005

Capillary feeding of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Animals↗
PubMed1991

Yeast dsRNA viruses: replication and killer phenotypes.

The cytoplasmic L-A dsRNA virus of Saccharomyces cerevisiae consists of a 4.5 kb dsRNA and the two gene products it encodes; the capsid (cap) and at least one copy of the capsid-polymerase (cap-pol) fusion protein. Virion cap-pol catalyses transcription of the plus (sense)-strand; this is extruded from the virus and serves as messenger for synthesis of cap and cap-pol. Nascent cap-pol binds to a specific domain in the plus strand to initiate encapsidation and then catalyses minus-strand synthesis to complete the replication cycle. Products of at least three host genes are required for replication, and virus copy number is kept at tolerable levels by the SKI antivirus system. S. cerevisiae killer viruses are satellite dsRNAs that use a similar encapsidation domain to parasitize the L-A replication machinery. They encode precursors of secreted polypeptide toxins and immunity (specific resistance) determinants and are self-selecting. Three unique killer types, K1, K2 and K28, are currently recognized. They are distinguished by an absence of cross-immunity and by toxin properties and lethal mechanisms; while K1 and K2 toxins bind to cell-wall glucan and disrupt membrane functions, K28 toxin binds to mannoprotein and causes inhibition of DNA synthesis.

DNA Topoisomerases, Type I↗
PubMed1992

DsRNA cloning and diagnosis of beet pseudo-yellows virus by PCR and nucleic acid hybridization.

DsRNA has been extracted from beet pseudo-yellows virus infected cucumber plants, purified to homogeneity, and cDNA clones to it produced. The clones are of insufficient sensitivity to detect infection-specific RNA in dot and northern blots of crude nucleic acid extracts. However, a knowledge of the sequence of these clones has been used to synthesize oligonucleotides that have been used for polymerase chain reaction amplification of specific sequences from both purified dsRNA and from infected plants and used as a previously unavailable highly sensitive diagnostic probe. The method of cDNA synthesis has been shown to be generally applicable to some other plant viral dsRNAs and should be of use in the production of cDNA clones when virion RNA is unavailable.

Base Sequence↗
PubMed2006

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function.

BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

Active Transport, Cell Nucleus↗
PubMed2005

The contributions of dsRNA structure to Dicer specificity and efficiency.

Dicer processes long double-stranded RNA (dsRNA) and pre-microRNAs to generate the functional intermediates (short interfering RNAs and microRNAs) of the RNA interference pathway. Here we identify features of RNA structure that affect Dicer specificity and efficiency. The data presented show that various attributes of the 3' end structure, including overhang length and sequence composition, play a primary role in determining the position of Dicer cleavage in both dsRNA and unimolecular, short hairpin RNA (shRNA). We also demonstrate that siRNA end structure affects overall silencing functionality. Awareness of these new features of Dicer cleavage specificity as it is related to siRNA functionality provides a more detailed understanding of the RNAi mechanism and can shape the development of hairpins with enhanced functionality.

Base Sequence↗
PubMed1994

dsRNA degrading nucleases are differentially expressed in tobacco anthers.

Nucleases, capable of digesting double-stranded RNAs are mainly confined to extracellular fractions of tobacco anthers and diffusate of mature pollen. dsRNAse activity is about 150-fold higher in anther fractions than in crude nuclease extracts from tobacco leaves. The level of dsRNAse activity varies during pollen development from the microspore stage to maturity. In the anther soluble fraction, dsRNAse activity reached a maximum (approx. 50 units/anther) at the end of microspore mitosis and then decreased continuously until the stage of almost mature anthers. In contrast, the nuclease activity associated with pollen increased continuously reaching a maximum (5 units/anther), during subsequent stages of pollen maturation. Gel electrophoretic analysis revealed four slowly migrating sugar-unspecific nucleases (active against DNA and RNA) and three faster migrating RNases which were all able to digest dsRNA. Competition experiments showed that the sugar-unspecific nucleases accounted for 95% of the total dsRNAse activity. Anther extracellular nucleases were further characterized after partial purification on NADP-agarose: dsRNAse activity had a pH optimum at 5.5, was strongly inhibited by NaCl and by 1 mM Zn2+ and was insensitive to EDTA which could stimulate activity in crude preparations. Analysis of the activity with defined substrates showed that ssRNA is more readily degraded than dsRNA and that both, endo- and exonucleolytic activities are detected.

Centrifugation, Density Gradient↗
PubMed2006

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Antibodies↗
PubMed1987

[Action of a yeast dsRNA preparation on the humoral immune response and the delayed hypersensitivity reaction].

The studies showed that yeast dsRNA in a dose of 2.5 mg/kg had a stimulating effect on the humoral immune response. When the preparation was administered soon after sensitization of experimental animals by the thymus-dependent antigen (sheep red blood cells, SRBC), there were observed a more than two-fold increase in the AFC count in the spleen and increased titers of hemolysins in blood serum. With the use of higher doses of the preparation (up to 10 mg/kg) the level of the immunostimulating effect remained unchanged. The efficacy of the preparation in CBA and C57BL/6 mice with high and low reactivity to the antigen was the same. Delayed-type hypersensitivity in the animals sensitized by SRBC was inhibited by yeast dsRNA in a dose of 10 mg/kg.

Animals↗
PubMed1995

Deamination of mammalian glutamate receptor RNA by Xenopus dsRNA adenosine deaminase: similarities to in vivo RNA editing.

Double-stranded RNA (dsRNA) adenosine deaminase (dsRAD) converts adenosines to inosines within dsRNA. A great deal of evidence suggests that dsRAD or a related enzyme edits mammalian glutamate receptor mRNA in vivo. Here we map the deamination sites that occur in a truncated glutamate receptor-B (gluR-B) mRNA after incubation with pure Xenopus dsRAD. We find remarkable similarities, as well as distinct differences, between the observed deamination sites and the sites reported to be edited within RNAs isolated from mammalian brain. For example, although deamination at the biologically relevant Q/R editing site occurs, it occurs much less frequently than editing at this site in vivo. We hypothesize that the similarities between the deamination and editing patterns exist because the deamination specificity that is intrinsic to dsRAD is involved in selecting editing sites in vivo. We propose that the observed differences are due to the absence of accessory factors that play indirect roles in vivo, such as binding to and occluding certain sites from dsRAD, or promoting the RNA structure required for correct and efficient editing. The work reported here also suggests that dsRAD is capable of much more selectivity than previously thought; a minimal number of deamination sites (average < or = 5) were found in each gluR-B RNA. We speculate that the observed selectivity is due to the various structural elements (mismatches, bulges, loops) that periodically interrupt the base paired region required for editing.

Adenosine Deaminase↗