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PubMed1986

Isolation and partial characterization of a monoclonal antibody specific for a naturally occurring double-stranded RNA.

A monoclonal antibody (Mab) against the double-stranded form of the cucumber mosaic virus-associated RNA 5, the sequence variant that induces the tomato necrosis disease (ds(n)CARNA 5), has been obtained. Specificity studies using a radioimmunoassay showed that the purified Mab recognizes neither DNA nor ssRNA of different sources including ss(n)CARNA 5, poly(I).poly(C), nor dsRNA of two other sequence variants of CARNA 5. Therefore, the Mab seems to recognize a conformational variation only present in the double helical structure of ds(n)CARNA 5.

Antibodies, Monoclonal↗
PubMed1989

Analysis of reassortment and superinfection during mixed infection of Vero cells with bluetongue virus serotypes 10 and 17.

The reassortment of genome segments during mixed infection of Vero cells with bluetongue virus (BTV) serotypes 10 and 17 was investigated, using non-selective conditions for analysis of the progeny of mixed infections. Reassortment was found to be an early event in the BTV replication cycle, indicating that progeny BTV genomes undergo a single round of reassortment. Non-random segregation of individual genome segments was observed in crosses at equal multiplicity of infection, and was confirmed in crosses performed at unequal multiplicity. Asynchronous infections showed that superinfection exclusion resulted in the failure of the superinfecting virus to contribute genome segments to reassortants if the second virus followed the first by more than 4 h. The significance of these results for the evolution and epidemiology of BTV is discussed.

Animals↗
PubMed1991

Cell-free translation and identification of the replicative form of Nudaurelia beta virus RNA.

Larvae of the pine emperor moth, Nudaurelia cytherea capensis, infected with Nudaurelia beta virus (N beta V) consistently contained one species of dsRNA. This dsRNA was the correct size to be the replicative form of the N beta V genome and, in Northern blots, it hybridized with 32P-end-labelled virion RNA. Other smaller dsRNAs were obtained in a non-reproducible manner but these had no sequences in common with the genomic probe; no dsRNAs were observed in extracts from virus-free larvae. Cell-free translation of N beta V RNA resulted in the synthesis of only one major polypeptide, of Mr about 71,000, which could not be precipitated by an anti-N beta V serum.

Animals↗
PubMed2002

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.

Animals↗
PubMed1989

Identification of virus-like particles in Eimeria stiedae.

When nucleic acid samples purified from sporozoites of Eimeria stiedae were analyzed by agarose gel electrophoresis, an ethidium-stainable band with an apparent electrophoretic mobility of 6.5 kb was consistently observed. The band was readily degradable upon RNAse treatment, and its susceptibility towards ribonuclease A on a decreasing ionic strength was suggestive of double-stranded RNA (dsRNA). Electron microscopy revealed spherical, probably icosahedral, virus-like particles (VLP) with a diameter of 35 nm in sporozoite lysates. The VLP were purified by CsCl buoyant density gradient centrifugation. Upon extraction, these particles yielded dsRNA molecules of a uniform length of 1.63 microns. The presence of the VLP was investigated in different Eimeria strains. All E. stiedae isolates contained the RNA virus, whereas the Eimeria intestinalis and Eimeria magna isolates tested did not. RNA/RNA hybridization experiments where the E. stiedae VLP dsRNA was probed to the genomes of the dsRNA viruses of Trichomonas vaginalis and Giardia intestinalis revealed a strong relatedness of the E. stiedae virus to the G. intestinalis virus, in contrast with the T. vaginalis virus, where no homology could be detected.

Animals↗
PubMedDate not supplied

Rapid isolation of double stranded RNA segments from disulphide crosslinked polyacrylamide gels.

A simple method was developed to isolate dsRNA segments from disulphide crosslinked polyacrylamide gels. The dsRNA preparations from the P4 killer virus strain 77 of Ustilago maydis containing 7 genomic segments with molecular sizes ranging from 0.36 to 6.7 kbp, the 20 kbp dsRNA associated with the '447' cytoplasmic male sterility in Vicia faba and the 23 kbp genomic dsRNA of citrus tristeza virus (CTV) were separated on disulphide crosslinked polyacrylamide gels. After UV visualisation, the dsRNA bands were excised from the gel and dissolved using 2-mercaptoethanol. The dsRNA were then purified from the solubilized fractions by specific adsorption on microgranular cellulose and elution with a small volume of water. The method is rapid, simple and convenient for the isolation of all the tested dsRNAs segments.

Cross-Linking Reagents↗
PubMed2004

Characterization of the chicken PKR: polymorphism of the gene and antiviral activity against vesicular stomatitis virus.

The double-stranded RNA-dependent protein kinase (PKR) is induced in mouse and human cells on treatment with interferon. In this study, we have cloned and determined the nucleotide sequences of chicken PKR cDNA in various chicken breeds. Chicken PKR was a 550-amino-acid protein as deduced from the cDNA open reading frame (ORF), and there were specific domains (two double-stranded RNA binding domains (DRBDs) and numerous kinase subdomains) characterized in RNA binding proteins and kinase families. Furthermore, it was suggested that chicken PKR was polymorphic. Transfected cell clones expressing chicken PKR mRNA were demonstrated to confer antiviral responses to vesicular stomatitis virus, except for Koshamo type-3 (KS-3). KS-3 PKR, which has an amino acid substitution at position 507 (Arg to Gln), showed amphibious antiviral responses. This specific amino acid substitution was considered to determine the antiviral function of chicken PKR in addition to essential domains as DRBDs and kinase subdomains.

Amino Acid Sequence↗
PubMed1984

Characterization of Nugget virus, a serotype of the Kemerovo group of orbiviruses.

The genome of Nugget virus, a serotype of the Kemerovo group of orbiviruses, consists of 10 segments of double-stranded RNA. The properties of the virus are consistent with its classification as an orbivirus , but the unusual patterns of separation of viral RNA and polypeptides compared with that reported for most other orbiviruses suggests the possibility of heterogeneity within the genus Orbivirus .

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism.

The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-alpha/beta or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-beta induction pathway have been analyzed. The transcription factor NF-kappa B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-kappa B by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

Animals↗
PubMed1983

Biochemical characterization of eel virus european.

The polypeptide and RNA compositions of purified virions of eel virus European were compared to those of three strains (VR-299, SP and AB) of infectious pancreatic necrosis virus (IPNV) isolated from trout. All three IPNV strains could be distinguished by the relative mobilities of either the virion polypeptides or the double-stranded RNA genome segments. The eel virus had a similar polypeptide profile to strain AB IPNV but differences between the two viruses in the migration of genome segments indicated each was unique.

Animals↗
PubMed2004

VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase.

Segment B of the bisegmented, double-stranded RNA genome of infectious bursal disease virus (IBDV) encodes the viral protein VP1. This has been presumed to represent the RNA-dependent RNA polymerase (RdRp) as it contains motifs that are typical for the RdRp of plus-strand RNA viruses. Here it is demonstrated that baculovirus-expressed wild-type but not motif A mutated VP1 acts as an RdRp on IBDV-specific RNA templates. Thus, on a plus-strand IBDV segment A cRNA template, minus-strand synthesis occurred in such a way that a covalently linked double-stranded RNA product was generated (by a 'copy-back' mechanism). Importantly, enzyme activity was observed only with templates that comprised the 3' non-coding region of plus-strand RNAs transcribed from IBDV segments A and B, indicating template specificity. RdRp activity was shown to have a temperature optimum of 37 degrees C and required magnesium ions for enzyme activity. Thus, it has been demonstrated unequivocally that VP1 represents the RdRp of IBDV.

Animals↗
PubMed1982

Stiffness of viroids and viroid-like RNA in solution.

The sedimentation coefficients of the potato spindle tuber viroid, four viroid-like RNAs from cadang-cadang-disease, circular RNA from velvet tobacco mottle virus, circular RNA from Solanum nodiflorum mottle virus and double stranded RNA5 from cucumber mosaic virus were measured in the analytical ultracentrifuge. The numbers of nucleotides of the RNA species varied between 246 and 670. The hydrodynamic models of rigid rods and flexible cylinders were applied for the interpretation of the sedimentation coefficients. Double-stranded RNA5 from cucumber mosaic virus with 335 basepairs fits the model of a rigid rod with an hydrated diameter of 29 A. Potato spindle tuber viroid and the four viroid-like RNA species of cadang-cadang-disease form a homologous series of flexible cylinders with a Kuhn's statistical length lambda-1 of 600 A. The circular RNA from the two viruses mentioned above are more flexibel than the viroids and viroid-like RNAs. The hydrodynamic interpretation is in accordance with thermodynamic data and secondary structure models. In two of the RNAs from cadang-cadang, cruciform structures would also be possible on the basis of the nucleotide sequence. The hydrodynamic data, however, favour clearly the extended structure over the cruciform.

Genes, Viral↗
PubMed1988

Studies on the infectivity of foot-and-mouth disease virus RNA using microinjection.

Foot-and-mouth disease virus (FMDV) RNA, isolated as virion RNA from purified virus particles or as total RNA from infected cells, has been microinjected into nuclei and cytoplasms of BHK cells. When injected directly into the nucleus FMDV RNA was not infectious, whereas cytoplasmic injection resulted in a high proportion of productive infections. Infectivity microinjection assays on dilution series of various FMDV RNAs showed that both single-stranded positive sense 35S RNA and double-stranded replicative form (Rf) RNA have an infectivity close to 1 p.f.u. per molecule, although only a minor fraction of BHK cells appeared able to support plaque formation following microinjection of Rf FMDV RNA. The infectivity of Rf FMDV RNA was not sensitive to inhibition by actinomycin D. The results are discussed in relation to the high virus particle to p.f.u. ratios observed for FMDV.

Animals↗
PubMed1986

Thermolabile L-A virus-like particles from pet18 mutants of Saccharomyces cerevisiae.

pet18 mutations in Saccharomyces cerevisiae confer on the cell the inability to maintain either L-A or M double-stranded RNAs (dsRNAs) at the nonpermissive temperature. In in vitro experiments, we examined the effects of pet18 mutations on the RNA-dependent RNA polymerase activity associated with virus-like particles (VLPs). pet18 mutations caused thermolabile RNA polymerase activity of L-A VLPs, and this thermolability was found to be due to the instability of the L-A VLP structure. The pet18 mutations did not affect RNA polymerase activity of M VLPs. Furthermore, the temperature sensitivity of wild-type L-A RNA polymerase differed substantially from that of M RNA polymerase. From these results, and from other genetic and biochemical lines of evidence which suggest that replication of M dsRNA requires the presence of L-A dsRNA, we propose that the primary effect of the pet18 mutation is on the L-A VLP structure and that the inability of pet18 mutants to maintain M dsRNA comes from the loss of L-A dsRNA.

Drug Stability↗
PubMed1986

Detection of double-stranded RNA by immunoblot electrophoresis.

A procedure is described which permits the serological detection of double-stranded RNA (dsRNA) which has been electrophoretically transferred from agarose gels to Zeta-Probe blotting membranes. Cucumber mosaic virus replicative forms of RNAs 1-3 and cassava (Manihot esculenta) clone 'Secundina' dsRNAs can be routinely detected from 1 g of leaf tissue. This procedure allows for the detection and size estimation of dsRNAs in crude nucleic acid preparations.

Electrophoresis, Agar Gel↗
PubMed1983

Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity.

Double-stranded (ds) RNA was isolated from either partially purified bluetongue virus (BTV) type 17 or BTV-17 infected cells. The RNA was denatured with either methylmercury hydroxide or dimethyl sulfoxide and translated in a reticulocyte cell-free system. The translation products were found to be similar to BTV-17 specific polypeptides in infected cells when analyzed by polyacrylamide gel electrophoresis and immunoprecipitation with BTV-17 specific polyclonal mouse ascitic fluid. The 10 dsRNA BTV-17 genome segments were resolved by preparative gel electrophoresis and the coding assignment of each segment was determined by translation in a cell-free system. Immunoprecipitation of VP3 from infected cell lysates by a type-specific neutralizing monoclonal antibody identified this polypeptide, which is coded for by genome segment 2, as being involved in virus neutralization. Immunoprecipitation of in vitro translation products directed by total BTV-17 RNA with a monoclonal antibody that reacts with all the serotypes of BTV and some other orbiviruses and precipitates VP7 directly demonstrated that gene segment 8 codes for this intergroup-specific polypeptide.

Animals↗
PubMed2002

The double-stranded RNA-activated kinase, PKR, can phosphorylate hepatitis D virus small delta antigen at functional serine and threonine residues.

Hepatitis D virus (HDV) encodes two proteins, the 24-kDa small delta antigen (S-HDAg) and 27-kDa large delta antigen (L-HDAg) in its single open reading frame. Both of them had been identified as nuclear phosphoproteins. Moreover, the phosphorylated form of S-HDAg was shown to be important for HDV replication. However, the kinase responsible for S-HDAg phosphorylation remains unknown. Therefore, we employed an in-gel kinase assay to search candidate kinases and indeed identified a kinase with a molecular mass of about 68 kDa. Much evidence demonstrated this kinase to be the double-stranded RNA-activated kinase, PKR. The immunoprecipitated endogenous PKR was sufficient to catalyze S-HDAg phosphorylation, and the kinase activity disappeared in the PKR-depleted cell lysate. The S-HDAg and PKR could be co-immunoprecipitated together, and both of them co-located in the nucleolus. The LC/MS/MS analysis revealed that the serine 177, serine 180, and threonine 182 of S-HDAg were phosphorylated by PKR in vitro. This result was consistent with previous phosphoamino acid analysis indicating that serine and threonine were phosphorylation targets in S-HDAg. Furthermore, serine 177 was also shown to be the predominant phosphorylation site for S-HDAg purified the from cell line. In dominant negative PKR-transfected cells, the level of phosphorylated S-HDAg was suppressed, but replication of HDV was enhanced. Other than human immunodeficiency virus type 1 trans-activating protein (Tat), S-HDAg is another viral protein phosphorylated by PKR that may regulates HDV replication and viral response to interferon therapy.

Cell Line↗
PubMed1994

The beta-interferon promoter responds to priming through multiple independent regulatory elements.

The induction of beta-interferon is markedly enhanced in some cell types by a pretreatment with type I interferon itself (priming). We show that induction in response to double-stranded RNA is completely dependent upon priming in HeLa cells. However, unprimed cells can be partially induced by Sendai virus. This indicates that Sendai virus can provide a signal that is different from, or additional to, that provided by double-stranded RNA. The requirement for priming cannot be localized to a specific sequence element in the beta-interferon promoter, suggesting that priming may induce an essential component for signal transduction in response to double-stranded RNA.

Base Sequence↗