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At least 1,279 records · Page 71Linked to original sources
PubMed1996

Geographic Distribution of Cytoplasmic Polyhedrosis Virus Infection in Douglas-Fir Tussock Moth Larvae, Orgyia pseudotsugata, in British Columbia

We have examined the geographic distribution and prevalence of insect-specific viral infections in Douglas-fir tussock moth populations in British Columbia. Nucleic acids extracted from field collected egg masses from 10 different locations in British Columbia were subjected to agarose gel electrophoresis and Southern blot analysis. Electrophoresis of nucleic acids after digestion with RNase A at high and low ionic strengths showed that the extra bands detected in agarose gels were double-stranded RNA (dsRNA). The dsRNA banding pattern was similar to the electropherotype of cytoplasmic polyhedrosis virus type 5. CPV infections were found in 27 of 170 DFTM larvae reared from field collected egg masses from four of 10 sites examined in three geographic regions of British Columbia. The CPV-infection rate for the DFTM larvae ranged from 10 to 86% depending on the location of the site.

Journal Article↗
PubMed1995

In vitro packaging of the single-stranded RNA genomic precursors of the segmented double-stranded RNA bacteriophage phi 6: the three segments modulate each other's packaging efficiency.

Bacteriophage phi 6 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. Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles. These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro. We have used this in vitro system for a detailed study of phi 6 RNA packaging. The reaction conditions for RNA packaging were optimized using a RNase protection assay. The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source. Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging. Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP. The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid. However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments. The packaging of the s and m segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments. In contrast, the packaging of the l segment is very inefficient alone, but is enhanced when packaged together with the m segment. We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the m segment creates the high-affinity binding site for the l segment. If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.

Bacteriophage phi 6↗
PubMed1998

RNA binding and modulation of PKR activity.

PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR.

Chromatography, Liquid↗
PubMed1993

Double-stranded RNA adenosine deaminase as a potential mammalian RNA editing factor.

Double-stranded RNA (dsRNA) adenosine deaminase, or DRADA, is a cellular enzyme that modifies adenosine residues to inosines in dsRNA by hydrolytic deamination, replacing A-U with mismatched I-U base pairs. Since it alters the base composition in its substrate RNA, one possible role played by DRADA is to participate in RNA editing. In this article, a brief review is given of characteristics of DRADA. Its possible involvement in RNA editing is also discussed in detail, including specific cases in which DRADA has been implicated as an RNA editing factor.

Adenosine Deaminase↗
PubMed1993

The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells.

The role of the interferon-induced double-stranded RNA (dsRNA)-activated human p68 protein kinase as an inhibitor of protein synthesis has been inferred from work with cell-free systems, but direct proof in animal cells is lacking. To document the action of p68 protein kinase in vivo, we have used an infection-transfection system where expression of p68 is driven by a vaccinia virus promoter regulated by the lacl repressor/operator controlling elements. In cultured cells infected with vaccinia virus and transfected with a plasmid containing the p68 gene, there is synthesis of p68 when lacl repressor is inhibited with isopropyl-beta-D-thiogalactoside. When infection-transfections are carried out with the p68 gene together with the luciferase (LUC) reporter gene, a strong inhibition of LUC expression developed with time postinfection. This inhibition was not observed with a mutant form of the kinase (Lys-->Arg at position 296) and it was reversed by antisense expression of the p68 gene. During inhibition of LUC expression the protein kinase was phosphorylated, possibly as a result of autophosphorylation activated by the dsRNA forms which are known to accumulate in vaccinia virus-infected cells. Inhibition of LUC expression was at the level of translation. Our findings demonstrate that expression and activation of the human p68 protein kinase in vivo potently inhibits protein synthesis.

Cells, Cultured↗
PubMed1993

RNA circularization reveals terminal sequence heterogeneity in a double-stranded RNA virus.

Double-stranded RNA viruses (dsRNA), termed LRV1, have been found in several strains of the protozoan parasite Leishmania. With the aim of constructing a full-length cDNA copy of the viral genome, including its terminal sequences, a protocol based on PCR amplification across the 3'-5' junction of circularized RNA was developed. This method proved to be applicable to dsRNA. It provided a relatively simple alternative to one-sided PCR, without loss of specificity inherent in the use of generic primers. LRV1 terminal nucleotide sequences obtained by this method showed a considerable variation in length, particularly at the 5' end of the positive strand, as well as the potential for forming 3' overhangs. The opposite genomic end terminates in 0, 1, or 2 TCA trinucleotide repeats. These results are compared with terminal sequences derived from one-sided PCR experiments.

Animals↗
PubMed1994

Characterization of rubella virus replication complexes using antibodies to double-stranded RNA.

A feature of the rubella virus (RV) replication cycle is the formation of cytoplasmic vesicle-containing structures known as replication complexes. Following detergent treatment of RV-infected cells, pre-embedding immunogold labeling electron microscopy using antiserum to double-stranded (ds) RNA was employed to characterize the replication complexes. Concentrations of gold particles were found associated with amorphous material located within the RV replication complex. Unlabeled long fine strands, 3-5 nm in width, were also frequently seen associated with this gold-labeled material. On some occasions gold-labeled vesicles within the replication complexes were also detected. The gold-labeled amorphous material was first detected in RV replication complexes at 12 hr postinfection, soon after the reported latent period of 8 hr. Concentrations of gold particles were not detected in mock-infected cells. The findings in this study indicate that the amorphous material is released from detergent-disrupted vesicles within the replication complex and that the vesicles contain the dsRNA. When cells were infected with the related Semliki Forest virus (SFV) and examined using the same antibody, similar gold-labeled material associated with unlabeled fine strands was also observed in SFV replication complexes. For both RV and SFV, the vesicles which line the inner membrane of the replication complexes contain the dsRNA which represent the viral replicative forms and replicative intermediates.

Animals↗
PubMed1994

HIV-1 TAR RNA has an intrinsic ability to activate interferon-inducible enzymes.

The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.

2',5'-Oligoadenylate Synthetase↗
PubMed1995

Protein-primed RNA synthesis in vitro by the virion-associated RNA polymerase of infectious pancreatic necrosis virus.

The 94-kDa virion-associated RNA-dependent RNA polymerase (RdRp) is present in infectious pancreatic necrosis virus in two forms: (i) as a free polypeptide (VP1) and (ii) as a genome linked protein (VPg) (J. G. Calvert et al., 1991, J. Gen. Virol. 72, 2563-2567). VP1 was guanylylated in vitro by incubating purified virus in the presence of [alpha2P]GTP. During further incubation in an in vitro RNA polymerase reaction mixture (in the presence of unlabeled GTP), the radiolabeled VP1-pG complex was "chased" via nascent RNA strands and replicative intermediates to a VP1-dsRNA complex. Labeled VP1-pG was recovered from the intermediate as well as from the final reaction products by digestion with RNase A and RNase V1, a dsRNA-specific nuclease. Analysis of the reaction products indicated that only the plus strands of the two genome segments were being synthesized in vitro which remained base-paired to their templates. The results suggest that in vitro transcription by the virion RdRp is primed by VP1 and then proceeds via an asymmetric, semiconservative, strand-displacement mechanism.

Animals↗
PubMed1995

Characterization of citrus tristeza virus subgenomic RNAs in infected tissue.

Citrus tristeza virus (CTV) specific RNAs extracted from infected citrus tissue were analyzed by Northern blot hybridization. RNAs were characterized by size and identified using cDNA probes specific to nine open reading frames (ORFs) identified by the analysis of sequence obtained from cDNA clones of the T36 isolate of CTV. Sequence specific cDNA probes identified the genomic RNA as well as subgenomic RNAs representing the p33, p65, p61, p27, p25, p18, p13, p20, and p23 ORFs in extracts of total or double-stranded RNA (dsRNA) isolated from infected tissue. A probe derived from the 3' terminal ORF (p23) hybridized to each of these subgenomic RNAs, indicating that the RNAs are 3' coterminal. The relative amounts of the different subgenomic RNAs varied widely. The RNAs for the p20 and p23 ORFs were the most abundant and surpassed the amount of the p25 or capsid protein specific subgenomic RNA. The number and sizes of the CTV subgenomic RNAs were the same in total RNA and dsRNA preparations. Propagation of T36 in seven different citrus hosts did not alter the pattern of subgenomic RNAs.

Citrus↗
PubMed1996

Immunolocalization of the dengue virus nonstructural glycoprotein NS1 suggests a role in viral RNA replication.

Although many workers have investigated the maturation and processing of the flavivirus nonstructural glycoprotein NS1 in infected cells, these studies have provided little insight into a possible function for NS1. In this study we investigated the subcellular localization of NS1 both by immunofluorescence and cryo-immuno electron microscopy of infected Vero and C6/36 cells. NS1 was found to be tightly associated with intracellular membranes, in particular with vesicle packets and large cytoplasmic vacuoles. Surprisingly, NS1 did not associate with mature virus particles, a finding that is inconsistent with the postulated role for this protein in virion assembly and/or maturation. However, dual-labeling experiments did reveal in both the confocal immunofluorescence and cryo-immuno EM studies the colocalization of NS1 with the viral dsRNA replicative form. Furthermore, localization of the dsRNA to the vesicle packets and cytoplasmic vacuoles seen in infected Vero and C6/36 cells, respectively, suggests that these structures may comprise the flavivirus replication complex. These findings provide further insights into the maturation pathway of NS1 and suggest a possible role for this protein in viral RNA replication.

Animals↗
PubMed1997

Stable protein-RNA interaction involves the terminal domains of bluetongue virus mRNA, but not the terminally conserved sequences.

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigated in vitro by means of a biochemical approach. By subjecting cytoplasmic extracts from virus-infected baby hamster kidney cells and in vitro synthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes most intimately associated with the labeled viral RNA. Competition binding studies indicated that NS2 has the greatest affinity for the 3' region of the viral transcripts. By analyzing the binding efficiency of NS2 to mutant RNA transcripts which lacked the fully conserved 5'- and/or 3'-terminal hexanucleotides, we have established that these sequences are not necessary for optimal binding. The specificity of the NS2-RNA interaction was investigated by competition experiments with unlabeled BTV-specific homologous and heterologous competitor RNAs as well as with viral double-stranded RNA (dsRNA). Although apparent differences in the ability of NS2 to bind to the different RNA transcripts were observed, it did not bind to the dsRNA.

Animals↗
PubMed1997

Preferential translation of reovirus mRNA by a sigma3-dependent mechanism.

We have characterized reovirus strains that differ in the degree to which they inhibit cellular protein synthesis and used them to investigate mechanisms regulating gene expression in infected cells. A previous genetic study associated distinct effects of reovirus strains on cellular translation with polymorphisms in viral protein sigma3. In cell extracts, sigma3 sequesters double-stranded RNA (dsRNA) and blocks activation of the dsRNA-activated protein kinase (PKR), an interferon-induced enzyme that inhibits translational initiation by phosphorylating elF-2alpha. We found that in infected cells, cellular protein synthesis is translationally regulated in a strain-specific manner. Using immunoprecipitation and indirect immunofluorescence we showed that the effect of a strain on cellular translation is not determined by the level of sigma3, but appears to result from differences in sigma3 localization. In cells infected with a strain that spares cellular translation, sigma3 is present throughout the cytoplasm, whereas in cells infected with inhibitory strains, sigma3 is restricted to perinuclear viral factories. Biochemical studies suggested that diffuse localization of sigma3 is a consequence of low affinity for capsid protein mu1. Our findings are consistent with a model in which the efficiency of cellular translation is determined by the cytoplasmic level of sigma3 that is not complexed with mu1.

Amino Acid Sequence↗
PubMed1999

Nascent flavivirus RNA colocalized in situ with double-stranded RNA in stable replication complexes.

Incorporation of bromouridine (BrU) into viral RNA in Kunjin virus-infected Vero cells treated with actinomycin D was monitored in situ by immunofluorescence using antibodies reactive with Br-RNA. The results showed unequivocally that nascent viral RNA was located focally in the same subcellular site as dsRNA, the putative template for flavivirus RNA synthesis. When cells were labeled with BrU for 15 min, the estimated cycle period for RNA synthesis, the nascent Br-RNA was not digested in permeabilized cells by RNase A under high-salt conditions, in accord with our original model of flavivirus RNA synthesis (Chu, P. W. G., and Westaway, E. G., Virology 140, 68-79, 1985). The model assumes that there is on average only one nascent strand per template, which remains bound until displaced during the next cycle of RNA synthesis. The replicase complex located by BrU incorporation in the identified foci was stable, remaining active in incorporating BrU or [32P]orthophosphate in viral RNA after complete inhibition of protein synthesis in cycloheximide-treated cells. These results are in accord with our proposal that dsRNA detected in foci previously located by immunofluorescence or by immunogold labeling of induced vesicle packets is functioning as the true replicative intermediate (Westaway et al., J. Virol. 71, 6650-6661, 1997; Mackenzie et al., Virology 245, 203-215, 1998). Implications are that the replicase complex is able to recycle in the same membrane site in the absence of continuing protein synthesis and that possibly apart from uncleaved NS3-NS4A, it has no requirement for a polyprotein precursor late in infection.

Animals↗
PubMed1999

Open reading frame in rotavirus mRNA specifically promotes synthesis of double-stranded RNA: template size also affects replication efficiency.

The 11 rotavirus mRNAs are capped, but not polyadenylated, have a high AU content, and serve as templates for the synthesis of double-stranded (ds)RNA. Earlier studies using a cell-free replication system showed that the 5'- and 3'-untranslated regions (UTRs) of the mRNAs have cis-acting signals that promote minus-strand synthesis. To identify additional factors that affect RNA replication, chimeric RNAs were made that consisted of portions of the gene 8 mRNA of SA11 rotavirus and of the gene for green fluorescent protein (gfp) or for the N protein of respiratory syncytial virus. Analysis of the chimeras in the cell-free replication system under noncompetitive conditions showed that the open reading frame (ORF) of viral mRNAs contains information that specifically promotes minus-strand synthesis. Results were also obtained indicating that a high AU content may increase the replication efficiency of RNAs and that, in general, an inverse correlation exists between replication efficiency and the length of the RNA template. Replication assays performed under competitive conditions showed that nonviral RNAs can interfere significantly with the replication of viral mRNAs, mostly likely by sequestering nonspecific RNA-binding proteins that are of limited concentration in the replication system and that are essential for dsRNA synthesis. In summary, rotavirus dsRNA synthesis is affected by many factors including cis-acting replication signals located in the 5'-UTR, 3'-UTR, and ORF of the mRNA as well as the size and possibly the AU content of the mRNA.

5' Untranslated Regions↗
PubMed2001

Analyzing the mechanisms of interferon-induced apoptosis using CrmA and hepatitis C virus NS5A.

The dsRNA-dependent protein kinase, PKR, is a key component of interferon (IFN)-mediated anti-viral action and is frequently inhibited by many viruses following infection of the cell. Recently, we have demonstrated that IFN and PKR can sensitize cells to apoptosis predominantly through the FADD/caspase-8 pathway (S. Balachandran, P. C. Roberts, T. Kipperman, K. N. Bhalla, R. W. Compans, D. R. Archer, and G. N. Barber. (2000b) J. Virol. 74, 1513-1523). Given these findings, it is thus plausible that rather than specifically target IFN-inducible genes such as PKR, viruses could also subvert the mechanisms of IFN action, in part, at locations that could block the apoptotic cascade. To explore this possibility, we analyzed whether the poxvirus caspase-8 inhibitor, CrmA, was able to inhibit IFN or PKR/dsRNA-mediated apoptosis. Our findings indicated that CrmA could indeed inhibit apoptosis induced by both viral infection and dsRNA without blocking PKR activity or inhibiting IFN signaling. In contrast HCV-encoded NS5A, a putative inhibitor of PKR, did not appear to inhibit cell death mediated by a number of apoptotic stimuli, including IFN, TRAIL, and etoposide. Our data imply that viral-encoded inhibitors of apoptosis, such as CrmA, can block the innate arms of the immune response, including IFN-mediated apoptosis, and therefore potentially constitute an alternative family of inhibitors of IFN action in the cell.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Generation and genetic characterization of avian reovirus temperature-sensitive mutants.

There currently is little known about the genetic and biological functions of avian reovirus (ARV), an atypical member of the family Reoviridae and the prototype of all nonenveloped viruses that induce syncytia formation. In this study, we created ARV temperature-sensitive (ts) mutants by chemical mutagenesis of ARV strain 138. We developed a novel efficiency of lysis (EOL) screening technique and used it and the classical efficiency of plating (EOP) assay to identify 17 ARV ts mutants. Pairwise mixed infections of these mutants and evaluation of recombinant progeny ts status led to their organization into seven recombination groups. This indicates that these new groups of mutants represent the majority of the ARV genome. To phenotypically characterize the ts mutants, progeny double-stranded RNA (dsRNA) produced at permissive and nonpermissive temperature was measured. Some mutants were capable of dsRNA synthesis at the restrictive temperature (RNA(+)), which indicates the effects of their ts lesions occur after RNA replication. Most mutants were RNA(-), which suggests their mutations affect stages in viral replication that precede progeny genome synthesis.

Animals↗
PubMed2001

A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique beta-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this beta-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this beta-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this beta-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a beta-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.

3' Untranslated Regions↗