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At least 595 records · Page 33Linked to original sources
PubMed2006

Mycovirus cryphonectria hypovirus 1 elements cofractionate with trans-Golgi network membranes of the fungal host Cryphonectria parasitica.

The mycovirus cryphonectria hypovirus 1 (CHV1) causes proliferation of vesicles in its host, Cryphonectria parasitica, the causal agent of chestnut blight. These vesicles have previously been shown to contain both CHV1 genomic double-stranded RNA (dsRNA) and RNA polymerase activity. To determine the cellular origins of these virus-induced membrane structures, we compared the fractionation of several cellular and viral markers. Results showed that viral dsRNA, helicase, polymerase, and protease p29 copurify with C. parasitica trans-Golgi network (TGN) markers, suggesting that the virus utilizes the fungal TGN for replication. We also show that the CHV1 protease p29 associates with vesicle membranes and is resistant to treatments that would release peripheral membrane proteins. Thus, p29 behaves as an integral membrane protein of the vesicular fraction derived from the fungal TGN. Protease p29 was also found to be fully susceptible to proteolytic digestion in the absence of detergent and, thus, is wholly or predominantly on the cytoplasmic face of the vesicles. Fractionation analysis of p29 deletion variants showed that sequences in the C terminal of p29 mediate membrane association. In particular, the C-terminal portion of the protein (Met-135-Gly-248) is sufficient for membrane association and is enough to direct p29 to the TGN vesicles in the absence of other viral elements.

Ascomycota↗
PubMed2006

Dynamics of dsRNA mycoviruses in black Aspergillus populations.

Approximately 10% of all examined 668 representatives of black Aspergillus species, independent of worldwide location, were infected with double-stranded RNA (dsRNA) mycoviruses. These isometric viruses (25-40 nm diameter) contained a variety of often multiple segments of different dsRNA sizes ranging from 0.8 to 4.4 kb in size. In one strain the virus shows clear visible effects on its host with non-sporulating sectors. We quantified the fitness costs of these and more 'cryptic' virus infections on mycelial growth rate and spore production, and on competitive ability with respect to other strains under different growth conditions. Mycovirus infection proved detrimental in all these measures. The reduced success in interference competition due to mycovirus infection belies co-evolution of mycovirus and host to a mutually beneficial symbiosis, like in killer virus systems in yeast and smut and agrees more to recent infections. For a stable virus infection frequency in the black Aspergillus population, fitness costs and spontaneous loss should be balanced with new infections. Implications of even small viral fitness effects combined with the observed transmission limits for host and mycovirus are discussed.

Aspergillus↗
PubMed1975

Comparison of the sensitivity to ultraviolet irradiation of reovirus 3 and some viruses of the Kemerovo group.

The kinetics of UV inactivation of the tick-borne Kemerovo (strain R-10) and Lipovnik (strain Lip-91) viruses which have been preliminarily classified as possible members of the Reovirus group was examined. Reovirus 3 and Sindbis virus served as reference double-stranded RNA and single-stranded RNA viruses, respectively. The parameters of UV inresembled those of Reovirus 3. This is consistent with their tentative classification as reovirus-like viruses.

Animals↗
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↗
PubMed2002

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus.

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.

Animals↗
PubMed1992

Antigenic and molecular analysis of influenza A(H3N2) virus strains isolated in 1985 in open and closed communities of northern Germany.

Antigenic and molecular analyses of influenza A(H3N2) virus strains isolated in 1985 during outbreaks in open and closed communities of North Germany were carried out. The data obtained have shown that 11 strains isolated in a closed orphanage were antigenically similar to each other. The electrophoretic mobilities of either HA, NP, M1 and NS1 polypeptides or of double stranded RNA segments were indistinguishable. Analysis of viruses isolated at the same time from open communities has revealed that they contained at least three groups of strains differing in homology of 3-5 RNA segments. These data support the idea that an outbreak of influenza in a community is caused by single virus strain, from which their slightly different variants of the virus arise during circulation among sensitive persons.

Adolescent↗
PubMed1998

The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase.

The parapoxvirus orf virus was resistant to type 1 (IFN-alpha) and type 2 (IFN-gamma) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon-resistance gene, and is referred to as the (putative) orf virus interferon-resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR-Tx) inhibited the activation (by autophosphorylation) of an interferon-inducible, double-stranded (ds) RNA-dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus-infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon-treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon-resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.

Animals↗
PubMed1993

Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase.

Deletion of the vaccinia virus K3L gene, a homologue of the alpha subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E. (1991) Virology 183, 419-422). Purified recombinant K3L gene product, pK3r, has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2 alpha (eIF-2 alpha)-specific protein kinase (PKR) in in vitro reactions. Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2 alpha. In the cell-free translation system, pK3r exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2 alpha. In addition, pK3r reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the alpha subunit of purified eIF-2. At 400 mM NaCl, in vitro translated [35S]methionine-radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR. This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2 alpha as the domain recognized by the eIF-2 alpha-specific protein kinases. In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3. Recombinant pK3 also prevents activation of the heme-sensitive eIF-2 alpha-specific protein kinase, eIF-2 alpha-PKh, in both cell-free translation systems as well as in partially purified preparations. This suggests some similarity between the eIF-2 alpha binding domains of the two eIF-2 alpha specific protein kinases.

Animals↗
PubMed1994

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗
PubMed1991

Expression of the terminal protein region of hepatitis B virus inhibits cellular responses to interferons alpha and gamma and double-stranded RNA.

Constructs expressing the core, surface, X, or polymerase proteins of hepatitis B virus were transfected into human cells. In transient assays, only the polymerase inhibited the responses to interferons alpha and gamma (IFN-alpha and -gamma). Stable expression of the polymerase was achieved in the cell line 2fTGH, which carries an IFN-inducible marker gene, by growth under conditions that select for inhibition of the response to IFN-alpha, but the clones grew poorly. When expressed alone, the terminal protein domain of the polymerase gene inhibited the response to IFN-alpha and the reverse transcriptase plus RNase H domains appeared to be toxic. Clones of cells expressing terminal protein alone, selected for the loss of response to IFN-alpha, grew normally and had no detectable response to IFN-alpha, IFN-gamma, or double-stranded RNA. Binding of IFN-alpha to these cells was not impaired but did not lead to activation of the E alpha subunit of the IFN-induced transcription factor E. These observations are of potential importance in relation to the pathogenesis of chronic hepatitis B virus infection and the resistance of such infection to IFN-alpha therapy.

Cell Line↗
PubMed1969

Comparison of the ribonucleic Acid subunits of reovirus, cytoplasmic polyhedrosis virus, and wound tumor virus.

Double-stranded ribonucleic acid (RNA) from intact cytoplasmic polynedrosis virus (CPV) and wound tumor virus (WTV) was analyzed by polyacrylamide gel electrophoresis. Using RNA from type 3 reovirus as a standard, it was calculated that CPV-RNA consisted of 9 subunits corresponding to a molecular weight of 12.7 x 10(6) and WTV-RNA consisted of 12 subunits corresponding to a molecular weight of 15.5 x 10(6).

Journal Article↗
PubMed1990

Translational control by the Epstein-Barr virus small RNA EBER-1. Reversal of the double-stranded RNA-induced inhibition of protein synthesis in reticulocyte lysates.

A role for the Epstein-Barr virus small RNA species EBER-1 in the regulation of protein synthesis has been investigated in the reticulocyte-lysate cell-free translation system. Recombinant EBER-1 was synthesized by in vitro transcription of a plasmid containing the viral gene and purified by CF11-cellulose chromatography and ribonuclease III treatment. When added to the reticulocyte lysate at 10-20 micrograms/ml or more, EBER-1 prevents the inhibition of protein synthesis caused by low concentrations of synthetic double-stranded RNA, poly(I).poly(C). This effect is eliminated by treatment of the recombinant EBER-1 with ribonuclease T1. Disruption of the secondary structure of EBER-1 by substitution of inosine for guanosine in the in-vitro-synthesized RNA impairs the ability of EBER-1 to prevent the poly(I).poly(C)-mediated inhibition of protein synthesis. These results suggest that high concentrations of EBER-1 regulate protein synthesis by blocking the activation of the double-stranded RNA-dependent eukaryotic initiation factor 2 alpha (eIF-2 alpha) protein kinase DAI (p68), and that this property is dependent on the secondary structure of the small RNA molecule.

Animals↗
PubMed2003

Transient expression of homologous hairpin RNA causes interference with plant virus infection and is overcome by a virus encoded suppressor of gene silencing.

Specific post-transcriptional gene silencing (PTGS) of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA (dsRNA) molecules. In plants, PTGS is part of a defense mechanism against virus infection. We have previously shown and patented that direct delivery to nontransgenic plants of dsRNA derived from viral sequences specifically interfere with virus infection. Here, we show that transient expression of constructs encoding hairpin RNA homologous to a rapidly replicating plant tobamovirus also interferes with virus multiplication in a sequence-dependent manner. A three-day lag period between delivery of hairpin RNA and virus into the same tissues completely block virus infectivity. Several hallmarks characteristic of PTGS were associated with viral interference mediated by hairpin RNA: high level of sequence identity between the hairpin RNA and the target RNA, presence of siRNAs in extracts derived from leaves infiltrated with hairpin RNA, and helper component-proteinase (HC-Pro) of potyviruses, a suppressor of PTGS, overcame interference. No evidence for a mobile silencing suppression signal induced by transient expression of HC-Pro was observed. The approach described here has the potential to be used as a versatile tool for studying the onset of PTGS in cases involving virus infection, in opposition to dsRNA-transgenic plants, which allow primarily for the study of PTGS maintenance.

Gene Silencing↗
PubMed1992

Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase.

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.

Base Sequence↗
PubMed1985

Structure and expression of the M2 genomic segment of a type 2 killer virus of yeast.

The M2 double-stranded (ds) RNA species encodes toxin and resistance functions in Saccharomyces cerevisiae strains with the K2 killer specificity. RNA sequence analysis reveals the presence of a large open reading frame on the larger heat-cleavage product of M2 dsRNA, which is translated in vitro to yield a 28 kd polypeptide as a major product. The postulated translation initiator AUG triplet is located within a stem and loop structure near the 5' terminus of the positive strand, which also contains plausible 18S and 5.8S ribosomal RNA binding sites. These features may serve to regulate the translation of the K2 toxin precursor. The M1 (from type 1 yeast killers) and M2 dsRNA species lack extensive sequence homology, although specific features are shared, which may represent structural elements required for gene expression and replication.

Amino Acid Sequence↗
PubMed2004

Putative replication intermediates in endornavirus, a novel genus of plant dsRNA viruses.

Oryza sativa endornavirus (OSV) belongs to a new genus (Endornavirus) and family (Endoviridae) with members containing large double-stranded RNA (dsRNA) replicons with plasmid-like properties. Analysis of products obtained from in vitro reaction of the OSV RNA-dependent RNA polymerase revealed a rapid increase of a population of the non-coding strand RNA molecules with a head-to-tail composition. Northern hybridization of total RNA from OSV-carrier cells with riboprobes specific for the coding strand RNA, revealed two types of RNA molecules (i) with a site specific nick and (ii) full-length unnicked molecules. Quantitative analyses of these RNAs showed about 50-fold higher amounts of full-length unnicked molecules in cultured cells in which the OSV copy number increases compared with those found in the seedling cells. Both the head-to-tail linked non-coding strand and the full-length coding strand molecules were also found in wild rice and broad beans infected with other endornaviruses indicating that the presence of these unique types of RNA molecules should be considered as a characteristic feature of Endoviridae .

Base Sequence↗