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Nucleotide sequence of the satellite of peanut stunt virus reveals structural homologies with viroids and certain nuclear and mitochondrial introns.

Peanut stunt virus-associated RNA 5 (PARNA 5), the satellite of a plant cucumovirus, is a linear RNA of 393 nucleotides with a 5' cap and a 3' hydroxyl group. Determination of its nucleotide sequence has revealed two consecutive open reading frames that together extend most of its length. Sequences at the 5' and 3' ends are homologous with those of the satellite of the related cucumber mosaic virus, and the double-stranded forms of both satellites contain an unpaired guanosine at the 3' end of the minus strand. However, little other homology exists between the two satellites. In contrast, PARNA 5 has several regions of 90% sequence homology with various plant viroids, including sequences of the conserved central region of most viroids. Such homologies suggest a common origin with viroids coupled with specific adaptation as a linear RNA. The presence within PARNA 5 of conserved intron sequences essential to proper RNA processing suggests a possible origin from plant introns and/or involvement of such sequences in the processing of PARNA 5 multimers to monomers at some stage of replication.

Base Sequence↗

Immunochemical detection of ds-RNA in healthy and virus-infected plants and specific detection of viral ds-RNA by hybridization to labelled complementary DNA.

Double-stranded (ds) RNA was detected in nucleic acid preparations from virus-free leaf tissues of several plant species by radial double-diffusion tests with antiserum raised against polyinosinic : polycytidylic acid [poly(I) : poly(C)]. No significant differences in the concentrations of ds-RNA were detected by such tests in virus-free French bean leaves and those infected with tobacco mosaic virus or tobacco ringspot virus. However, virus-specific ds-RNAs as well as genomic RNAs of both viruses were readily detected and estimated quantitatively by hybridization to 3H-labelled complementary DNA probes. It is concluded that it is not feasible to screen plant material infected with single-stranded RNA viruses by immunochemical tests for virus-associated ds-RNAs.

Fabaceae↗

Natural occurrence of 2-5A in interferon-treated EMC virus-infected L cells.

Until now the interferon-mediated 2'-5' adenine oligonucleotide inhibitors (2-5A) of cell-free protein synthesis have not been detected in intact cells. Here we report their natural occurrence in interferon-treated, EMC virus-infected mouse L cells in amounts consistent with the idea that they play a part in the inhibition of virus growth.

Adenine Nucleotides↗

Enhancement of the interferon-induced double-stranded RNA-dependent protein kinase activity by Sindbis virus infection and heat-shock stress.

In extracts of FL cells that were infected with Sindbis virus or treated with heat-shock stress, dsRNA-dependent phosphorylation of 77K protein was markedly increased. The 77K phosphoprotein was indistinguishable from the autophosphorylated and activated form of interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) by two-dimensional gel electrophoresis, and was immunologically related to P68 (Galabru, J. and Hovanessian, A., J. Biol. Chem. 262, 15538 (1987], the HeLa cell counterpart of PK-I. Immunoblotting experiments using monoclonal antibody against PK-I revealed that control cell extracts contained a substantial amount of PK-I protein, although they showed no measurable PK-I activity even when dsRNA was added. The amount of PK-I protein did not increase during a transient dsRNA-dependent enhancement of PK-I activity caused by Sindbis virus infection and heat-shock stress. This implies that the conversion of PK-I protein from a dsRNA-unresponsive form to a responsive form may be important in the regulation of PK-I activity. A similar mode of PK-I regulatory mechanism was operative in the early stages of IFN treatment, although after a prolonged treatment a net synthesis of the PK-I protein did take place.

Cells, Cultured↗

RNA-dependent RNA polymerases, viruses, and RNA silencing.

Most viruses have RNA genomes that are replicated and transcribed into messenger RNA by viral RNA-dependent RNA polymerases (RdRps), usually in concert with other viral and host factors. Many, if not most, eukaryotes also encode putative RdRps that have been implicated in sequence-specific, RNA-triggered gene silencing. Although the viral and cellular RdRps have no sequence homology, they share functional similarities such as copying messenger RNA templates and intercellular spread of the amplified sequences. Better understanding of viral and host RdRps will improve our ability to control viruses and to use RNA silencing and viruses as tools for research, biotechnology, and medicine.

Animals↗

Specific site of action for single-strand specific nuclease on the double-stranded circular DNA intermediates of an avian RNA tumor virus.

Circular viral DNA intermediates obtained from the quail tumor line, QT6, at 1 day after infection, were opened at one specific location by the single-strand specific nuclease, S1, of Aspergillus oryzae. This site was no longer accessible to the S1 nuclease when circles were first opened at another location with a restriction endonuclease.

Alpharetrovirus↗

Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome.

The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2 (Ld), of the ascomycete fungus Ophiostoma novo-ulmi has been determined. One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins of 178 and 182 amino acids, respectively. The large ORF contains 12 UGA codons which code for tryptophan in ascomycete mitochondria and has a codon bias typical of mitochondrial genes, consistent with the localization of Ld dsRNAs within the mitochondria. The amino acid sequence contains motifs characteristic of RNA-dependent RNA polymerases (RdRps). This putative RdRp was shown to be related to putative RdRps of mitochondrial dsRNAs of another ascomycete and a basidiomycete fungus and also to a putative RdRp encoded by the mitochondrial genome of Arabidopsis thaliana. In multiple sequence alignments, the fungal mitochondrial dsRNA-encoded RdRp-like proteins formed a cluster, ancestrally related to the RdRps of the yeast 20S and 23S RNA replicons and of the positive-stranded RNA bacteriophages of the Leviviridae family, but distinct from RdRps of other families and genera of fungal RNA viruses and related plant and animal RNA viruses. Northern blot analysis with RNA 3a strand-specific probes indicated that nucleic acid extracts of Ld contain more single-stranded (positive-stranded) RNA than dsRNA, consistent with an evolutionary relationship between RNA 3a and positive-stranded RNA phages.

Amino Acid Sequence↗

Isolation of a dsRNA virus from Dipodascus (Endomyces) magnusii.

Virus-like particles (VLPs) of 40 nm diameter were isolated from the yeast-like fungus Dipodascus magnusii. These VLPs copurify with several linear double-stranded RNA molecules of different size. We have found some polymorphism in both the length and the number of these dsRNAs among six D. magnusii strains. Analysis of CsCl gradient-purified VLPs on PAGE/SDS electrophoresis showed one major protein component with an apparent molecular weight of 75 kDa.

Fungi↗

From interferon induction to fungal viruses.

Viruses of fungi (mycoviruses) were first discovered in diseased mushrooms. However the finding that the antiviral and interferon-inducing activities of extracts of apparently healthy isolates of a number of Penicillium species were due to the presence of double-stranded (ds) RNA arising from mycovirus infections sparked off an explosion of interest in what has now become a distinct area of virology. Two families of dsRNA mycoviruses are now established: the Totiviridae and the Partitiviridae which comprise isometric viruses with genomes of one or two dsRNA segments respectively. Virus isolates in both families often contain additional satellite dsRNAs, which may in some fungi (e.g. Saccharomyces cerevisiae, Ustilago maydis) code for "killer" proteins which are toxic to other sensitive strains of the same or closely related species. In Endothia parasitica, which causes chestnut blight disease, dsRNA is associated with hypovirulence and is enclosed in lipid-rich vesicles. In Ophiostoma (Ceratocystis) ulmi, which causes Dutch elm disease, dsRNA is associated with the mitochondria and, in some diseased isolates of the fungus, specific dsRNA segments are associated with reduction of cytochrome oxidase and respiratory deficiency, resulting in slow growth and abnormal morphology.

Fungi↗

Analyses and conservation of sequences among the cognate L3 segments of the five United States bluetongue viruses.

We determined the complete nucleotide sequences of the cognate L3 double-stranded RNA (ds-RNA) segments of bluetongue virus (BTV) serotypes 2, 11, and 13 encoding the major viral inner capsid protein, VP3. Each cognate L3 segment was 2772 nucleotides long and contained a single open reading frame (ORF) with an initiation codon at nucleotides #18-20 and a termination codon at nucleotides #2721-2723. This ORF can encode the 901-amino acid VP3 protein (103 kDa) with a calculated isoelectric point of 6. Phylogenetic analyses using both the nucleotide and the deduced amino acid sequences of the L3 cognate gene of the five US BTV serotypes indicated that the BTV-2 serotype recently isolated in Florida was more distantly related than BTV-10, 11, 13 or 17. The five US BTV serotypes were derived apparently from two distinct gene pools, findings consistent with their current geographic distribution in North America.

Amino Acid Sequence↗

Identification of rice black streaked dwarf virus in different cereal crops with dwarfing symptoms in China.

This report describes isolation of virus particles from plants of rice, maize, wheat and sorghum with symptoms of dwarfing collected from two provinces of China, purification of double-stranded RNA (dsRNA) from the virus particles, and synthesis of full-length cDNAs of genome segments 9 (S9) and 10 (S10) by reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis showed that the S9 sequences of the Chinese isolates and a Japanese rice black-streaked disease virus (RBSDV) isolate were very similar (89.1-89.6% homology at nucleotide level and 92.3-92.9% and 95.8-98.6% homology at amino acid level for ORF1 and ORF2, respectively). Analogical similarity was found also for the S10 sequences of the isolates under comparison: 93.0-95.4% homology at nucleotide level and 96.2-97.0% homology at amino acid level. However, there was a relatively lower similarity for S9 and S10 segments ofthe Chinese isolates and an Italian maize rough dwarf virus (MRDV) isolate. The phylogenetic analysis indicated that the Chinese isolates that infect rice, maize, wheat and sorghum and cause similar symptoms could represent the same virus species, RBSDV.

Amino Acid Sequence↗

Nucleic acid and structural proteins of infectious bursal disease virus isolates belonging to serotypes I and II.

The nucleic acid and structural proteins of infectious bursal disease virus serotype I (six isolates) were compared with those of serotype II (two isolates). Five of the serotype I isolates originated from chickens, whereas both serotype II isolates were from turkeys. The growth curves of representative isolates of both serotypes were similar, but the latent period and virus yield were different. The seven isolates tested had two segments of double-stranded genomic RNA. The RNA migration patterns of viruses belonging to each serotype were similar, but differences were noticed between the two serotypes. There were differences in the molecular weights of viral proteins (VP) 3 and 4 from the two serotypes, and serotype II isolates lacked VP-2.

Animals↗

The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro.

Gene expression by a viral-like double-stranded RNA genetic element associated with reduced virulence (hypovirulence) of the chestnut blight fungus was recently shown to involve an autoproteolytic event which resulted in the release of an encoded protease, designated p29, from a polyprotein during translation. Mutational analysis of p29, described in this report, revealed that residues Cys-162 and His-215 are essential for autocatalytic cleavage. The results were also consistent with previous predictions that cleavage occurs between Gly-248 and Gly-249. Interestingly, p29 bears a striking resemblance to the potyvirus-encoded protease HC-Pro. Both proteases autocatalytically cleave at glycine dipeptides. In addition, there is a significant degree of similarity in the amino acid sequences flanking the essential Cys and His residues of the two proteases and in the spacing of these residues from their respective cleavage sites.

Amino Acid Sequence↗

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.

Apoptosis↗