Variability and inheritance of double-stranded RNA viruses in Phaffia rhodozyma

 The present survey demonstrates polymorphism in both the length and the number of double-stranded RNAs (dsRNAs) among six Phaffia rhodozyma strains. Strains with one-, three- and four-types of dsRNA molecules were found, while two strains proved to be dsRNA-free. Elongated icosahedral virus-like particles (VLPs) 34×26 nm in size were detected in strains carrying four- or three-types of dsRNAs. One 3.7-kb dsRNA molecule was found not to form part of the VLP genome. Transmission of the VLPs of strain ATCC 24203 was followed through the basidiospores during the sexual cycle. Cytoplasmic inheritance was observed. Received: 24 February/3 June 1996     

The Gene Product Specified by a Double Stranded RNA in the Flax Rust (<Emphasis Type="Italic">Melampsora lini</Emphasis>) is Expressed during Rust Growth

Strain SP6 of the flax rust (Melampsora lini) contains 11 double-stranded RNAs (dsRNAs) of unknown function. A large open-reading frame (B3ORF1) in dsRNA B3 encodes a polypeptide of 614 amino acids, and using an antiserum raised against the B3ORF1-glutathione S-transferase fusion protein prepared from a bacterial expression system, we have detected the presence of a 67 kDa polypeptide in rust urediospores. This polypeptide, identical in size to that of the predicted translation product of B3ORF1, was not detected in spores from either a fungal strain lacking the B3 dsRNA or an isogenic strain containing no dsRNA. These data indicate that B3ORF1 present in the flax rust B3 dsRNA is expressed in vivo which warrants farther investigation in search for its function during rust development.     

Searching for a New Putative Cryptic Virus in Pinus sylvestris L

Double-stranded RNAs (dsRNAs) were detected in different pine populations in Germany and Hungary. Two dsRNA species of 1.5 and 1.58 kbp, respectively, persisted in the same trees for at least 2 years and their presence was not associated with any symptoms. The dsRNAs were found to sediment in the VLP (virus-like particles) fraction and to be protected by protein(s) against RNase A digestion at low salt. cDNA cloning and sequencing of the smaller segment (dsRNA2) led to the identification of a putative RNA-dependent RNA-polymerase (RdRp) containing the GDD, as well as three other, conserved motifs. Sequence comparison with different RNA viruses and phylogenetic analysis indicates that the putative RdRp from pine shows highest similarity to the homologous proteins of Beet cryptic virus 3 and of a cryptic virus of Pyrus pyrifolia. On the basis of these results we suggest that the 1.5 and 1.58 kbp dsRNAs in P. sylvestris may represent the genomic segments of a new plant cryptic virus, Cryptoviruses have not yet been reported to occur in Gymnosperms.     

Molecular characterisation of two novel double-stranded RNA elements from <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

The incomplete sequences of two large, 10–12 kbp, double-stranded RNAs (dsRNAs) found in the TW-2 isolate of the saprophytic fungus, Phlebiopsis gigantea (Pg) are reported. Both PgV-TW2 dsRNA1 and dsRNA2 potentially encode fusion proteins which are apparently expressed by a translational frameshifting mechanism. The C-terminal region of both predicted proteins was 21% identical and contained the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses and had highest similarity with members of the family Totiviridae, but possibly do not form virions. The remainder of the N-terminal protein sequences predicted from the PgV-TW2 dsRNA1 and dsRNA2 sequences and the 3′-terminal nucleotide sequences of both dsRNAs had no homology with one another or any sequence in the database suggesting that individually both may be members of novel families of mycoviruses. The nucleotide sequence data reported in this article has been assigned the accession numbers AM111O96 and AM111097 for PgV-TW2 dsRNA1 and PgV-TW2 dsRNA2 respectively.     

Double stranded RNA in natural isolates ofNeurospora

Thirty-six wild type isolates ofNeurospora were surveyed for the presence of dsRNA. The survey identified seven strains which contain dsRNA molecules. These seven strains are all from different geographic locations. The sizes of the dsRNAs range from 500 bp to 18 kb and a total of seven distinct dsRNA species was identified. Cross homologies of some of the dsRNAs were apparent. There was homology between the 9.0 kb dsRNA and genomic DNA prepared from all strains in the survey, indicating a possible cellular rather than viral origin for this dsRNA species. None of the other dsRNAs hybridized with genomic DNA suggesting a viral origin for these dsRNAs.     

Sequences of three dsRNAs associated with La France disease of the cultivated mushroom (Agaricus bisporus)

Summary La France disease of the cultivated mushroom, Agaricus bisporus, is known to be associated with the presence of a number of dsRNA segments. The nucleotide sequences of the dsRNAs M2 (1.3 kb), M1 (1.55 kb) and L3 (2.8 kb), invariably associated with the disease, were determined. Putative coding sequences for proteins with molecular weights of 38, 40 and 87 kDa were found for M2, M1 and L3 dsRNAs, respectively. The average G+C content of these dsRNAs was 43%, close to that of A. bisporus nuclear DNA. The nucleotide sequences, as well as the amino acid sequences, appear to be unique, as no matching sequences could be found among databases. S3 dsRNA (0.39 kb), which is occasionally found in large amounts in diseased mushrooms, is an internally deleted variant of M2 dsRNA and is largely composed of the non-coding ends of that dsRNA.     

Physicochemical properties of two distinct types of virus-like particles from Helminthosporium victoriae

Two serologically distinct types of virus-like particles (VLPs) were isolated from Helminthosporium victoriae. The first, which was isolated from three normal and two diseased isolates, sedimented at a rate of 190 S when centrifuged in linear-log sucrose density gradients. The second, which was found only in the two diseased isolates, had a sedimentation coefficient of 145 S. No VLPs were detected in two other normal isolates of the fungus. The 190 and 145 S VLPs both were polyhedral and 35 to 40 nm in diameter and had typical nucleoprotein absorbancy spectra. The 190 S type of VLP was electrophoretically distinct from the 145 S VLP when electrophoresed on either polyacrylamide gels or cellulose acetate strips. Equilibrium density gradient centrifugation in cesium chloride revealed a single density component for the 190 S VLPs with a buoyant density of 1.4325 g/cm³ The 145 S VLPs could be resolved into two to four components with buoyant densities of 1.3813 to 1.4138 g/cm³. SDS polyacrylamide gel electrophoresis of the 190 S VLPs revealed two major proteins of molecular weight 83,000 and 88,000 present in equimolar amounts. The 145 S type of VLP had three major proteins with molecular weights of 92,000, 97,000, and 106,000; these were also present in equimolar amounts. Both the 145 and 190 S VLPs contained double-stranded RNA (dsRNA). The 190 S type of VLP had a single species of dsRNA with a molecular weight of 3.0 × 10⁶. There were four classes of dsRNA associated with the 145 S VLPs, and their molecular weights were 2.4, 2.2, 2.1, and 2.0 × 10⁶.     

Gene-protein assignments within the yeast Yarrowia lipolytica dsRNA viral genome

Summary Some strains of the yeast Yarrowia lipolytica possess virus-like particles (VLPs) which encapsidate a double-stranded RNA (dsRNA) genome designated L_y. We report here that these VLPs have two associated polypeptides of molecular weights 83 kd (VL_y-P1) and 77 kd (VL_y-P2). Denatured Ly-dsRNA was used to program a cell-free rabbit reticulocyte translation system, resulting in the appearance of four major products, viz. Ly-P1 (83 kd); L_y-P2 (77 kd); L_y-P3 (74 kd) and L_y-P4 (68 kd). The in vivo viral-associated protein VL_y-P1 co-migrated on SDS-polyacrylamide gels with the in vitro product L_y-P1 and, similarly, VL_y-P2 co-migrated with L_y-P2. Peptide mapping data confirm the identity of the in vivo products (VL_y-P1 and VL_y-P2) and their in vitro counterparts. The conclusion made is that VL_y-P1 and VL_yP2 are almost identical primary translation products of the L_y genome, derived from a single or multiple species of L_y-dsRNA. RNA blot hybridizations using L_1A M₁ and separately, L_2A M₂ probes prepared from appropriate K1 and K2 Saccharomyces cerevisiae killer strains, failed to show any detectable homology to L_y-dsRNA, substantiating the uniqueness of the Ly genome with respect to the K1 and K2 S. cerevisiae dsRNA killer systems.     

Assignment of linkage groups to the electrophoretically-separated chromosomes of the fungus Podospora anserina

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.     

Genome Organization and Expression of the <Emphasis Type="Italic">Penicillium stoloniferum</Emphasis> Virus F

The complete sequences of three double-stranded (ds) RNAs (referred to F1, F2 and F3) of Penicillium stoloniferum virus F (PsV-F) were established. The F1 dsRNA was 1677 bp in length, and it contained one open reading frame (ORF) of 538 amino acids (molecular weight of 63 kDa, referred to P63), The F2 dsRNA was 1500 by in length, and also it contained one ORF of 420 amino acids (molecular weight of 46 kDa, referred to P46). The F3 dsRNA was 677 bp in length, but contained a small ORF with unknown function. A sequence motif of (5′-CGTAAAA-3′) was found only at the 5′ termini of the F1 and F2 dsRNAs, and a sequence motif of (5′-TAAAAAAAAA-3′) was found at the 3′ termini of all three dsRNA segments. The predicted amino acid sequence of F1 showed 38–48% sequence homology with the putative dsRNA-dependent RNA polymerases (RdRp) of dsRNA viruses, but the predicted amino acid of F2 showed no homology. Phylogenetic analysis using the RdRp sequences of the various Partitiviruses and Alphacryptoviruses revealed that PsV-F clustered well with Partitiviruses, but showed remote relationship with PsV-S. Near full-length and positive-sense single-stranded (ss) RNAs derived from the Fl, F2 and F3 dsRNAs were detected from the PsV-infected host cell. The expressed proteins of P63 and P46 showed a positive reaction against PsV-F antiserum, indicating P63 and P46 as RdRp and capsid protein, respectively. These results suggest that PsV-F can be a member of Partitivirus, but it is quite distinct from PsV-S electrophoretically, serologically and genetically, though both viruses coexist in the same cell.     

A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata

Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617 bp), dsRNA 2 (2794 bp), dsRNA 3 (2576 bp) and dsRNA 4 (1420 bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration (3 μg/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed (0.3 μg/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35–1.40 g/cm³ depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447 nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3′-poly (A) tails ranging from 33 to 50 nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1).     

The 3-phosphoglycerate kinase gene of the yeastYarrowia lipolytica de-represses on gluconeogenic substrates

We have isolated the 3-phosphoglycerate kinase (PGK) gene of the yeastYarrowia lipolytica by probing a genomic library with a PCR fragment amplified with primers deduced from two highly conserved regions of various PGKs. It is a unique sequence encoding a polypeptide of 417 residues with extensive homology to other PGKs, especially to that ofAspergillus nidulans (76% identity). The expression of theY. lipolytica PGK1 gene proved to be higher on gluconeogenic substrates than on glycolytic ones. Haploid strains harboring a disrupted allele were able to grow on mixtures of a gluconeogenic carbon source and of a glycolytic one, but required proline supplementation in the presence of glucose, and were inhibited by glycerol.     

Viral double-stranded RNAs of <Emphasis Type="Italic">Eimeria</Emphasis> spp. of the domestic fowl: analysis of genetic relatedness and divergence among various strains

Genetic relatedness was examined among putative viral double-stranded RNA (dsRNA) genomes of Eimeria acervulina, E. maxima, and E. necatrix using Northern hybridization. No cross-hybridization was found among these dsRNAs, revealing little sequence homology, if any. In addition, dsRNAs from eight E. acervulina strains collected from different localities in the United States were also examined for genetic relatedness. The putative viral dsRNAs from these eight strains, including the Guelph strain, hybridized with one another to varying degrees, indicating that they are related but divergent. Received: 10 February 2000 / Accepted: 17 March 2000     

Purification and some properties of a double-stranded DNA containing virus-like particle from Uronema gigas, a filamentous eucaryotic green alga

A scheme has been developed for the recovery and purification of about 50% of the 390-nm-diameter virus-like particles (VLPs) present in filtered medium in which Uronema gigas was grown for 6 to 10 weeks. Purification was assayed by electron microscopy of negatively stained samples. Yield of VLPs was low, for example 150 mug from 8 liters of medium. The purified VLP had the following properties:A260/A280 = 1.14 to 1.21;s(20,w) = 6340 +/- 300 S; buoyant density in sucrose, 1.30 g/ml. One major polypeptide (MW = 45,000) and up to nine minor polypeptides (MW = 26,000 to 64,000) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The nucleic acid associated with the VLP was double-stranded DNA with a buoyant density of 1.719 g/ml in cesium chloride and 1.436 g/ml in cesium sulfate. Molecules of the DNA were linear with a length of 4 to 36 mum (MW = 8 to 72 x 10(6)). No modal length was observed. These properties distinguish this VLP from other groups of viruses with dsDNA genomes.     

A very small viral double-stranded RNA

UmV is a double-stranded RNA (dsRNA) virus of the corn fungal pathogen Ustilago maydis. UmV has no infectious cycle. Some UmV subtypes have viral dsRNAs encoding secreted toxins that kill sensitive cells of the same species and related species. There are three viral subtypes, P1, P4 and P6, which differ in the specificity of their secreted killer toxins. Each has three size classes of dsRNA: H (heavy), M (medium) and L (light). The L segments of UmV are unique in being derived from one end of the larger M segments. We have sequenced P1 L and placed it at the 3 end of the P1 M1 plus strand. In their overlapping regions, these dsRNAs are identical in sequence. In vitro translation of P1 M1 results in a peptide whose size is consistent with its being encoded by the non-L region of M1. P1 L is a very small dsRNA of 355 bp. It has no long open reading frames and produces no detectable in vitro translation product. The sequence of P1 L suggests that it is derived by a process unique among dsRNA viruses: replication and packaging of the 3 end fragment of a processed mRNA.     

Double-stranded RNA molecules in Brassica are inherited biparentally and appear not to be associated with mitochondria

Summary The large RNAs previously detected in mitochondrial preparations of Brassica plants are shown here to be double-stranded molecules that lack homology to the mitochondrial genome and are synthesized in a DNA-independent manner. In crosses between nuclear isogenic lines, the dsRNAs were transmitted through both seed and pollen, whereas the mtDNA, as expected, was transmitted only from the seed or the maternal parent. In addition, the Brassica dsRNAs did not co-purify with mitochondria during Percoll gradient centrifugation. In identical gradients, however, the maize autonomously replicating species S/Ru-a and S/Ru-b co-purified precisely with mitochondria. The results suggest that, unlike the maize RNAs, the Brassica dsRNAs are not physically or genetically associated with mitochondria and, more generally, that the occurrence of dsRNA in mitochondria may be less widespread than recent reports indicate.     

Complexity of dsRNA Mycovirus Isolated from Fusarium graminearum

Fusarium graminearum is the causal agent of a serious scab disease of small grains in Korea. We screened 827 isolates of F. graminearum from diseased barley and maize and tested for the presence of double-stranded RNA (dsRNA) mycovirus. Of them, 19 isolates contained various sizes of dsRNAs. A dsRNA associated with pronounced morphological changes including reduction in mycelial growth, increase in dark orange to red pigmentation, reduced sporulation and virulence was previously observed in nine dsRNA-containing Fusarium isolates (Chu et al., Appl Env Microbiol 68, 2529-2534, 2002). Ten additional isolates were found infected with dsRNA mycoviruses. These mycoviruses contain 2-4 different segments of dsRNAs with the size-range of approximately 1.7-10 kbp in length. The presence of dsRNAs did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100%. Interestingly, dsRNA mycovirus found in F. graminearum isolates, JB33 and JNKY19, that show the pattern of mixed infection of two different viruses were transmitted to all progeny conidia and ascospores. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs.     

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S–S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible. Electron microscopy showed that either C115A or C201A mutation disrupted de novo VLP formation significantly. As shown in micrographs and thermal decay curves, β-mercaptoethanol-treated WT-VLPs remained intact, merely resulting in lower tolerance to thermal disruption than native WT-VLPs. This thiol reduction broke disulfide linkages inside the pre-fabricated VLPs, but it did not disrupt the appearance of icosahedrons. Small dissociated capsomers from EGTA-treated VLPs were able to reassemble back to icosahedrons in the presence of calcium ions, but additional treatment with β-mercaptoethanol during EGTA dissociation resulted in inability of the capsomers to reassemble into the icosahedral form. These results indicated that Cys115 and Cys201 were essential for capsid formation of DGNNV icosahedron structure in de novo assembly and reassembly pathways, as well as for the thermal stability of pre-fabricated particles.