Virus-like particle capsid proteins encoded by different L double-stranded RNAs of Saccharomyces cerevisiae: their roles in maintenance of M double-stranded killer plasmids.

The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.     

Killer systems in Saccharomyces cerevisiae: three distinct modes of exclusion of M2 double-stranded RNA by three species of double-stranded RNA, M1, L-A-E, and L-A-HN.

M1 and M2 double-stranded RNAs (dsRNAs) code for the K1R1 and K2R2 killer toxin and resistance functions, respectively. Natural variants of a larger dsRNA (L-A) carry various combinations of the [EXL], [HOK], and [NEX] genes, which affect the K1 and K2 killer systems. Other dsRNAs, the same size as L-A, called L-B and L-C, are often present with L-A. We show that K1 killer strains have [HOK] and [NEX] but not [EXL] on their L-A (in disagreement with Field et al., Cell 31:193-200, 1982). These strains also carry other L-size molecules detectable after heat-curing has eliminated L-A. The exclusion of M2 dsRNA observed on mating K2 strains with K1 strains is due to the M1 dsRNA (not the L-A dsRNA as claimed by Field et al.) in the K1 strains. Four independent mutants of a [KIL-k2] [NEX-o] [HOK-o] strain were selected for resistance to [EXL] exclusion of M2 ([EXLR] phenotype). The [EXLR] phenotype showed non-Mendelian inheritance in each case, and these mutants had simultaneously each acquired [HOK]. The mutations were located on L-A and not on M2, and did not confer resistance to M1 exclusion of M2.     

A Study of the Transmission and Structure of Double Stranded Rnas Associated with the Killer Phenomenon in SACCHAROMYCES CEREVISIAE

Killer strains contain two double stranded RNAs, L and M. The M dsRNA appears to be necessary for production of a toxin and for resistance to that toxin. Mutant strains have been found that are defective in their ability to kill and in their resistance to toxin. These sensitive, non-killer strains have altered dsRNA composition. One class has no M dsRNA. Another class of sensitive, non-killers called suppressives has no M dsRNA but instead has smaller dsRNAs called S. In diploids resulting from a cross of a wild-type killer by a suppressive the transmission of the M dsRNA is suppressed by the S dsRNA. When a suppressive is crossed by a strain with no M dsRNA, the diploids and all four meiotic spores have the S dsRNA characteristic of the parental suppressive strain. Suppressive strains do not suppress each other. Intercrosses between two different suppressives yields diploids with both parental S dsRNAs. These two S dsRNAs are transmitted to all 4 meiotic progeny. Another class of mutants has been found which is defective for one of the traits but retains the other. One type, temperature-sensitive killers, has a normal dsRNA composition but is unable to kill at 30°. The other type, immunity-minus, has a complex dsRNA pattern. The immunity-minus strain is extremely unstable during mitotic growth and segregates several different types of non-killers. Analysis of the dsRNAs from wild type and the mutants by electron microscopy shows that the L, M, and S dsRNAs are linear. All strains regardless of killer phenotype appear to have the same size L dsRNA.     

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (K1 or K2 toxin). Based on their densities on CsCl gradients, L-A- and M1-containing particles can be separated. Using this method, we detected a new type of M1 dsRNA-containing VLP (M1-H VLP, for heavy) that has a higher density than those previously reported (M1-L VLP, for light). M1-H and M1-L VLPs are present together in the same strains and in all those we tested. M1-H, M1-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and M1-L VLPs have one dsRNA molecule per particle, M1-H VLPs contain two M1 dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the M1-L particles to be used later for dsRNA synthesis. We show that M1-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of M1 dsRNA synthesis is in fractions lighter than M1-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).     

Conservative replication of double-stranded RNA in Saccharomyces cerevisiae by displacement of progeny single strands.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) molecules, L and M, encapsulated in virus-like particles. After cells are transferred from dense (13C 15N) to light (12C 14N) medium, only two density classes of dsRNA are found, fully light (LL) and fully dense (HH). Cells contain single-stranded copies of both dsRNAs and, at least for L dsRNA, greater than 99% of these single strands are the positive protein-encoding strand. Single-stranded copies of L and M dsRNA accumulate rapidly in cells arrested in the G1 phase. These results parallel previous observations on L dsRNA synthesis and are consistent with a role of the positive single strands as intermediates in dsRNA replication. We propose that new positive strands are displaced from parental molecules and subsequently copied to produce the completely new duplexes.     

Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon

Endogenous, 14 kb double-stranded RNAs (dsRNAs) have been found in two ecospecies of cultivated rice (temperate japonica rice and tropical japonica rice, Oryza sativa L.) and in wild rice (O. rufipogon, an ancestor of O. sativa). A comparison of the nucleotide and deduced amino acid sequences of the core regions of the RNA-dependent RNA polymerase domains found in these three dsRNAs suggested that these dsRNAs probably evolved independently within each host plant from a common ancestor. These dsRNAs were introduced into F1 hybrids by crossing cultivated rice and wild rice. Unusual cytoplasmic inheritance of these dsRNAs was observed in some F1 hybrids; the evolutionarily related dsRNAs were incompatible for each other, and the resident dsRNA of an egg cell from cultivated rice was excluded by the incoming dsRNA of a pollen cell from wild rice. Coexisting dsRNAs in the F1 hybrids segregated away from each other in the F2 plants. However, the total amount of these dsRNAs in the host cells remained constant (ca. 100 copies/cell). The stringent regulation of the dsRNA copy number may be responsible for their unusual inheritance.     

Translational analysis of the killer-associated virus-like particle dsRNA genome of S. cerevisiae: M dsRNA encodes toxin

The M species (medium sized) dsRNA (1.1–1.4 × 10⁶ daltons) isolated from a toxin-producing yeast killer strain (K⁺R⁺) and three related, defective interfering (suppressive) S species dsRNAs of the yeast killer-associated cytoplasmic multicomponent viral-like particle system were analyzed by in vitro translation in a wheat germ cell-free protein synthesis system. Heat-denatured M species dsRNA programmed the synthesis of two major polypeptides, M-P1 (32,000 daltons) and M-P2 (30,000 daltons). M-P1 has been shown by the criteria of proteolytic peptide mapping and cross-antigenicity to contain the 12,000 dalton polypeptide corresponding to the in vivo produced killer toxin, thus establishing that it is the M species dsRNA which carries the toxin gene. An M species dsRNA obtained from a neutral strain (K^?R⁺) also programmed the in vitro synthesis of a polypeptide identical in molecular weight to M-P1, thus indicating that the cytoplasmic determinant of the mutant neutral phenotype is either a simple point mutation in the dsRNA toxin gene or a mutation in a dsRNA gene which is required for functional toxin production. In vitro translation of each of the three different suppressive S dsRNAs resulted in the production of a polypeptide (S-P1) of approximately 8000 daltons instead of the 32,000 dalton M-P1 polypeptide programmed by M dsRNA. This result is consistent with the heteroduplex analysis of these dsRNAs by Fried and Fink (1978), which shows retention of M dsRNA ends, accompanied by large internal deletions in each of the S dsRNAs translated.     

Virus-like particles and double stranded RNA from killer and non-killer strains of Saccharomyces cerevisiae.

Virus-like particles and DsRNA found in extracts of killer, non-killer and suppressive non-killer strains were co-precipitated from cell extracts using an antibody prepared against purified virus-like particles isolated from a non-killer strain having only the higher molecular weight L dsRNA. The relative amount of virus-like particles correlated roughly with the amount of dsRNA: those strains with high concentrations of dsRNA had the most particles. When a preparation of particles was subjected to sucrose gradient velocity centrifugation, particles containing the S and M dsRNA could be separated from those containing the L dsRNA. These experiments taken together suggest that the L, M and S dsRNAs are separately encapsulated by the same protein coat.     

Molecular characterization of totiviruses in Xanthophyllomyces dendrorhous

ABSTRACT: BACKGROUND: Occurrence of extrachromosomal dsRNA elements has been described in the red-yeast Xanthophyllomyces dendrorhous, with numbers and sizes that are highly variable among strains with different geographical origin. The studies concerning to the encapsidation of viral-like particles and dsRNA-curing have suggested that some dsRNAs are helper viruses, while others are satellite viruses. However, the nucleotide sequences and functions of these dsRNA are still unknown. In this work, the nucleotide sequences of four dsRNAs of the strain UCD 67-385 of X. dendrorhous were determined, and their identities and genome structures are proposed. Based on this molecular data, the dsRNAs of different strains of X. dendrorhous were analyzed. RESULTS: The complete sequences of L1, L2, S1 and S2 dsRNAs of X. dendrorhous UCD 67-385 were determined, finding two sequences for L1 dsRNA (L1A and L1B). Several ORFs were uncovered in both S1 and S2 dsRNAs, but no homologies were found for any of them when compared to the database. Instead, two ORFs were identified in each L1A, L1B and L2 dsRNAs, whose deduced amino acid sequences were homologous with a major capsid protein (5'-ORF) and a RNA-dependent RNA polymerase (3'-ORF) belonging to the Totivirus family. The genome structures of these dsRNAs are characteristic of Totiviruses, with two overlapped ORFs (the 3'-ORF in the -1 frame with respect to the 5'-ORF), with a slippery site and a pseudoknot in the overlapped regions. These structures are essential for the synthesis of the viral polymerase as a fusion protein with the viral capsid protein through -1 ribosomal frameshifting. In the RNase protection analysis, all the dsRNAs in the four analyzed X. dendrorhous strains were protected from enzymatic digestion. The RT-PCR analysis revealed that, similar to strain UCD 67-385, the L1A and L1B dsRNAs coexist in the strains VKM Y-2059, UCD 67-202 and VKM Y-2786. Furthermore, determinations of the relative amounts of L1 dsRNAs using two-step RT-qPCR revealed a 40-fold increment of the ratio L1A/L1B in the S2 dsRNA-cured strain compared to its parental strain. CONCLUSIONS: Three totiviruses, named as XdV-L1A, XdV-L1B and XdV-L2, were identified in the strain UCD 67-385 of X. dendrorhous. The viruses XdV-L1A and XdV-L1B were also found in other three X. dendrorhous strains. Our results suggest that the smaller dsRNAs (named XdRm-S1 and XdRm-S2) of strain UCD 67-385 are satellite viruses, and particularly that XdRm-S2 is a satellite of XdV-L1A.     

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.     

豇豆单孢锈菌中病毒样颗粒中双链RNA的存在

从北京西郊清华园附近田间豇豆上采集的豇豆单孢锈菌(Uormyces vignal Barcl)夏孢子。萌发后提取双链RNA,电泳分析可测出300—8000碱基对的三组双链RNA。从萌发的孢子中通过差迷离心提取病毒样颗粒,可获得二种类型的病毒样颗粒,一种直径为35—40nm的等轴颗粒。另一种为长短不等的棒状颗粒,用提纯物提取核酸电泳分析与直接从孢子中提取的双链RNA有相同的核酸带,从而证明这些双链RNA存在于病毒样颗粒中。     

Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum , a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH₂ terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C . parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.     

Inheritance of dsRNAs in the rice blast fungus, Magnaporthe grisea

The 1.6 and 1.8 kbp dsRNAs have been found in the rice blast fungus, Magnaporthe grisea strain MG01. These dsRNA molecules are located in cytoplasm of the fungal cells and maintained stably during vegetative growth. Three crosses between dsRNA free and dsRNA containing strains including a parental cross, sib-mating and back cross were made to follow the inheritance of dsRNAs during sexual reproduction. Approximately 10% of ascospore progenies (11 out of 105) contained dsRNAs from all three crosses. These data indicate that dsRNAs of M. grisea are inherited at a low frequency and not in a Mendelian fashion.     

Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus.

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.     

A viral double-stranded RNA up regulates the fungal virulence of Nectria radicicola

Double-stranded RNAs (dsRNAs) are widespread in plant pathogenic fungi, but their functions in fungal hosts remain mostly unclear, with a few exceptions. We analyzed dsRNAs from Nectria radicicola, the causal fungus of ginseng root rot. Four distinct sizes of dsRNAs, 6.0, 5.0, 2.5, and 1.5 kbp, were detected in 24 out of the 81 strains tested. Curing tests of individual dsRNAs suggested that the presence of 6.0-kbp dsRNA was associated with high levels of virulence, sporulation, laccase activity, and pigmentation in this fungus. The 6.0-kbp dsRNA-cured strains completely lost virulence-related phenotypes. This 6.0-kbp dsRNA was reintroduced by hyphal anastomosis to a dsRNA-cured strain marked with hygromycin resistance, which resulted in the restoration of virulence-related phenotypes. These results strongly suggest that 6.0-kbp dsRNA up regulates fungal virulence in N. radicicola. Sequencing of several cDNA clones derived from 6.0-kbp dsRNA revealed the presence of a RNA-dependent RNA polymerase (RDRP) gene. Phylogenetic analysis showed that this gene is closely related to those of plant cryptic viruses. Biochemical analyses suggested that the 6.0-kbp dsRNA may regulate fungal virulence through signal-transduction pathways involving cyclic AMP-dependent protein kinase and protein kinase C.     

Yeast killer mutants with altered double-stranded ribonucleic acid

Killer strains of Saccharomyces cerevisiae contain two species of double-stranded ribonucleic acid (dsRNA) with molecular weights estimated at 2.5 x 10(6) (L) and 1.4 x 10(6) (M). The M component appears to have a high adenine content. All mutants of killer which are defective for both the toxin and immunity functions lack the M dsRNA. One of these mutants has a novel dsRNA with a molecular weight of 5 x 10(5). Another class of killer mutants contains strains which are defective for either the toxin or the immunity function. They include temperature-sensitive killers, superkillers, and immunity-minus strains. The dsRNA profile of temperature-sensitive killers resembles that of the standard killer. The superkiller has 2.5 times more of the M dsRNA (1.4 x 10(6) daltons) than does the standard killer. Immunity-minus killers have, in addition to the two dsRNAs species of standard killer, a novel dsRNA with a molecular weight of 2.5 x 10(5). The data are consistent with the hypothesis that the M RNA controls toxin production. In addition, the two RNAs, L and M, seem to be regulated together. When the M RNA is missing, the amount of L is either greatly elevated or greatly reduced.