Presence of non-suppressive, M2-related dsRNAs molecules in Saccharomyces cerevisiae strains isolated from spontaneous fermentations

Total dsRNA extractions in five killer K2 strains of Saccharomyces cerevisiae isolated from spontaneous fermentations revealed the presence of a novel dsRNA fragment (which we named NS dsRNA) of approximately 1.30 kb, together with L and M2 dsRNAs. NS dsRNA appeared to be encapsidated in the same kind of viral particles as L and M2 dsRNA. Northern blot hybridization experiments indicated that NS dsRNA was derived from M2 dsRNA, likely by deletion of the internal A+U-rich region. However, unlike S dsRNAs (suppressive forms derived from M1 dsRNA in K1 killers), NS dsRNA did not induce exclusion of the parental M2 dsRNA when the host strain was maintained for up to 180 generations of growth.     

Killer toxin-secreting double-stranded RNA mycoviruses in the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii.

Killer toxin-secreting strains of the yeasts Hanseniaspora uvarum and Zygosaccharomyces bailii were shown to contain linear double-stranded RNAs (dsRNAs) that persist within the cytoplasm of the infected host cell as encapsidated virus-like particles. In both yeasts, L- and M-dsRNAs were associated with 85-kDa major capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was capsid protein, whereas the additional Z-dsRNA (2.8 kb), present only in the wild-type Z. bailii killer strain, was shown to be encapsidated by a 35-kDa coat protein. Although Northern (RNA) blot hybridizations indicated that L-dsRNA from Z. bailii is a LA species, additional peptide maps of the purified 85-kDa capsid from Z. bailii and the 88- and 80-kDa major coat proteins from K1 and K28 killer viruses of Saccharomyces cerevisiae revealed distinctly different patterns of peptides. Electron microscopy of purified Z. bailii viruses (ZbV) identified icosahedral particles 40 nm in diameter which were undistinguishable from the S. cerevisiae killer viruses. We demonstrated that purified ZbVs are sufficient to confer the Z. bailii killer phenotype on transfected spheroplasts of a S. cerevisiae nonkiller strain and that the resulting transfectants secreted even more killer toxin that the original ZbV donor strain did. Curing experiments with ZbV-transfected S. cerevisiae strains indicated that the M-dsRNA satellite from Z. bailii contains the genetic information for toxin production, whereas expression of toxin immunity might be dependent on Z-dsRNA, which resembles a new dsRNA replicon in yeasts that is not dependent on an LA helper virus to be stably maintained and replicated within the cell.     

Encapsidation of yeast killer double-stranded ribonucleic acids: dependence of M on L.

Virus-like particles containing either L or M double-stranded ribonucleic acid (dsRNA) were isolated from a killer toxin-producing strains of Saccharomyces cerevisiae (K+ R+). At least 95% of M- and 87% of L-dsRNA were recovered in virus-like particle-containing fractions. The major capsid polypeptides (ScV-P1) of both L and M virus-like particles were shown to be identical, and 95% of the cellular ScV-P1 was found in the virus-like particle-containing fractions. Since L-dsRNA encodes ScV-P1, provision of this protein for encapsidation of M-dsRNA defines at least one functional relationship between these dsRNA genomes and associates the L-dsRNA with the killer character. If encapsidation of M-dsRNA is essential for its replication or expression, then L-dsRNA plays an essential role in maintenance or expression of the killer phenotype. The relationship between the L- and M-dsRNA genomes would be analogous to that between a helper and a defective virus. The presence of only minor quantities or uncomplexed dsRNA and ScV-P1 suggests that their production is stringently coupled.     

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.     

Long internal inverted repeat in a yeast viral double-stranded RNA.

The Saccharomyces cerevisiae viruses are non-infectious double-stranded (ds) RNA viruses present in most laboratory strains of yeast. Their genome consists of one or more dsRNAs separately encapsidated in particles composed mainly of one polypeptide, which has a Mr of 88 kdaltons in the best-studied viral subtype. A large viral dsRNA (L1, of 4.7 kb) encodes the capsid polypeptide. We have determined the sequences of a number of cDNA clones homologous to portions of L1 and mapped them by a novel heteroduplex technique. Several of these clones originate from a region of L1 2.3-2.5 kb from the 5' end of the plus strand that contains stop codons in all three reading frames in the plus strand. We therefore suspect that the capsid polypeptide gene lies in the 5' 2.3-2.6 kb of the plus strand. One of the cloned cDNAs has an inverted repeat of 170 bp that appears to be present in its parental RNA. The inverted repeat in L1 is the longest known inverted repeat in a viral dsRNA and the only known non-terminal inverted repeat. It might serve the function of creating two mRNAs from one viral dsRNA.     

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.     

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.     

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.     

Yeast viral double-stranded RNAs have heterogeneous 3′ termini

The yeast virus, ScV, is communicated only by mating. It has two separately encapsidated dsRNAs. One of these, L, codes for the major capsid polypeptide. The other, M, codes for a polypeptide toxic to yeasts without ScV-M particles. Defective interfering particles containing fragments of M (S) displace ScV-M when they arise. We have shown that five independently isolated S dsRNAs are all derived by internal deletion of M. The 3′ ends of all the ScV dsRNAs are markedly heterogeneous. For instance, half of the first 35 nucleotides at one 3′ end of M and S are variable. Conserved sequences at the 3′ ends of M and S are AAACACCCAUCA_OH and AUUUCUUUAUUUUUCA_OH. Conserved sequences at the 3′ ends of L are UAAAAAUUUUUCA_OH and AAAAAUXCA_OH, where X is variable. We propose that the sequence AUUUUUCA_OH is a recognition sequence for the capsid-associated single-stranded RNA polymerase activity. Since all the viral RNAs have pppGp 5′ termini, their 3′ termini probably extend one nucleotide beyond the terminal pppGp.     

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).     

Translation of the L-species dsRNA genome of the killer-associated virus-like particles of Saccharomyces cerevisiae.

Virus-like particles containing the L (P1)-species of double-stranded RNA (dsRNA) were isolated from Saccharomyces cerevisiae, and the translational activity of the virus-like particle-derived dsRNA was analyzed in the wheat germ cell-free system. Denaturation of the dsRNA immediately prior to in vitro translation resulted in the synthesis of one major and at least three minor polypeptides, whereas undenatured dsRNA, as expected, did not stimulate [35S]methionine incorporation into polypeptides, but actually slightly inhibited endogenous activity. The major in vitro translation product of the denatured L-dsRNA was shown to be identical with the major L-dsRNA containing virus-like particle capsid polypeptide on the basis of three criteria: co-electrophoresis on sodium dodecyl sulfate polyacrylamide gels, immunoprecipitation, and tryptic peptide analysis. We have therefore established that the L-dsRNA genome encodes the major virus-like particle capsid polypeptide. This result adds considerable support to the hypothesis that the L-dsRNA genome acts as a helper genome to the smaller (1.6 x 10(6) dalton) M-dsRNA genome in killer strains of yeast by providing the M-dsRNA containing virus-like particles with their major coat protein.     

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.     

Multiple L double-stranded RNA species of Saccharomyces cerevisiae: evidence for separate encapsidation.

The L double-stranded (ds) RNA component of Saccharomyces cerevisiae may contain up to three dsRNA species, each with a distinct sequence but with identical molecular weights. These dsRNAs have been separated from each other by denaturation and polyacrylamide gel electrophoresis. The 3' terminal sequences of the major species, LA dsRNA, were determined. Secondary structural analysis supported the presence of two stem and loop structures at the 3' terminus of the LA positive strand. In strain T132B NK-3, both the LA and LC species are virion encapsidated. Two distinct classes of virions were purified from this strain, each with a different RNA polymerase activity and with distinct protein components. The heavy virions harbored LA dsRNA, whereas the LC dsRNA species co purified with the light virion peak. Thus, LA and LC dsRNAs, when present in the same cell, may be separately encapsidated.     

Relatedness of the double-stranded RNAs present in yeast virus-like particles.

The relatedness of several double-stranded RNAs (dsRNA's) present in the virus-like particles of yeast was examined by T1 fingerprint analysis. The dsRNA's examined were L, the dsRNA encoding the capsid polypeptide of yeast virus-like particles; M, which appears to code for a toxic polypeptide and for resistance to the effects of the toxin; and two S dsRNA's present in particles analogous to the defective interfering particles of animal viruses. S3, a dsRNA of 0.46 X 10(6) daltons, was derived entirely from M, a dsRNA of 1.2 X 10(6) daltons. S1, a dsRNA of 0.92 X 10(6) daltons, was a duplication of S3. This conclusion has also been reached independently by heteroduplex mapping techniques (H. M. Fried and G. R. Fink, personal communication). S1 and S3, at least in one yeast strain, were unstable in sequence, apparently due to the accumulation of sequence variants of the same molecular weight. L was a species of 3 X 10(6) daltons, unrelated in sequence to M, S1, or S3. S1, S3, and M had a 3' T1 dodecanucleotide in common.