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.     

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.     

嗜杀酵母的生物学功能及其应用

嗜杀酵母能够分泌毒素蛋白,杀死敏感酵母。嗜杀酵母对自身分泌的嗜杀毒素具有免疫力。嗜杀酵母的嗜杀特性与两种双链线状RNA(dsRNA)有关,即编码产生毒素蛋白的M-dsRNA和编码自身和M-dsRNA外壳蛋白的L-dsRNA。嗜杀毒素破坏细胞跨膜化学质子梯度,造成ATP和钾离子泄漏,导致细胞死亡。应用嗜杀酵母可避免野生型酵母污染,净化发酵体系,改善发酵产物品质;嗜杀毒素也可作为抵制病原酵母和类酵母微生物的抗真菌剂。     

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.     

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.     

嗜杀酿酒酵母毒素蛋白及其杀伤质粒的研究

Killer toxin from \%Saccharomyces cerevisiae\% SK was isolated by ultrafiltration of culture supernatants and purified by poly(ethylene glycol). The toxin migrates as one single protein band on SDS-PAGE and its molecular weight is 15kD. The SK toxin has the greatest lethal effect on the sensitive yeast strain in the lat lag phase. Extraction and purification of killer heretity factor(dsRNA) from SK found that M dsRNA plasmid and L dsRNA plasmid have different molecular lengths being 1.7kb and 4.0kb.     

Structural organization of double-stranded RNA from killer yeasts Saccharomyces cerevisiae by the thermal denaturation method

The thermal denaturation method for studying the structural organization of double-stranded RNA (dsRNA) from virus-like particles of killer yeasts Saccharomyces cerevisiae was used. High resolution derivative denaturation profiles of total dsRNA and its L- and M-types were obtained. Comparative analysis of these data with those on phage DNA denaturation demonstrated that the processes of denaturation of dsRNA and phage DNA were identical in quality. Increase of thermostability, interval of thermal denaturation and width of local helix-to-coil transitions in dsRNA as compared with phage DNA are caused by the differences of corresponding thermodynamic parameters. Derivative denaturation profiles of L- and M-types of yeasts dsRNA were shown to have certain identical local transitions. Low melting transition, consisting of three local thermalites, is due to the denaturation of AU-rich region (about 200 n.b.p.) in M-dsRNA.     

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.     

酿酒酵母两种不同嗜杀菌的比较研究

以酿酒酵母两种不同类型的嗜杀菌株ＳＫ４（Ｋ１型）和ＥＲＲ１（Ｋ２型）为材料,分析了不同嗜杀酵母的嗜杀特性,两株嗜杀酵母具有相互杀死作用,其嗜杀活性与菌体生长有关。ＳＫ４和ＥＲＲ１的嗜杀质粒的比较表明：Ｍ１－ｄｓＲＮＡ质粒和Ｍ２－ｄｓＲＮＡ质粒分子量分别为１．７ｋｂ和１．５ｋｂ,两株菌的Ｌ－ｄｓＲＮＡ质粒均为４．０ｋｂ。用高温和紫外线处理嗜杀酵母,嗜杀活性随之消失,消除菌中的Ｍ－ｄｓＲＮＡ质粒也相应     

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.     

A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.     

A glycosylated protoxin in killer yeast: models for its structure and maturation

The type 1 killer phenotype in S. cerevisiae, mediated by secretion of an 11.5 kilodalton (kd) protein toxin, is cytoplasmically determined by the 1.9 kb M1-dsRNA plasmid. Maintenance of M1-dsRNA is dependent on the 4.5 kb L1-dsRNA because L1 encodes the capsid protein of the virus-like particles that separately encapsidate both dsRNA species. We have shown that in vitro translation of denatured M1-dsRNA produces M1-P1, a 32 kd protein containing the toxin peptides. We now demonstrate the presence of an unstable, 42 kd, membrane-associated, glycosylated protoxin in killer cells, probably derived from M1-P1 by cotranslational processing, and glycosylation. In vitro cotranslational processing of M1-P1, derived both from in vivo mRNAs and from denatured M1-dsRNA, produces a product resembling protoxin. Processing involves loss of 1.6 kd of protein, presumably an N-terminal leader peptide, and glycosylation. This information, together with data on in vitro expression of suppressive deletion mutants of M1-dsRNA, allows construction of testable models for the functional sequence of M1-P1 and for its maturation to toxin.     

Virus-like Particles in USTILAGO MAYDIS: Mutants with Partial Genomes

Mutants with partial genomes for the virus-like particles of U. maydis were recovered following treatment with nitrosoguanidine. Examination of the properties retained by progeny of genetic crosses indicates that the 2.9 x 10⁶ dalton component of double-stranded RNA contains the information for capsid formation and dsRNA replication. Other components appear to contain the information for killer function and immunity to killer. The use of such mutants for studies on the evolution of viruses with segmented genomes is discussed.     

The dsRNA ofTrichomonas vaginalis is associated with virus-like particles and does not correlate with metornidazole resistance

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains ofTrichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA -positive strains to contain virus-like particles 32 –38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist. After this paper had been accepted for publication the occurrence of virus-like particles in dsRNA-positive trichomonads was reported by others (Wang A.L., Wang C.C.: The double stranded RNA inTrichomonas vaginalis may originate from virus-like particles.Proc. Nat. Acad. Sci. USA 83, 7956–7961, 1986).     

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.     

In vitro generation and type-specific neutralization of a human papillomavirus type 16 virion pseudotype.

We report a system for generating infectious papillomaviruses in vitro that facilitates the analysis of papillomavirus assembly, infectivity, and serologic relatedness. Cultured hamster BPHE-1 cells harboring autonomously replicating bovine papillomavirus type 1 (BPV1) genomes were infected with recombinant Semliki Forest viruses that express the structural proteins of BPV1. When plated on C127 cells, extracts from cells expressing L1 and L2 together induced numerous transformed foci that could be specifically prevented by BPV neutralizing antibodies, demonstrating that BPV infection was responsible for the focal transformation. Extracts from BPHE-1 cells expressing L1 or L2 separately were not infectious. Although Semliki Forest virus-expressed L1 self-assembled into virus-like particles (VLPs), viral DNA was detected in particles only when L2 was coexpressed with L1, indicating that genome encapsidation requires L2. Expression of human papillomavirus type 16 (HPV16) L1 and L2 together in BPHE-1 cells also yielded infectious virus. These pseudotyped virions were neutralized by antiserum to HPV16 VLPs derived from European (114/K) or African (Z-1194) HPV16 variants but not by antisera to BPV VLPs, to a poorly assembling mutant HPV16 L1 protein, or to VLPs of closely related genital HPV types. Extracts from BPHE-1 cells coexpressing BPV L1 and HPV16 L2 or HPV16 L1 and BPV L2 were not infectious. We conclude that (i) mouse C127 cells express the cell surface receptor for HPV16 and are able to uncoat HPV16 capsids; (ii) if a papillomavirus DNA packaging signal exists, then it is conserved between the BPV and HPV16 genomes; (iii) functional L1-L2 interaction exhibits type specificity; and (iv) protection by HPV virus-like particle vaccines is likely to be type specific.     

Mak mutants of yeast: mapping and characterization.

Killer strains of Saccharomyces cerevisiae are those carrying a 1.5 x 10(6)-dalton double-stranded (ds) ribonucleic acid (RNA) (M) in virus-like particles and secreting a protein toxin. Most yeast (koller or not) also carry a 3 x 10(6)-dalton dsRNA (L). We have mapped mutations in eight of the chromosomal genes needed for maintaining M (mak genes). The mak genes are widely distributed on the yeast map, with no multigene complexes. We show that mutants defective in these and other mak genes lose M dsRNA, but not L dsRNA. The mak3-1 mutation results in markedly decreased cellular levels of L dsRNA, but mak3-1 stains do not lose L dsRNA completely. Mutation of mak16 results in temperature-sensitive growth, whereas mutations in mak13, mak15, mak17, mak20, mak22, and mak27 result in slow growth at any temperature. No effect of mak mutations on mating, meiosis, sporulation, germination, homothallism, or ultraviolet sensitivity has been found. The specificity of mak mutations is discussed.