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[URE3] and [PSI(+)] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild-type [URE3] cells with ssa2-10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2-10 allele is compatible with propagation of [PSI(+)]. However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of [PSI(+)] cells., (Copyright 2003 John Wiley & Sons, Ltd.)

Just as nucleic acids can carry out enzymatic reactions, proteins can be genes. These heritable infectious proteins (prions) follow unique genetic rules that enable their identification: reversible curing, inducible "spontaneous generation," and phenotype surprises. Most prions are based on self-propagating amyloids, depend heavily on chaperones, show strain phenomena and, like other infectious elements, show species barriers to transmission. A recently identified prion is based on obligatory self-activation of an enzyme in trans. Although prions can be detrimental, they may also be beneficial to their hosts.

The [URE3] prion is an inactive, self-propagating, filamentous form of the Ure2 protein, a regulator of nitrogen catabolism in yeast. The N-terminal "prion" domain of Ure2p determines its in vivo prion properties and in vitro amyloid-forming ability. Here we determined the overall structures of Ure2p filaments and related polymers of the prion domain fused to other globular proteins. Protease digestion of 25-nm diameter Ure2p filaments trimmed them to 4-nm filaments, which mass spectrometry showed to be composed of prion domain fragments, primarily residues approximately 1-70. Fusion protein filaments with diameters of 14-25 nm were also reduced to 4-nm filaments by proteolysis. The prion domain transforms from the most to the least protease-sensitive part upon filament formation in each case, implying that it undergoes a conformational change. Intact filaments imaged by cryo-electron microscopy or after vanadate staining by scanning transmission electron microscopy (STEM) revealed a central 4-nm core with attached globular appendages. STEM mass per unit length measurements of unstained filaments yielded 1 monomer per 0.45 nm in each case. These observations strongly support a unifying model whereby subunits in Ure2p filaments, as well as in fusion protein filaments, are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone, surrounded by the corresponding C-terminal moieties.

Known prions (infectious proteins) are self-propagating amyloids or conformationally altered proteins, but in theory an enzyme necessary for its own activation could also be a prion (or a gene composed of protein). We show that yeast protease B is such a prion, called [beta].[beta] is infectious, reversibly curable, and its de novo generation is induced by overexpression of the pro-protease. Present in normal cells but masked by the functionally redundant protease A, [beta] is advantageous during starvation and necessary for sporulation. We propose that other enzymes whose active, modified, form is necessary for their maturation might also be prions.

Prions are "infectious" proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PSI(+)] prion form its function is compromised. When Rnq1 is aggregated in its [PIN(+)] prion form, it promotes the de novo appearance of [PSI(+)]. Heritable variants (strains) of [PSI(+)] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PIN(+)] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PSI(+)]. Unlike [PSI(+)] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PIN(+)] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PSI(+)], or different [PIN(+)] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PIN(+)] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PSI(+)] appearance, [PIN(+)] enhances the de novo appearance of [URE3]. Surprisingly, [PSI(+)] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.

The [URE3] prion of Saccharomyces cerevisiae is a self-propagating inactive amyloid form of the Ure2 protein. Ure2p residues 1-65 constitute the prion domain, and the remaining C-terminal portion regulates nitrogen catabolism. We have examined the URE2 genes of wild-type isolates of S. cerevisiae and those of several pathogenic yeasts and a filamentous fungus. We find that the normal function of the S. cerevisiae Ure2p in nitrogen regulation is fully complemented by the Ure2p of Candida albicans, Candida glabrata, Candida kefyr, Candida maltosa, Saccharomyces bayanus, and Saccharomyces paradoxus, all of which have high homology in the C-terminal nitrogen regulation domain. However, there is considerable divergence of their N-terminal domains from that of Ure2p of S. cerevisiae. [URE3(Sc)] showed efficient transmission into S. cerevisiae ure2Delta cells if expressing a Ure2p of species within Saccharomyces. However, [URE3(Sc)] did not seed self-propagating inactivation of the Ure2p's from the other yeasts. When overexpressed as a fusion with green fluorescent protein, residues 5-47 of the S. cerevisiae prion domain are necessary for curing the [URE3] prion. Residues 11-39 are necessary for an inactivating interaction with the full-length Ure2p. A nearly identical region is highly conserved among many of the yeasts examined in this study, despite the wide divergence of sequences found in other parts of the N-terminal domains.

The structure of the yeast L-A virus was determined by X-ray crystallography at 3.4 A resolution. The L-A dsRNA virus is 400 A in diameter and contains a single protein shell of 60 asymmetric dimers of the coat protein, a feature common among the inner protein shells of dsRNA viruses and probably related to their unique mode of transcription and replication. The two identical subunits in each dimer are in non-equivalent environments and show substantially different conformations in specific surface regions. The L-A virus decaps cellular mRNA to efficiently translate its own uncapped mRNA. Our structure reveals a trench at the active site of the decapping reaction and suggests a role for nearby residues in the reaction.

The [URE3] infectious protein (prion) of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The C-terminal domain of Ure2p controls nitrogen catabolism by complexing with the transcription factor, Gln3p, whereas the asparagine-rich N-terminal "prion" domain is responsible for amyloid filament formation (prion conversion). On filament formation, Ure2p is inactivated, reflecting either a structural change in the C-terminal domain or steric blocking of its interaction with Gln3p. We fused the prion domain with four proteins whose activities should not be sterically impeded by aggregation because their substrates are very small: barnase, carbonic anhydrase, glutathione S-transferase, and green fluorescent protein. All formed amyloid filaments in vitro, whose diameters increased with the mass of the appended enzyme. The helical repeat lengths were consistent within a single filament but varied with the construct and between filaments from a single construct. CD data suggest that, in the soluble fusion proteins, the prion domain has no regular secondary structure, whereas earlier data showed that in filaments, it is virtually all beta-sheet. In filaments, the activity of the appended proteins was at most mildly reduced, when substrate diffusion effects were taken into account, indicating that they retained their native structures. These observations suggest that the amyloid content of these filaments is confined to their prion domain-containing backbones and imply that Ure2p is inactivated in [URE3] cells by a steric blocking mechanism.

Yeast infectious protein (prion) forms of the Ure2 and Sup35 proteins determine the nonchromosomal genes [URE3] and [PSI], and these are, therefore, the basis for a kind of epigenetic phenomena. In many systems, introduction of multiple copies of a DNA gene, or dsRNA copies of its sequence, results in the epigenetic silencing of that gene. In parallel with these homology effects, which act at the level of DNA or RNA, elevated copy number of the Ure2 and Sup35 proteins increases the frequency of their own "silencing" by prion formation. Both [URE3] and [PSI] appear to be due to self-propagating-amyloid formation of Ure2p and Sup35p, respectively. Another prion, [Het-s] of the filamentous fungus, Podospora anserina, is necessary for a normal cellular function, heterokaryon incompatibility. Since these prions are nonchromosomal genes, they are proteins acting as genes, a parallel to the fact that nucleic acids can catalyze enzymatic reactions.

The inactivity of Ure2p, caused by either a ure2 mutation or the presence of the [URE3] prion, increases DAL5 transcription and thus enables Saccharomyces cerevisiae to take up ureidosuccinate (USA+). Rtg2p regulates transcription of glutamate-repressible genes by facilitation of the nuclear entry of the Rtg1 and Rtg3 proteins. We find that rtg2 Delta cells take up USA even without the presence of [URE3]. Thus, the USA+ phenotype of rtg2 Delta strains is not the result generation of the [URE3] prion but is a regulatory effect. Because rtg1 Delta or rtg3 Delta mutations or the presence of glutamate do not produce the USA+ phenotype, this is a novel function of Rtg2p. The USA+ phenotype of rtg2 Delta strains depends on GLN3, is caused by overexpression of DAL5, and is blocked by mks1 Delta, but not by overexpression of Ure2p. These characteristics suggest that Rtg2p acts in the upstream part of the nitrogen catabolism regulation pathway.

TheL-A virus (LAV) particle is a specialized compartment for the transcription and replication of double-stranded RNA. It is 390 A in diameter and infects yeast. The particle is formed by a capsid containing 120 copies of a 680-residue gene product arranged with T = 1 icosahedral symmetry, approximately two copies of an RNA-directed RNA polymerase, and a 4.6-kb linear, duplex RNA. LAV crystals diffracting to at least 4.5-A resolution were grown in a combination of polyethylene glycol 8000, ethylene glycol, and lithium chloride. Following crystallization the reservoir solution was replaced by a 2x concentrated reservoir solution in order for ethylene glycol to function as a cryoprotectant even though initial crystals would not grow at sufficiently high concentrations of ethylene glycol for cryoprotection. A complete data set was collected to 6-A resolution from a frozen crystal obtained with this procedure. The crystals belong to space group P2(1). The unit cell dimensions are a = 406.7 A, b = 403.3 A, c = 572.5 A, beta = 90.3 degrees with two virus particles in the unit cell. The particle orientation was determined with the rotation function and the particle center was estimated on the basis of packing considerations., (Copyright 2001 Academic Press.)

The [URE3] prion (infectious protein) of yeast is a self-propagating, altered form of Ure2p that cannot carry out its normal function in nitrogen regulation. Previous data have shown that Ure2p can form protease-resistant amyloid filaments in vitro, and that it is aggregated in cells carrying the [URE3] prion. Here we show by electron microscopy that [URE3] cells overexpressing Ure2p contain distinctive, filamentous networks in their cytoplasm, and demonstrate by immunolabeling that these networks contain Ure2p. In contrast, overexpressing wild-type cells show a variety of Ure2p distributions: usually, the protein is dispersed sparsely throughout the cytoplasm, although occasionally it is found in multiple small, focal aggregates. However, these distributions do not resemble the single, large networks seen in [URE3] cells, nor do the control cells exhibit cytoplasmic filaments. In [URE3] cell extracts, Ure2p is present in aggregates that are only partially solubilized by boiling in SDS and urea. In these aggregates, the NH(2)-terminal prion domain is inaccessible to antibodies, whereas the COOH-terminal nitrogen regulation domain is accessible. This finding is consistent with the proposal that the prion domains stack to form the filament backbone, which is surrounded by the COOH-terminal domains. These observations support and further specify the concept of the [URE3] prion as a self-propagating amyloid.

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1--80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97--354), at 2.3 A resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97--354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

It was believed that only proteins could carry out enzymatic reactions, and only nucleic acids could mediate inheritance. In recent years, the work of Cech and Altman and others has shown that nucleic acids can catalyze reactions. Now it has been shown that, in yeast, proteins can mediate inheritance. The infectious protein (prion) concept arose from studies of the transmissible spongiform encephalopathies (TSEs) of mammals (1), and several lines of evidence suggest that TSEs are indeed caused by infectious forms of the PrP protein, but the absence of definitive proof has left substantial doubt and disagreement on this point (2-6). The ease of genetic manipulation of yeast offers experimental possibilities not yet available even in the mouse system. This enabled the discovery of yeast prions (7), and has facilitated the rapid characterization of these systems. The parallels between the yeast and mammalian systems are striking. Moreover, because both of the yeast prion systems appear to involve self-propagating amyloid forms of the respective proteins, these systems may also serve as models for the broader class of diseases for which amyloid accumulation is a central feature. The discovery of the [HET-s] prion of the filamentous fungus Podospora, another genetically manipulable system, adds a new dimension to prion studies (8).

The [URE3] nonchromosomal genetic element is an infectious form (prion) of the Ure2 protein, apparently a self-propagating amyloidosis. We find that an insertion mutation or deletion of HSP104 results in inability to propagate the [URE3] prion. Our results indicate that Hsp104 is a common factor in the maintenance of two independent yeast prions. However, overproduction of Hsp104 does not affect the stability of [URE3], in contrast to what is found for the [PSI(+)] prion, which is known to be cured by either overproduction or deficiency of Hsp104. Like Hsp104, the Hsp40 class chaperone Ydj1p, with the Hsp70 class Ssa1p, can renature proteins. We find that overproduction of Ydj1p results in a gradual complete loss of [URE3]. The involvement of protein chaperones in the propagation of [URE3] indicates a role for protein conformation in inheritance.

In wild-type cells, the 3' poly(A) structure is necessary for translation of mRNA and for mRNA stability. The superkiller 2 (ski2), ski3, ski6, ski7, and ski8 mutations enhance the expression of the poly(A)(-) mRNAs of yeast RNA viruses. Ski2p is a DEVH-box RNA helicase and Slh1p resembles Ski2p. Both repress L-A double-stranded RNA (dsRNA) virus copy number, further suggesting that their functions may overlap. We find that slh1Delta ski2Delta double mutants are healthy (in the absence of viruses) and show normal rates of turnover of several cellular mRNAs. The slh1Delta ski2Delta strains translate electroporated nonpoly(A) mRNA with the same kinetics as polyA(+) mRNA. Thus, the translation apparatus is inherently capable of efficiently using nonpoly(A) mRNA even in the presence of normal amounts of competing poly(A)(+) mRNA, but is normally prevented from doing so by the combined action of the nonessential proteins Ski2p and Slh1p.

Infectious proteins (prions) can arise de novo as well as by transmission from another individual. De novo prion generation is believed responsible for most cases of Creutzfeldt-Jakob disease and for initiating the mad cow disease epidemic. However, the cellular components needed for prion generation have not been identified in any system. The [URE3] prion of Saccharomyces cerevisiae is an infectious form of Ure2p, apparently a self-propagating amyloid. We now demonstrate a protein required for de novo prion generation. Mks1p negatively regulates Ure2p and is itself negatively regulated by the presence of ammonia and by the Ras-cAMP pathway. We find that in mks1Delta strains, de novo generation of the [URE3] prion is blocked, although [URE3] introduced from another strain is expressed and propagates stably. Ras2(Val19) increases cAMP production and also blocks [URE3] generation. These results emphasize the distinction between prion generation and propagation, and they show that cellular regulatory mechanisms can critically affect prion generation.

Two infectious proteins (prions) of Saccharomyces cerevisiae have been identified by their unusual genetic properties: (1) reversible curability, (2) de novo induction of the infectious prion form by overproduction of the protein, and (3) similar phenotype of the prion and mutation in the chromosomal gene encoding the protein. [URE3] is an altered infectious form of the Ure2 protein, a regulator of nitrogen catabolism, while [PSI] is a prion of the Sup35 protein, a subunit of the translation termination factor. The altered form of each is inactive in its normal function, but is able to convert the corresponding normal protein into the same altered inactive state. The N-terminal parts of Ure2p and Sup35p (the "prion domains") are responsible for prion formation and propagation and are rich in asparagine and glutamine residues. Ure2p and Sup35p are aggregated in vivo in [URE3]- and [PSI]-containing cells, respectively. The prion domains can form amyloid in vitro, suggesting that amyloid formation is the basis of these two prion diseases. Yeast prions can be cured by growth on millimolar concentrations of guanidine. An excess or deficiency of the chaperone Hsp104 cures the [PSI] prion. Overexpression of fragments of Ure2p or certain fusion proteins leads to curing of [URE3]., (Copyright 2000 Academic Press.)

Self-propagating abnormal proteins, prions, have been identified in yeast; asparagine/glutamine-rich 'prion domains' within these proteins can inactivate the linked functional domains; new prion domains and reporters have been used to make 'synthetic prions', leading to discoveries of new natural prions.

[URE3] and [PSI] are two non-Mendelian genetic elements discovered over 25 years ago and never assigned to a nucleic acid replicon. Their genetic properties led us to propose that they are prions, altered self-propagating forms of Ure2p and Sup35p, respectively, that cannot properly carry out the normal functions of these proteins. Ure2p is partially protease-resistant in [URE3] strains and Sup35p is aggregated specifically in [PSI] strains supporting this idea. Overexpression of Hsp104 cures [PSI], as does the absence of this protein, suggesting that the prion change of Sup35p in [PSI] strains is aggregation. Strains of [PSI], analogous to those described for scrapie, have now been described as well as an in vitro system for [PSI] propagation. Recently, two new potential prions have been described, one in yeast and the other in the filamentous fungus, Podospora.

Genetic evidence showed two non-Mendelian genetic elements of Saccharomyces cerevisiae, called [URE3] and [PSI], to be prions of Ure2p and Sup35p, respectively. [URE3] makes cells derepressed for nitrogen catabolism, while [PSI] elevates the efficiency of weak suppressor tRNAs. The same approach led to identification of the non-Mendelian element [Het-s] of the filamentous fungus Podospora anserina, as a prion of the het-s protein. The prion form of the het-s protein is required for heterokaryon incompatibility, a normal fungal function, suggesting that other normal cellular functions may be controlled by prions. [URE3] and [PSI] involve a self-propagating aggregation of Ure2p and Sup35p, respectively. In vitro, Ure2p and Sup35p form amyloid, a filamentous protein structure, high in beta-sheet with a characteristic green birefringent staining by the dye Congo Red. Amyloid deposits are a cardinal feature of Alzheimer's disease, non-insulin-dependent diabetes mellitus, the transmissible spongiform encephalopathies, and many other diseases. The prion domain of Ure2p consists of Asn-rich residues 1 to 80, but two nonoverlapping fragments of the molecule can, when overproduced, induce the de nova appearance of [URE3]. The prion domain of Sup35 consists of residues 1 to 114, also rich in Asn and Gln residues. While runs of Asn and Gln are important for [URE3] and [PSI], no such structures are found in PrP or the Het-s protein. Either elevated or depressed levels of the chaperone Hsp104 interfere with propagation of [PSI]. Both [URE3] and [PSI] are cured by growth of cells in millimolar guanidine HCl. [URE3] is also cured by overexpression of fragments of Ure2p or fusion proteins including parts of Ure2p.

The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation. Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation. Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa(+)) when cells grow on a poor nitrogen source such as proline. We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline. Overexpression of Mks1p does not affect cellular levels of Ure2p. An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline. Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia. These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH(3) dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5. Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth.

Ure2p of Saccharomyces cerevisiae normally functions in blocking utilization of a poor nitrogen source when a good nitrogen source is available. The non-Mendelian genetic element [URE3] is a prion (infectious protein) form of Ure2p, so that overexpression of Ure2p induces the de novo appearance of infectious [URE3]. Earlier studies defined a prion domain comprising Ure2p residues 1 to 64 and a nitrogen regulation domain included in residues 66 to 354. We find that deletion of individual runs of asparagine within the prion domain reduce prion-inducing activity. Although residues 1 to 64 are sufficient for prion induction, the fragment from residues 1 to 80 is a more efficient inducer of [URE3]. In-frame deletion of a region around residue 224 does not affect nitrogen regulation but does eliminate prion induction by the remainder of Ure2p. Larger deletions removing the region around residue 224 and more of the C-terminal part of Ure2p restore prion-inducing ability. A fragment of Ure2p lacking the original prion domain does not induce [URE3], but surprisingly, further deletion of residues 151 to 157 and 348 to 354 leaves a fragment that can do so. The region from 66 to 80 and the region around residue 224 are both necessary for this second prion-inducing activity. Thus, each of two nonoverlapping parts of Ure2p is sufficient to induce the appearance of the [URE3] prion.

We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses in Saccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-alpha. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3' RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3' untranslated regions (3'UTRs) differing in length. In a wild-type strain, increasing the length of the 3'UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Delta ski2Delta and ski1/xrn1Delta ski7Delta mutants were viable but temperature sensitive for growth.

The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in extracts of [URE3] strains, these 180- to 220-angstrom-diameter filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3].

The [URE3] nonchromosomal genetic element is a prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. Ure2p1-65 is the prion domain of Ure2p, sufficient to propagate [URE3] in vivo. We show that full length Ure2p-green fluorescent protein (GFP) or a Ure2p1-65-GFP fusion protein is aggregated in cells carrying [URE3] but is evenly distributed in cells lacking the [URE3] prion. This indicates that [URE3] involves a self-propagating aggregation of Ure2p. Overexpression of Ure2p1-65 induces the de novo appearance of [URE3] by 1,000-fold in a strain initially [ure-o], but cures [URE3] from a strain initially carrying the [URE3] prion. Overexpression of several other fragments of Ure2p or Ure2-GFP fusion proteins also efficiently cures the prion. We suggest that incorporation of fragments or fusion proteins into a putative [URE3] "crystal" of Ure2p poisons its propagation.

Mak21-1 mutants are unable to propagate M1 double-stranded RNA, a satellite of the L-A double-stranded RNA virus, encoding a secreted protein toxin lethal to yeast strains that do not carry M1. We cloned MAK21 using its map location and found that Mak21p is homologous to a human and mouse CAATT-binding protein and open reading frames in Schizosaccharomyces pombe and Caenorhabditis elegans. Although the human protein regulates Hsp70 production, Mak21p is essential for growth and necessary for 60 S ribosomal subunit biogenesis. mak21-1 mutants have decreased levels of L-A coat protein and L-A double-stranded RNA. Electroporation with reporter mRNAs shows that mak21-1 cells cannot optimally express mRNAs which, like L-A viral mRNA, lack 3'-poly(A) or 5'-cap structures but can normally express mRNA with both cap and poly(A). The virus propagation phenotype of mak21-1 is suppressed by ski2 or ski6 mutations, each of which derepresses translation of non-poly(A) mRNA.

We mapped and cloned SKI6 of Saccharomyces cerevisiae, a gene that represses the copy number of the L-A double-stranded RNA virus, and found that it encodes an essential 246-residue protein with homology to a tRNA-processing enzyme, RNase PH. The ski6-2 mutant expressed electroporated non-poly(A) luciferase mRNAs 8- to 10-fold better than did the isogenic wild type. No effect of ski6-2 on expression of uncapped or normal mRNAs was found. Kinetics of luciferase synthesis and direct measurement of radiolabeled electroporated mRNA indicate that the primary effect of Ski6p was on efficiency of translation rather than on mRNA stability. Both ski6 and ski2 mutants show hypersensitivity to hygromycin, suggesting functional alteration of the translation apparatus. The ski6-2 mutant has normal amounts of 40S and 60S ribosomal subunits but accumulates a 38S particle containing 5'-truncated 25S rRNA but no 5.8S rRNA, apparently an incomplete or degraded 60S subunit. This suggests an abnormality in 60S subunit assembly. The ski6-2 mutation suppresses the poor expression of the poly(A)- viral mRNA in a strain deficient in the 60S ribosomal protein L4. Thus, a ski6 mutation bypasses the requirement of the poly(A) tail for translation, allowing better translation of non-poly(A) mRNA, including the L-A virus mRNA which lacks poly(A). We speculate that the derepressed translation of non-poly(A) mRNAs is due to abnormal (but full-size) 60S subunits.

The L-A double-stranded RNA virus of yeast encodes its major coat protein, Gag, and a Gag-Pol fusion protein made by a -1 ribosomal frameshift, a coding strategy used by many retroviruses. We find that cells expressing only Gag from one plasmid and only Gag-Pol (in frame) from a separate plasmid can support the propagation of M1 double-stranded RNA, encoding the killer toxin. We use this system to separately investigate the functions of Gag and the Gag part of Gag-Pol. L-A contains two fusion protein molecules per particle, and although N-terminal acetylation of Gag is essential for viral assembly, it is completely dispensable for function of Gag-Pol. In general, the requirements on Gag for viral assembly and propagation are more stringent than on the Gag part of Gag-Pol. Finally, we directly show that it is Gag that instructs the incorporation of Gag-Pol into the viral particles.

The genetic properties of the non-Mendelian element, [URE3], suggest that it is a prion (infectious protein) form of Ure2p, a mediator of nitrogen regulation in Saccharomyces cerevisiae. Into a ure2Delta strain (necessarily lacking [URE3]), we introduced a plasmid overproducing Ure2p. This induced the frequent "spontaneous generation" of [URE3], with properties identical to the original [URE3]. Altering the translational frame only in the prion-inducing domain of URE2 shows that it is Ure2 protein (and not URE2 RNA) that induces appearance of [URE3]. The proteinase K-resistance of Ure2p is unique to [URE3] strains and is not seen in nitrogen regulation of normal strains. The prion-inducing domain of Ure2p (residues 1-65) can propagate [URE3] in the absence of the C-terminal part of the molecule. In contrast, the C-terminal part of Ure2p cannot be converted to the prion (inactive) form without the prion-inducing domain covalently attached. These experiments support the prion model for [URE3] and extend our understanding of its propagation.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3' end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1 kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.

Saccharomyces cerevisiae is host to the dsRNA viruses L-A (including its killer toxin-encoding satellite, M) and L-BC, the 20S and 23S ssRNA replicons, and the putative prions, [URE3] and [PSI]. review the genetic and biochemical evidence indicating that [URE3] and [PSI] are prion forms of Ure2p and Sup35p, respectively. Each has an N-terminal domain involved in propagation or generation of the prion state and a C-terminal domain responsible for the protein's normal function, nitrogen regulation, or translation termination, respectively. The L-A dsRNA virus expression, replication, and RNA packaging are reviewed. L-A uses a -1 ribosomal frameshift to produce a Gag-Pol fusion protein. The host SK12, SK13 and SK18 proteins block translation of nonpoly(A) mRNAs (such as viral mRNA). Mutants deficient in 60S ribosomal subunits replicate L-A poorly, but not if cells are also ski-. Interaction of 60S subunits with the 3' polyA is suggested. SKI1/XRN1 is a 5'--> 3' exoribonuclease that degrades uncapped mRNAs. The viral Gag protein decapitates cellular mRNAs apparently to decoy this enzyme from working on viral mRNA.

L-A and L-BC are two double-stranded RNA viruses present in almost all strains of Saccharomyces cerevisiae. L-A, the major species, has been extensively characterized with in vitro systems established, but little is known about L-BC. Here we report in vitro template-dependent transcription, replication, and RNA recognition activities of L-BC. The L-BC replicase activity converts positive, single-stranded RNA to double-stranded RNA by synthesis of the complementary RNA strand. Although L-A and L-BC do not interact in vivo, in vitro L-BC virions can replicate the positive, single-stranded RNA of L-A and its satellite, M1, with the same 3' end sequence and stem-loop requirements shown by L-A virions for its own template. However, the L-BC virions do not recognize the internal replication enhancer of the L-A positive strand. In a direct comparison of L-A and L-BC virions, each preferentially recognizes its own RNA for binding, replication, and transcription. These results suggest a close evolutionary relation of these two viruses, consistent with their RNA-dependent RNA polymerase sequence similarities.

[URE3] is a non-Mendelian genetic element that mimics recessive mutations in the chromosomal URE2 gene making cells derepressed for nitrogen catabolic enzymes. [PSI] is a non-Mendelian enhancer of readthrough of translational termination similar in its effects to some mutations in the chromosomal SUP35 gene. Three lines of evidence led to the proposal that both [URE3] and [PSI] are prions, infectious proteins analogous to the scrapie agent mediating transmissible spongiform encephalopathies of mammals. 1) Both [PSI] and [URE3] are reversibly curable. 2) [PSI] propagation requires SUP35 and [URE3] propagation requires URE2 with recessive chromosomal mutants having the same phenotypes as the presence of the respective dominant non-Mendelian element. 3) Overproduction of Sup35p and Ure2p increases the frequency of cells acquiring [PSI] or [URE3], respectively.

The genetic properties of the [URE3] non-Mendelian element of Saccharomyces cerevisiae suggest that it is a prion (infectious protein) form of Ure2p, a regulator of nitrogen catabolism. In extracts from [URE3] strains, Ure2p was partially resistant to proteinase K compared with Ure2p from wild-type extracts. Overexpression of Ure2p in wild-type strains induced a 20- to 200-fold increase in the frequency with which [URE3] arose. Overexpression of just the amino-terminal 65 residues of Ure2p increased the frequency of [URE3] induction 6000-fold. Without this "prion-inducing domain" the carboxyl-terminal domain performed the nitrogen regulation function of Ure2p, but could not be changed to the [URE3] prion state. Thus, this domain induced the prion state in trans, whereas in cis it conferred susceptibility of the adjoining nitrogen regulatory domain to prion infections.

Chromosomal mutants (maintenance of frame = mof) in which the efficiency of -1 ribosomal frameshifting is increased can be isolated using constructs in which lacZ expression is dependent upon a -1 shift of reading frame. We isolate a new mof mutation, mof9, in Saccharomyces cerevisiae and show that it is complemented by both single and multi-copy 5 S rDNA clones. Two independent insertion mutations in the rDNA locus (rDNA::LEU2 and rDNA::URA3) also display the Mof- phenotype and are also complemented by single and multi-copy 5 S rDNA clones. Mutant 5 S rRNAs expressed from a plasmid as 20-50% of total 5 S rRNA in a wild-type host also induced the Mof- phenotype. The increase in frameshifting is greatest when the lacZ reporter gene is expressed on a high copy, episomal vector. No differences were found in 5 S rRNA copy number or electrophoretic mobilities in mof9 strains. Both mof9 and rDNA::LEU2 increase the efficiency of +1 frameshifting as well but have no effect on readthrough of UAG or UAA termination codons, indicating that not all translational specificity is affected. These data suggest a role for 5 S rRNA in the maintenance of frame in translation.