The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Corresponding author: Michael J. Leibowitz, UMDNJ-Robert Wood Johnson Medical School, Department of Molecular Genetics and Microbiology, 675 Hoes Lane, Piscataway, NJ 08854-5635., leibowit{at}umdnj.edu (E-mail)

Communicating editor: S. SANDMEYER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are "healed" in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10^-4–10^-5 and reappears with variegated phenotypic expression with a frequency of 10^-3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

KILLER virus of Saccharomyces cerevisiae is a cytoplasmically inherited dsRNA virus (reviewed by WICKNER 1996 ). The viral genome consists of the replication-competent L-A double-stranded RNA (dsRNA) helper genome, which is present in most yeast strains, and the M satellite dsRNA, which is present only in killer yeasts and which depends on L-A for replication functions. Different alleles of L-A dsRNA are found in different strains, including L-A-H and L-A-HN (HANNIG et al. 1985 ). Cells harboring the M dsRNA segment of this virus secrete a protein toxin that kills uninfected yeast cells and are themselves resistant to the toxin. Wild-type infected cells have the phenotypes of K⁺ (killing) and R⁺ (resistance), while cells lacking M dsRNA are K^-R^- in phenotype. Both the toxin and the resistance substance are processed products derived from preprotoxin, the primary translation product of the only long open reading frame on viral transcripts of M dsRNA. Distinct M dsRNA species, denoted M₁ and M₂, encode different preprotoxin proteins, resulting in different specificities of killing and resistance. Type 1 and type 2 killer yeasts (encoded by their respective infecting M dsRNA species) are resistant to toxin of the same type but are sensitive to the other toxin. When different M dsRNA species are introduced into the same cell, they display incompatibility (termed "exclusion"), so that one (generally M₁) will persist while the other is lost. Yeast cells harboring killer-virus-related deletion mutants derived from L-A dsRNA, denoted X (ESTEBAN and WICKNER 1988 ), and derived from M dsRNA, denoted S (FRIED and FINK 1978 ; THIELE et al. 1984 ), have been described.

Mutations in viral and host genes resulting in altered replication, expression, or regulation of the virus have been characterized extensively (WICKNER 1996 ). The most puzzling of the mutants characterized have been the diploid-dependent mutants, which harbor the cytoplasmically inherited [KIL-d] element (WICKNER 1976 ; TALLOCZY et al. 1998 ). These mutants were isolated after ethyl methanesulfonate mutagenesis of virus-infected haploid strain A364A (WICKNER 1976 ). The primary mutant isolates displayed various mitotically stable defective phenotypes (K^-R⁺, K⁺R^-, K^-R^-, or intermediate "weak" phenotypes, denoted by the w superscript). There is an increased tendency of these mutants to lose M dsRNA during vegetative growth, resulting in occasional K^-R^- segregants. When a [KIL-d] strain harboring M dsRNA is crossed with a wild-type nonkiller lacking M, the resulting diploids are K⁺R⁺ in phenotype. When such diploids are subjected to meiotic sporulation, the spore clones display different mitotically stable killer phenotypes (K*R*), many of which are defective in killing and resistance, and show increased mitotic instability of M dsRNA. These spore clones show the same behavior on repeated backcrosses with wild-type nonkiller haploids, such that defective phenotypes expressed in the haploid state are "healed" by mating and "reset" to variegated mitotically stable phenotypes upon meiosis (WICKNER 1976 ). Although K⁺R⁺ spore clones are recovered among meiotic progeny harboring [KIL-d], such clones, upon mating with a wild-type partner and meiotic sporulation, result in K*R* variegated haploid progeny. Thus, the genetic element resulting in this unusual genetic behavior is cytoplasmically inherited (segregates to all four meiotic progeny). Since, in the presence of [KIL-d], the defective expression of M dsRNA is seen only in haploids, while the wild-type phenotype is expressed in diploids, this cytoplasmic determinant was termed "the diploid-dependent element" (WICKNER 1976 ). Similarly, when two [KIL-d] haploids are crossed with each other, the resulting diploids are K⁺R⁺ in phenotype and show resetting to variegated K*R* defective phenotypes upon meiosis (WICKNER 1976 ). The "healing" of the defective phenotypes upon mating indicates that [KIL-d] exerts epigenetic effects on M dsRNA, which remains competent to code for preprotoxin.

We have previously shown that [KIL-d] does not map on M or L-A dsRNA. This was demonstrated by curing a [KIL-d] strain of M₁ and L-A-HN dsRNA to yield a K^-R^- strain, which upon crossing with a strain harboring M₂ and L-A-H yielded type 2 killer diploids. Meiotic sporulation of these diploids resulted in type 2 killer progeny showing variegated defective phenotypes (K₂*R₂*). Ethidium bromide curing similarly proved [KIL-d] not to map on mitochondrial DNA. Moreover, the [KIL-d] element can be transmitted along with M dsRNA from one haploid strain to another by cytoplasmic transfer (cytoduction). However, [KIL-d] does not exert its phenotypic effect upon expression of M dsRNA in the recipient until that strain has gone through a cycle of mating and meiotic sporulation, resulting in variegated defective phenotypes in the spore clones. The defects in these clones are, again, healed by mating (TALLOCZY et al. 1998 ). The failure of [KIL-d] to exert its epigenetic effects on M expression until a cycle of mating and meiosis has occurred suggests that nuclear events are involved in the epigenetic regulatory process. This process is not simply the result of cell type regulation, since neither cell type switching mediated by expression of the HO (homothallism) gene nor transformation of either cell type (a or ) [KIL-d] strain with a plasmid expressing the opposite MAT allele alters the epigenetic effect on M dsRNA (TALLOCZY et al. 1998 ).

It has been observed that [KIL-d] haploids with defective phenotypes spontaneously revert to the wild-type K⁺R⁺ phenotype at a frequency of 10^-4 to 10^-5 (WICKNER 1976 ; TALLOCZY et al. 1998 ). In this report we show that such revertants of [KIL-d] spontaneously "backrevert" at even higher frequencies than the reversion rate. This backreversion results in cells with mitotically stable variegated defective phenotypes typical of [KIL-d]. [KIL-d] is shown not to reside on killer-virus-related dsRNAs or on the 2µ DNA plasmid and not to depend on Hsp104 protein for its maintenance. We also show that [KIL-d] specifically alters gene expression from viral M dsRNA but not from M-derived cDNA.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genetic and molecular methods:
Strains of S. cerevisiae used in this study are listed in Table 1. All [KIL-d] strains are derivatives of strain M89, which carries the [KIL-d30] element (WICKNER 1976 ). Media and growth conditions were as described previously (TALLOCZY et al. 1998 ), including standard yeast genetics methods (KAISER et al. 1994 ). Yeast transformation was done using lithium acetate (KIM and POWERS 1991 ) in the presence of 25 mM dithiothreitol in 10 mM HEPES buffer, pH 7.5, or by electroporation using a Gene Pulser II (Bio Rad, Hercules, CA). All plasmids were purified from transformed Escherichia coli DH5 (GIBCO-BRL, Gaithersburg, MD) using miniprep (QIAprep spin plasmid kit; QIAGEN, Valencia, CA) or midiprep (Plasmid Midi kit; QIAGEN) protocols. Curing of M dsRNA was by growth at 38° (WICKNER 1974 ; WEINSTEIN et al. 1993 ). Loss of M dsRNA was demonstrated by lack of the expected 318-bp product by single-tube RT-PCR of M from a miniprep of RNA prepared as described (TALLOCZY et al. 1998 ), using primers 3179 and 3180 (10 sec at 94°, 1 min at 50°, 2.5 min at 68°). However, RT-PCR analysis indicated that strain M1080 retained a small fragment of M dsRNA sequence detected by this assay; this fragment was absent from isolate M1082. The nature of the M-related sequence in strain M1080 is unknown. In all genetic experiments, the two "cured" isolates showed identical behavior. Oligonucleotide primers used for PCR are listed in Table 2.

Killer phenotype assays:
Killer (K) and resistance (R) phenotypes were assayed as described previously (LEIBOWITZ and WICKNER 1978 ; WICKNER and LEIBOWITZ 1979 ), using diploid tester strains M984 (sensitive), M21 (K1 killer), and M1052 (K2 killer). When strains contained episomal plasmids with selectable markers, R phenotype testing was performed on synthetic complete (H) medium lacking the selected marker, buffered to pH 4.7 with sodium citrate, and supplemented with methylene blue (0.003%) rather than on standard 4.7 MB plates. When phenotypes were tested under galactose-induction conditions, K phenotype was determined on modified 4.7 MB medium in which glucose was replaced by galactose (2%) plus either glycerol (3%) or raffinose (3%). Resistance was determined on synthetic complete medium lacking leucine, buffered to pH 4.7 with sodium citrate, supplemented with methylene blue (0.003%) and with glucose replaced by galactose (2%), glycerol (3%), and raffinose (3%).

Plasmids:
Disruption of the HSP104 gene in various strains was performed by lithium acetate transformation with SstI/SmaI-digested plasmid pBCKS⁺hsp104::LEU2 or SstI/BamHI-digested pBCKS⁺hsp104::URA3 (CHERNOFF et al. 1995 ). Disruption of HSP104 was confirmed by colony PCR with HSP104-specific primers 13861 and 13662 for LEU2 disruptants and primers 13662 and 13860 for URA3 disruptants (10 sec at 94°, 1 min at 66°, 2.5 min at 68°). Plasmid pFL44LHSP104 (provided by M. Boguta, Institute of Biochemistry and Biophysics, Warsaw) is a high-copy episomal plasmid expressing HSP104. Plasmid pBIS-GALkFLP(URA3) expresses a galactose-inducible mutant flp recombinase, whose expression cures (destabilizes) the 2µ plasmid (TSALIK and GARTENBERG 1998 ). After 72 hr of induction by galactose (performed in liquid-defined medium lacking uracil and glucose and containing 2% galactose and 3% glycerol), colonies were grown on defined medium plates containing 5-fluoroorotic acid (0.1%) to select for plasmid loss. Absence of 2µ plasmid in individual colonies was demonstrated by failure to amplify a 783-bp DNA fragment by PCR using primers 12853 and 12854 from yeast colonies (10 sec at 94°, 1 min at 58°, 1.5 min at 68°).

M cDNA expression vectors:
Plasmid pDG2-5 (expressing preprotoxin) was constructed from plasmid pL315, which consisted of the large HindIII/BamHI fragment of pRS722 (2µ origin, ampicillin resistance, and LEU2 genes) and the yeast galactokinase promoter (GAL1-10) with a downstream SalI/PstI/BamHI polylinker (provided by D. Y. Thomas, Institute de Recherce en Biotechnologie, Montreal). Plasmid pL315 was linearized by digestion with HindIII and BamHI, and the 1100-bp BamHI/HindIII M₁-1 fragment (BOONE et al. 1986 ) from pUC8-KT-Authentic 5' and the 620-bp BamHI M₁-2 fragment from pEH-2 (GEORGOPOULOS et al. 1986 ) were ligated into pL315 to produce a full-length M₁ sense clone (minus the poly A-U tract). Plasmid pDG3-1 is cL434, which contains M₁-1, denoted KT-MD1 (SKIPPER et al. 1984 ), cloned into the HindIII site of pL315 in the antisense orientation, with the downstream HindIII site at base 1749 destroyed. Wild-type yeast cells lacking M dsRNA and harboring pDG2-5 were K⁺₁R⁺₁ on galactose and K^-R^- on glucose-containing media, while these cells with pDG3-1 were K^-R^- on either carbon source.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

[KIL-d] revertants backrevert to variegated phenotypes:
Reversion of [KIL-d] strain M89 (K^-R⁺) by apparent loss of [KIL-d] to yield strains with the wild-type K⁺R⁺ phenotype (strains M1009 and M1010) occurs with a frequency of 10^-4 to 10^-5 (WICKNER 1976 ; TALLOCZY et al. 1998 ). When these two revertant isolates were crossed with various wild-type nonkillers (Table 3), the resulting diploids were wild-type killers, but upon meiosis these diploids yielded variegated defective haploid progeny at a frequency ranging from 0 to 27.6% (excluding the few spores that failed to inherit M dsRNA). Two of these defective progeny spore clones yielded K⁺R⁺ diploids upon crossing with a wild-type nonkiller and upon meiosis yielded spore clones with variegated phenotypic defects (data not shown), indicating that [KIL-d] had apparently reappeared in the revertants. Three other spore clones that were not defective (K⁺R⁺) yielded 100% K⁺R⁺ meiotic progeny upon a cross with a wild-type nonkiller, consistent with the apparent loss of [KIL-d] in the original revertants. Crosses of revertants M1009 and M1010 with various [KIL-d] strains yielded K⁺R⁺ diploids and variegated defective meiotic progeny (Table 3), confirming that the revertants did not carry a genetic element suppressing or excluding [KIL-d].

To test for the frequency of such backrevertants, single colonies isolated from revertants M1009 and M1010, which had been stored at -70° in 15% glycerol, were plated on YPAD plates and tested for killer phenotype. Among single colonies of these two revertants, K^- and K^W colonies appeared with a frequency of 7.3–10.1 x 10^-3, and these colonies yielded K⁺R⁺ diploids upon crossing with a wild-type nonkiller strain. This result indicates that the revertants contained apparent backrevertants with variegated phenotypic expression of killer virus. Colonies that had lost M dsRNA appeared with varying frequency, as reported (WICKNER 1976 ). Colonies that harbored genetically stable cytoplasmically inherited mutations resulting in the K^-R⁺ (neutral) phenotype were also noted among these colonies; the spontaneous appearance of such mutations has been noted in wild-type strains (BEVAN and SOMERS 1969 ).

To exclude the variable of long storage of the revertants before detection of the backrevertants, three K⁺R⁺ single colonies of both M1009 and M1010 revertants were isolated. Each of these was grown in liquid medium and plated for single colonies, which were replica plated and tested for their killer phenotype. Variegated defective segregants arose with a frequency of 1.2–5.2 x 10^-3 among all six sets of single colonies derived from K⁺R⁺ colonies of the parent strains. Upon crossing with a wild-type nonkiller, these defective segregants yielded K⁺R⁺ diploids, which upon meiosis resulted in variegated defective haploid spore clones (Table 4). As noted above, single colonies that had lost M dsRNA or contained stable neutral mutants were also recovered with comparable frequencies, among both the single colony isolates of the reisolated revertant colonies (data not shown) and the meiotic segregants obtained from crosses of the backrevertants with wild-type nonkillers. Except for these segregants, which had lost M dsRNA or harbored neutral mutations, defective spore clones from these crosses yielded wild-type killer phenotype diploids upon backcrossing with wild-type nonkillers, as is characteristic of haploid cells harboring [KIL-d]. Thus, revertants of [KIL-d] backrevert to variegated defective phenotypes with a frequency of 10^-3. These backrevertants display the [KIL-d] genetic trait. This frequency of backreversion is underestimated since the screening procedure used would not have detected K⁺ [KIL-d] segregants.

[KIL-d] epigenetic regulation is virus specific:
To determine whether the epigenetic effects of [KIL-d] were virus specific, we compared its effect on the expression of M cDNA (plasmid pDG2-5) and viral M dsRNA. Two haploid [KIL-d] K^-R⁺ strains, M1079 and M1081, generated by crosses of different wild-type nonkillers with strain M89, were grown at 38° to isolate K^-R^- segregants M1080 and M1082. These "heat-cured" strains lack intact M dsRNA, as evidenced by failure to detect M dsRNA using gel electrophoresis and ethidium bromide staining of extracted RNA and by production of K^-R^- diploids upon crossing of these "cured" strains with wild-type nonkillers.

When strains M1080 and M1082 (which lack intact M dsRNA) were transformed with plasmid pDG2-5, 24 transformants of each strain were all K^-R^- in phenotype on glucose and K⁺R⁺ on galactose plates, consistent with the absence of [KIL-d] or its failure to affect expression of M cDNA. However, when the parental [KIL-d] strains M1079 and M1081 (which harbor M dsRNA) were transformed with pDG2-5, 24 transformants of each were K^-R⁺ on glucose plates, confirming that [KIL-d] was present (Fig 1). Furthermore, these transformants were K⁺R⁺ on galactose plates, indicating that even in cells in which [KIL-d] affected expression of M dsRNA, this element had no effect on M cDNA. As expected, control transformation of all four strains with the antisense cDNA construct pDG3-1 failed to alter the phenotype of the recipients.

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Figure 1. [KIL-d] does not affect M cDNA expression. M1081 and M1079 [KIL-d] strains transformed with galactose-inducible M cDNA expression vector (pDG2-5) were tested for virus-related killer phenotypes on galactose and glucose medium. Zones of clearing (due to killing of the lawn of sensitive cells) indicating the K+ phenotype are evident on galactose but not on glucose medium. All tested transformants were R+ on both media.

To prove that [KIL-d] does not require a cycle of mating and meiosis to establish regulation of the M cDNA, as is required for regulation of M dsRNA expression after cytoduction (TALLOCZY et al. 1998 ), pDG2-5-transformed [KIL-d] strains, M1079 and M1081, were crossed with the leu2 wild-type nonkillers M1102 and M831. These crosses yielded diploids that were K⁺R⁺ on both glucose and galactose medium. Sporulation of these diploids, followed by dissection of tetrads and spore germination on H-Leu medium (Table 5), revealed that all surviving spore clones were leucine prototrophs, indicating that they carried pDG2-5. All of these clones were K⁺R⁺ on galactose, but they showed variegated killer phenotypes on glucose. All defective phenotype spore clones yielded K⁺R⁺ diploids (on either medium) upon crossing with wild-type nonkillers, indicating that [KIL-d] was present. When pDG2-5-transformed strain M1079 was crossed with leu2 wild-type nonkillers M1102 and M739 and similarly sporulated with germination on rich YPAD medium, some spore clones remained leucine prototrophs, indicating retention of pDG2-5, while other clones were auxotrophic, indicating plasmid loss. Both auxotrophic and prototrophic clones showed variegated killer phenotypes on glucose. However, the prototrophs were K⁺R⁺ on galactose, while auxotrophs showed the same variegated defective phenotype on galactose that they displayed on glucose plates (Table 5). When pDG2-5-transformed strain M1079 was crossed with LEU2 wild-type nonkiller M422 and spore clones were germinated on YPAD, inheritance of [KIL-d] was demonstrated by the variegated defective phenotype on glucose. Thus, even after a cycle of mating, with the characteristic phenotypic healing, meiotic sporulation, and the characteristic generation of variegated defective expression of M dsRNA, [KIL-d] failed to exert its epigenetic effect on M cDNA in the same cells in which this effect on M dsRNA expression was evident.

[KIL-d] is distinct from multiple cytoplasmic genetic elements:
We have shown previously that [KIL-d] is not lost when strains harboring it are cured of various cytoplasmic elements, including mitochondrial DNA and M and L-A dsRNA (TALLOCZY et al. 1998 ). Since strains heat cured (WICKNER 1974 ) of M dsRNA may harbor M-related sequences, we tested the possibility that [KIL-d] resides on a virus-related RNA that escaped heat curing. Therefore, we determined whether mak3 and mak10 mutant strains, which are unable to maintain L-A, M, and related dsRNAs (SOMMER and WICKNER 1982 ; WICKNER and TOH-E 1982 ), could maintain [KIL-d] (Table 6). Strain M1104 ([KIL-d] K^-R^W) was used as a cytoduction donor into strains M1105 (mak3), M1106 (mak10), and M1107 (mak10). As expected, all cytoductants (which are unable to maintain killer viral dsRNA) were K^-R^- in phenotype. These cytoductants, in turn, were used as cytoduction donors into nonkiller strains M1109 and M1108, again yielding K^-R^- cytoductants. These final cytoductants were crossed with type 2 killer strain M707. All diploids isolated were type 2 killers, and upon meiotic sporulation produced type 2 killer spore clones showing the variegated defects typical of [KIL-d] (Table 6). Thus, unlike L-A, M, and related dsRNAs, [KIL-d] can be maintained by mak3 and mak10 mutants. Moreover, [KIL-d] is cytoplasmically transmissible through multiple cytoductions in the absence of phenotypic expression.

Plasmid pBIS-GALkFLP(URA3) was transformed into three [KIL-d] strains and, after galactose induction of the mutant flp recombinase, the presence of 2µ DNA was determined by PCR. As indicated in Table 7, neither transformation with the plasmid nor loss of 2µ DNA resulted in alteration of the killer phenotype, indicating that the yeast DNA plasmid does not harbor [KIL-d].

Another group of cytoplasmically inherited genetic elements in yeast consists of the [PSI] and [URE3] prions (WICKNER 1994 , WICKNER 1997 ). We had previously shown that growth in the presence of guanidinium hydrochloride, which reversibly cures the [PSI] and [URE3] prions, did not increase the rate of reversion of [KIL-d] (TALLOCZY et al. 1998 ). Since disruption or overexpression of the gene encoding chaperone protein Hsp104 prevents maintenance of [PSI] (CHERNOFF et al. 1995 ), we tested the effect of altering Hsp104 expression levels on maintenance of [KIL-d]. HSP104 was disrupted (confirmed by PCR analysis) by insertion of LEU2 in [KIL-d] strains M1081 (three isolates), M1079 (one isolate), and strain M1098 (two isolates) and by insertion of URA3 in strain M1098 (two isolates). All confirmed hsp104 disruptants remained K^-R⁺ in phenotype. When strain M1098 ([KIL-d] K^-R⁺) was transformed with plasmid pFL44HSP104, transformants (34 tested) were unaltered in phenotype despite the presence of this high-copy plasmid expressing HSP104. Thus, like the [URE3] prion (Y. O. CHERNOFF and S. W. LIEBMAN, cited in WICKNER and CHERNOFF 1999 ) but unlike [PSI], neither disruption nor high-level expression of HSP104 alters the maintenance of [KIL-d]. Thus, the genetic properties of [KIL-d] are not identical to those of either [PSI] or [URE3].

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Prion-like behavior of [KIL-d]:
The relatively high rate of spontaneous reversion of [KIL-d] and even higher rate of apparent backreversion to variegated defective phenotypes are unlike the properties of known cytoplasmic nucleic acids. While yeast also may spontaneously generate mitotic segregants lacking nucleic acid plasmids or viruses, such segregants do not regenerate the lost elements. We have shown previously that [KIL-d] is not lost when cells are cured of mitochondrial DNA or the L-A or M viral dsRNA genomes (TALLOCZY et al. 1998 ). Here we demonstrate that cells incapable of maintaining virus-related dsRNA or 2µ DNA can maintain [KIL-d]. Thus, [KIL-d] does not map on any of these cytoplasmic nucleic acid genomes.

The reappearance of [KIL-d] with variegated phenotypes resembles the [PSI] prion. [PSI] reappears spontaneously after loss (LUND and COX 1981 ), and this reappearance occurs with higher frequency and results in variegated phenotypes when resulting from high levels of expression of SUP35 (CHERNOFF et al. 1993 ; DERKATCH et al. 1996 ), which encodes the prion protein. Chaperone proteins, which function in protein folding, might be expected to play a role in the conformation changes leading to prion formation. Although [KIL-d] differs from [PSI] in being insensitive to disruption or high-copy expression of the HSP104 gene, which encodes such a chaperone protein, the [URE3] prion, like [KIL-d], does not require HSP104 for its maintenance (Y. O. CHERNOFF and S. W. LIEBMAN, cited in WICKNER and CHERNOFF 1999 ). [KIL-d] is a cytoplasmically inherited genetic element with some genetic properties resembling prions of yeast.

[KIL-d] specifically regulates expression of viral M dsRNA:
The results presented here indicate that the epigenetic effects of [KIL-d] are specific for viral M dsRNA and are not exerted on M cDNA. When [KIL-d] and M dsRNA are both introduced into a wild-type yeast cell by cytoduction, a cycle of mating and meiotic sporulation are required before [KIL-d] can exert its effects on viral gene expression (TALLOCZY et al. 1998 ). Even after the mating and meiotic sporulation of cells harboring [KIL-d], M dsRNA, and M cDNA, [KIL-d] fails to alter expression of M cDNA in the same cells in which viral M dsRNA is epigenetically regulated, proving the viral specificity of [KIL-d]. We cannot exclude the possibility that very high levels of expression of presumably capped mRNA from M cDNA may bypass regulation of expression by [KIL-d]. However, [KIL-d] can affect expression of M dsRNA in the same cells in which M cDNA expression in not affected. Note that cDNA is transcribed in the nucleus by cellular DNA-dependent RNA polymerase II, and the resulting mRNA is capped and transported into the cytoplasm. On the other hand, viral M dsRNA transcription by RNA-dependent RNA polymerase in cytoplasmic virions yields apparently uncapped mRNA, whose translation appears to be coupled to transcription (BARBONE and LEIBOWITZ 1991 ). Moreover, variation of viral mRNA level cannot explain the phenotypic variegation seen in meiotic segregants harboring [KIL-d] and M dsRNA, in which K^-R⁺ and K⁺R^- phenotypes (and others) are observed.

We show here that [KIL-d] has properties unlike known nucleic acid replicons that rather resemble certain properties of the known yeast prions. On that basis, we call this unique element "prion-like," although the test of the hypothesis that [KIL-d] is a prion requires the identification of the gene encoding the prion-forming protein. We speculate that the specific interaction of [KIL-d] with the M dsRNA viral genome could provide insights into the controversial role of cryptic viruses (CAUGHEY and CHESEBRO 1997 ; CHESEBRO 1997 ) in human neurological diseases (COHEN and PRUSINER 1998 ; MASTRIANNI et al. 1999 ) caused by prions, if the hypothesis that [KIL-d] is a prion is correct.

	ACKNOWLEDGMENTS

We thank L. Neigeborn (Rutgers University), J. Dinman, M. Hampsey, and T. Goss Kinzy for helpful discussions and critical reading of this manuscript; M. Gartenberg for sharing the method for curing 2µ DNA before publication; and R. B. Wickner (National Institutes of Health) for discussion of experiments involving mak mutants. R. Mazar was supported in part by a Henry Rutgers Honors Fellowship from Rutgers University. F. Ramos was a student in the Graduate Science Careers Program of UMDNJ and Rutgers University, which was supported by an Initiative for Minority Student Development Award from the National Institutes of Health (5R25 GM55145).

Manuscript received November 15, 1999; Accepted for publication February 28, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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