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

Esteban, Rosa; Wickner, Reed B.

doi:10.1128/mcb.6.5.1552

Cited by 78 publications

(57 citation statements)

References 40 publications

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“…Without heat denaturation, the product did not hybridize with plusor minus-strand DNA (lanes 4 and 5). A longer incubation (135 min) at 30°C allowed the particles to synthesize ssRNA (lane 6), and without heat denaturation, this ssRNA hybridized with minus-strand DNA (lane 8) but not with plusstrand DNA (lane 7). These results clearly indicate that the newly synthesized strand of dsRNA was the minus strand of L-A dsRNA and that the ssRNA produced by L-A dsRNAsynthesizing particles after prolonged incubation was plusstrand L-A ssRNA.…”

Section: Resultssupporting

confidence: 50%

“…A similar model has been proposed for M dsRNA replication, except for the formation of particles with two M dsRNA molecules per particle (7).…”

mentioning

confidence: 99%

“…L-A VLPs prepared from stationary-phase cells have one L-A dsRNA molecule per particle and have an RNA polymerase activity whose product is plus-strand L-A singlestranded RNA (ssRNA) which is extruded from the VLPs (4,7,11). We call these "mature" or "main-peak" L-A VLPs.…”

mentioning

confidence: 99%

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L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Fujimura¹,

Wickner²

1987

Mol. Cell. Biol.

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Previously, we found that log-phase cells of Saccharomyces cerevisiae contain a new type of viruslike particles containing only plus-strand L-A single-stranded RNA (ssRNA). These particles synthesize minus-strand RNA in an in vitro RNA polymerase reaction to produce L-A double-stranded RNA (dsRNA). The major class of particles contains L-A dsRNA and synthesizes plus-strand L-A ssRNA by a conservative mechanism (9). In this paper, we show that mutations in maklO or the petl8 locus, which result in temperature-dependent replication of L-A dsRNA in vivo, also result in instability of the L-A dsRNA-containing (major class) viruslike particles in vitro. The L-A dsRNA (minus-strand)-synthesizing particles isolated by CsCl density gradient centrifugation synthesize plus-strand L-A ssRNA after completion of dsRNA (minus-strand) synthesis and have the same major coat protein as that of the major-class particles. Furthermore, the density of the dsRNA-synthesizing particles from wild-type cells shifts to that of the major-class dsRNA-containing particles as a result of the in vitro RNA polymerase reaction. Thus, L-A dsRNA-synthesizing particles undergo functional and structural maturation in vitro.Most strains of the yeast Saccharomyces cerevisiae harbor a 4.5-kilobase double-stranded RNA (dsRNA) called L-A dsRNA. L-A dsRNA is found as intracellular noninfectious viruslike particles (VLPs) whose major coat protein is encoded by L-A dsRNA itself. L-A VLPs are transmitted by cytoplasmic mixing such as occurs during mating (1,12,19).L-A dsRNA requires the products of the chromosomal genes MAK3, MAKIO, and PET18 for its replication or maintenance (18,19,25), and petl8 mutations cause structural instability of L-A dsRNA-containing VLPs (10). These genes also affect maintenance of another dsRNA, called M (1.5 or 1.8 kb, depending on the subspecies), which encodes the killer toxin, a secreted protein lethal to other strains of the same species (3,20,24). A set of six chromosomal genes, called SKI genes, negatively control L-A dsRNA replication (2, 21). L-A dsRNA itself also carries genetic activities ([HOK], [NEX], and [EXL]) in several different combinations, which are defined by their effects on M dsRNA replication (18,19,23,25).Judging by in vivo density transfer experiments, L-A dsRNA replication seems to be conservative (14, 17), similar to that of reoviruses (27) but unlike that of Penicillium stoloniforum virus S, which is semiconservatively replicated (5, 6). In vivo pulse-chase experiments indicate a sequential replication: first plus-strand synthesis, followed by minusstrand synthesis (15).L-A VLPs prepared from stationary-phase cells have one L-A dsRNA molecule per particle and have an RNA polymerase activity whose product is plus-strand L-A singlestranded RNA (ssRNA) which is extruded from the VLPs (4, 7, 11). We call these "mature" or "main-peak" L-A VLPs. Previously, we reported a new type of L-A VLP of lighter density whose in vitro RNA polymerase reaction product was L-A dsRNA (9). These particles were present whe...

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Section: Resultssupporting

confidence: 50%

“…A similar model has been proposed for M dsRNA replication, except for the formation of particles with two M dsRNA molecules per particle (7).…”

mentioning

confidence: 99%

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L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Fujimura¹,

Wickner²

1987

Mol. Cell. Biol.

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“…The smaller dsRNAs S1-and S2-were encapsidated in the different X. dendrorhous strains supporting the hypothesis that they are satellite elements. The satellite dsRNAs in the viral system of S. cerevisiae are encapsidated independently of helper virus, generating virions with one L-A molecule or two M molecules [5][6][7][21][22][23]; in some case can encapsidated 1 to 8 X dsRNA, a molecule of 0.52 kb spontaneously arose in a non killer strain [24]. More analyses are necessary to evaluate if this exclusion mechanism of dsRNA encapsidation exists in totiviruses of X. dendrorhous, or if the fractions showing multiple dsRNAs in the sucrose gradients correspond to virions that encapsidated more than one type of dsRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…Some strains of S. cerevisiae have an additional dsRNA molecule of 1.6-1.8 kbp called M-dsRNA, that encoded fort a killer toxin and self-immunity; this ''satellite virus'' depends on the viral proteins encoded by L-A (the ''helper virus'') for its encapsidation and replication [6]. The ScV-L-A virions can encapsidated independently one L-A or two M-dsRNAs, and their protein composition is estimated in 1-2 CP-Pol and 120 CP proteins [7]. The CP protein participate in cap-snatching mechanism in which m(7)Gp from cap structure of cellular mRNAs is transferred to viral transcripts, required for its stability and efficient translation [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous

et al. 2015

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Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

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Internal and terminal cis-acting sites are necessary for in vitro replication of the L-A double-stranded RNA virus of yeast.

1989

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Empty particles of the L‐A dsRNA virus of Saccharomyces cerevisiae bind to added viral (+) strands and convert them to dsRNA (RNA replication) in an in vitro reaction that is dependent on host factors. X dsRNA (530 bp long) is a deletion derivative of L‐A dsRNA (4.5 kb). By modifying our cDNA clone of X and testing template activity of T7 RNA polymerase transcripts, we have found that both the 3′ end 30 bases and an internal site on the (+) strand are necessary for optimal replication [in vitro (‐) strand synthesis]. Changing any one of the 3′ terminal three bases eliminates template activity, but the 3′ terminal five bases of M1 (a satellite virus of L‐A) can replace the 3′ terminal four bases of X. A subterminal stem‐loop structure is also important for template activity. The internal site that enhances replication is approximately 400 bp from the 3′ end and is distinct from the site necessary for binding of (+) strands to the empty viral particles.

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

Cited by 78 publications

References 40 publications

L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

L-A double-stranded RNA viruslike particle replication cycle in Saccharomyces cerevisiae: particle maturation in vitro and effects of mak10 and pet18 mutations.

Characterization of virus-like particles and identification of capsid proteins in Xanthophyllomyces dendrorhous

Internal and terminal cis-acting sites are necessary for in vitro replication of the L-A double-stranded RNA virus of yeast.

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