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W2069061056 abstract "20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3′-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3′-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5′-GGGGC... GCCCC-OH). The fifth G from the 3′-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3′-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3′-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3′-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3′-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons. 20 S RNA virus is a persistent positive strand RNA virus found in Saccharomyces cerevisiae. The viral genome encodes only its RNA polymerase, p91, and resides in the cytoplasm in the form of a ribonucleoprotein complex with p91. We succeeded in generating 20 S RNA virus in vivo by expressing, from a vector, genomic strands fused at the 3′-ends to the hepatitis delta virus antigenomic ribozyme. Using this launching system, we analyzed 3′-cis-signals present in the genomic strand for replication. The viral genome has five-nucleotide inverted repeats at both termini (5′-GGGGC... GCCCC-OH). The fifth G from the 3′-end was dispensable for replication, whereas the third and fourth Cs were essential. The 3′-terminal and penultimate Cs could be eliminated or modified to other nucleotides; however, the generated viruses recovered these terminal Cs. Furthermore, extra nucleotides added at the viral 3′-end were eliminated in the launched viruses. Therefore, 20 S RNA virus has a mechanism(s) to maintain the correct size and sequence of the viral 3′-end. This may contribute to its persistent infection in yeast. We also succeeded in generating 20 S RNA virus similarly from antigenomic strands provided active p91 was supplied from a second vector in trans. Again, a cluster of four Cs at the 3′-end in the antigenomic strand was essential for replication. In this work, we also present the first conclusive evidence that 20 S and 23 S RNA viruses are independent replicons. Positive strand RNA viruses encode RNA-dependent RNA polymerases in their genomes and utilize them to synthesize viral RNA in conjunction with other viral or host proteins (1Buck K.W. Adv. Virus Res. 1996; 47: 159-251Crossref PubMed Google Scholar). The antigenomic (negative) strand RNA is an intermediate of replication and serves as a template to synthesize progeny positive strands. Because viral replication takes place within the host cell and because there are plenty of cellular RNAs in these cells, the viruses must find efficiently not only the positive strands but also the negative strands during replication. Therefore, both the positive and negative strands bear cis-acting signals for replication, including those necessary to interact with the polymerase machinery. To analyze and characterize these signals, it is essential to develop appropriate in vivo or in vitro systems in which changes in the signals can be tested. 20 S and 23 S RNAs are positive strand RNA viruses found in Saccharomyces cerevisiae and belong to the genus Narnavirus (2Wickner R.B. Esteban R. Hillman B.I. Van Regenmortel M.H.V. Fauquet C. Bishop D.H.L. Carsten E. Estes M. Lemon S.M. Maniloff J. Mayo M. McGeoon D. Pringle C. Wickner R.B. Virus Taxonomy: Seventh Report of the International Committee on Taxonomy of Viruses. Academic Press, Inc., New York2000: 651-656Google Scholar). These viruses were initially identified as RNA species induced in cells under nitrogen starvation conditions (3Kadowaki K. Halvorson H.O. J. Bacteriol. 1971; 105: 826-830Crossref PubMed Google Scholar, 4Wejksnora P.J. Haber J.E. J. Bacteriol. 1978; 134: 246-260Crossref PubMed Google Scholar). Most laboratory strains carry 20 S RNA, and fewer strains harbor 23 S RNA along with 20 S RNA. They are compatible in the same host cell. Because all known strains that carry 23 S RNA also carry 20 S RNA, it has not been clear whether 23 S RNA virus can replicate without 20 S RNA virus. Typical of fungal viruses, they are not infectious and have no extracellular pathways of transmission. They are transmitted horizontally through mating or vertically from mother to daughter cells. No curing methods to eliminate these viruses from yeast are known so far. Furthermore, 20 S and 23 S RNA viruses do not confer any phenotypic changes on the host. This makes their genetic manipulation difficult. The 20 S and 23 S RNA genomes are small (2514 and 2891 nucleotides, respectively), and each RNA encodes only a single protein: a 91-kDa protein (p91) and a 104-kDa (p104), respectively (5Rodríguez-Cousiño N. Esteban L.M. Esteban R. J. Biol. Chem. 1991; 266: 12772-12778Abstract Full Text PDF PubMed Google Scholar, 6Matsumoto Y. Wickner R.B. J. Biol. Chem. 1991; 266: 12779-12783Abstract Full Text PDF PubMed Google Scholar, 7Esteban L.M. Rodríguez-Cousiño N. Esteban R. J. Biol. Chem. 1992; 267: 10874-10881Abstract Full Text PDF PubMed Google Scholar, 8Rodríguez-Cousiño N. Solórzano A. Fujimura T. Esteban R. J. Biol. Chem. 1998; 273: 20363-20371Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Both proteins contain four amino acid motifs well conserved among RNA-dependent RNA polymerases (9Esteban R. Rodríguez-Cousiño N. Esteban L.M. Prog. Nucleic Acid Res. Mol. Biol. 1993; 46: 155-182Crossref PubMed Scopus (19) Google Scholar). When yeast cells are grown at 37 °C, the cells accumulate double-stranded RNAs called W and T (10Wesolowski M. Wickner R.B. Mol. Cell. Biol. 1984; 4: 181-187Crossref PubMed Scopus (52) Google Scholar), the double-stranded forms of 20 S and 23 S RNA genomes, respectively (8Rodríguez-Cousiño N. Solórzano A. Fujimura T. Esteban R. J. Biol. Chem. 1998; 273: 20363-20371Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). They are not replication intermediates, but by-products (11Fujimura T. Solórzano A. Esteban R. J. Biol. Chem. 2005; 280: 7398-7406Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Because these viruses do not have genes for capsid proteins, their genomes are not encapsidated into intracellular viral particles (12Widner W.R. Matsumoto Y. Wickner R.B. Mol. Cell. Biol. 1991; 11: 2905-2908Crossref PubMed Scopus (21) Google Scholar, 13Esteban L.M. Fujimura T. García-Cuéllar M.P. Esteban R. J. Biol. Chem. 1994; 269: 29771-29777Abstract Full Text PDF PubMed Google Scholar, 14García-Cuéllar M.P. Esteban L.M. Fujimura T. Rodríguez-Cousiño N. Esteban R. J. Biol. Chem. 1995; 270: 20084-20089Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Instead, the RNAs form ribonucleoprotein complexes with their cognate RNA-dependent RNA polymerases at a 1:1 stoichiometry and reside in the cytoplasm (15Solórzano A. Rodríguez-Cousiño N. Esteban R. Fujimura T. J. Biol. Chem. 2000; 275: 26428-26435Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The viral genomes lack poly(A) tails at the 3′-ends and have perhaps no 5′-cap structures (8Rodríguez-Cousiño N. Solórzano A. Fujimura T. Esteban R. J. Biol. Chem. 1998; 273: 20363-20371Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), thus resembling degradation intermediates of mRNAs. How these viruses can survive as persistent viruses in the host cytoplasm without their RNA genomes being digested by exonucleases involved in mRNA degradation is therefore interesting. Recently, we succeeded in generating 23 S RNA virus in vivo from a vector containing the entire cDNA sequence of the viral genome (16Esteban R. Fujimura T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2568-2573Crossref PubMed Scopus (36) Google Scholar). Using this launching system, we began reverse genetics to investigate cis-acting signals for replication in 23 S RNA virus. 20 S and 23 S RNA genomes share five-nucleotide inverted repeats at the 5′ and 3′ termini (5′-GGGGC... GCCCC-OH) (8Rodríguez-Cousiño N. Solórzano A. Fujimura T. Esteban R. J. Biol. Chem. 1998; 273: 20363-20371Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The 23 S RNA genome contains a bipartite cis-signal in the 3′-region that consists of the cluster of the terminal four Cs and a mismatched pair of purines present in a stem structure adjacent to the 3′-end (17Fujimura T. Esteban R. J. Biol. Chem. 2004; 279: 13215-13223Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Although the 3′-terminal and penultimate Cs are dispensable for launching 23 S RNA virus, the generated viruses recover these Cs. This indicates that the virus has an efficient 3′-terminal repair mechanism(s). Subsequently, we found that the bipartite 3′-cis-signal for replication is also essential for formation of ribonucleoprotein complexes in vivo with its RNA-dependent RNA polymerase, p104 (18Fujimura T. Esteban R. J. Biol. Chem. 2004; 279: 44219-44228Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). This indicates the importance of complex formation for 23 S RNA virus and suggests that p104 protects the viral 3′-ends from degradation by binding to the 3′-cis-signal. In this work, we describe the generation of 20 S RNA virus in vivo from a vector, a system similar to the one developed previously for 23 S RNA virus (16Esteban R. Fujimura T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2568-2573Crossref PubMed Scopus (36) Google Scholar). In contrast to the previous work, however, we succeeded in producing 20 S RNA virus not only from positive strands but also from negative strands if, in the latter case, active p91 was provided in trans from a second vector. Using these systems, we modified the viral 3′-ends and found that the clusters of the 3′-terminal four Cs in both the positive and negative strands are 3′-cis-signals for replication. We also provide, for the first time, conclusive evidence that 23 S RNA virus does not require 20 S RNA virus for replication; thus, they are independent replicons. Strains and Media—20 S RNA negative strains 2928-4 and 2928-5 were obtained in this work (see Fig. 1) from strain 2928 L-A-o (a ura3 trp1 his3, 20 S RNA, 23 S RNA-o, L-A-o) (16Esteban R. Fujimura T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2568-2573Crossref PubMed Scopus (36) Google Scholar). These strains were used to analyze launching of 20 S RNA virus from plasmids. Cells were grown in rich YPAD medium (1% yeast extract, 2% peptone, 0.04% adenine sulfate, and 2% glucose) or synthetic medium deprived of tryptophan, uracil, or both (19Wickner R.B. Cell. 1980; 21: 217-226Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Nitrogen starvation in 1% potassium acetate was performed as described previously (4Wejksnora P.J. Haber J.E. J. Bacteriol. 1978; 134: 246-260Crossref PubMed Google Scholar). Northern Hybridization—Cells were broken with glass beads (15Solórzano A. Rodríguez-Cousiño N. Esteban R. Fujimura T. J. Biol. Chem. 2000; 275: 26428-26435Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and RNA was extracted from cell lysates once with phenol and twice with phenol/chloroform and precipitated with ethanol. RNA was separated on an agarose gel, blotted onto a neutral nylon membrane (Hybond-N, Amersham Biosciences), and hybridized with a 32P-labeled 20 S RNA or 23 S RNA positive or negative strand-specific probe (20Fujimura T. Esteban R. Esteban L.M. Wickner R.B. Cell. 1990; 62: 819-828Abstract Full Text PDF PubMed Scopus (98) Google Scholar). We analyzed 5–10 independent transformants in each experiment. For the sake of simplicity, only a representative of each experiment is presented in the figures. Plasmids—The standard 20 S RNA virus-launching plasmid pRE740 contains the entire 20 S RNA cDNA sequence (2514 bp) downstream of the PGK1 promoter in a pI2 derivative (21Wickner R.B. Icho T. Fujimura T. Widner W.R. J. Virol. 1991; 65: 155-161Crossref PubMed Google Scholar). The hepatitis delta virus (HDV) 4The abbreviations used are: HDV, hepatitis delta virus; RT, reverse transcription; RACE, rapid amplification of cDNA ends. antigenomic ribozyme was directly fused to the 3′-end of the 20 S RNA cDNA genome (see Fig. 2A). There are 42 nucleotides between the major transcription start site from the PGK1 promoter and the 20 S RNA sequence. Transcripts from this vector have 20 S RNA positive strand polarity. The template plasmid pRE805 is similar to pRE740, but the orientation of the 20 S RNA cDNA is reversed. Therefore, transcripts from the PGK1 promoter have the negative strand polarity of the viral genome. In addition, the TRP1 marker of this plasmid was disrupted at the unique EcoRV site by insertion of a 1.1-kb URA3 fragment. 20 S RNA positive and negative strand-specific probes were made by T7 and T3 runoff transcription from pRE449 predigested with appropriate restriction enzymes. pRE449 contains a 1262-bp BamHI-SmaI fragment (nucleotides 1253–2514) of the 20 S RNA cDNA sequence between the BamHI and SamI sites of the pBluescript KS+ vector (Stratagene). 23 S RNA positive strand-specific probes were made from plasmid pALI38 as described (15Solórzano A. Rodríguez-Cousiño N. Esteban R. Fujimura T. J. Biol. Chem. 2000; 275: 26428-26435Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In vitro mutagenesis was performed as described (22Esteban R. Fujimura T. Wickner R.B. EMBO J. 1989; 8: 947-954Crossref PubMed Scopus (69) Google Scholar). All mutations introduced were confirmed by DNA sequencing. Reverse Transcription (RT)-PCR—To prove that 20 S RNA virus was generated from plasmids, a DNA fragment of 842 bp was amplified by RT-PCR from lysates using internal primers RE171 (5′-CGCTTCTGCGATCGTAGATG-3′) and ALI-3 (5′-TAAAACTGTATGCAGCAG-3′). The lysates were prepared from 20 S RNA virus-generated cells from which the launching plasmids had been cured or from cells containing endogenous 20 S RNA virus. In either case, the lysate was pre-treated with DNase I before RT-PCR. The RT-PCR products were then digested with SmaI and analyzed on agarose gels. 3′-Rapid Amplification of cDNA Ends (RACE)—Total RNA isolated by launching plasmid-cured cells was poly(A)-tailed using poly(A) polymerase (Invitrogen). The poly(A)-tailed RNA was denatured with hydroxymethylmercuric hydroxide as described (23Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar), and cDNA was synthesized using oligonucleotide RE156 (5′-GACTCGAGTCGAGGATCCTTTTTTTTTTTTTTTTT-3′) and SuperScript II RNase H– reverse transcriptase (Invitrogen). After cDNA synthesis, RNA was digested with RNase A, and the unincorporated primer was eliminated using a Sephadex G-50 mini spin column (Worthington). cDNA containing the 3′-ends of 20 S RNA positive strands was PCR-amplified for 30 cycles using Taq polymerase (Promega) and primers RE157 (5′-GACTCGAGTCGAGGATCC-3′) and PG6 (5′-CGAATCGTCGCCAGTAG-3′). For amplification of the negative strand 3′-ends, we used oligonucleotide RE157 and oligonucleotide RE233 (5′-GCGTCGAAAGACGACAGC-3′). The amplified products from positive strand 3′-ends were digested with HindIII and BamHI and ligated into the pBluescript KS+ vector. In the case of negative strand 3′-ends, PCR products were digested with SalI and BamHI and ligated into the pBluescript KS+ vector. 20 S and 23 S RNA Viruses Are Independent Replicons—23 S RNA virus can be generated in vivo from an expression plasmid containing the entire viral cDNA sequence. We wished to establish a similar launching system for 20 S RNA virus to carry out reverse genetics. However, most laboratory strains harbor 20 S RNA virus. During the course of 23 S RNA virus-launching experiments, we happened to obtain cells that had lost endogenous 20 S RNA virus. Strain 2928 L-A-o was transformed with the standard 23 S RNA-launching plasmid pRE637 (16Esteban R. Fujimura T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2568-2573Crossref PubMed Scopus (36) Google Scholar). When transformants were analyzed, all of them generated 23 S RNA virus from the plasmid, and one of them (transformant 1) had apparently lost its endogenous 20 S RNA virus (Fig. 1A). We eliminated the launching plasmid from transformant 1 by isolating single colonies on nonselective YPAD agar plates. When the plasmid-cured colonies were analyzed, we found two types of segregants; the majority of colonies (10 of 12) contained only 23 S RNA virus generated from the plasmid, and the rest harbored neither 20 S nor 23 S RNA virus. Both types of segregants retained the genetic markers of the original strain. The segregants carrying 23 S RNA virus maintained the virus stably and did not produce virus-free colonies anymore. This suggests that the loss of endogenous 20 S RNA virus preceded the generation of 23 S RNA virus from the launching plasmid. We confirmed the curing of 20 S RNA virus in these segregants by three criteria. First, we did not detect 20 S RNA by Northern blotting (Fig. 1B). Even when the gels were overloaded, we found no signal for 20 S RNA. The conditions used should have allowed us to detect 20 S RNA if the virus were present at as low as one copy/cell. Second, we could not amplify 20 S RNA cDNA from the lysates of these cells by RT-PCR (data not shown). Finally, as will be shown below, we could generate 20 S RNA virus tagged with a silent mutation from a launching plasmid using these 20 S RNA-negative segregants as hosts. We confirmed that the virus retained the mutation and was not the result of amplification of any residual endogenous 20 S RNA virus. Generated 20 S RNA virus could be maintained stably and was also induced normally under nitrogen starvation conditions in these cells. Therefore, the 20 S RNA-negative segregants have no genetic defects to harbor 20 S RNA virus. We designated segregants 4 and 5 shown in Fig. 1B as 2928-4 and 2928-5, respectively. These segregants showed the same doubling time as the original strain, and we noticed no phenotypic changes in them. Strain 2928-5 carries only 23 S RNA virus derived from plasmid pRE637, but no 20 S RNA virus. Strain 2928-4 harbors none of the narnaviruses and can be used successfully as a host to generate 23 S RNA virus from the 23 S RNA-launching plasmid (17Fujimura T. Esteban R. J. Biol. Chem. 2004; 279: 13215-13223Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Although it has been reported that some industrial yeast strains (Saccharomyces diastaticus) appear to harbor only 23 S RNA-like molecules (24López V. Gil R. Carbonell J.V. Navarro A. Yeast. 2002; 19: 545-552Crossref PubMed Scopus (12) Google Scholar), these results are the first conclusive evidence that 23 S RNA virus does not require 20 S RNA virus for replication. Therefore, 20 S and 23 S RNA viruses are independent replicons. Launching of 20 S RNA Virus from Expression Plasmids—We constructed a launching plasmid (pRE740) to generate 20 S RNA virus in yeast by inserting the entire 20 S RNA cDNA sequence (2514 bp) downstream of the constitutive PGK1 promoter (Fig. 2A). Positive strands of 20 S RNA can be transcribed from the promoter. To generate transcripts in vivo with the precise viral 3′-end sequence at the 3′ termini, an 82-nucleotide HDV antigenomic ribozyme cDNA sequence (25Perrota A.T. Been M.D. Nature. 1991; 350: 434-436Crossref PubMed Scopus (309) Google Scholar) was directly fused to the 3′-end of the 20 S RNA sequence. The plasmid has the TRP1 gene as a selectable marker. We transformed strains 2928-4 and 2928-5 with pRE740. Transformants were transferred to 1% potassium acetate to induce 20 S RNA, and the RNA was extracted from the induced cells. By Northern hybridization, we detected single-stranded 20 S RNA positive strands in all of the transformants. The amounts of positive strands among them were similar (Fig. 2B, lanes 1–4). We also found small amounts of W, the double-stranded form of 20 S RNA, in all transformants. Furthermore, we detected single-stranded 20 S RNA negative strands with a negative strand-specific probe (data not shown). These results indicate that 20 S RNA negative strands are synthesized from the positive strand transcripts in vivo and suggest the generation of 20 S RNA virus from the plasmid. To confirm this, we took two experimental approaches. First, we cured the plasmid from the transformants by isolating single colonies in nonselective YPAD agar plates. More than 70% of the plasmid-cured colonies retained 20 S RNA virus. Once generated, the virus replicated autonomously and could be maintained stably in the cells for >100 generations (so far examined) in the absence of the plasmid. The generated virus could be induced under nitrogen starvation conditions, and the viral RNA could be seen directly by ethidium bromide staining on agarose gels. The amount of induced RNA and the positive/negative strand ratios were similar to those found in endogenous 20 S RNA virus. We did not notice any effect of 23 S RNA virus on the efficiency of 20 S RNA virus generation (Fig. 2B, compare lanes 1 and 2 with lanes 3 and 4). Second, we introduced a unique SmaI site into the 20 S RNA cDNA sequence in pRE740 by changing the T at position 1476 to G (numbered from the 20 S RNA 5′-end) (Fig. 2C). This marking did not alter the amino acid sequence of the encoded p91 protein. We transformed strains 2928-4 and 2928-5 with the SmaI-tagged plasmid. Transformants from both strains generated 20 S RNA virus. After curing the plasmid, RNA was extracted from both strains, and an 842-bp 20 S RNA cDNA fragment encompassing the SmaI site was amplified by RT-PCR. We also amplified the 842-bp fragment from endogenous 20 S RNA virus present in the original 2928 L-A-o strain as a control. As shown in Fig. 2C (lower panel), the amplified cDNA fragment from generated 20 S RNA virus in strain 2928-4 (lane 1) or 2928-5 (lane 2) was completely digested with SmaI, whereas the control from endogenous 20 S RNA virus was fully resistant to the enzyme (lane C). Therefore, these results demonstrate that 20 S RNA virus was generated from the launching plasmid and also confirm that the host strains 2928-4 and 2928-5 do not carry any endogenous 20 S RNA virus. p91 Is Essential—20 S RNA encodes a single protein, p91. p91 has the four amino acid motifs well conserved among RNA-dependent RNA polymerases. When one of the motifs (460GDD462) was changed to 460EFD462 in the launching plasmid, the modified vector failed to produce 20 S RNA virus (Fig. 2B, lane 5), indicating that active p91 is essential for replication. The presence of 23 S RNA virus in the host could not rescue the modified plasmid to generate 20 S RNA virus (data not shown). This indicates that p104 encoded by 23 S RNA cannot substitute p91 for replication of 20 S RNA virus and is consistent with the fact that 20 S and 23 S RNA viruses are independent replicons. p91 and p104 share an eight-amino acid sequence (R(V/I)CGDDLI) surrounding the GDD motif with only one mismatch. When the mismatched amino acid (Val458) in p91 was replaced with Ile, thus making the stretch identical to the one found in p104, the plasmid with the modified p91 sequence produced 20 S RNA virus without any deleterious effects (Fig. 2B, lane 6). Previously, we found that 23 S RNA virus can also tolerate the reverse modification (Ile to Val) in the eight-amino acid sequence in p104 (16Esteban R. Fujimura T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2568-2573Crossref PubMed Scopus (36) Google Scholar). A Cluster of Four Cs at the 3′-End of the Positive Strand Is Essential for Replication—The transcription termination site for the FLP gene of the 2μ plasmid is located 0.7 kb downstream of the 20 S RNA genome in the vector (26Hartley J.L. Donelson J.E. Nature. 1980; 286: 860-865Crossref PubMed Scopus (242) Google Scholar). When the ribozyme GGG sequence 3′ to the cleavage site was substituted with AAA in the standard launching plasmid pRE740, the modified plasmid failed to generate 20 S RNA virus (data not shown). Therefore, it is important to generate transcripts in vivo with the precise viral 3′ termini for virus launching. 20 S RNA has five-nucleotide inverted repeats at both termini (5′-GGGGC... GCCCCOH). We examined the role of the 3′-terminal nucleotides of the 20 S RNA positive strand in replication by modifying each nucleotide with A in the vector. As shown in Fig. 3, changing the 3′-terminal or penultimate C did not affect the generation of 20 S RNA virus. On the contrary, a modification at the third or fourth C did not produce the virus. Therefore, the third and fourth Cs from the 3′-end are essential for replication. In good agreement, deletions of up to two Cs (but not three) generated 20 S RNA virus (Fig. 3) (data not shown). We were interested in the 3′-terminal sequences of 20 S RNA viruses generated from these modified vectors. We cured the launching plasmids from the cells, and the viral RNAs were isolated. The 3′-terminal sequences of these RNAs were then amplified by 3′-RACE, and 8–10 independently isolated clones were sequenced. As shown in TABLE ONE, the substitution of the terminal (pRE757) or penultimate (pRE758) C with A at the viral 3′-end was corrected to the wild-type C in the generated viruses. Furthermore, viruses launched from 20 S RNA cDNA lacking two Cs at the 3′-end (pRE759) recovered these nucleotides. Therefore, these results indicate that 20 S RNA virus, like 23 S RNA virus, has an efficient 3′-end repair mechanism(s) in vivo. The major transcription start site in the launching plasmid is located at position –42, relative to the 5′ terminus of the 20 S RNA genome. These extra nucleotides were not present in the generated viruses as judged from the 3′-sequences of the negative strands (TABLE ONE). We also added extra nucleotides between the 3′-end of 20 S RNA and the ribozyme sequence and examined their effects on virus launching. As shown in Fig. 3, the addition of a single U (lane 6), C(lane 7), or A (data not shown) did not affect the generation of 20 S RNA virus. However, the addition of a single G reduced virus launching (lane 8). Likewise, the addition of three Gs (lane 10), but not three Cs (lane 9), severely affected 20 S RNA virus generation. Extra Gs at the 3′-end reduced the efficiency of virus launching by severalfold as judged by counting virus-positive and virus-negative colonies after curing the plasmids. Once the plasmids were cured, however, the amount of 20 S RNA in the cells was similar to that generated from the wild-type cDNA without extra nucleotides at the 3′-end. We isolated viral RNA from plasmid-cured cells and analyzed the 3′-ends of the positive strands by 3′-RACE. As shown in TABLE ONE, the launched viruses examined eliminated the extra nucleotides present at the 3′-ends in the vectors. Therefore, these results clearly indicate that 20 S RNA virus has a mechanism(s) to correctly maintain the size and sequence of the 3′-end of the positive strand and is thus capable of repairing limited damage of the 3′-end.TABLE ONE3′-RACE: 3′-ends of viral RNA generated from positive strand mutantsLaunching plasmidaModifications of or extra nucleotides added at the 3′-end of the 20 S RNA positive strand in the launching vectors are underlined.ClonesPositive strandbExtra sequences found in the viral 3′-ends cloned are indicated by parentheses.ClonesNegative strandbExtra sequences found in the viral 3′-ends cloned are indicated by parentheses.pRE757 (...GCCCA-OH)87...GCCCC-OH33...GCCCC-OH1...GCC-OHpRE758 (...GCCAC-OH)87...GCCCC-OH32...GCCCC-OH1...GCCCC(G)-OH1...GCCCC(G)-OHpRE759 (...GCC_-OH)87...GCCCC-OH21...GCCCC-OH1...GCCCC(G)-OH1...GCCCC(G)-OHpRE794 (...GCCCCU-OH)97...GCCCC-OHNDcNot determined.1...GCCCC(G)-OH1...GCCCC(U)-OHpRE800 (...GCCCCG-OH)107...GCCCC-OHND1...GCCCC(C)-OH2...GCC-OHpRE795 (...GCCCCCCC-OH)107...GCCCC-OHND3...GCCC-OHpRE793 (...GCCCCGGG-OH)108...GCCCC-OHND1...GCCCC(G)-OH1...GCC-OHa Modifications of or extra nucleotides added at the 3′-end of the 20 S RNA positive strand in the launching vectors are underlined.b Extra sequences found in the viral 3′-ends cloned are indicated by parentheses.c Not determined. Open table in a new tab The Fifth G from the 3′-End in the Positive Strand Is Dispensable for Replication—When the fifth G from the 3′-end was changed to C (5G→ C, numbered from the 3′-end) in the vector, the modified plasmid failed to generate 20 S RNA virus (Fig. 4, lane 1), thus suggesting that this nucleotide is essential for replication. Alternatively, because the fifth G is part of a structure adjacent to the 3′-end, the inability of this plasmid to generate the virus may be a secondary effect due to a perturbation of the stem structure caused by the modification. Because the corresponding nucleotide in the 23 S RNA genome 3′-end is dispensable for its replication (17Fujimura T. Esteban R. J. Biol. Chem. 2004; 279: 13215-13223Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), we were interested in this resul" @default.
W2069061056 created "2016-06-24" @default.
W2069061056 creator A5020879855 @default.
W2069061056 creator A5051003882 @default.
W2069061056 creator A5068143450 @default.
W2069061056 date "2005-10-01" @default.
W2069061056 modified "2023-10-02" @default.
W2069061056 title "Launching of the Yeast 20 S RNA Narnavirus by Expressing the Genomic or Antigenomic Viral RNA in Vivo" @default.
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