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. 2003 Feb;77(3):2116-23.
doi: 10.1128/jvi.77.3.2116-2123.2003.

Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae

Vitantonio Pantaleo  1 , Luisa Rubino, Marcello Russo
Affiliations

Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae

Vitantonio Pantaleo et al. J Virol. 2003 Feb.

Abstract

Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3' end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3' end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5' terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of CIRV genomic and DI RNAs and plasmids used in this work. (A) The ORF1 sequence of the CIRV genome (middle drawing) was cloned in plasmid pA36K, and the ORF2 sequence, containing a tyrosine codon in place of the ORF1 amber codon was cloned in plasmid YE95K. The plasmids contain the ADH1 promoter and terminator, the 2μm origin of replication, and the selectable markers HIS3 and LEU2, respectively. NCR, noncoding regions. (B) Diagrammatic representation of plasmid pBDI-7, containing the complete DI-7 sequence cloned under the control of GAL1 promoter and ADH1 terminator. I, II, and III indicate the three discontinuous sequence blocks derived from the viral genome. Plasmid pBDI-7 has the ARS1CEN4 origin of replication and TRP1 as a selectable marker. The 5′ terminus of DI RNA (underlined) was fused to the GAL1 promoter sequence, whose major transcription initiation sites are indicated by an arrow. The 3′ terminus (underlined) was fused to the TRSVs ribozyme, which leaves 19 nonviral nucleotides after self-cleavage at the position indicated by an arrowhead. (C) Dimeric head-to-tail DI-7 RNA was cloned in plasmid pBDI-7dim, which has the features of pBDI-7. The junction sequence is indicated.
FIG. 2.
FIG. 2.
Analysis of the expression of p36, p95, and DI-7 RNA in yeast. (A) Western blot analysis of protein extracts from yeasts transformed with plasmid pA36K (lane 1), plasmid YE95K (lane 2), or both (lane 3). The blot was probed with anti-36K antiserum, which recognizes both p36 and p95. (B) Northern blot analysis of RNA extracts from yeasts transformed with plasmid pBDI-7 (lane 1) or untransformed (lane 2) and from Nicotiana benthamiana-infected tissues (lane 3), showing the positions of genomic and subgenomic RNAs (G, sg1, and sg2, respectively) and monomeric and dimeric DI-7 RNA. The blot was probed with a 32P-labeled riboprobe recognizing the 3′ noncoding region of the CIRV genome.
FIG. 3.
FIG. 3.
Fluorescence light microscope visualization of mitochondria in yeast cells grown in SM containing 3% glycerol-2% galactose (upper panels) or 3% glycerol-0.1% dextrose (lower panels). Cells were immunolabeled by using antibodies to the inner (YHM2) or outer (TOM40) mitochondrial membrane proteins, which were detected with rhodamine-labeled secondary antibodies.
FIG. 4.
FIG. 4.
Northern blot analysis of extracts from yeasts transformed with plasmid pBDI-7 and expressing or not expressing p36 or p95 grown in SM containing 3% glycerol-0.1% dextrose (second preculture and GAL1 repression) or containing 3% glycerol-2% galactose (GAL1 induction) at 26°C (A and C) or 30°C (B). The blots in panels A and B were probed with a DI-7 RNA-specific probe; the blots in panel C were probed with a probe specific to TRSVs ribozyme sequence.
FIG. 5.
FIG. 5.
Northern blot analysis of RNA extracts from 36K+95K+ (lanes 2, 4, and 6) and 36K+95K− (lanes 1, 3, and 5) cells transformed with pBDI-7 (lanes 1 and 2), pBDI-7dim (lanes 3 and 4), and pBI-7ΔG (lanes 5 and 6), showing synthesis of monomeric DI-7 RNA from the dimeric template (lane 4), which is indistinguishable from the progeny of the monomeric template (lane 2), and absence of progeny from the mutant pBI-7ΔG (lane 6).
FIG. 6.
FIG. 6.
Detection of negative-strand DI-7 RNA in 36K+95K+ cells grown in GAL1 repression medium. (A) Ethidium bromide-stained gel of PCR products obtained by amplifying cDNA to positive-strand (lane 3) or negative-strand (lane 4) DI-7 RNA from 36K+95K+ cells. Lanes 1 and 2, control samples with no cDNA or no primers added, respectively. The marker lane contains DNA Molecular Weight Marker VI (Roche). (B) Two-cycle RNase protection assay of RNA extracts from 36K−95K− (lane 3) and 36K+95K+ (lane 4) cells. Lanes 1 and 2, results of two-cycle RNase protection assay of positive in vitro transcripts only (lane 1) or of a mixture of positive and negative in vitro transcripts (lane 2). Hybridization was with a 32P-labeled probe of 0.26 kb which was electrophoresed as a size marker.
FIG. 7.
FIG. 7.
Localization of bromo-RNA in 36K+95K+ cells after BrUTP incorporation. Cells were immunolabeled with mouse BrUTP-specific antibodies (middle panels, green) or rabbit antibodies against the mitochondrial outer membrane protein TOM40 (upper panels, red). Nascent RNA is present only in 36K+95K+ cells. The merged images (lower panels) show that the distribution of BrUTP-labeled RNA coincides only partially with the mitochondrial pattern.
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