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Istituto di Virologia Vegetale del CNR, Sezione di Bari, c/o Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi, Bari, Italy
Correspondence
Luisa Rubino
l.rubino{at}ba.ivv.cnr.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary figures showing immunofluorescent analysis of UTL-7A yeast cells transformed with plasmids expressing CymRSV proteins are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The basic similarity in the replication strategy of positive-strand RNA viruses has encouraged the use of any simple virus of this group as a model to analyse the factors involved in replication. Members of the plant virus genus Tombusvirus (family Tombusviridae) have proved to be good models for studying virus–host interactions (Russo et al., 1994
; White & Nagy, 2004). Tombusviruses contain a positive-sense, single-stranded RNA genome of 
4·8 kb with five open reading frames (ORFs). The genomic RNA acts as mRNA for translation of a protein of 33–36 kDa from ORF1 and a readthrough product of 92–95 kDa (ORF2). These two proteins constitute the viral replicase and are the only viral products essential for replication. The 41 kDa protein (ORF3) is translated from a subgenomic RNA. A second subgenomic RNA is synthesized for translation of two nested ORFs (4 and 5) encoding proteins of 22 and 19 kDa (p19), required for cell-to-cell movement and systemic invasion, respectively. In addition, p19 has been shown to act as a suppressor of RNA silencing (Voinnet et al., 1999). Infections caused by tombusviruses are normally associated with small defective interfering (DI) RNAs that are deletion mutants of the viral genome. DI RNAs do not encode any proteins and depend for replication on the viral replicase supplied in trans by the viral genome. However, as DI RNA molecules contain all cis-acting elements required for replication, they represent a useful model for studying tombusvirus replication (Russo et al., 1994; White & Nagy, 2004).
Cymbidium ringspot virus (CymRSV) and Carnation Italian ringspot virus (CIRV) are two well-characterized tombusviruses. Their differential trait resides in the targeting signal present in the 33 kDa (p33; CymRSV) or 36 kDa (p36; CIRV) protein, which addresses the replication complex to the peroxisomal membrane (CymRSV) or to the outer mitochondrial membrane (CIRV) (Burgyan et al., 1996). Both targeting signals are characterized by two transmembrane domains and accompanying short sequences responsible for the specificity of the target (Rubino & Russo, 1998; Weber-Lotfi et al., 2002; Navarro et al., 2004). Targeted organelles are transformed in conspicuous structures (multivesicular bodies) where the limiting membrane is stimulated to abnormal growth, accounting for the formation of flask-shaped vesicles open to the cytoplasm in which the viral RNA replication takes place (Russo et al., 1983, 1987; Di Franco et al., 1984). Interestingly, by exchanging only the genome segments containing the targeting signals, the site of the replication complex becomes peroxisomal for CIRV and mitochondrial for CymRSV (Burgyan et al., 1996; Rubino & Russo, 1998).
Replication of tombusviruses can be studied in Saccharomyces cerevisiae cells by using DI RNA molecules as the replication template and replicase proteins derived from CIRV (Pantaleo et al., 2003) or Cucumber necrosis virus (CNV; Panavas & Nagy, 2003). Analysis of yeast cells in which the replication of DI RNA is supported by CIRV replicase proteins has shown that the membrane-associated complex, in which the viral replicase proteins accumulate and progeny DI RNA is synthesized, derives from mitochondria (Pantaleo et al., 2003, 2004). Following previous analysis of the expression of CymRSV p33 in S. cerevisiae (Navarro et al., 2004), the present study has identified and characterized the replication complex in yeast cells where DI RNA is synthesized following expression of CymRSV p33 and p92 replicase proteins. The way in which the replicase proteins may be sorted to the replication site, whether directly from the cytosol or indirectly via the endoplasmic reticulum (ER), is also discussed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Navarro et al., 2004) to obtain clone pEL26-33K.
The p92-coding sequence was amplified by PCR from the same full-length infectious clone or from a clone in which the stop codon of p33 was mutated to a tyrosine codon and inserted between the alcohol dehydrogenase gene (ADH1) promoter and the terminator of vector YE, a 2 µm plasmid containing LEU2 as a selectable marker (Pantaleo et al., 2003). This process yielded clones YE92Kwt and YE92Ktyr.
Plasmids pEL26-Myc33K and YEHA92K contained the same CymRSV p33 and p92 proteins, tagged at their 5' termini with Myc (EQKLISEEDL; Evan et al., 1985) and haemagglutinin (HA) (MYPYDVPDYAG; Kolodziej & Young, 1991), respectively. Plasmids pEL26-Myc92Kwt and pEL26-Myc92Ktyr contained the wild-type or tyrosine Myc-tagged version of p92 cloned in the vector pEL26.
The CymRSV DI RNA sequence (DI-3, 481 nt; Burgyan et al., 1992) was used as replication template. It was cloned between the galactose-inducible (GAL1) promoter and the ADH1 terminator into the low-copy-number centromeric plasmid vector pBMI3S containing TRP1 as a selectable marker (Ishikawa et al., 1997; Pantaleo et al., 2003). As a negative control of replication, a mutated version of DI-3 was used, in which the G residue at position –4 from the 3' end was mutated to A (DI RNA
G) (Pantaleo et al., 2003).
Yeast strains and growth conditions.
The yeast strains used were UTL-7A (MATa leu2-3,112 ura3-52 trp1) (Erdmann et al., 1989) and UTL-7Apex19 (same as UTL-7A but pex19 : : LEU2 (Götte et al., 1998). Yeasts were transformed by using the lithium acetate/polyethylene glycol method (Ito et al., 1983), plated on selective medium (SM) containing 0·67 % yeast nitrogen base supplemented with appropriate amino acids and 2 % glucose, and incubated at 30 °C for 2–3 days. UTL-7A and UTL-7Apex19 transformants were initially grown at 30 °C in an oleate-based medium (SYO) as described previously (Navarro et al., 2004), then at 26 °C in SYO containing 1 % galactose to induce DI RNA transcription and finally in the same medium without galactose.
Immunofluorescence.
Rabbit polyclonal antisera to yeast peroxisomal Fox3p, ER Kar2p and Golgi Emp47p protein markers were kindly provided by B. Distel (University of Amsterdam, The Netherlands), M. Rose (Princeton University, USA) and H. D. Schmitt (Max-Planck-Institut für biophysikalische Chemie, Germany), respectively. Mouse monoclonal antibody (mAb) against the mitochondrial marker CoxIII was from Molecular Probes. mAbs against the Myc epitope, and mAbs and rabbit polyclonal antibodies against the HA epitope and the mitochondrial outer-membrane marker Tom40p were from Santa Cruz Biotechnology. Rhodamine-conjugated goat anti-rabbit and Alexa 488-conjugated anti-mouse secondary antibodies were from Molecular Probes. Fixation of yeast cells and immunofluorescent staining were done according to Redding et al. (1991).
Detection of nascent and progeny RNA.
Semi-intact cells were prepared according to Schlenstedt et al. (1993). Labelling of newly synthesized viral RNA by BrUTP incorporation and in situ hybridization were done as described by Restrepo-Hartwig & Ahlquist (1999). Cells were then processed for immunofluorescence as above. Antibodies against BrUTP and digoxigenin (DIG) were from Sigma and Roche, respectively.
Protein and RNA analysis.
Proteins were extracted and examined by Western blotting as described previously (Pantaleo et al., 2003; Navarro et al., 2004). Total RNA was extracted by using acid phenol (Leeds et al., 1991). One microgram of RNA was denatured with formamide and formaldehyde, run on a formaldehyde-permeated agarose gel, transferred to a nylon membrane and hybridized with DIG-labelled probes consisting of the last 300 nt of DI-3 in positive and negative orientations.
Microscopy.
Fluorescent cells were viewed and photographed by using an Axioplan 2 imaging fluorescence microscope (Zeiss). Image acquisition was done with an ApoTome imaging system, allowing the generation of optical sections through fluorescent samples. Images were taken with a CCD camera (AxioCam) and the information was visualized with the software package AxioVision (Zeiss). For electron microscopy, cells were fixed with 2 % glutaraldehyde in 0·1 M cacodylate buffer (pH 7·2), washed extensively with the same buffer, treated briefly with lyticase and post-fixed with 4 % potassium permanganate. Cells were then bulk stained with uranyl acetate, dehydrated with ethanol and embedded in Spurr's resin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Using this plasmid, expression and activity of p33 could then be investigated under growth conditions favourable to biogenesis of peroxisomes (Elgersma et al., 1996). The replicase protein p92 was expressed with the plasmid YE92K under the control of the constitutive promoter ADH1. Cells were transformed with plasmids expressing the replicase proteins and DI-3 RNA (clone pBDI-3) in different combinations.
Transcription of DI-3 RNA was induced first by growing yeasts in SYO containing galactose. An aliquot of this culture was then subinoculated into SYO without galactose and the rest was pelleted and frozen. After 24 h further growth, RNA was extracted from all cultures. Northern blot analysis of RNA extracts showed the presence of positive-strand DI-3 RNA only in samples in which both p33 and p92 replicase proteins were expressed (Fig. 1a, lanes 8 and 16). No RNA band was detected if one or both replicase proteins were absent (Fig. 1a, lanes 2–7 and 10–15) or if the putative replication template was DI-3 RNAG with the G
A mutation at position –4 from the 3' end (Fig. 1a, lanes 1 and 9). Above the DI-3 RNA monomer, a minor band corresponding to the size of the DI-3 dimer was detected (Fig. 1a, lane 16).
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G or replicable DI RNA were analysed for the presence of negative-strand DI RNA. Two bands corresponding to DI-3 RNA monomers and dimers were detected only for the clone expressing p33, p92 and DI RNA (Fig. 1b
, lanes 1 and 2).
To test for the possibility of expressing both p33 and p92 proteins from a single clone, the wild-type p92 sequence containing the p33 amber stop codon was cloned into plasmid YE (YE92Kwt) and transformed into yeast cells together with plasmid pBDI-3. DI-3 RNA replicated in these cells, indicating that the stop codon of p33 was read through, as in infected plants, producing a functional p92 (Fig. 1c, lane 1). However, the level of replication was lower than that in cells in which p33 and p92 were supplied from separate plasmids (Fig. 1c, lane 3). These results were in agreement with those of Panavas & Nagy (2003), who showed that a readthrough mechanism exists in yeast, but contrasted with those reported for CIRV replicase proteins in strain YPH499 (Pantaleo et al., 2003). Therefore, the experiments with CIRV were repeated to test the possibility that DI RNA replication could also be sustained by the CIRV replicase proteins p36 and p95 expressed by a plasmid carrying the wild-type p36/p95 gene alone. It was found that the readthrough product p95 was formed correctly along with p36, although below the limit of detection in Western blots, as it supported the replication of DI RNA (M. Russo & L. Rubino, unpublished results).
p33 and p92 co-localize to peroxisomes
Expression and localization of p33 in yeast strain UTL-7A, grown in oleate-containing medium, were extensively investigated in a previous work, where it was found that p33 accumulated in one or two large bodies consisting of aggregates of peroxisomes mixed with membranes and mitochondria (Navarro et al., 2004). These observations were repeated and extended here to compare the cytological effect of p33 with that of the sole protein p92 or of both proteins together, in the presence or absence of DI RNA. Peroxisomes, mitochondria, ER and Golgi were identified by using the immunological markers Fox3p, CoxIIIp, Kar2p and Emp47p, respectively. The distribution of markers of mitochondria, ER and Golgi in cells of all transformants was indistinguishable from that of control cells (see Supplementary Fig. S1a, available in JGV Online) and is shown only for the transformant competent for DI RNA replication (see Supplementary Fig. S1b, available in JGV Online). Mitochondria were visualized as a branched tubular network near the cortex of the cell. The ER appeared mainly as the perinuclear membrane with some extensions into the cytoplasm up to the periphery of the cell. Finally, the Golgi apparatus had a punctate cytoplasmic pattern. As for peroxisomes, the cytology of transformants showed essentially two different patterns. Cells expressing p33 alone (Fig. 2c, e) or together with p92 (Fig. 2a, b, h) showed structures corresponding to peroxisomes consisting of one or two prominent bodies occupying a large part of the cell, whereas the appearance and distribution of these organelles in cells expressing p92 only (Fig. 2d, f) had the typical punctate pattern made up of a few small scattered bodies, as in cells expressing DI RNA only (Fig. 2g) or in control cells transformed with empty vectors (see Supplementary Fig. S1a). Western blot analysis showed that the expression level of p92 was somewhat lower than that of p33 (Fig. 1e); however, we were not able to say whether this was sufficient to explain the different structural changes in peroxisomes in yeast expressing p33 or p92. As, in these experiments, p33 and p92 were expressed by two different plasmids with different promoters (CTA1 and ADH1, respectively), p92 was also expressed by using plasmid pEL26. In addition, p33- and p92-coding sequences were tagged with the epitope Myc at the N terminus to analyse their subcellular distribution. Yeast cells were transformed with these clones separately and analysed by immunofluorescence using anti-Myc in conjunction with each organelle marker as primary antibodies. It was confirmed that CymRSV p33 localized largely to peroxisomal aggregates (Fig. 3a, upper row), in line with previous findings using this protein fused to the green fluorescent protein (Navarro et al., 2004). Expression of p92 resulted in punctate structures, most of which coincided with scattered peroxisomes (Fig. 3a, lower row). These results indicated that p92 is targeted to peroxisomes as p33, but, although entirely containing the p33 sequence, does not alter the distribution of these organelles.
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, lane 1) and Northern blot analysis demonstrated that they were fully competent for the replication of DI-3 RNA (Fig. 1d). Concurrent with biochemical analysis, yeasts were fixed and processed for double-labelled immunofluorescence using anti-Myc and anti-HA antibodies. Signals corresponding to Myc-tagged p33 and HA-tagged p92 largely coincided (Fig. 3b, upper row). These observations strongly indicated the co-localization of replicase proteins p33 and p92. To investigate whether co-localization of p33 and p92 was dependent on DI RNA replication, cells transformed with plasmids expressing the replicase proteins and plasmid pBMI3S, i.e. without the DI RNA sequence, were analysed. The distribution of p33 and p92 was independent of transcription and replication of DI RNA (Fig. 3b, lower row). The site of co-localization of p33 and p92 appeared morphologically similar to the aggregates of peroxisomes described above in cells expressing p33 (Fig. 2a–c, e and h; Fig. 3a, upper row). To verify these results, samples were double-labelled with antibodies to either Myc or HA and Fox3p. As shown in Fig. 3(c), the localization of both p33 (upper row) and p92 (lower row) correlated with the peroxisomal marker.
Nascent RNA and progeny RNA do not co-localize
To investigate the site of DI RNA replication, spheroplasts were prepared from cells expressing the replicase proteins and DI RNA grown in the absence of galactose, in which the presence of DI RNA was dependent only on replication and not on DNA-driven transcription. Spheroplasts were incubated with BrUTP for 10 min and then fixed and double-labelled with antibodies to BrUTP and Fox3p. Incorporated BrUTP was detected in cytoplasmic sites, which largely coincided with peroxisome aggregates also labelled with anti-Fox3p antibodies (Fig. 4a, upper row). Nascent viral RNA was never found associated with the ER or mitochondria (see Supplementary Fig. S2a, available in JGV Online). No incorporation of BrUTP was detected in yeast cells expressing the replicase proteins and the non-replicable form of DI RNA (Fig. 4a, middle row) or in cells expressing DI RNA, but not the replicase proteins (Fig. 4a, lower row). To visualize the distribution of DI-3 RNA replication progeny, in situ hybridization was performed with DIG-labelled oligonucleotides as described previously (Restrepo-Hartwig & Ahlquist, 1999; Pantaleo et al., 2004). As shown in Fig. 4(b, upper row), DI RNA progeny accumulated at the site of peroxisomal aggregates and was diffused throughout the cytoplasm, but not in the ER or mitochondria (see Supplementary Fig. S2b, available in JGV Online). No DI RNA-specific signal was found in control cells (Fig. 4b, middle and lower rows). In conclusion, these data suggest that replication of DI RNA occurs at the site of peroxisome aggregations and that progeny RNA is then released into the surrounding cytoplasm.
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). By contrast, sections of cells also expressing the replicase proteins contained a much higher number of peroxisomes (20–30) of different sizes, which were tightly appressed to the point of no longer displaying a defined morphology (Fig. 5b). Several such peroxisomes were partially encircled by membranous elements resembling ER strands, but differing because of the higher electron density (Fig. 5c). These electron-dense membranes were also occasionally apposed to mitochondria, but only when these organelles were intermingled with peroxisome clusters. A few cells also displayed stacks of membranes often massively surrounding lipid droplets (Fig. 5d). Despite extensive analysis, in no cases were vesiculated structures observed resembling the multivesicular bodies typical of tombusvirus infections in plant cells (Russo et al., 1987). It was concluded that the major cytological features, i.e. peroxisomal aggregates and membrane proliferation, were due to the expression of the replicase proteins, probably p33 alone, as suggested by the light-microscope observations reported above.
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40 kDa required for peroxisome biogenesis (Götte et al., 1998). It interacts with the majority of peroxisomal membrane proteins (PMPs), escorting them from the cytosol to the peroxisomal membrane (Jones et al., 2004). The S. cerevisiae mutant UTL-7Apex19 (in which PEX19 is deleted; Götte et al., 1998) is unable to synthesize Pex19p and lacks peroxisomes, so that both peroxisomal matrix and membrane proteins are mislocalized in the cytosol or, occasionally, in other organelles (Hettema et al., 2000; Sacksteder et al., 2000; Rottensteiner et al., 2003). The competence of UTL-7Apex19 cells to support CymRSV replicase-mediated DI RNA replication was tested. As cells of this mutant cannot be selected for leucine auxotrophy, the plasmid YE92Ktyr (carrying the LEU2 selectable marker) could not be used. Therefore, cells were co-transformed with plasmid pBDI-3 and plasmid pEL26-Myc92Kwt, which contained the wild-type p92 sequence including the amber stop codon of p33 and the Myc tag fused in frame at the N terminus. As a control, UTL-7A cells were transformed with the same plasmids. Northern blot analysis showed that replication of DI-3 RNA also took place in UTL-7Apex19 cells, although at a level at least five times lower than that of the control wild-type UTL-7A cells (Fig. 6a). Immunofluorescence analysis showed that p33 (and presumably the readthrough product, p92) accumulated in elongated structures identified as ER strands as a result of their reactivity with the anti-Kar2p antibody (Fig. 6b, upper row). No co-localization with mitochondria was observed (Fig. 6b, lower row). These results were in line with a previous analysis where it was shown that a CymRSV p33–GFP fusion protein expressed in strain UTL-7Apex19 cells mislocalized to the ER instead of to the peroxisomal membrane (Navarro et al., 2004). Given the low level of DI RNA replication, no attempt was made to establish the site of replication by BrUTP incorporation. However, this is likely to coincide with the accumulation of the replicase proteins, i.e. ER strands in this case.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, which is not, as it is unable to synthesize Pex19p, a farnesylated protein required for peroxisome biogenesis. Evidence that DI RNA replicated in UTL-7A cells was given by the presence of dimeric molecules in addition to unit-length monomers of positive and negative polarities, and by BrUTP incorporation into nascent RNA progeny only when both replicase proteins p33 and p92 were expressed. Expression of p33 and p92 could be achieved from two separate plasmids (provided that the p33 stop codon was mutated to a tyrosine codon) or from only one plasmid encoding the entire p92-coding sequence, including the p33 termination codon. Since in S. cerevisiae, as in higher eukaryotes, several anti-codon mutations of tRNA genes produce nonsense suppressors capable of reading stop codons as sense codons (Beier & Grimm, 2001), the complete set of CymRSV replicase proteins was synthesized, leading to DI RNA replication. Visual inspection of RNA blots indicated that the DI RNA progeny derived from the expression of CymRSV replicase from a single plasmid accumulated less than in transformants in which both p33 and p92 were overexpressed from separate plasmids. As suggested by Panavas & Nagy (2003), suppression of the amber stop codon may not be as efficient as the readthrough mechanism operating in plant cells infected with tombusviruses. Fluorescence microscopy was used to analyse the cytological modifications occurring in transformants expressing either p33 or p92, or both, co-expressed with replicable or non-replicable DI RNA. Aggregates of peroxisomes constituted the major cytological alteration of UTL-7A cells expressing p33, regardless of whether this protein was expressed alone or with p92 and DI RNA template. There was a clear co-localization of p33 and the peroxisomal marker. By contrast, expression of p92 only did not perturb the size and distribution of peroxisomes, which remained the only site of localization of this protein.
When expressed together, p33 and p92 always co-localized on peroxisomal aggregates in the presence or absence of DI RNA, thus indicating that template RNA is not required for accumulation of p33 and p92 on peroxisomal membranes. However, co-expression of CymRSV replicase proteins and template RNA may be required for the formation of an active replication complex, as shown for the assembly of the CNV replication complex, which was stimulated by co-expression of template RNA and replicase proteins (Panaviene et al., 2004).
BrUTP incorporation into the nascent DI RNA strand occurred at the site of accumulation of p33 and p92 in UTL-7A cells, providing evidence of the involvement of peroxisomes in the replication of DI RNA sustained by the CymRSV replicase. A similar association of CNV replicase proteins p33 and p92 with peroxisomal membranes was shown to take place in yeast cells together with template RNA (Panavas et al., 2005a). DI RNA progeny do not accumulate at the sites of replication, instead being diffused throughout the cytoplasm, similar to the accumulation of RNA progeny in yeast cells expressing CIRV (Pantaleo et al., 2004) and Brome mosaic virus (BMV) (Restrepo-Hartwig & Ahlquist, 1999) replicase proteins. Time-course experiments with CNV replicase-directed DI RNA replication in yeast have confirmed that the majority of RNA progeny does not accumulate at the site of synthesis and is distributed throughout the cytoplasm (Panavas et al., 2005a). Incidentally, it is also worth noting that this indicates that the organelles involved in tombusvirus replication depend only on the replicase rather than on the RNA replication template. In fact, the same DI RNA (DI-3 RNA) derived from CymRSV genome is replicated at the level of either mitochondrial aggregates in the case of CIRV (Pantaleo et al., 2004) or peroxisomes in the case of CymRSV (this paper).
Electron-microscopy observations of yeast cells in which there was active DI RNA replication did not show cytological alterations comparable to the typical multivesicular bodies of CymRSV-infected plant cells. Similar negative results were obtained with BMV (Schwartz et al., 2002) and Flock house virus (FHV) (Miller et al., 2003). In particular, the typical vesiculated mitochondria found in FHV-infected insect cells (Miller et al., 2001) were absent in the corresponding yeast cells, where very few vesicles were found on severely damaged and hardly recognizable mitochondria. For BMV, better visualization of vesicle accumulation in the perinuclear space was obtained when nuclei were extracted, thus permitting the observation of many such organelles (Schwartz et al., 2002). In plant cells infected with CymRSV and other tombusviruses (Russo et al., 1983), the peroxisomal membrane undergoes profound modifications consisting of dilations that close on themselves, thus yielding enclaves where the bulk of vesicles accumulate. The scarcity of membranous structures related to virus replication in yeasts, as compared with the plant host, may simply be due to the short life (24–48 h) of individual yeast cells, which may not permit the production and accumulation of replication-related vesicles. Alternatively, the space between closely appressed peroxisomes might constitute the protected environment for CymRSV DI RNA replication.
As peroxisomes were shown to be the sites of DI RNA replication directed by CNV (Panavas et al., 2005a) and CymRSV (this paper) replicase proteins, the low but consistent DI RNA replication in strain UTL-7Apex19 is difficult to reconcile with the absence of peroxisomes (Götte et al., 1998). However, it was shown that a mutant strain of the yeast Pichia pastoris, unable to express Pex19p, contained tiny vesicular and tubular structures, possibly derived from the ER, identified as precursors of peroxisomes (Snyder et al., 1999). If also present in S. cerevisiae strain UTL-7Apex19, these structures may be candidates for DI RNA replication sites when biogenesis of peroxisomes is blocked at a very early stage because of the absence of Pex19p.
It is an open question where CymRSV p33 and p92 proteins are synthesized, whether on free or membrane-bound ribosomes, and how they are targeted to the peroxisomal membrane, whether directly or indirectly. Most PMPs are synthesized in the cytosol and are targeted directly to peroxisomes; others are synthesized on ER-bound polysomes and localized in the ER before being targeted to peroxisomes (Baerends et al., 2000; Titorenko & Rachubinski, 2001). Direct insertion of PMPs is mediated by the cytosolic chaperone Pex19p, itself an integral PMP (Hettema et al., 2000; Jones et al., 2004), which recognizes and binds specific signals in the PMPs (Rottensteiner et al., 2004). Another peroxisomal protein (Pex3p) is required for correct sorting of this class of PMP (Hettema et al., 2000), acting as a docking factor in the peroxisomal membrane (Fang et al., 2004). Other PMPs are targeted indirectly to the peroxisomal membrane, i.e. they are first sorted to the ER and then to peroxisomes. For instance, overexpression of Pex3p (Baerends et al., 1996; Kammerer et al., 1998) and Pex15p (Elgersma et al., 1997) results in profound proliferation of ER membrane, which is taken as an indication that these proteins are sorted first to the ER and then to peroxisomes. A similar route is suggested for Pex2p and Pex16p (Titorenko & Rachubinski, 1998). It was shown previously that CymRSV p33 and Pex19p failed to interact in a yeast two-hybrid assay system (Navarro et al., 2004). Furthermore, definite targeting of p33 to the ER and DI RNA replication were shown to take place in the mutant UTL-7Apex19, which does not synthesize Pex19p and lacks peroxisomes (Navarro et al., 2004; this paper). These results may be taken as an indication that the p33 protein does not follow the direct sorting to peroxisomes via the chaperone activity of Pex19p, but is targeted first to the ER. A similar route was suggested for CNV p33 (Panavas et al., 2005a). Interestingly, PEX19 is not among the 96 identified yeast genes whose absence affects the replication of tomato bushy stunt virus DI RNA (Panavas et al., 2005b). However, by using an algorithm developed for yeast PMPs (Rottensteiner et al., 2004), three putative binding sites of p33 for Pex19p have been predicted (S. Lorenzen & H. Rottensteiner, personal communication). Clearly, further experimental data are required to establish the route of p33 to peroxisomes.
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ACKNOWLEDGEMENTS |
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Received 8 July 2005;
accepted 11 November 2005.
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