Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective...

Cytological analysis of Saccharomyces cerevisiaecells supporting cymbidium ringspot virus defectiveinterfering RNA replication

Beatriz Navarro, Marcello Russo, Vitantonio Pantaleo and Luisa Rubino

Correspondence

Luisa Rubino

[email protected]

Istituto di Virologia Vegetale del CNR, Sezione di Bari, c/o Dipartimento di Protezione dellePiante e Microbiologia Applicata, Universita degli Studi, Bari, Italy

Received 8 July 2005

Accepted 11 November 2005

The replicase proteins p33 and p92 ofCymbidium ringspot virus (CymRSV) were found to support

the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast

strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the

membranous vesicular environment in which CymRSV RNA replication takes place in infected plant

cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins

localized to peroxisomes, independently of one another and of the presence of the replication

template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic

reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the

number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in

yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative

polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis,

whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the

cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested

that replication in these cells was targeted to the endoplasmic reticulum.

INTRODUCTION

Replication of positive-strand RNA viruses takes place inassociation with host-cell membranes, but different virusesrecruit diverse intracellular membranes for the assemblyof their replication complexes. Study of the targeting signalsof viral proteins interacting with host-cell membranes, aswell as details of the formation, maintenance and func-tioning of the virus replication complex, is of importance forcomprehension of the virus life cycle and, therefore, forenvisaging a possible antiviral therapy. Positive-strand RNAviruses constitute a large group of viruses, including agentsof important plant, animal and human diseases.

The basic similarity in the replication strategy of positive-strand RNA viruses has encouraged the use of any simplevirus of this group as a model to analyse the factors involvedin replication. Members of the plant virus genusTombusvirus(family Tombusviridae) have proved to be good modelsfor 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 fiveopen reading frames (ORFs). The genomic RNA acts asmRNA for translation of a protein of 33–36 kDa from ORF1

and a readthrough product of 92–95 kDa (ORF2). Thesetwo proteins constitute the viral replicase and are the onlyviral products essential for replication. The 41 kDa protein(ORF3) is translated from a subgenomic RNA. A secondsubgenomic RNA is synthesized for translation of two nestedORFs (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 asuppressor of RNA silencing (Voinnet et al., 1999). Infectionscaused by tombusviruses are normally associated with smalldefective interfering (DI) RNAs that are deletion mutants ofthe viral genome. DI RNAs do not encode any proteins anddepend for replication on the viral replicase supplied in transby the viral genome. However, as DI RNA molecules containall cis-acting elements required for replication, they repre-sent a useful model for studying tombusvirus replication(Russo et al., 1994; White & Nagy, 2004).

Cymbidium ringspot virus (CymRSV) and Carnation Italianringspot virus (CIRV) are two well-characterized tombus-viruses. Their differential trait resides in the targeting signalpresent in the 33 kDa (p33; CymRSV) or 36 kDa (p36;CIRV) protein, which addresses the replication complexto the peroxisomal membrane (CymRSV) or to the outermitochondrial membrane (CIRV) (Burgyan et al., 1996).Both targeting signals are characterized by two transmem-brane domains and accompanying short sequences respon-sible for the specificity of the target (Rubino & Russo, 1998;

Supplementary figures showing immunofluorescent analysis of UTL-7Ayeast cells transformed with plasmids expressing CymRSV proteins areavailable in JGV Online.

0008-1325 G 2006 SGM Printed in Great Britain 705

Journal of General Virology (2006), 87, 705–714 DOI 10.1099/vir.0.81325-0

Weber-Lotfi et al., 2002; Navarro et al., 2004). Targeted orga-nelles are transformed in conspicuous structures (multi-vesicular bodies) where the limiting membrane is stimulatedto abnormal growth, accounting for the formation of flask-shaped vesicles open to the cytoplasm in which the viralRNA replication takes place (Russo et al., 1983, 1987; DiFranco et al., 1984). Interestingly, by exchanging onlythe genome segments containing the targeting signals, thesite of the replication complex becomes peroxisomal forCIRV and mitochondrial for CymRSV (Burgyan et al., 1996;Rubino & Russo, 1998).

Replication of tombusviruses can be studied in Saccharomycescerevisiae cells by using DI RNA molecules as the replicationtemplate and replicase proteins derived from CIRV (Pantaleoet al., 2003) or Cucumber necrosis virus (CNV; Panavas &Nagy, 2003). Analysis of yeast cells in which the replication ofDI RNA is supported by CIRV replicase proteins has shownthat the membrane-associated complex, in which the viralreplicase proteins accumulate and progeny DI RNA is synthe-sized, derives from mitochondria (Pantaleo et al., 2003, 2004).Following previous analysis of the expression of CymRSVp33 in S. cerevisiae (Navarro et al., 2004), the present studyhas identified and characterized the replication complex inyeast cells where DI RNA is synthesized following expres-sion of CymRSV p33 and p92 replicase proteins. The way inwhich the replicase proteins may be sorted to the replicationsite, whether directly from the cytosol or indirectly via theendoplasmic reticulum (ER), is also discussed.

METHODS

Plasmids. To express the CymRSV p33 protein, the coding sequencewas amplified by PCR from a CymRSV full-length infectious cloneand cloned between the oleate-inducible catalase A gene (CTA1)promoter and the terminator of vector pEL26, a 2 mm plasmidcontaining URA3 as a marker (Elgersma et al., 1996; Navarro et al.,2004) to obtain clone pEL26-33K.

The p92-coding sequence was amplified by PCR from the samefull-length infectious clone or from a clone in which the stop codonof p33 was mutated to a tyrosine codon and inserted between thealcohol dehydrogenase gene (ADH1) promoter and the terminator ofvector YE, a 2 mm plasmid containing LEU2 as a selectable marker(Pantaleo et al., 2003). This process yielded clones YE92Kwt andYE92Ktyr.

Plasmids pEL26-Myc33K and YEHA92K contained the same CymRSVp33 and p92 proteins, tagged at their 59 termini with Myc (EQKLISEEDL;Evan et al., 1985) and haemagglutinin (HA) (MYPYDVPDYAG;Kolodziej & Young, 1991), respectively. Plasmids pEL26-Myc92Kwtand pEL26-Myc92Ktyr contained the wild-type or tyrosine Myc-taggedversion 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 asa selectable marker (Ishikawa et al., 1997; Pantaleo et al., 2003). As anegative control of replication, a mutated version of DI-3 was used, inwhich the G residue at position 24 from the 39 end was mutated to A(DI RNADG) (Pantaleo et al., 2003).

Yeast strains and growth conditions. The yeast strains usedwere UTL-7A (MATa leu2-3,112 ura3-52 trp1) (Erdmann et al., 1989)and UTL-7Apex19D (same as UTL-7A but pex19D : : LEU2 (Gotteet al., 1998). Yeasts were transformed by using the lithium acetate/polyethylene glycol method (Ito et al., 1983), plated on selectivemedium (SM) containing 0?67 % yeast nitrogen base supplementedwith appropriate amino acids and 2 % glucose, and incubated at30 uC for 2–3 days. UTL-7A and UTL-7Apex19D transformants wereinitially grown at 30 uC in an oleate-based medium (SYO) as de-scribed previously (Navarro et al., 2004), then at 26 uC in SYO con-taining 1 % galactose to induce DI RNA transcription and finally inthe same medium without galactose.

Immunofluorescence. Rabbit polyclonal antisera to yeast peroxi-somal Fox3p, ER Kar2p and Golgi Emp47p protein markerswere kindly provided by B. Distel (University of Amsterdam, TheNetherlands), M. Rose (Princeton University, USA) and H. D. Schmitt(Max-Planck-Institut fur biophysikalische Chemie, Germany), respec-tively. Mouse monoclonal antibody (mAb) against the mitochondrialmarker CoxIII was from Molecular Probes. mAbs against the Mycepitope, and mAbs and rabbit polyclonal antibodies against the HAepitope and the mitochondrial outer-membrane marker Tom40pwere from Santa Cruz Biotechnology. Rhodamine-conjugated goatanti-rabbit and Alexa 488-conjugated anti-mouse secondary antibodieswere from Molecular Probes. Fixation of yeast cells and immuno-fluorescent staining were done according to Redding et al. (1991).

Detection of nascent and progeny RNA. Semi-intact cells wereprepared according to Schlenstedt et al. (1993). Labelling of newlysynthesized viral RNA by BrUTP incorporation and in situ hybridi-zation 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 Sigmaand Roche, respectively.

Protein and RNA analysis. Proteins were extracted and examinedby 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 withformamide and formaldehyde, run on a formaldehyde-permeatedagarose gel, transferred to a nylon membrane and hybridized withDIG-labelled probes consisting of the last 300 nt of DI-3 in positiveand negative orientations.

Microscopy. Fluorescent cells were viewed and photographed byusing an Axioplan 2 imaging fluorescence microscope (Zeiss). Imageacquisition was done with an ApoTome imaging system, allowingthe generation of optical sections through fluorescent samples.Images were taken with a CCD camera (AxioCam) and the informa-tion was visualized with the software package AxioVision (Zeiss).For electron microscopy, cells were fixed with 2 % glutaraldehyde in0?1 M cacodylate buffer (pH 7?2), washed extensively with the samebuffer, treated briefly with lyticase and post-fixed with 4 % potas-sium permanganate. Cells were then bulk stained with uranyl acet-ate, dehydrated with ethanol and embedded in Spurr’s resin.

RESULTS

Replication of CymRSV DI RNA in yeast strainUTL-7A

Replicase protein p33 was expressed in yeast strain UTL-7Awith the plasmid pEL26, under the control of the oleate-inducible CTA1 promoter (pEL26-33K; Navarro et al., 2004).Using this plasmid, expression and activity of p33 couldthen be investigated under growth conditions favourable to

706 Journal of General Virology 87

B. Navarro and others

biogenesis of peroxisomes (Elgersma et al., 1996). The repli-case protein p92 was expressed with the plasmid YE92K underthe control of the constitutive promoter ADH1. Cells weretransformed with plasmids expressing the replicase proteinsand DI-3 RNA (clone pBDI-3) in different combinations.

Transcription of DI-3 RNA was induced first by growingyeasts in SYO containing galactose. An aliquot of this culturewas then subinoculated into SYO without galactose and therest was pelleted and frozen. After 24 h further growth, RNAwas extracted from all cultures. Northern blot analysis ofRNA extracts showed the presence of positive-strand DI-3RNA only in samples in which both p33 and p92 replicaseproteins were expressed (Fig. 1a, lanes 8 and 16). No RNAband was detected if one or both replicase proteins wereabsent (Fig. 1a, lanes 2–7 and 10–15) or if the putative repli-cation template was DI-3 RNADG with the GRA muta-tion at position 24 from the 39 end (Fig. 1a, lanes 1 and9). Above the DI-3 RNA monomer, a minor band corres-ponding to the size of the DI-3 dimer was detected (Fig. 1a,lane 16).

Clones containing both replicase proteins and non-replicableDI RNADG or replicable DI RNA were analysed for the pres-ence of negative-strand DI RNA. Two bands correspondingto DI-3 RNA monomers and dimers were detected only forthe clone expressing p33, p92 and DI RNA (Fig. 1b, lanes 1and 2).

To test for the possibility of expressing both p33 and p92proteins from a single clone, the wild-type p92 sequencecontaining the p33 amber stop codon was cloned intoplasmid YE (YE92Kwt) and transformed into yeast cellstogether with plasmid pBDI-3. DI-3 RNA replicated in thesecells, 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 thanthat in cells in which p33 and p92 were supplied from sepa-rate plasmids (Fig. 1c, lane 3). These results were in agree-ment with those of Panavas & Nagy (2003), who showed thata readthrough mechanism exists in yeast, but contrasted withthose reported for CIRV replicase proteins in strain YPH499(Pantaleo et al., 2003). Therefore, the experiments with CIRV

Fig. 1. Replication of DI-3 RNA in yeaststrain UTL-7A expressing the CymRSV repli-case proteins p33 and p92. (a, b) Northernblot analysis of RNA extracts from cells trans-formed with plasmids expressing replicable(+) or non-replicable (+*) DI RNA or emptyvector (”) and expressing (+) or not (”) p33and p92 replicase proteins. Cells were grownin the presence (a, lanes 1–8; b, lanes 1 and3) or absence (a, lanes 9–16; b, lanes 2 and4) of galactose. Blots in (a) and (b) wereprobed to detect positive- and negative-strand DI RNA sequences, respectively. (c)Northern blot analysis of positive-strand RNAextracted from yeast cells expressing replic-able DI RNA and wild-type p92 (p92wt; lane1), the p92 mutant with the tyrosine codonsubstituted for the p33 stop codon (p92tyr;lane 2) or both p33 and p92tyr (lane 3). (d)Northern blot analysis of RNA extracts fromyeast cells expressing (+) (lane 1) or notexpressing (”) (lane 2) Myc-tagged p33 andHA-tagged p92 together with DI RNA. Blotsin (c) and (d) were probed to detect positive-strand DI RNA sequences. The positions ofdimeric (dim) and monomeric (mon) DI RNAmolecules in (a–d) are indicated. (e, f)Western blot analysis of extracts from cellsexpressing (+) or not (”) p33 and p92 native(e) or epitope-tagged (f) proteins. (e) wasprobed with an anti-p33 antiserum and (f)with mixed antibodies to the Myc and HAepitopes. The positions of p33 and p92 areindicated. Molecular size markers (kDa) areshown on the left.

http://vir.sgmjournals.org 707

CymRSV DI RNA replication in yeasts

were repeated to test the possibility that DI RNA replicationcould also be sustained by the CIRV replicase proteins p36and p95 expressed by a plasmid carrying the wild-type p36/p95 gene alone. It was found that the readthrough productp95 was formed correctly along with p36, although belowthe limit of detection in Western blots, as it supported thereplication of DI RNA (M. Russo & L. Rubino, unpublishedresults).

p33 and p92 co-localize to peroxisomes

Expression and localization of p33 in yeast strain UTL-7A,grown in oleate-containing medium, were extensivelyinvestigated in a previous work, where it was found thatp33 accumulated in one or two large bodies consisting ofaggregates of peroxisomes mixed with membranes andmitochondria (Navarro et al., 2004). These observationswere repeated and extended here to compare the cytologicaleffect of p33 with that of the sole protein p92 or of bothproteins together, in the presence or absence of DI RNA.Peroxisomes, mitochondria, ER and Golgi were identifiedby using the immunological markers Fox3p, CoxIIIp, Kar2pand Emp47p, respectively. The distribution of markers ofmitochondria, ER and Golgi in cells of all transformants wasindistinguishable from that of control cells (see Supple-mentary Fig. S1a, available in JGV Online) and is shownonly for the transformant competent for DI RNA replication(see Supplementary Fig. S1b, available in JGV Online).Mitochondria were visualized as a branched tubular networknear the cortex of the cell. The ER appeared mainly as theperinuclear membrane with some extensions into the cyto-plasm up to the periphery of the cell. Finally, the Golgiapparatus had a punctate cytoplasmic pattern. As for per-oxisomes, the cytology of transformants showed essentiallytwo different patterns. Cells expressing p33 alone (Fig. 2c, e)or together with p92 (Fig. 2a, b, h) showed structurescorresponding to peroxisomes consisting of one or twoprominent bodies occupying a large part of the cell, whereasthe appearance and distribution of these organelles in cellsexpressing p92 only (Fig. 2d, f) had the typical punctate

pattern made up of a few small scattered bodies, as in cellsexpressing DI RNA only (Fig. 2g) or in control cells trans-formed with empty vectors (see Supplementary Fig. S1a).Western blot analysis showed that the expression level of p92was somewhat lower than that of p33 (Fig. 1e); however, wewere not able to say whether this was sufficient to explainthe different structural changes in peroxisomes in yeastexpressing p33 or p92. As, in these experiments, p33 and p92were expressed by two different plasmids with differentpromoters (CTA1 and ADH1, respectively), p92 was alsoexpressed by using plasmid pEL26. In addition, p33- andp92-coding sequences were tagged with the epitope Myc atthe N terminus to analyse their subcellular distribution.Yeast cells were transformed with these clones separately andanalysed by immunofluorescence using anti-Myc in con-junction with each organelle marker as primary antibodies.It was confirmed that CymRSV p33 localized largely toperoxisomal aggregates (Fig. 3a, upper row), in line withprevious findings using this protein fused to the green fluor-escent protein (Navarro et al., 2004). Expression of p92resulted in punctate structures, most of which coincidedwith scattered peroxisomes (Fig. 3a, lower row). Theseresults indicated that p92 is targeted to peroxisomes as p33,but, although entirely containing the p33 sequence, does notalter the distribution of these organelles.

To analyse the expression and distribution of CymRSVreplicase proteins when expressed together, the Myc and HAtags were fused in frame to the N terminus of p33 and p92and expressed with plasmids pEL26 and YE, respectively.Yeast transformants were prepared that expressed theseproteins together with DI RNA. Western blot analysisshowed that the tagged proteins could be detected by usingthe respective anti-Myc and anti-HA mAbs (Fig. 1f, lane 1)and Northern blot analysis demonstrated that they werefully competent for the replication of DI-3 RNA (Fig. 1d).Concurrent with biochemical analysis, yeasts were fixed andprocessed for double-labelled immunofluorescence usinganti-Myc and anti-HA antibodies. Signals correspondingto Myc-tagged p33 and HA-tagged p92 largely coincided

Fig. 2. Expression of p33 modifies the size and distribution of peroxisomes. One or two large bodies co-localized with theperoxisomal marker Fox3p in transformants expressing p33 alone (c and e) or together with p92 (a, b and h). Cells notexpressing p33 (d, f and g) displayed punctate structures identical to cells transformed with empty vectors (seeSupplementary Fig. S1a, available in JGV Online). The distribution of mitochondrial (CoxIIIp), ER (Kar2p) and Golgi (Emp47p)protein markers was unaffected in all transformants and is shown only for the transformant expressing p33, p92 and DI RNA(see Supplementary Fig. S1b, available in JGV Online). Bars, 1 mm.



(Fig. 3b, upper row). These observations strongly indicatedthe co-localization of replicase proteins p33 and p92. Toinvestigate whether co-localization of p33 and p92 was

dependent on DI RNA replication, cells transformed withplasmids expressing the replicase proteins and plasmidpBMI3S, i.e. without the DI RNA sequence, were analysed.The distribution of p33 and p92 was independent of tran-scription and replication of DI RNA (Fig. 3b, lower row).The site of co-localization of p33 and p92 appearedmorphologically similar to the aggregates of peroxisomesdescribed above in cells expressing p33 (Fig. 2a–c, e and h;Fig. 3a, upper row). To verify these results, samples weredouble-labelled with antibodies to either Myc or HA andFox3p. As shown in Fig. 3(c), the localization of both p33(upper row) and p92 (lower row) correlated with theperoxisomal marker.

Nascent RNA and progeny RNA do notco-localize

To investigate the site of DI RNA replication, spheroplastswere prepared from cells expressing the replicase proteinsand DI RNA grown in the absence of galactose, in whichthe presence of DI RNA was dependent only on replicationand not on DNA-driven transcription. Spheroplasts wereincubated with BrUTP for 10 min and then fixed anddouble-labelled with antibodies to BrUTP and Fox3p.Incorporated BrUTP was detected in cytoplasmic sites,which largely coincided with peroxisome aggregates alsolabelled with anti-Fox3p antibodies (Fig. 4a, upper row).Nascent viral RNA was never found associated with the ERor mitochondria (see Supplementary Fig. S2a, available inJGV Online). No incorporation of BrUTP was detected inyeast cells expressing the replicase proteins and the non-replicable form of DI RNA (Fig. 4a, middle row) or in cellsexpressing DI RNA, but not the replicase proteins (Fig. 4a,lower row). To visualize the distribution of DI-3 RNAreplication progeny, in situ hybridization was performedwith DIG-labelled oligonucleotides as described previously(Restrepo-Hartwig & Ahlquist, 1999; Pantaleo et al., 2004).As shown in Fig. 4(b, upper row), DI RNA progenyaccumulated at the site of peroxisomal aggregates and wasdiffused throughout the cytoplasm, but not in the ER ormitochondria (see Supplementary Fig. S2b, available in JGVOnline). No DI RNA-specific signal was found in controlcells (Fig. 4b, middle and lower rows). In conclusion, thesedata suggest that replication of DI RNA occurs at the site ofperoxisome aggregations and that progeny RNA is thenreleased into the surrounding cytoplasm.

Electron microscopy fails to identify featuresassociated with DI RNA replication

Thin-sectioned yeast transformants were examined withthe aim of identifying fine-structural details of DI RNAreplication sites. Cells expressing only DI RNA showed thepresence of five to ten round to ovoid peroxisomal profilesintermingled with mitochondria (Fig. 5a). By contrast, sec-tions of cells also expressing the replicase proteins containeda much higher number of peroxisomes (20–30) of differ-ent sizes, which were tightly appressed to the point of nolonger displaying a defined morphology (Fig. 5b). Several

Fig. 3. p33 and p92 localize to peroxisomes. (a) Upper row:p33 localizes to and modifies the structure of peroxisomes;lower row: p92 localizes to peroxisomes without altering theirsize and distribution. Both proteins were tagged with the Mycepitope. Cells were immunolabelled by using anti-Myc and anti-Fox3p antibodies. (b) p33 and p92 co-localize in the presence(upper row) or absence (lower row) of DI RNA. Cells wereimmunolabelled with anti-Myc and anti-HA. (c) p33 and p92co-localize to peroxisomes. Cells were immunolabelled by usinganti-Myc and anti-Fox3p (upper row) or anti-HA and anti-Fox3p(lower row) antibodies, respectively. Images of each label andtheir superimposition are shown. Bars, 1 mm.



Fig. 4. Distribution of DI RNA progeny in UTL-7A cells. Newly synthesized DI RNA localized predominantly in association withperoxisomes (a, upper row), whereas DI RNA progeny accumulated in the cytoplasm (b, upper row). Cells were double-labelled by using anti-BrUTP (a) or anti-DIG (b) and anti-Fox3p antibodies. No significant BrUTP signal was detected incontrol cells expressing non-replicable DI RNA (middle rows) or replicable DI RNA in the absence of replicase proteins (lowerrows). Images of each label and their superimposition are shown. Bars, 1 mm.

Fig. 5. Expression of p33 in UTL-7A cellssignificantly increases the size and numberof peroxisomes. (a) A cell transformed withempty vectors. (b–d) Cells expressing p33together with p92 and DI RNA transcriptsdisplaying clusters of peroxisomes (b, c),many partially encircled by electron-densetubular structures (c, arrows) and accumula-tions of membranes (d) free in the cytoplasmor encircling lipid droplets. P, Peroxisomes;M, mitochondria; N, nucleus; L, lipid drop-lets. Bar, 0?25 mm.



such peroxisomes were partially encircled by membranouselements resembling ER strands, but differing because ofthe higher electron density (Fig. 5c). These electron-densemembranes were also occasionally apposed to mitochon-dria, but only when these organelles were intermingled withperoxisome clusters. A few cells also displayed stacks ofmembranes often massively surrounding lipid droplets(Fig. 5d). Despite extensive analysis, in no cases werevesiculated structures observed resembling the multivesi-cular bodies typical of tombusvirus infections in plant cells(Russo et al., 1987). It was concluded that the major cyto-logical features, i.e. peroxisomal aggregates and membraneproliferation, were due to the expression of the replicaseproteins, probably p33 alone, as suggested by the light-microscope observations reported above.

DI-3 RNA replication in the absence ofperoxisomes

Pex19p is an oleic-inducible, farnesylated protein of~40 kDarequired for peroxisome biogenesis (Gotte et al., 1998).It interacts with the majority of peroxisomal membraneproteins (PMPs), escorting them from the cytosol to theperoxisomal membrane (Jones et al., 2004). The S. cerevisiaemutant UTL-7Apex19D (in which PEX19 is deleted; Gotteet al., 1998) is unable to synthesize Pex19p and lacksperoxisomes, so that both peroxisomal matrix and mem-brane proteins are mislocalized in the cytosol or, occasion-ally, in other organelles (Hettema et al., 2000; Sackstederet al., 2000; Rottensteiner et al., 2003). The competenceof UTL-7Apex19D cells to support CymRSV replicase-mediated DI RNA replication was tested. As cells of thismutant cannot be selected for leucine auxotrophy, theplasmid YE92Ktyr (carrying the LEU2 selectable marker)could not be used. Therefore, cells were co-transformed withplasmid pBDI-3 and plasmid pEL26-Myc92Kwt, whichcontained the wild-type p92 sequence including the amberstop codon of p33 and the Myc tag fused in frame at the Nterminus. As a control, UTL-7A cells were transformed withthe same plasmids. Northern blot analysis showed thatreplication of DI-3 RNA also took place in UTL-7Apex19Dcells, although at a level at least five times lower than thatof the control wild-type UTL-7A cells (Fig. 6a). Immuno-fluorescence analysis showed that p33 (and presumably thereadthrough product, p92) accumulated in elongated struc-tures identified as ER strands as a result of their reactivitywith the anti-Kar2p antibody (Fig. 6b, upper row). No co-localization with mitochondria was observed (Fig. 6b, lowerrow). These results were in line with a previous analysiswhere it was shown that a CymRSV p33–GFP fusion proteinexpressed in strain UTL-7Apex19D cells mislocalized to theER instead of to the peroxisomal membrane (Navarro et al.,2004). Given the low level of DI RNA replication, no attemptwas made to establish the site of replication by BrUTPincorporation. However, this is likely to coincide with theaccumulation of the replicase proteins, i.e. ER strands inthis case.

DISCUSSION

In the present work, we employed a yeast system to studythe replication of CymRSV-derived DI RNA template sup-ported by CymRSV replicase proteins. Two yeast strainswere used: UTL-7A, which is particularly favourable for theanalysis of peroxisome biogenesis, and UTL-7Apex19D,which is not, as it is unable to synthesize Pex19p, a farnesyl-ated protein required for peroxisome biogenesis. Evidencethat DI RNA replicated in UTL-7A cells was given by thepresence of dimeric molecules in addition to unit-lengthmonomers of positive and negative polarities, and by BrUTPincorporation into nascent RNA progeny only when bothreplicase proteins p33 and p92 were expressed. Expression ofp33 and p92 could be achieved from two separate plasmids(provided that the p33 stop codon was mutated to a tyrosinecodon) 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 suppres-sors capable of reading stop codons as sense codons (Beier &Grimm, 2001), the complete set of CymRSV replicaseproteins was synthesized, leading to DI RNA replication.Visual inspection of RNA blots indicated that the DI RNAprogeny derived from the expression of CymRSV replicasefrom a single plasmid accumulated less than in transfor-mants in which both p33 and p92 were overexpressed fromseparate plasmids. As suggested by Panavas & Nagy (2003),suppression of the amber stop codon may not be as effi-cient as the readthrough mechanism operating in plant cellsinfected with tombusviruses.

Fluorescence microscopy was used to analyse the cytologicalmodifications occurring in transformants expressing either

Fig. 6. DI RNA replication in UTL-7Apex19D cells. (a) Northernblot analysis of RNA extracted from UTL-7Apex19D (lane 1)and UTL-7A (lane 2) cells expressing DI RNA and p92wt.Lanes 1 and 2 were loaded with 5 and 1 mg, respectively. Thepositions of dimeric (dim) and monomeric (mon) DI RNA mole-cules are indicated. (b) UTL-7Apex19D cells expressing DIRNA and p92wt were immunolabelled by using anti-Myc andanti-Kar2p antibodies (upper row) and anti-Myc and anti-Tom40p (lower row) antibodies. Images of each label and theirsuperimposition are shown. Bars, 1 mm.



p33 or p92, or both, co-expressed with replicable or non-replicable DI RNA. Aggregates of peroxisomes constitutedthe major cytological alteration of UTL-7A cells expressingp33, regardless of whether this protein was expressed aloneor with p92 and DI RNA template. There was a clear co-localization of p33 and the peroxisomal marker. By con-trast, expression of p92 only did not perturb the size anddistribution of peroxisomes, which remained the only site oflocalization of this protein.

When expressed together, p33 and p92 always co-localizedon peroxisomal aggregates in the presence or absence ofDI RNA, thus indicating that template RNA is not requiredfor accumulation of p33 and p92 on peroxisomal mem-branes. However, co-expression of CymRSV replicase pro-teins and template RNA may be required for the formationof an active replication complex, as shown for the assemblyof the CNV replication complex, which was stimulatedby co-expression of template RNA and replicase proteins(Panaviene et al., 2004).

BrUTP incorporation into the nascent DI RNA strandoccurred at the site of accumulation of p33 and p92 inUTL-7A cells, providing evidence of the involvement ofperoxisomes in the replication of DI RNA sustained by theCymRSV replicase. A similar association of CNV replicaseproteins p33 and p92 with peroxisomal membranes wasshown to take place in yeast cells together with templateRNA (Panavas et al., 2005a). DI RNA progeny do notaccumulate at the sites of replication, instead being diffusedthroughout the cytoplasm, similar to the accumulation ofRNA progeny in yeast cells expressing CIRV (Pantaleo et al.,2004) and Brome mosaic virus (BMV) (Restrepo-Hartwig &Ahlquist, 1999) replicase proteins. Time-course experimentswith CNV replicase-directed DI RNA replication in yeasthave confirmed that the majority of RNA progeny doesnot accumulate at the site of synthesis and is distributedthroughout the cytoplasm (Panavas et al., 2005a). Inciden-tally, it is also worth noting that this indicates that theorganelles involved in tombusvirus replication depend onlyon the replicase rather than on the RNA replicationtemplate. In fact, the same DI RNA (DI-3 RNA) derivedfrom CymRSV genome is replicated at the level of eithermitochondrial 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 whichthere was active DI RNA replication did not show cyto-logical alterations comparable to the typical multivesicularbodies of CymRSV-infected plant cells. Similar negativeresults were obtained with BMV (Schwartz et al., 2002) andFlock house virus (FHV) (Miller et al., 2003). In particular,the typical vesiculated mitochondria found in FHV-infectedinsect cells (Miller et al., 2001) were absent in the corres-ponding yeast cells, where very few vesicles were found onseverely damaged and hardly recognizable mitochondria.For BMV, better visualization of vesicle accumulation in theperinuclear space was obtained when nuclei were extracted,thus permitting the observation of many such organelles

(Schwartz et al., 2002). In plant cells infected with CymRSVand other tombusviruses (Russo et al., 1983), the peroxi-somal membrane undergoes profound modifications con-sisting of dilations that close on themselves, thus yieldingenclaves where the bulk of vesicles accumulate. The scarcityof membranous structures related to virus replication inyeasts, as compared with the plant host, may simply bedue to the short life (24–48 h) of individual yeast cells,which may not permit the production and accumulation ofreplication-related vesicles. Alternatively, the space betweenclosely appressed peroxisomes might constitute the pro-tected environment for CymRSV DI RNA replication.

As peroxisomes were shown to be the sites of DI RNA repli-cation directed by CNV (Panavas et al., 2005a) and CymRSV(this paper) replicase proteins, the low but consistent DIRNA replication in strain UTL-7Apex19D is difficult toreconcile with the absence of peroxisomes (Gotte et al.,1998). However, it was shown that a mutant strain of theyeast Pichia pastoris, unable to express Pex19p, containedtiny vesicular and tubular structures, possibly derived fromthe ER, identified as precursors of peroxisomes (Snyder et al.,1999). If also present in S. cerevisiae strain UTL-7Apex19D,these structures may be candidates for DI RNA replicationsites when biogenesis of peroxisomes is blocked at a veryearly stage because of the absence of Pex19p.

It is an open question where CymRSV p33 and p92 pro-teins are synthesized, whether on free or membrane-boundribosomes, and how they are targeted to the peroxisomalmembrane, whether directly or indirectly. Most PMPs aresynthesized in the cytosol and are targeted directly to peroxi-somes; others are synthesized on ER-bound polysomes andlocalized in the ER before being targeted to peroxisomes(Baerends et al., 2000; Titorenko & Rachubinski, 2001).Direct insertion of PMPs is mediated by the cytosolicchaperone Pex19p, itself an integral PMP (Hettema et al.,2000; Jones et al., 2004), which recognizes and binds specificsignals in the PMPs (Rottensteiner et al., 2004). Anotherperoxisomal protein (Pex3p) is required for correct sortingof this class of PMP (Hettema et al., 2000), acting as adocking factor in the peroxisomal membrane (Fang et al.,2004). Other PMPs are targeted indirectly to the peroxi-somal membrane, i.e. they are first sorted to the ER and thento peroxisomes. For instance, overexpression of Pex3p(Baerends et al., 1996; Kammerer et al., 1998) and Pex15p(Elgersma et al., 1997) results in profound proliferation ofER membrane, which is taken as an indication that theseproteins are sorted first to the ER and then to peroxi-somes. A similar route is suggested for Pex2p and Pex16p(Titorenko & Rachubinski, 1998). It was shown previouslythat CymRSV p33 and Pex19p failed to interact in a yeasttwo-hybrid assay system (Navarro et al., 2004). Further-more, definite targeting of p33 to the ER and DI RNAreplication were shown to take place in the mutant UTL-7Apex19D, which does not synthesize Pex19p and lacksperoxisomes (Navarro et al., 2004; this paper). These resultsmay be taken as an indication that the p33 protein does not



follow the direct sorting to peroxisomes via the chaperoneactivity of Pex19p, but is targeted first to the ER. A similarroute was suggested for CNV p33 (Panavas et al., 2005a).Interestingly, PEX19 is not among the 96 identified yeastgenes whose absence affects the replication of tomato bushystunt virus DI RNA (Panavas et al., 2005b). However, byusing an algorithm developed for yeast PMPs (Rottensteineret al., 2004), three putative binding sites of p33 for Pex19phave been predicted (S. Lorenzen & H. Rottensteiner, per-sonal communication). Clearly, further experimental dataare required to establish the route of p33 to peroxisomes.

ACKNOWLEDGEMENTS

The authors wish to thank Professor G. P. Martelli for advice andcritical reading of the manuscript, Mrs A. Antonacci for valuable

technical help and the Istituto di Tecnologie Biomediche del CNR,Sezione di Bari, Italy, for use of the fluorescence microscope. Thisresearch was partially supported by MIUR, Project Cluster CO3, Legge488/92, ‘Studi di geni di interesse biomedico e agroalimentare’.

REFERENCES

Baerends, R. J. S., Rasmussen, S. W., Hilbrands, R. E. & 7 otherauthors (1996). The Hansenula polymorpha PER9 gene encodes aperoxisomal membrane protein essential for peroxisome assembly

and integrity. J Biol Chem 271, 8887–8894.

Baerends, R. J. S., Faber, K. N., Kiel, J. A. K. W., van der Klei, I. J.,Harder, W. & Veenhuis, M. (2000). Sorting and function ofperoxisomal membrane proteins. FEMS Microbiol Rev 24, 291–301.

Beier, H. & Grimm, M. (2001). Misreading of termination codons ineukaryotes by natural nonsense suppressor tRNAs. Nucleic Acids Res29, 4767–4782.

Burgyan, J., Dalmay, T., Rubino, L. & Russo, M. (1992). Thereplication of cymbidium ringspot tombusvirus defective interfering-

satellite RNA hybrid molecules. Virology 190, 579–586.

Burgyan, J., Rubino, L. & Russo, M. (1996). The 59-terminal region

of a tombusvirus genome determines the origin of multivesicularbodies. J Gen Virol 77, 1967–1974.

Di Franco, A., Russo, M. & Martelli, G. P. (1984). Ultrastructure andorigin of cytoplasmic multivesicular bodies induced by carnationItalian ringspot virus. J Gen Virol 65, 1233–1237.

Elgersma, Y., Vos, A., van der Berg, M., van Roermund, C. W. T.,van der Sluijs, P., Distel, B. & Tabak, H. F. (1996). Analysis of thecarboxyl-terminal peroxisomal targeting signal 1 in a homologous

context in Saccharomyces cerevisiae. J Biol Chem 271, 26375–26382.

Elgersma, Y., Kwast, L., van den Berg, M., Snyder, W. B.,Distel, B., Subramani, S. & Tabak, H. F. (1997). Overexpressionof Pex15p, a phosphorylated peroxisomal integral membrane pro-tein required for peroxisome assembly in S. cerevisiae, causes

proliferation of the endoplasmic reticulum membrane. EMBO J 16,7326–7341.

Erdmann, R., Veenhuis, D., Mertens, D. & Kunau, W.-H. (1989).Isolation of peroxisome-deficient mutants of Saccharomyces cerevi-siae. Proc Natl Acad Sci U S A 86, 5419–5423.

Evan, G. I., Lewis, G. K., Ramsay, G. & Bishop, J. M. (1985). Isolationof monoclonal antibodies specific for human c-myc proto-oncogeneproduct. Mol Cell Biol 5, 3610–3616.

Fang, Y., Morrell, J. C., Jones, J. M. & Gould, S. J. (2004). PEX3

functions as a PEX19 docking factor in the import of class I

peroxisomal membrane proteins. J Cell Biol 164, 863–875.

Gotte, K., Girzalsky, W., Linkert, M., Baumgart, E., Kammerer, S.,

Kunau, W.-H. & Erdmann, R. (1998). Pex19p, a farnesylated protein

essential for peroxisome biogenesis. Mol Cell Biol 18, 616–628.

Hettema, E. H., Girzalsky, W., van den Berg, M., Erdmann, R. &

Distel, B. (2000). Saccharomyces cerevisiae Pex3p and Pex19p are

required for proper localization and stability of peroxisomal mem-

brane proteins. EMBO J 19, 223–233.

Ishikawa, M., Janda, M., Krol, M. A. & Ahlquist, P. (1997). In vivo

DNA expression of functional brome mosaic virus RNA replicons in

Saccharomyces cerevisiae. J Virol 71, 7781–7790.

Ito, H., Fukuda, Y., Murata, K. & Kimura, A. (1983). Transformation of

intact yeast cells treated with alkali cations. J Bacteriol 153, 163–168.

Jones, J. M., Morrell, J. C. & Gould, S. J. (2004). PEX19 is a

predominantly cytosolic chaperone and import receptor for class 1

peroxisomal membrane proteins. J Cell Biol 164, 57–67.

Kammerer, S., Holzinger, A., Welsch, U. & Roscher, A. A. (1998).

Cloning and characterization of the gene encoding the human

peroxisomal assembly protein Pex3p. FEBS Lett 429, 53–60.

Kolodziej, P. A. & Young, R. A. (1991). Epitope tagging and protein

surveillance. Methods Enzymol 194, 508–519.

Leeds, P., Peltz, S. W., Jacobson, A. & Culbertson, M. R. (1991). The

product of the yeast UPF1 gene is required for rapid turnover of

mRNAs containing a premature translational termination codon.

Genes Dev 5, 2303–2314.

Miller, D. J., Schwartz, M. D. & Ahlquist, P. (2001). Flock house virus

RNA replicates on outer mitochondrial membranes in Drosophila

cells. J Virol 75, 11664–11676.

Miller, D. J., Schwartz, M. D., Dye, B. T. & Ahlquist, P. (2003).Engineered retargeting of viral RNA replication complexes to an

alternative intracellular membrane. J Virol 77, 12193–12202.

Navarro, B., Rubino, L. & Russo, M. (2004). Expression of the

Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces

cerevisiae and molecular dissection of the peroxisomal targeting

signal. J Virol 78, 4744–4752.

Panavas, T. & Nagy, P. D. (2003). Yeast as a model host to study

replication and recombination of defective interfering RNA of

Tomato bushy stunt virus. Virology 314, 315–325.

Panavas, T., Hawkins, C. M., Panaviene, Z. & Nagy, P. D. (2005a).

The role of the p33 : p33/p92 interaction domain in RNA replication

and intracellular localization of p33 and p92 proteins of Cucumber

necrosis tombusvirus. Virology 338, 81–95.

Panavas, T., Serviene, E., Brasher, J. & Nagy, P. D. (2005b).Yeast genome-wide screen reveals dissimilar sets of host genes

affecting replication of RNA viruses. Proc Natl Acad Sci U S A 102,

7326–7331.

Panaviene, Z., Panavas, T., Serva, S. & Nagy, P. D. (2004). Purifica-

tion of the Cucumber necrosis virus replicase from yeast cells: role of

coexpressed viral RNA in stimulation of replicase activity. J Virol 78,

8254–8263.

Pantaleo, V., Rubino, L. & Russo, M. (2003). Replication of

Carnation Italian ringspot virus defective interfering RNA in

Saccharomyces cerevisiae. J Virol 77, 2116–2123.

Pantaleo, V., Rubino, L. & Russo, M. (2004). The p36 and p95

replicase proteins of Carnation Italian ringspot virus cooperate in

stabilizing defective interfering RNA. J Gen Virol 85, 2429–2433.

Redding, K., Holcomb, C. & Fuller, R. S. (1991). Immunolocalization

of Kex2 protease identifies a putative late Golgi compartment in the

yeast Saccharomyces cerevisiae. J Cell Biol 113, 527–538.



Restrepo-Hartwig, M. & Ahlquist, P. (1999). Brome mosaic virusRNA replication proteins 1a and 2a colocalize and 1a indepen-dently localizes on the yeast endoplasmic reticulum. J Virol 73,10303–10309.

Rottensteiner, H., Stein, K., Sonnenhol, E. & Erdmann, R. (2003).Conserved function of Pex11p and the novel Pex25p and Pex27p inperoxisome biogenesis. Mol Biol Cell 14, 4316–4328.

Rottensteiner, H., Kramer, A., Lorenzen, S., Stein, K., Landgraf, C.,Volkmer-Engert, R. & Erdmann, R. (2004). Peroxisomal membraneproteins contain common Pex19p-binding sites that are an integralpart of their targeting signals. Mol Biol Cell 15, 3406–3417.

Rubino, L. & Russo, M. (1998). Membrane targeting sequences intombusvirus infections. Virology 252, 431–437.

Russo, M., Di Franco, A. & Martelli, G. P. (1983). The fine structureof Cymbidium ringspot virus infections in host tissues. III. Role ofperoxisomes in the genesis of multivesicular bodies. J Ultrastruct Res82, 52–63.

Russo, M., Di Franco, A. & Martelli, G. P. (1987). Cytopathology inthe identification and classification of tombusviruses. Intervirology28, 134–143.

Russo, M., Burgyan, J. & Martelli, G. P. (1994). Molecular biology ofTombusviridae. Adv Virus Res 44, 381–428.

Sacksteder, K., Jones, J. M., South, S. T., Li, X., Liu, Y. & Gould, S. J.(2000). PEX19 binds multiple peroxisomal membrane proteins, ispredominantly cytoplasmic, and is required for peroxisome mem-brane synthesis. J Cell Biol 148, 931–944.

Schlenstedt, G., Hurt, E., Doyle, V. & Silver, P. A. (1993).Reconstitution of nuclear protein transport with semi-intact yeastcells. J Cell Biol 123, 785–798.

Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J. &Ahlquist, P. (2002). A positive-strand RNA virus replication com-plex parallels form and function of retrovirus capsids. Mol Cell 9,505–514.

Snyder, W. B., Faber, K. N., Wenzel, T. J., Koller, A., Luers, G. H.,Rangell, L., Keller, G. A. & Subramani, S. (1999). Pex19p interactswith Pex3p and Pex10p and is essential for peroxisome biogenesis inPichia pastoris. Mol Biol Cell 10, 1745–1761.

Titorenko, V. I. & Rachubinski, R. A. (1998). Mutants of the yeastYarrowia lipolytica defective in protein exit from the endoplasmicreticulum are also defective in peroxisome biogenesis. Mol Cell Biol18, 2789–2803.

Titorenko, V. I. & Rachubinski, R. A. (2001). The life cycle of theperoxisome. Nat Rev Mol Cell Biol 2, 357–368.

Voinnet, O., Pinto, Y. M. & Baulcombe, D. C. (1999). Suppression ofgene silencing: a general strategy used by diverse DNA and RNAviruses of plants. Proc Natl Acad Sci U S A 96, 14147–14152.

Weber-Lotfi, F., Dietrich, A., Russo, M. & Rubino, L. (2002). Mito-chondrial targeting and membrane anchoring of a viral replicase inplant and yeast cells. J Virol 76, 10485–10496.

White, K. A. & Nagy, P. D. (2004). Advances in the molecular biologyof tombusviruses: gene expression, genome replication, and recom-bination. Prog Nucleic Acid Res Mol Biol 78, 187–226.



Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective...

Documents

Transcript of Cytological analysis of Saccharomyces cerevisiae cells supporting cymbidium ringspot virus defective...