Dipartimento di Protezione delle Piante, Via Amendola 165/A, Università degli Studi, and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, 70126 Bari, Italy1
Author for correspondence: Luisa Rubino. Fax +39 80 5442911. e-mail csvvlr02{at}area.ba.cnr.it
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Abstract |
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Introduction |
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Carnation Italian ringspot virus (CIRV) is one of several tombusviruses which have been studied (Di Franco et al., 1984 ; Rubino et al., 1995
). In particular, early cytopathological investigations have shown that CIRV-infected cells contain vesiculated structures (multivesicular bodies, MVB) composed primarily of a large number of vesicles derived from the proliferation of the outer membrane of mitochondria (Di Franco et al., 1984
). CIRV-induced MVBs are similar to structures induced by other tombusviruses except that these latter structures develop from proliferation of the limiting membrane of peroxisomes (Russo et al., 1987
). Whichever organelle gives rise to MVB, vesicles are thought to be the site of virus replication (Rubino & Russo, 1998
). Recently, it was proposed that the CIRV 36K protein contains a signal that directs the virus replicase to mitochondria, where it is anchored to the outer lamella of the limiting membrane by two transmembrane segments (Rubino & Russo, 1998
).
It is an open question how proliferation of the mitochondrial membrane takes place in CIRV-infected cells, i.e. if and how the 36K protein is involved in the generation of the membranous vesicles. A good model system for analysis of membrane proliferation and vesicle transport is represented by the yeast Saccharomyces cerevisiae (Wright et al., 1988 ; Kaiser & Scheckman, 1990
; Pryer et al., 1992
). This prompted us to use yeast cells to express the CIRV 36K protein to allow study of its cytological effects in the absence of a productive virus infection.
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Methods |
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Construction of GFP+36K plasmids.
To construct a vector expressing the 36K protein fused to the green fluorescent protein (GFP), a clone of a yeast codon-optimized form of the GFP (pyEGFP) gene in pUC19 was used (Cormack et al., 1997 ). The coding sequence for the 36K protein was fused to either the 5' or 3' end of the GFP gene. For the first construct (p36K-GFP), a HindIII restriction site was engineered at the stop codon of the 36K gene in the clone p36K by site-directed mutagenesis. The fragment containing the sequence between the HindIII sites in the linker and at the end of the viral gene was purified and ligated into the HindIII site of clone pyEGFP. A clone containing 36K and GFP in the correct orientation was chosen by sequencing. This contained four additional amino acids between the 36K and GFP protein sequences encoded by the nucleotides between the HindIII site and the start codon of GFP.
To produce a plasmid containing the 36K gene positioned at the 3' end of the GFP sequence, the clone pyEGFP was digested with PflMI, which cuts 21 nt upstream of the protein stop codon, treated with T4 DNA polymerase to generate a blunt end and then digested with HindIII (in the linker, upstream from the GFP start codon); the clone p36K was first digested with NcoI, made blunt-ended with Klenow enzyme and digested with HindIII (in the linker, upstream from the 36K start codon). Finally, the HindIIIPflMI fragment from pyEGFP was cloned into p36K to produce pGFP-36K. In this clone, 7 aa were deleted in the 3' terminal region of GFP.
Yeast transformation.
Plasmids p36K, pyEGFP, p36K-GFP and pGFP-36K were digested with HindIII and EcoRI (present only at the ends of the vector linker sequence), and the resulting fragments were ligated into the plasmid pYES2 containing the galactose-activated GAL1 promoter (Invitrogen) digested with the same restriction enzymes. Clones in vector pYES2 maintain the same nomenclature as those in pUC18.
For expression, the S. cerevisiae strain YPH499 (MATa, ura3-52, lys2-801, ade2-101, trp1-delta63, his3-delta200, leu2-delta1; Sikorski & Hieter, 1989 ) was used throughout. Transformation was done using frozen cells. Briefly, the yeast was cultured on YPD (1% yeast extract, 2% bactopeptone, 2% dextrose, pH 4·85·0) plates at 30 °C for 23 days to form a continuous layer. Cells were then scraped from one plate with a spatula, transferred to an Eppendorf tube containing 1 ml TES (1 M sorbitol, 10 mM TrisHCl, 3% ethylene glycol, pH 8·3), pelleted by centrifugation (6000 r.p.m. for 30 s) and washed three times with TES. The washed cell pellet was resuspended in 750 µl TES to which 45 µl DMSO was added. The mixture was then divided into 100 µl aliquots in 1·5 ml Eppendorf tubes. These were snap-frozen in liquid nitrogen for a few seconds and stored at -70 °C for at least 30 min. About 1 µg DNA in 2030 µl TE (10 mM TrisHCl, 1 mM EDTA, pH 8·0) was pipetted on top of the frozen cells which were then allowed to thaw for a few seconds at room temperature. The tubes were then incubated at 37 °C for 5 min with gentle shaking every 30 s. One millilitre of TPEG buffer (200 mM TrisHCl, 40% PEG 1000, pH 8·3) was added and the cells were incubated for 1 h at 30 °C on a rotary agitator (150 r.p.m.). Finally, the cells were sedimented and resuspended in 300 µl TN buffer (10 mM TrisHCl, 150 mM NaCl, pH 8·3). The cell suspension (200 µl) was spread on minimal selective medium (SM) plates containing 0·67% Difco yeast nitrogen base without amino acids, 2% dextrose, amino acids at 40 µg/ml, adenine at 10 µg/ml, 2% agar and no uracil. Red colonies were streaked and maintained on SM plates.
Culture of transformants and Western blot analysis.
A single colony was collected, inoculated in 10 ml SM containing 2% dextrose and incubated for 24 h at 30 °C at 150 r.p.m. One millilitre of this culture was used to inoculate 10 ml SM containing 3% glycerol and 0·1% dextrose, followed by incubation for a further 24 h. Finally, 1 ml of this second culture was used to inoculate 10 ml YP (1% yeast extract, 2% bactopeptone, pH 4·85·0) containing 3% glycerol and 2% galactose and the mixture was incubated for 16 h.
Cells were converted to spheroplasts using the protocol of Ausubel et al. (1995) except that it was adapted to a smaller scale by using 5 ml of a 10 ml culture (A600 of 0·6), and by performing washings, resuspension and treatment with Zymolyase (20 U/ml) in a volume of 500 µl. To extract proteins, the final spheroplast pellet was resuspended in 1 ml lysis buffer (50 mM TrisHCl, pH 7·4, 15 mM MgCl2, 10 mM KCl, 0·3 M sorbitol, 0·1%
-2-mercaptoethanol, 5 µg/ml leupeptin, 2 µg/ml aprotinin) and incubated for 2 min at room temperature. The cell lysate was centrifuged at 500 g for 3 min at 4 °C to remove large debris and intact cells, and the supernatant was centrifuged at 12000 g for 15 min at 4 °C, saving the pellet (P12) and supernatant (S12). The P12 pellet was examined as it was or after treatment with one of the following reagents for 30 min on ice: 100 mM Na2CO3 (pH 11·5), 4 M urea or 1 M KCl (Schaad et al., 1997
). After treatment, the solution was centrifuged at 12000 g for 15 min at 4 °C to generate a supernatant and a pellet fraction. Immunoblot analysis was performed as previously described (Rubino et al., 1995
) using an enhanced chemiluminescent assay (Amersham).
Light and electron microscopy.
Microscopic observations of cells expressing GFP were routinely done with a Nikon ECLIPSE E400 epifluorescence microscope equipped with FITC filters (excitation 450490, dichroic minor 505, band pass 520). Photography was with a Nikon ECLIPSE E800 epifluorescence microscope equipped with GFP(R)-Long Pass (excitation 460500, dichroic minor 505, long pass 510) or GFP(R)-Band Pass (excitation 460500, dichroic minor 505, band pass 510560). To stain cells with MitoTracker (Molecular Probes), cells were incubated with 0·5 µM MitoTracker, washed three times with PBS (10 mM potassium phosphate buffer, 0·14 M NaCl, pH 7·4) and observed using a TRITC filter set (excitation 510560, dichroic minor 575, band pass 590). Cells were immobilized on glass slides by mixing 5 µl cell suspension with 5 µl 1% low-melting-point agarose maintained at 45 °C and covering the mixture with a cover-slip. Images were collected with a Power HAD 3CCD videocamera (Sony), using Visiol@b 200 (Biocom) and processed with Adobe Photoshop.
For electron microscopy, cells from a galactose-induced 10 ml liquid culture were sedimented, fixed with 4% (v/v) glutaraldehyde, post-fixed with 4% (w/v) potassium permanganate and embedded in Spurrs resin as described (Yaffe, 1995 ). For immunogold labelling, cells were fixed and embedded in LR White resin (Wright & Rine, 1989
). The primary anti-36K or pre-immune sera were used at a dilution of 1/1000, and the gold (10 µm)-labelled goat anti-rabbit antiserum (Amersham) at 1/20.
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Results |
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Prominent features of cells transformed with vector only were a nucleus surrounded by a double membrane interrupted by several pores, a vacuole containing very electron-dense material and several strands of endoplasmic reticulum. The plasmalemma lined a cell wall in which one or more scars indicated where daughter cells separated during budding. Profiles of mitochondria were rounded or elongated and were regularly distributed in the cytoplasm, never showing a preferential site of accumulation. Up to 1012 mitochondrial profiles were counted per section. Cristae lay mostly perpendicular to the mitochondrial surface (Fig. 4a). Cells transformed with pyEGFP showed no difference compared to those transformed with the vector only (Fig. 4b
).
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Discussion |
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The subcellular distribution of fluorescence in cells expressing unfused GFP or 36K-fused GFP is very distinct. Cells expressing unfused GFP show equal distribution of fluorescence throughout the cytoplasm and nucleus. Given its small size (ca. 27 kDa), GFP probably enters the nucleoplasm by diffusion through the nuclear pores. Conversely, it is excluded from vacuoles, as it lacks the specific sorting signal possessed by yeast proteins targeted to this organelle (Banta et al., 1988 ). When fused to the CIRV 36K protein, GFP acquired a strong localization signal, since fluorescence in p36K-GFP and pGFP-36K cells was not diffuse, but associated with cytoplasmic structures. Under the electron microscope, these structures appeared distinct, depending upon whether they were induced by the construct having the viral protein at the N or the C terminus. With p36K-GFP, the abnormal structures were composed of accumulations of mitochondria and a few membranous strands, whereas with pGFP-36K, membranous strands were much more abundant than mitochondria. Structures induced by the unfused 36K were identical to those elicited by p36K-GFP. No definitive explanation can now be provided concerning the significance of these structures. One possibility could be that massive membrane flow is activated by the 36K protein leading to an increase in the number of mitochondria. It is worth noting that transformation of yeast cells with the DNA encoding the cymbidium ringspot tombusvirus (CymRSV) 33K protein (known to induce the formation of MVBs from peroxisomes; Burgyan et al., 1996
) fused to GFP did not lead to any particular localization of the reporter protein (unpublished results). Since under the growing conditions we have used, peroxisomes are not detectable in yeast, it can be deduced that no localization of GFP fused to a viral protein takes place in the absence of the targeted organelle. These findings suggest that the localization of the GFP fused to CIRV 36K is specifically due to interactions with mitochondria. The different behaviour of the p36K-GFP and pGFP-36K constructs may be ascribed to the fact that, in the latter construct, the targeting signal, usually located towards the N terminus of the protein, is obscured by the presence of GFP upstream of it. The formation of true MVBs similar to those formed in CIRV-infected plant cells was not really expected since the expression of the ORF 1-encoded protein is not the only factor leading to the synthesis of MVBs. In fact, transgenic plant cells constitutively expressing the CymRSV 33K protein do not contain peroxisome-derived MBVs, but only proliferating membranes and extremely altered peroxisomes, where the 33K protein accumulates, and no alterations of any other organelle (Bleve-Zacheo et al., 1997
). Therefore, we believe that the constitutive or transient expression of tombusvirus ORF 1-encoded proteins may be useful to study the interactions of this protein with cell organelles, including the analysis of host cell factors composing the active replicase, of which the CIRV 36K and CymRSV 33K are part.
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Acknowledgments |
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References |
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Received 1 June 1999;
accepted 13 September 1999.