The open reading frame 1-encoded (‘36K’) protein of Carnation Italian ringspot virus localizes to mitochondria

L. Rubino1, F. Weber-Lotfi2, A. Dietrich2, C. Stussi-Garaud2 and M. Russo1

Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Centro di Studio del CNR sui Virus e le Virosi delle Colture Mediterranee, Via Amendola 165/A, I-70126 Bari, Italy1
Institut de Biologie Moléculaire des Plantes, CNRS and Université Louis Pasteur, Strasbourg, France2

Author for correspondence: Marcello Russo. Fax +39 080 5442911. e-mail csvvmr01{at}area.ba.cnr.it


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The localization of the 36 kDa (‘36K’) protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K–GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.


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Carnation Italian ringspot virus (CIRV) is a member of the genus Tombusvirus in the family Tombusviridae, a family of positive-stranded RNA viruses. Its 4760 nt genome is not polyadenylated, lacks a 5' cap structure and encodes five functional open reading frames (ORFs). The genome-length RNA is translated into a 36 kDa (K) protein encoded by ORF 1. Readthrough of the amber stop codon of the 36K protein results in the translation of a 95 kDa protein (ORF 2), which contains the eight conserved motifs (PI–PVIII) of RNA-dependent RNA polymerases of supergroup II of the positive-strand RNA viruses (Koonin, 1991 ). ORF 3 encodes the 41 kDa capsid protein. ORFs 4 and 5 overlap in different reading frames and encode two proteins of 22 and 19 kDa in size, respectively, which are involved in virus movement and symptom expression in infected plants. ORF 3 is expressed from a subgenomic RNA of 2·1 kb, whereas ORFs 4 and 5 are expressed from a subgenomic RNA of 0·9 kb, which is bifunctional and serves as a template for the synthesis of both 22K and 19K proteins (Rubino et al., 1995 ).

The major cytopathological feature of cells infected by CIRV are multivesicular bodies (MVBs) deriving from modified mitochondria whose outer membrane undergoes progressive vesiculation so as to become totally disorganized (Di Franco et al., 1984 ). The single-membraned vesicles are rounded to ovoid, develop as invaginations of the mitochondrial outer membrane to which they remain connected, measure up to 200 nm in diameter, and contain granular material or a network of tiny fibrils. A short neck region connects the interior of the vesicle to the cytoplasm. These vesicles have been suggested to be the sites of virus replication due to the nature of the fibrillar content, which consists of double-stranded RNA (Di Franco et al., 1984 ), and due to the fact that virus replicase is recognized by replicase-specific antibodies in membranous components sedimenting at 30000 g (Rubino & Russo, 1998 ). Transformation of mitochondria into MVBs is determined by ORF 1 (Burgyan et al., 1996 ), which contains a signal whereby virus replicase is directed to the mitochondria where it is likely to be anchored to the outer lamella of the mitochondrial limiting membrane by two transmembrane segments (Rubino & Russo, 1998 ).

The localization of the CIRV 36K protein has now been investigated in CIRV-infected plant cells and cells expressing this protein transiently.

Nicotiana benthamiana plants were inoculated with CIRV in vitro RNA transcripts (Burgyan et al., 1996 ). At various intervals after inoculation, tissue pieces were sampled and processed for immunoelectron microscopy as previously described (Bleve-Zacheo et al., 1997 ). Repeated attempts to detect the 36K protein in CIRV-infected tissues by immunogold labelling of thin sections were unsuccessful, for MVB-labelling in infected cells was scanty and, in any case, not significantly different from labelling in cells treated with pre-immune primary antiserum (data not shown). Since the same antiserum proved to be effective in the detection of denatured 36K protein on Western blot, further attempts to detect directly the native protein in cell sections was discontinued and a different approach was pursued in order to localize the 36K protein.

CIRV-infected leaf tissue was extracted as described by Gualberto et al. (1995) . Extracts were centrifuged at 3000 g at 4 °C for 10 min. The supernatant was recovered and centrifuged sequentially at 10000, 30000 and 100000 g for 30 min to obtain different pellet fractions (P10, P30 and P100). Aliquots of each pellet (50 µg of protein, determined according to Bradford, 1976 ) were examined by Western blot analysis (Rubino & Russo, 1998 ) and the rest were fixed with 4% glutaraldehyde and processed for electron microscopy (Martelli & Russo, 1984 ). The 36K protein was detected in all three fractions, P10, P30 and P100, by Western blot analysis. In contrast, a marker protein for mitochondria, heat shock protein 70 (hsp70; Hase et al., 1984 ), was found only in the P10 pellet (data not shown). Electron microscope observations showed that the P10 sediment contained both intact and mildly vesiculated (initial MVB stage) mitochondria and a few peroxisomes, the P30 pellet contained peroxisomes, thylakoids and heavily vesiculated mitochondria (advanced MVB stage), and the P100 pellet contained vesicles of various sizes, some of which could have derived from the disruption of MVBs during extraction (data not shown). When the cellular material sedimenting at 10000 g was further fractionated through a discontinuous Percoll gradient (Gualberto et al., 1995 ), the 36K protein was found to be associated with fractions at the 21–45% and 13–21% Percoll interface (Fig. 1a, lanes 1 and 2), which also reacted with antibodies to hsp70 (Fig. 1b, lanes 1 and 2). Electron microscope analysis of the heavier fraction showed the presence of mitochondria with a few peripheral vesicles (initial MVB stage) (Fig. 1c) together with intact mitochondria, whereas developed MVBs (Fig. 1d) and chloroplast remnants prevailed in the lighter fraction. Fractions from uninfected leaf tissues had contents comparable to each other, except for the absence of MVBs (data not shown). Taken together, fractionation experiments showed that in CIRV-infected tissues the 36K protein is associated with subcellular fractions containing MVBs in addition to normal mitochondria and some peroxisomes.



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Fig. 1. Western blot analysis (a, b) and electron micrographs (c, d) of mitochondria banding at the 21–45% (c) and 13–21% (d) Percoll interfaces, respectively, upon fractionation of a resuspended P10 pellet from CIRV-infected plants on a discontinuous gradient. Lanes 1 and 2 (a, b) contain the cellular material from infected plants banding at the 21–45% and 13–21% interfaces, respectively. Blots were exposed to antibodies recognizing the CIRV 36K protein (a) or against the mitochondrial marker hsp70 (b). The numbers on the right indicate the size in kDa of pre-stained marker proteins (Bio-Rad). Bar, 200 nm.

 
For further localization and targeting studies, the 36K protein was expressed in different cell systems so as to avoid interference by other viral products and to allow use of the green fluorescent protein (GFP) reporter gene.

First, constitutive expression of the 36K protein in transgenic N. benthamiana plants was attempted by using previously established methods for the transformation of this plant species with sequences derived from Cymbidium ringspot virus (Rubino et al., 1992 , 1993 ). Although several kanamycin-resistant rooted-explants were obtained, PCR analysis of genomic DNA showed that in all these plantlets the viral transgene was not intact, but contained large deletions in one or more regions. Western blot analysis failed to detect the presence of the full or truncated ORF 1-encoded protein. This was taken as an indication that expression and targeting of the 36K protein to the mitochondria is not compatible with the survival of stable transformants.

Subsequently, transient expression of the 36K protein was obtained in tobacco leaf tissues or protoplasts and suspension-cultured tobacco BY2 cells. Three plasmids were used, pRTL2-GFP, COXIV-GFP and pRTL2-36K/GFP. Plasmid pRTL2-GFP was constructed by cloning the GFP-encoding sequence into the vector pRTL2 (Carrington & Freed, 1990 ). Plasmid COXIV-GFP (Menand et al., 1998 ) contained the sequence encoding the transit peptide of yeast cytochrome oxidase subunit IV (Hurt et al., 1985 ) fused in-frame with the GFP gene. To construct plasmid pRTL2-36K/GFP, expressing the 36K protein fused to GFP, two NcoI sites were engineered into the full-length cDNA clone of CIRV so as to contain the start and stop codons of the 36K protein, respectively. The fragment between the two engineered NcoI sites was isolated and cloned in-frame with the GFP sequence in the plasmid pRTL2-GFP.

N. tabacum BY2 cells were cultured according to Nagata et al. (1992) . Three days after subculturing, cells were collected under light vacuum on Whatman 540 filter paper discs. The discs were transferred to Petri dishes containing solid culture medium supplemented with 0·15 M mannitol and 0·15 M sorbitol. Cells were allowed to plasmolyse for 5 h prior to transfection by biolistic bombardment as previously described (Russel et al., 1992 ; Menand et al., 1998 ) using a helium-driven device. Protoplasts were obtained from N. tabacum or N. benthamiana leaves as described by Nagy & Maliga (1976) and transfected in the presence of PEG (Negrutiu et al., 1987 ). Cells were observed using either an epifluorescent microscope as previously described (Rubino et al., 2000 ) or a confocal laser scanning microscope (Zeiss Axiovert 100M) equipped with an LSM510 confocal unit and an argon laser. Filters were used to detect GFP (excitation 488, dichroic mirror 505, emission band pass 505–545 nm), a mitochondrial-specific dye MitoTracker (Molecular Probes) (excitation 543, dichroic mirror 545, emission long pass 560 nm) and chlorophyll (excitation 633, dichroic mirror 635, emission long pass 650 nm).

Protoplasts and BY2 cells transfected with plasmid pRTL2-GFP both showed GFP expression throughout the cytoplasm and nucleus with no preferential localization (data not shown). Conversely, when cells were transfected with plasmid COXIV-GFP, green fluorescence was localized to discrete spots 1–5 µm in size (data not shown). Finally, when cells were transfected with the GFP fused to the 36K protein, fluorescence was also concentrated in spots, which were sometimes very similar in size to those found in the case of the COXIV leader sequence but were often larger (data not shown). The green-fluorescing spots in cells transfected with plasmids pRTL2-36K/GFP and COXIV-GFP fluoresced red when the samples were treated with MitoTracker according to Koehler et al. (1997) and viewed in the red channel (data not shown).

For transient expression in intact leaves, an Agrobacterium-mediated procedure was adopted. The expression cassette was excised from plasmids pRTL2-GFP and pRTL2-36K/GFP and inserted into the binary vector pGA482. The resulting constructs were introduced by electroporation into A. tumefaciens strain C58C1 ATHV. Colonies were selected for growth in the presence of kanamycin (50 mg/l) and rifampicin (25 mg/l). Recombinant clones were cultured essentially as described by Kapila et al. (1997) . One single colony was inoculated in 50 ml of LB containing 50 mg/l kanamycin and incubated for 24 h at 28 °C with shaking (250 r.p.m.) (OD600=ca. 2·0). This culture (25 ml) was then centrifuged at 4500 g for 5 min at 15 °C, resuspended gently in 5 ml of LB medium containing 10 mM MES pH 5·6, 50 mg/l kanamycin and 20 µM acetosyringone, and diluted into 200 ml of the same medium. The suspension was incubated overnight (ca. 10 h) at 28 °C with shaking (250 r.p.m.) to OD600=ca. 1·5. The whole culture was centrifuged as before and resuspended to a final OD600=ca. 3·0 in a medium containing MS salts (Murashige & Skoog, 1962 ), 10 mM MES pH 5·6, 20 g/l sucrose and 200 µM acetosyringone. The bacterial suspension was kept for 1·5–2 h at room temperature and then infiltrated into N. benthamiana leaves. To do so, either detached leaves were vacuum infiltrated and incubated in a Petri dish as in Kapila et al. (1997) or the Agrobacterium suspension was forced through the stomata into the intercellular space of undetached leaves with a 2 ml syringe (without a needle). Whole plants or detached leaves were maintained for 24–72 h in a growth chamber at 25 °C with a 14 h light/10 h dark photoperiod and 70% relative humidity. Expression and differential localization of unfused and 36K-fused GFP were clearly discernible even at a low magnification with an epifluorescence microscope (data not shown), which permitted a precise selection of tissue pieces for further analysis by confocal and electron microscopy. Expression of the 36K–GFP protein in the analysed tissues was confirmed by Western blotting (data not shown). Using confocal microscopy under the imaging conditions for GFP, the 36K–GFP protein was shown to localize to irregularly shaped structures ca. 4–6 µm in size (Fig. 2a). The same structures fluoresced red when the cells were stained with MitoTracker and viewed in the red channel (Fig. 2b). The localization of the 36K protein, shown as yellow-orange fluorescence, was evident in the merged images of cells expressing red and green fluorescence (Fig. 2d). Chloroplasts fluoresced in a blue-violet colour under the experimental conditions used (Fig. 2c, d).



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Fig. 2. Confocal microscopy of Agrobacterium-infiltrated mesophyll tissue expressing 36K–GFP fusion protein. The same cells viewed in the GFP (a), MitoTracker (b) and chlorophyll (c) channels, respectively, are shown. A merged vision of the three channels (d) is also shown. Bar, 5 µm.

 
Mitochondria of thin-sectioned Agrobacterium-infiltrated tissues expressing only GFP had an apparently normal appearance (Fig. 3a). In contrast, cells expressing the 36K–GFP fusion protein were characterized by the presence of severely misshapen and clumped mitochondria (Fig. 3bg). Mitochondrial stroma was more electron-opaque and the cristae were less numerous and larger and irregularly shaped (Fig. 3b, c). The intermembrane space was dilated and electron-lucent (Fig. 3b, c) or contained cytoplasmic material (Fig. 3ce). The outer mitochondrial membrane was invariably lined, completely or in part, with an electron-dense amorphous material (Fig. 3bg), which was also present in the cytoplasm in discrete accumulations and which was not found in CIRV-infected cells. Peroxisomes, chloroplasts and nuclei had a normal appearance.



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Fig. 3. Electron micrographs of cells expressing unfused GFP (a) or GFP fused to the 36K protein (bg). Mitochondria in (a) show a normal appearance; those in (b)–(g) show various levels of alterations consisting of dilations of the intermembrane space, which may appear electron-lucent (b, c) or contain cytoplasm (be). All mitochondria in (b)–(g) are surrounded by electron-dense material, which is also present in the cytoplasm (c). Bar, 200 nm.

 
Immunogold labelling failed to establish whether the 36K protein was present in the electron-dense material, for the number of gold particles per unit area in sections probed with an antiserum recognizing this protein did not differ substantially from the labelling observed in cells treated with pre-immune serum (data not shown). Similarly, no significant accumulation of gold particles was detected on the mitochondria surrounded by the electron-dense material. As immunogold labelling also failed to detect the 36K protein in CIRV-infected cells (see above), it is possible that the antiserum used recognizes the denatured but not the native 36K polypeptide.

In conclusion, the study shows that the CIRV 36K protein localizes to mitochondria and that it is likely to contain a signal to promote the overgrowth of the mitochondrial outer membrane. Similar to the expression of 36K in yeast cells (Rubino et al., 2000 ), no MVBs resembling those formed in infected tissues were detected. Unless the 36K protein is not fully functional when fused to GFP, this is in line with the likelihood that expression of this protein is not the only factor responsible for vesiculation of mitochondria because, during infection, the complete replicase (ORF 2) is also expressed and replication of viral RNA takes place. Nevertheless, transient expression of the CIRV 36K protein may be a useful system to further analyse the mechanism of its association with cell membranes.


   Acknowledgments
 
The authors wish to thank Professor G. P. Martelli for helpful discussion and critical reading of the manuscript and Mrs Antonella Antonacci for the skilful technical assistance given throughout this work. L.R. is particularly grateful to Dr M. Castellano for introducing her to electron microscopy methods. This work was supported by funding from the Italian/French Galileo program. The confocal microscope equipment has been co-financed by the Centre National de la Recherche Scientifique (CNRS), the Université Louis Pasteur (ULP, Strasbourg), the Association pour la Recherche contre le Cancer (ARC) and the Région Alsace.


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Received 10 July 2000; accepted 20 September 2000.