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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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Additional figures are available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Villanueva et al., 2005; Martelli & Boudon-Padieu, 2006). The initial assembly step of the replication complex requires targeting of the viral replicase to the specific cell membrane where it anchors. Targeting and anchoring depend on signals in the viral proteins which allow specific association with organelle membranes. Following this, membranes undergo drastic morphological changes resulting in the production of a number of flask-shaped vesicles.
The association of viral replicase proteins with the limiting membrane of peroxisomes and mitochondria has been studied in detail with members of the genus Tombusvirus (family Tombusviridae). The genome of these viruses consists of a single RNA molecule of approximately 4800 nt containing five open reading frames (ORFs) (Russo et al., 1994; White & Nagy, 2004). The two 5'-proximal ORFs code for two proteins required for the replication of genomic RNA and other small molecules such as satellite and defective interfering (DI) RNAs. These replicase proteins have a size ranging from 33 (p33) to 36 (p36) kDa and 92 (p92) to 95 (p95) kDa, depending on the viral species, and are translated directly from genomic RNA. Internal ORFs code for the capsid (ORF 3), movement (ORFs 4 and 5) and gene silencing suppressor (ORF 5) proteins, respectively, and are expressed via the synthesis of two subgenomic RNAs. The tombusviruses Cymbidium ringspot virus (CymRSV) and Carnation Italian ringspot virus (CIRV) have been widely used for the analysis of targeting signals of the replicase, taking advantage of the diversity of the intracellular localization of their replication complex. In fact, replication of CymRSV takes place in vesicles derived from the peroxisomal membrane (Russo et al., 1983, 1987), whereas CIRV replicates in vesicles derived from the outer membrane of mitochondria (Di Franco et al., 1984; Russo et al., 1987). By exchanging genome fragments between infectious cDNA clones of the two viruses, it was shown that the signals dictating the site of replication were present in the protein encoded by ORF 1 (i.e. p33 or p36, for CymRSV and CIRV, respectively) (Burgyan et al., 1996). These signals consisted of short stretches of hydrophilic amino acids plus two hydrophobic transmembrane domains (Weber-Lotfi et al., 2002; Navarro et al., 2004). Peroxisomes also proved to be involved in the assembly of the CymRSV replication complex in yeast cells (Navarro et al., 2006). However, when a yeast strain genetically deficient for peroxisome biogenesis was used, the replicase proteins (p33 and p92) and DI RNA were targeted to the ER, which became the site of DI RNA replication (Navarro et al., 2006).
In the present work, the association of the CymRSV DI RNA replication complex with ER membranes was further analysed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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63 his3-200 leu2-1) (Sikorski & Hieter, 1989) and UTL-7A (MATa leu2-3,112 ura3-52 trp1) (Erdmann et al., 1989). UTL-7A transformants were previously described and grown accordingly (Navarro et al., 2006). YPH499 cells were transformed using the lithium acetate–polyethylene glycol method (Ito et al., 1983) and grown as described previously (Pantaleo et al., 2003).
Plasmids.
pALDsRedAKL and pKat84 express constitutively (ADH1 promoter) the red fluorescent protein DsRed containing the peroxisomal matrix targeting signal AKL (Wang et al., 2001; Navarro et al., 2004), and the Pex27p peroxisomal membrane protein fused to the green fluorescent protein (GFP) (Rottensteiner et al., 2003), respectively. CymRSV p33 and p92 proteins were expressed constitutively with vectors pA and YE (Pantaleo et al., 2003; Navarro et al., 2006), respectively. These proteins were tagged at their N termini with Myc and haemagglutinin (HA) epitopes, respectively; in addition, p92 was mutated to carry a tyrosine codon in place of the p33 stop codon (Navarro et al., 2006). Alternatively, both p33 and p92 were expressed using only one plasmid (YE) by readthrough of the p33 stop codon from the p92 wild-type (wt) coding sequence (p92wt) (Navarro et al., 2006). DI-3 RNA (Burgyan et al., 1992) was cloned in the vector pBMI3S under the control of the galactose-inducible GAL1 promoter (Ishikawa et al., 1997; Pantaleo et al., 2003). GFP and fusion protein p33–GFP were expressed using the galactose-inducible vector pYES2 (Invitrogen) (Navarro et al., 2004).
For the intracellular detection of DI RNA progeny, the BamHI–BglII fragment, encoding six MS2 recognition hairpins, was excised from plasmid pSL-MS2-6 (Bertrand et al., 1998) and cloned between blocks II and III of DI-3 RNA (Burgyan et al., 1992), where a BamHI site had been introduced by site-directed mutagenesis (QuikChange kit; Stratagene) to give clone DI-3/MS2. The GFP-tagged MS2 coat protein containing a nuclear localization signal (NLS) was expressed with plasmid pG14-MS2-GFP (Bertrand et al., 1998).
Miscellaneous techniques.
Immunofluorescence, electron microscopy and immunoelectron microscopy methods were detailed in previous papers (Navarro et al., 2004, 2006). For simultaneous detection of p33 and p92, ultrathin sections of LR White-embedded cells were treated with mouse anti-Myc (p33 tag) and rabbit anti-HA (p92 tag) antibodies and then with goat anti-mouse and anti-rabbit antibodies labelled with 10 and 15 nm gold particles, respectively. RNA was extracted as described by Schmitt et al. (1990) and analysed by Northern blotting as described by Navarro et al. (2006). Protein extraction and Western blot analysis were as previously described (Pantaleo et al., 2003; Navarro et al., 2004). Carbonate treatment was as described by Rubino & Russo (1998); proteinase K digestion and analysis of digests using a discontinuous Tris-Tricine system electrophoresis (Schagger & von Jagow, 1987) were as described by Weber-Lotfi et al. (2002).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Flock House virus (FHV) (YPH500; Price et al., 1996) and CIRV (YPH499; Pantaleo et al., 2003). Previous investigations on CymRSV replicase-driven DI RNA replication (Navarro et al., 2006) used UTL-7A, a strain in which peroxisomes are induced and increase in size and number in medium containing oleate as sole carbon source (Erdmann et al., 1989). By contrast, strain YPH499 does not grow on an oleate medium, which suggests that its cells do not contain mature peroxisomes (Cavero et al., 2003).
To verify this point YPH499 cells were transformed with plasmids pALDsRedAKL, expressing a red-fluorescing protein with a peroxisomal matrix targeting signal (Wang et al., 2001), or pKat84, expressing the peroxisomal membrane protein Pex27p fused to GFP (Rottensteiner et al., 2003). UTL-7A cells transformed with the same plasmids were used as control (Navarro et al., 2004). Fluorescence microscope observations showed that DsRedAKL was not targeted to any organelle of YPH499 cells, for it was diffused in the cytosol, whereas Pex27p–GFP localized to scattered spots, often just one, in some cells, probably representing a peroxisome (Fig. 1, upper row). In UTL-7A cells, as expected, both DsRedAKL and Pex27p–GFP highlighted numerous spots, identified as peroxisomes (Fig. 1, lower row). Shifting to oleate medium increased only slightly the growth of YPH499 cells and did not alter the peroxisomal content, in contrast with UTL-7A cells whose peroxisomes enlarge and proliferate in this medium (Götte et al., 1998; Navarro et al., 2004) (see Supplementary Fig. S1, available in JGV Online).
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It was previously shown that in UTL-7A cells, expression of p33 fused to GFP was localized to peroxisomal aggregates and induced membrane proliferation (Navarro et al., 2004). A similar experiment was done in YPH499 cells, by expressing the fusion protein p33–GFP with vector pYES2 in the absence of other viral products. Cells growing in the presence of galactose showed accumulation of GFP in large bodies around the nucleus and in patches scattered in the cytoplasm, which reacted with antibodies to the ER marker protein Kar2p (Fig. 2a, upper row), but not to the Golgi apparatus (Emp47p) (Fig. 2a, middle row) or mitochondrial (Tom40p) (Fig. 2a, lower row) markers. Control cells expressing unfused GFP showed the expected pattern of green fluorescence diffused in the cytoplasm (see Supplementary Fig. S2a, available in JGV Online). Co-expression of p33 and Pex27p–GFP showed that the large accumulation of p33 was not accompanied by peroxisome proliferation (Fig. 2b). In conclusion, CymRSV p33 seems to be associated with ER membranes in YPH499 cells and does not alter the peroxisomal pattern.
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), as compared to control cells (see Supplementary Fig. S2b), and was not associated with mitochondria or Golgi (see Supplementary Fig. S3a, b). When mouse anti-Myc antibodies were used in conjunction with rabbit anti-HA antibodies, it was shown that the p92 accumulation largely coincided with p33 (Fig. 3b). Results on the distribution of p33 also suggest that p92 accumulates on the perinuclear and cortical ER. No alteration of the peroxisome pattern was induced by coexpression of p33 and p92 (see Supplementary Fig. S3c).
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) showed the presence of the nucleus, short ER strands, mitochondria and vacuoles. Golgi cisternae, which are not organized in stacks in S. cerevisiae (Rossanese et al., 1999), are not morphologically distinguishable in these cells. Cells expressing p33 and p92 alone or together with DI RNA (see below) showed cytoplasmic accumulations of membranes either scattered in the cytoplasm or appressed in stacks to the nuclear membrane (Fig. 4b–f). Altogether, these membrane accumulations recall the ER-derived structures identified in fluorescence microscopy as sites of accumulation of p33 and p92 and, when present, of DI RNA (see below).
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) and were absent in other parts of the cell or in control cells transformed with empty vectors (Fig. 5a). The accumulation of p92, as visualized by 15 nm gold particles, was much lower than for p33, in line with the immunofluorescence results. The localization of the two proteins on ER membranous strands coincided.
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). This result could indicate association of the viral proteins with cell membranes.
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, both p33 and p92 were mostly recovered in the pellet after this treatment. This finding would be consistent with the presence of two hydrophobic membrane-spanning domains which were previously shown to promote the integration in the peroxisomal membrane of plant and yeast cells, leaving most of the sequence upstream and downstream the integrated segment exposed in the cytoplasm (Rubino & Russo, 1998; Navarro et al., 2004).
If the same type of integration of p33 into the ER membrane occurred, only the parts of the protein exposed in the cytoplasm would be accessible for a protease treatment in the absence of detergent. To verify this point, the membrane pellet fraction from transformed YPH499 cells expressing p33 and p92 was digested with proteinase K in the presence or absence of Triton X-100. As control, similar protein extracts from transformant yeast strain UTL-7A cells, which were previously shown to contain a membrane-embedded protease-resistant fragment of p33 (Navarro et al., 2004), were used. As shown in Fig. 6(c) (left panel), no protease-resistant fragment was detected by anti-p33 antiserum in the YPH499 cell extracts with or without detergent treatment. Conversely, UTL-7A extracts contained two small resistant fragments of approximately 14 and 12 kDa, which were no longer detected if the membranes were treated with Triton X-100 (Fig. 6c, right panel). Altogether these results indicate that p33 and p92 (i) were present in the P30 fraction because they are associated with membranes; (ii) are peripheral but not membrane-integral proteins; (iii) are associated with the cytoplasmic side of the ER.
DI RNA replication and localization of the replication complex in YPH499 cells
To see whether p33 and p92 targeted to ER membranes in YPH499 yeast cells were competent to form a replication complex capable of replicating in trans DI RNA molecules, cells were cotransformed to express the tagged versions of the replicase proteins (Myc–p33 and HA–p92) under the control of constitutive promoter ADH1, and to express DI RNA under the control of the galactose-inducible GAL1 promoter (Navarro et al., 2006). Northern blot analysis of RNA extracts from transformed YPH499 cells showed that robust DI RNA replication took place only in the presence of both replicase proteins p33 and p92 (Fig. 7a). These proteins were expressed either by two separate plasmids (lane 4) or by only one plasmid expressing wt p92 (lane 3), whereas no progeny RNA was detected when p92 (lane 1) or p33 (lane 2) were omitted. As in previous experiments (Pantaleo et al., 2004; Rubino et al., 2004; Navarro et al., 2006), the progeny DI RNA was composed of monomers and dimers (Fig. 7a, lanes 3 and 4).
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) which had been successfully applied to study tombusvirus replication in yeast cells by Panavas et al. (2005). Briefly, six copies of the MS2 phage coat protein (CP) recognition hairpins were cloned between blocks II and III of DI RNA in both positive and negative orientation. Yeast cells were transformed to coexpress mutant DI-3 RNA (DI-3/MS2), the MS2 CP fused to GFP containing NLS and the CymRSV p92wt. Only cells expressing both p33 and p92 would be able to replicate DI-3/MS2, switching the localization of GFP from the nucleus to the site of DI RNA replication. Transformed yeasts were grown as described previously (Pantaleo et al., 2003; Rubino et al., 2004) and divided in two aliquots, from one of which RNA was extracted and the other was fixed for immunofluorescence analysis. Northern blot analysis of RNA extracts showed that mutant DI RNA molecules containing the MS2 recognition sequence in both positive (Fig. 7b, lane 1) or negative (Fig. 7b, lane 3) orientations were replication competent, but at a level much lower than the wt (Fig. 7b, lane 4). As expected, no DI RNA replication occurred in the absence of CymRSV replicase proteins (Fig. 7b, lane 2). In the immunofluorescence analysis, a small number of cells showed negative-strand DI RNA localized mainly over the nuclear membrane, whereas positive-strand DI RNA progeny partially colocalized with the ER marker Kar2p (Fig. 7c), indicating the ER as the site of DI RNA replication.
Mutational analysis of putative ER targeting signals
Two putative ER targeting signals are present near the N terminus of CymRSV p33, consisting of dibasic motifs, namely lysine (K)–arginine (R) at positions 5 and 6 (K5R6) and KK at positions 11 and 12 (K11K12) (McCartney et al., 2005). To evaluate the role of these regions in the ER targeting of p33 and assembly of active replication complex, each pair was substituted with glycine (G) residues (mutants G5G6 and G11G12, respectively). Yeasts were transformed to express these p33 mutants alone or together with p92 and DI RNA and analysed for the ability to replicate DI RNA. Northern blot analysis of RNA extracts from these transformants did not reveal a significant reduction in the amount of replicated DI RNA (see Supplementary Fig. S4, available in JGV Online).
Immunofluorescence analysis of mutant G5G6-expressing cells showed that p33 colocalized with portions of perinuclear ER (Fig. 8a) which, however, did not show the conspicuous enlargement as in cells expressing wt p33 (Fig. 3a), resulting from the apposition to the nucleus of proliferated membranes (compare Fig. 8c with Fig. 4b–f). In addition, large spots present in the cytoplasm reacted with anti-Myc but not anti-Kar2p antibodies (Fig. 8a). Conversely, accumulation of p33 mutant G11G12 was more similar to wt p33, because of the presence of large perinuclear bodies and smaller cytoplasmic accumulations reacting with anti-Myc and Kar2p antibodies (Fig. 8b). In fact, corresponding electron microscope analysis showed limited membrane proliferation apposed to the nucleus (Fig. 8d). The cytoplasmic electron-dense bodies (triangles in Fig. 8c, d) likely represent aggregates of p33 corresponding to the green-fluorescent spots of Fig. 8 (a, b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 1987; Navarro et al., 2006). The working hypothesis was that replication could be retargeted to an alternative membrane system, as in other cases where such retargeting was experimentally obtained by modifying the structure of the viral replicase proteins (Miller et al., 2003) or by altering the ratio of the components constituting the active viral replicase (Schwartz et al., 2004).
For CymRSV, replication retargeting was investigated in a host where biogenesis of peroxisomes is impaired. In this respect, preliminary experiments with yeast strain UTL-7Apex19 suggested the capability of the CymRSV replication complex to localize to the ER instead of peroxisomes, which are absent in these cells (Götte et al., 1998; Navarro et al., 2006). In the present study strain YPH499 was used, whose cells are unable to form mature peroxisomes. Nevertheless, DI RNA replication based on the CymRSV replicase was still very efficient because the replicase proteins p33 and p92 were targeted to and associated with alternative membranes such as the ER. However, the affinity of CymRSV replicase for ER membranes was not consistent with integration, rather indicating a peripheral association. In fact, no protease-resistant fragment was detected, suggesting that the protein is completely exposed to the cytoplasm with no luminal protrusion. Incidentally, the detection of two fragments in UTL-7A extracts, which contrasts with the detection of only one fragment of smaller size in previous studies (Navarro et al., 2004), is likely due to the better resolution in the Tris–Tricine electrophoresis system (Schagger & von Jagow, 1987). The peripheral association of CymRSV with ER membranes recalls that of BMV protein 1a with the same membranes (den Boon et al., 2001), but whereas in the latter case the finding is consistent with the absence of hydrophobic sequences capable of spanning the membrane, this is not the case of CymRSV p33 (and p92) which contains two such sequences participating in the formation of a peroxisomal targeting signal (Rubino & Russo, 1998; Navarro et al., 2004). Thus, targeting and association of CymRSV p33 with the ER probably do not involve the two transmembrane domains.
Other studies have demonstrated that determinants of ER membrane association of viral proteins include amphipathic helices and transmembrane hydrophobic sequences for animal viruses such as poliovirus (van Kuppeveld et al., 1996; Paul et al., 1994), hepatitis C virus (Brass et al., 2002) and a plant virus (tomato ringspot virus; Zhang et al., 2005). The involvement of hydrophobic amphipathic helices and/or transmembrane segments was also predicted on the basis of sequence similarities for other viruses, i.e. cowpea mosaic virus (Carette et al., 2002), BMV (den Boon et al., 2001) and tobacco mosaic virus (dos Reis Figueira et al., 2002).
Using a prediction method for monotopic proteins inserted and anchored parallel to the membrane (in-plane membrane anchors, IPM; Sapay et al., 2006), no such sequence segments were found in CymRSV p33. Further computer analysis of CymRSV p33, performed using the SOPM computer program for secondary structure prediction (Combet et al., 2000) and the ANTHEPROT program (Deleage et al., 2001) for prediction and projection of the putative amphipathic helices, showed the occurrence of an amphipathic helix between residues 52 and 64, which may be involved in the peripheral interactions of p33 with the ER membranes.
In plant cells infected by several tombusviruses, p33 is targeted to the peroxisomal membrane, where it interacts with p92, viral RNA and perhaps host factors to form an active replication complex as shown experimentally for tomato bushy stunt virus (TBSV) (Panaviene et al., 2004; Rajendran & Nagy, 2004) and elicits the formation of the characteristic flask-shaped vesicles constituting the multivesicular bodies (MVB) (Martelli et al., 1988). The involvement of ER in the formation of MVB was suggested by Russo et al. (1983) and demonstrated by McCartney et al. (2005). The latter authors demonstrated the occurrence of an indirect ER targeting signal operating in a peroxisome-to-ER pathway, followed by retrieval of ER membranes to peroxisomes. We suggest that the pair of positively charged residues (K5R6) in the N-terminal region identified by McCartney et al. (2005) is also likely to contribute to the formation of an ER targeting signal in yeast cells in the absence of peroxisomes, indicating that the preliminary localization of p33 to the peroxisomal membrane is not a necessary step for targeting to ER. Rather, the ER membranes could be the initial target for CymRSV replicase proteins prior to trafficking to peroxisomes, in the absence of which the ER-to-peroxisome pathway would be blocked. However, this event does not affect virus replication since the ER membranes were shown to be fully competent for the assembly of the virus replication complex, in alternative to the peroxisomal membrane.
Finally, it is worth noting the adaptability of plus-strand RNA viruses to use diverse types of membranes if alterations are forced in the structure of their replicase (Burgyan et al., 1996; Miller et al., 2003; Schwartz et al., 2004) or when the organelle content of the host cell is modified (this paper). Furthermore, we show that replicase proteins launched by non-replicating mRNAs, which cannot themselves evolve or adapt at the amino acid level, have enough plasticity to adjust without any change in their structural properties. Consequently, in wt virus infections, where mRNA and protein evolution could occur, adaptation may be even greater. This fact may not support an antiviral strategy based on the manipulation of the structure of a particular membrane by pharmacological or genetic means, as suggested by Lee & Ahlquist (2003).
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ACKNOWLEDGEMENTS |
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Received 21 November 2006;
accepted 7 January 2007.
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