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Short Communication |
Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, Via Amendola 165/A, 70126 Bari, Italy
Correspondence
Marcello Russo
csvvmr01{at}area.ba.cnr.it
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ABSTRACT |
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Supplementary figures showing a representative Northern blot of DI RNA and mean relative accumulation of DI RNA are available in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, 2001; Weber-Lotfi et al., 2002). When expressed in the yeast Saccharomyces cerevisiae, p36 and p95 support the replication of defective interfering (DI) RNA, and the absence of either abolishes replication (Pantaleo et al., 2003). In the present investigation, the yeast system was utilized in an attempt to define the role of each of these viral proteins in stabilizing, targeting and replicating the DI RNA template.
To analyse the distribution and expression of p36 and p95 independently of each other, the c-Myc and HA tags were fused in-frame to the N terminus of each. To do so, p36 and p95 coding sequences inserted in plasmids pA36K and YE95K (Pantaleo et al., 2003) were mutated by PCR to contain, at their 5' termini, the c-Myc and HA coding sequences, respectively. Both also contained the ADH1 promoter and terminator, the 2 µm origin of replication and either the HIS3 or the LEU2 selectable marker. S. cerevisiae (strain YPH499) cells were co-transformed with these plasmids, separately or together, or with empty vectors, by using the lithium acetate/polyethylene glycol method (Ito et al., 1983). Transformants (designated p36+p95+, p36–p95+, p36+p95– and p36–p95–) were grown at 26 °C in selective medium containing 3 % glycerol/0·1 % glucose to mid-exponential phase (OD600 0·6–0·8). The cells were then fixed with formaldehyde (Restrepo-Hartwig & Ahlquist, 1999) and immunolabelled with anti-HA or anti-c-Myc mAbs (Santa Cruz Biotechnology) and polyclonal antisera to Tom40p (Baker et al., 1990), Kar2p (Rose et al., 1989) or Emp47p (Schröder et al., 1995), which are marker proteins for mitochondria, endoplasmic reticulum (ER) and the Golgi apparatus, respectively. Fluorescence images were obtained with a Leica TCS SP2 confocal laser-scanning microscope using a x63 objective lens and FITC and TRITC lasers.
Fig. 1(a) shows a representative sample of p36+p95+ cells illustrating the exclusive mitochondrial localization of both p36 and p95. Identical results were obtained with cells expressing either protein alone (not shown). To evaluate the competence of the tagged proteins to replicate a DI RNA, other transformants were prepared expressing, in addition to the replicase proteins, a DI RNA of 481 nt (Burgyan et al., 1992). The DI RNA sequence was cloned into plasmid pB3MI3S containing the galactose-inducible GAL1 promoter and TRP1 as a selectable marker (Ishikawa et al., 1997; Pantaleo et al., 2003). These transformants were first induced with 2 % galactose and then grown in a repressive medium containing glucose (Pantaleo et al., 2003). DI RNA replication was evaluated by Northern blot analysis (Pantaleo et al., 2003) and in situ hybridization using a mixture of specific digoxigenin (DIG)-labelled oligonucleotide probes complementary to the positive-strand DI RNA. Detection was with an anti-DIG mAb according to the method of Restrepo-Hartwig & Ahlquist (1999). Northern blot analysis showed that N-terminally tagged p36 and p95 supported DI RNA replication to a level equivalent to that supported by wild-type proteins (supplementary Fig. S1). In situ hybridization showed that DI RNA progeny was mostly associated with the mitochondrial marker Tom40p (Fig. 1b, upper row, lanes 1–3) but was never associated with the ER or Golgi apparatus (Fig. 1b, upper row, lanes 4–6 and 7–9, respectively). Unlike replicase proteins (Fig. 1a), sometimes the accumulation of DI RNA did not coincide with the Tom40p signal (Fig. 1b, upper row, lane 3, arrows), suggesting that the newly synthesized DI RNA gradually accumulated in clusters separated from, but still proximal to, the mitochondria.
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; Chen & Ahlquist, 2000; Pantaleo et al., 2003). Western blot analysis showed that p36 and p95 sedimented in the pellet with the mitochondrial protein YHM2 (Cho et al., 1998), regardless of whether they were expressed singly or together (Fig. 2a). Northern blot analysis showed that about one-third of the total amount of DI RNA was in the membrane-enriched pellet (Fig. 2b, lane 4) and the rest was in the supernatant (Fig. 2b, lane 3), as estimated with Quantity One software (Bio-Rad). This confirmed that part of the DI RNA progeny was no longer associated with mitochondria, consistent with the immunofluorescence microscopy results, where part of the DI RNA was not associated with membranes (Fig. 1b
, lane 3). At the short photographic exposure of the autoradiograph of Fig. 2(b), no signal other than that in lanes 3 and 4 was detected. However, a longer exposure revealed a weak signal in both pellet and supernatant of samples p36+p95– and p36–p95+, but no signal at all in sample p36–p95– (not shown).
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; Russo et al., 1994; Havelda & Burgyan, 1995; Pantaleo et al., 2003). In this way the fate of template RNA could be discriminated from that of progeny RNA. p36–p95–, p36+p95–, p36–p95+ and p36+p95+ yeast cells were grown, disrupted, fractionated as above and analysed by Northern blotting to monitor the expression and localization of DI RNA. No DI RNA transcripts were found in extracts of cells grown when both viral proteins were absent (Fig. 2c, lanes 5 and 6), whereas a weak DI RNA signal was observed in both supernatant and pellet in the absence of either p36 or p95 only (Fig. 2c, lanes 1 and 2, and 7 and 8, respectively). When both proteins were expressed, a much larger amount of non-replicable DI RNA template was observed, particularly in association with the pellet (Fig. 2c, lane 4). A possible conclusion is that, although template RNA stabilization and targeting may be mediated by either p36 or p95, these proteins have a synergistic effect when co-expressed. Indeed, RNA-binding domains were predicted in CIRV p36 and p95 by sequence comparison with the related Tomato bushy stunt virus (TBSV) (Rajendran & Nagy, 2003). However, it was not possible to establish from the available data whether some host factors participated in binding and recruiting the template DI RNA in yeast cells.
To analyse further the contributions of p36 and p95 to DI RNA stabilization to the CIRV replicase membrane-bound complex, experiments were done in which the two viral proteins were modulated. The ADH1 promoter in plasmids pA36K and YE95K was substituted by the yeast CUP1 promoter, which results in rapid induction after exposure to copper, the required level of induction depending primarily on the copper resistance of the host (Mascorro-Gallardo et al., 1996). Since preliminary observations indicated no adverse effect on yeast cell growth in terms of doubling time at a copper concentration up to 500 µM, concentrations no higher than 250 µM were used in all experiments that followed (Fig. 3 and supplementary Fig. S2). The influence of increasing p36 or p95 concentration on the stability of DI RNA transcripts was analysed after repression of the GAL1 promoter. As shown in Fig. 3(a), lanes 6–10, transcripts were detected in extracts of cells expressing p95 even in the absence of added copper, possibly because of the activity of the CUP1 promoter in the presence of a trace amount of copper in the growth medium. Conversely, when p36 was modulated, DI RNA transcripts (Fig. 3a, lanes 1–5) were detected only at the highest copper concentration, albeit only at a concentration of 
50 %, the amount associated with the lowest quantity of p95 (no added copper) (Fig. 3a, compare lanes 5 and 6). This was taken as an indication that more p36 than p95 molecules are necessary for the efficient stabilization of DI RNA.
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, upper left panel). Northern blot analysis showed that the accumulation of positive-strand DI RNA was detectable only at copper concentrations of 250 µM (Fig. 3b, lower left panel), although viral proteins were synthesized at lower copper concentrations (Fig. 3b, left panels). In ADHp36–CUPp95 cells, there was a progressive increase in p95 expression starting from no copper (Fig. 3b, upper middle panel), associated with a progressive increase in DI RNA accumulation (Fig. 3b, lower middle panel). This suggested that the RdRp activity of p95 increases up to 200-fold if compared with the activity in the absence of copper (Fig. 3b, lower middle panel, compare lanes 10 and 6). In contrast, in CUPp36–ADHp95 cells, the ADH1-promoter-mediated level of p95 was unable to support DI RNA replication unless p36 was expressed at 250 µM copper (Fig. 3b, right panels), thus reproducing what was observed in CUPp36–CUPp95 cells (Fig. 3b, left panels). In conclusion, a higher level of synthesis of p36 than of p95 is required for DI RNA replication in yeast, in line with the finding that in plant cells about 20 times as much p36 as p95 is synthesized (Lupo et al., 1994).
This paper, together with our previous results (Pantaleo et al., 2003), confirms that two replicase proteins are required for replication of tombusviruses (Russo et al., 1994). Whereas p95 function is explained by the presence of polymerase motifs, the function of p36 remains unclear, but when p36 is expressed together with p95, it dramatically increases the recruitment and stabilization of DI RNA transcripts. However, p36 exerts its activity only after reaching a threshold below which the protein is unable to stabilize template RNA. This is in line with recent in vitro studies on TBSV showing the ‘all-or-none’ behaviour of the ORF1 protein (p33) of this virus (Rajendran & Nagy, 2003). CIRV replicase proteins are directly involved in the stabilization and recruitment of template RNA, similar to replication proteins 1a and 2a of Brome mosaic virus (BMV) but with some important differences. First, the two BMV proteins are totally different from each another and play a different role in replication, since 1a contains the RNA capping and helicase domains, whereas 2a has the conserved domain of RdRps. Second, only protein 1a is able to stabilize template RNA and to target it, together with 2a, to the replication site (the ER) (Restrepo-Hartwig & Ahlquist, 1996, 1999; Chen & Ahlquist, 2000). In contrast, CIRV p36 is part of p95 and does not contain any conserved motif allowing identification of its role. We thus suggest that the tombusvirus ORF1 product plays a key role in stabilizing and targeting template RNA to the replication site in conjunction with the ORF2 product. However, it remains to be established whether recruitment of template RNA is done solely by viral proteins or requires the intervention of host factors. In particular, for the latter aspect, the use of the yeast system may be of help.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Burgyan, J., Dalmay, T., Rubino, L. & Russo, M. (1992). The replication of cymbidium ringspot tombusvirus defective interfering-satellite RNA hybrid molecules. Virology 190, 579–586.[CrossRef][Medline]
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Schröder, S., Schimmoller, F., Singer-Kruger, B. & Riezman, H. (1995). The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP. J Cell Biol 131, 895–912.
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Received 1 March 2004;
accepted 22 April 2004.
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