The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA

Vitantonio Pantaleo, Luisa Rubino and Marcello Russo

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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The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an ‘all-or-none’ behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

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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Carnation Italian ringspot virus (CIRV) is a member of the genus Tombusvirus (family Tombusviridae). The 4·8 kb genome is a linear single-stranded, monopartite RNA molecule of positive polarity containing five ORFs. The 5'-proximal ORF (ORF1) encodes a 36 kDa protein (p36) and ORF2 encodes a 95 kDa protein (p95), translated by readthrough of the p36 stop codon. p36 contains a signal targeting to the outer mitochondrial membrane and p95 additionally contains the conserved motifs of RNA-dependent RNA polymerases (RdRps) (Rubino et al., 1995, 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+, p36p95+, p36+p95 and p36p95) 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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Fig. 1. CIRV replicase proteins p36 and p95 colocalize to mitochondria and DI RNA progeny accumulates predominantly but not exclusively at the sites of localization of p36 and p95. (a) Cells expressing p36 and p95 were fixed and immunolabelled using either anti-HA (upper row) or anti-c-Myc mouse antibodies (lower row) and yeast mitochondrial protein Tom40p (left), ER protein Kar2p (middle) or Golgi protein Emp47p (right) rabbit antibodies followed by anti-mouse antibodies conjugated to Alexa 488 and anti-rabbit antibodies conjugated to Rhodamine Red-X (Molecular Probes). (b) Cells expressing DI RNA with (upper row) or without (lower row) p36 and p95 were hybridized with a mixture of DIG-labelled oligodeoxynucleotides complementary to four regions of DI RNA and processed for double-labelled immunofluorescence using primary antisera against DIG and Tom40p, Kar2p or Emp47p and secondary antibodies as above. Arrows point to DI RNA accumulations not coinciding with the mitochondrial marker Tom40p (upper row, left). In both panels, selected images of each label and their superimposition are shown (overlay). Bar, 1 µm.

 
The occurrence of some DI RNA in areas distinct from mitochondria suggests that these progeny molecules may no longer be associated with membranes, in contrast to those still engaged in replication. To verify this point, p36p95+, p36+p95, p36p95 and p36+p95+ yeast cells, which also express DI RNA, were first induced with galactose and then grown for 16 h in the presence of glucose (thus blocking the GAL1 promoter). The cells were then spheroplasted, lysed and fractionated by centrifugation at 10 000 g into a membrane-enriched pellet and supernatant (Russell et al., 1991; 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 p36p95+, but no signal at all in sample p36p95 (not shown).



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Fig. 2. Analysis of p36, p95 and DI RNA distribution by cell fractionation. Yeast cells expressing either p36 or p95, or both, and replicating (a, b) or non-replicating (c) DI RNA were extracted. The extracts were centrifuged to yield pellet (Pell.) and supernatant (Sup.) fractions and analysed by Western (a) or Northern (b, c) blotting. Immunoblot analysis was done with antibodies against the c-Myc or HA tags and the mitochondrial marker YHM2; Northern blot analysis was with a 32P-labelled riboprobe complementary to DI RNA. Proteins accumulated essentially with the pellet fraction (a), whereas a consistent portion of replicating DI RNA was also detected in the supernatant (b). Non-replicating DI RNA was stabilized by p36 and p95, especially when both were synthesized (c). The consistent accumulation in the pellet suggested that the plasmid-DNA-based transcripts are driven to mitochondrial membranes on their passage to the cytoplasm from the nucleus. Autoradiographs were exposed for 4 h (b) or 3 days (c).

 
To avoid the disturbing effect of the strong signal resulting from the DI RNA progeny, the stabilizing and targeting effect of the simultaneous expression of p36 and p95 was evaluated in the absence of replication by using non-replicating template RNA in which the G residue at position –4 from the 3' end was mutated to A (Dalmay et al., 1993; 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. p36p95, p36+p95, p36p95+ 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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Fig. 3. Effect of increasing expression of p36 and/or p95 on the stability of non-replicating DI RNA (a) and on the replication of wild-type DI RNA (b). Protein synthesis was regulated through the constitutive ADH1 promoter or the CUP1 promoter, which was induced with tenfold increasing concentrations of copper (CuSO4), and was analysed by Western blotting (upper rows). DI RNA was detected by Northern blot analysis (lower rows). (a) Only the highest level of p36 expressed had a detectable effect on DI RNA transcripts (left panel), whereas p95 exerted its stabilizing effect proportionally, beginning with barely detectable amounts (right panel). (b) Replication of DI RNA was detected only at the highest expression of p36, regardless of the level of p95, whether modulated by the CUP1 promoter (left panel) or regulated at constant level by the ADH1 promoter (right panel). When the p36 concentration was kept constant, accumulation of DI RNA depended on the p95 level (middle panel). Autoradiographs were exposed for 3 days (a) or 4 h (b). For quantification analysis, see supplementary Fig. S2.

 
To evaluate the relative contribution of p36 and p95 to replication, three combinations of yeast transformants were analysed, all expressing DI RNA under the control of the GAL1 promoter. p36 and p95 were (i) both under the control of the CUP1 promoter (designated CUPp36–CUPp95); or (ii) one was under the control of CUP1 and the other was under the control of the ADH1 promoter (ADHp36–CUPp95 or CUP36–ADH95). In this way, both proteins or either could be modulated by copper concentration. Western blot analysis of protein extracts from CUPp36–CUPp95 yeast cells showed that p36 and p95 levels increased simultaneously (Fig. 3b, 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.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Drs L. Palmieri, M. D. Rose and H. D. Schmitt for gifts of antibodies to YHM2, Kar2 and Emp47, respectively; Dr P. Ahlquist for a gift of plasmid pSAL1, source of the CUP1 promoter sequence; Mrs C. Capobianco for skilful technical assistance with confocal microscopy; and Professor G. P. Martelli, Drs B. Navarro and L. Stavolone for advice and critical reading of the manuscript. This research was partially supported by MIUR, Project Cluster CO3, Legge 488/92, ‘Studi di geni di interesse biomedico e agroalimentare’.


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Received 1 March 2004; accepted 22 April 2004.