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Short Communication |
Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia (CSIC), Av. de los Naranjos s/n, 46022 Valencia, Spain
Correspondence
Vicente Pallás
vpallas{at}ibmcp.upv.es
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ABSTRACT |
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; Van Der Heijden et al., 2001), the 2a polymerase and the movement protein (MP) of Cucumber mosaic virus (Hwang et al., 2005), the MP and coat protein (CP) of AMV (Sanchez-Navarro et al., 2006), Maize streak virus (Liu et al., 2001) and Cowpea mosaic virus (Pouwels et al., 2004), the helper component protein and CP of Lettuce mosaic virus (Roudet-Tavert et al., 2002) or CP dimerization of AMV (Choi & Loesch-Fries, 1999; Tenllado & Bol, 2000) and Cowpea chlorotic mottle virus (Zhao et al., 1995). Determination of these interactions has been mainly performed by in vitro approaches, such as overlay assays, co-immunoprecipitation and glutathione S-transferase pull-down, or by in vivo techniques as such the two hybrid system or fluorescence resonance energy transfer.
Recently, bimolecular fluorescence complementation analysis (BiFC) has become a useful alternative technique to study protein–protein interactions in living cells (Hu et al., 2002). BiCF is based on the complementation between two non-fluorescent fragments of the enhanced yellow fluorescent protein (EYFP), when each is fused to a different candidate protein where the reconstitution of fluorescence takes place only when the proteins fused to these fragments interact. This approach allows the direct real-time visualization of the protein complex under physiological conditions. Until now, BiFC has been mainly used to study interactions among transcription factors from animal, plant and fungal origin in living eukaryotic and bacterial cells (Hu et al., 2002; Walter et al., 2004; Bracha-Drori et al., 2004; Hynes et al., 2004; Wilson et al., 2004; Hoff & Kück, 2005). In the present work, we used BiCF to study the putative CP dimerization capacity of Prunus necrotic ringspot virus (PNRSV) and to map the interaction domain of the PNRSV CP.
PNRSV is a positive-strand RNA plant virus with a tripartite genome that belongs to the genus Ilarvirus. Ilarviruses have the same genome organization, encoding functionally similar translation products, as those of AMV and members of the genera Bromovirus, Cucumovirus and Oleavirus, which belong to the family Bromoviridae. RNAs 1 and 2 encode the replicase subunits P1 and P2, respectively. RNA 3 is translated into the MP, whereas the CP is synthesized from a subgenomic RNA 4. Both MP and CP have RNA-binding properties (Pallás et al., 1999; Aparicio et al., 2003; Herranz & Pallas, 2004; Herranz et al., 2005). Binding of the CP to the 3' non-translated (3'-NTR) region of viral RNAs is a crucial requirement to establish the infection of AMV and ilarviruses (reviewed by Bol, 2005).
PNRSV and AMV are phylogenetically closely related (Sánchez-Navarro & Pallás, 1997; Codoñer et al., 2004; Codoñer & Elena, 2006). Most studies on the implication of CP in the viral cycle have been conducted in AMV, whereas there are relatively few experimental data reporting PNRSV CP structural properties and biological functions. PNRSV CP has the capacity to bind to the 3'-NTR of its RNA 4 (Pallás et al., 1999) and can substitute all the AMV CP functions in the replication cycle of a chimeric AMV RNA 3 (Sánchez-Navarro et al., 1997). The RNA-binding domain of PNRSV CP is located at the basic N-terminal region of the CP. We previously demonstrated that CP binding to the 3'-NTR regulates the conformation of the RNA and that the replicase complex of AMV recognizes the 3'-NTR of PNRSV RNA 3, suggesting a similar regulatory mechanism at the 3'-NTR level in AMV and the genera Ilarvirus (Aparicio et al., 2001, 2003). In AMV, CP is involved in the asymmetric plus-strand RNA accumulation, viral RNA translation, virion formation, cell-to-cell movement and the systemic spread of the virus (reviewed by Bol, 2005). In solution, AMV CP occurs as dimers (Kruseman et al., 1971) and CP dimerization is required to obtain wild-type replication level in protoplasts (Choi et al., 2003) to efficiently stimulate RNA translation (Neeleman et al., 2004) and systemic movement (Tenllado & Bol, 2000).
To characterize further the PNRSV CP structural domains, in the present study, we mapped the PNRSV CP region involved in CP–CP interaction by the BiCF approach in both bacteria and intact plant tissues.
To perform BiFC analysis in bacteria, we designed a bacterial-expression strategy that enables the co-expression of bait and prey in the same plasmid. For this purpose, we chose the pETDuet plasmid (Novagen) as the bacterial protein expression vector. This plasmid has two bacteriophage T7 transcription promoters, two multicloning sites (MCS) and a single T7 terminator for the co-expression of two target open reading frames (ORFs). Fig. 1(a) shows a schematic representation of the steps followed to create the fusion constructs. Firstly, fragments corresponding to the N terminus (aa 1–154) and C terminus (aa 155–238) of the EYFP were PCR-amplified and cloned into the MCS to generate pNY : : CY construct (Fig. 1a). Then, viral ORFs were amplified by PCR and fused to the NYFP (NY) and/or CYFP (CY) sequences. All fusion proteins contain a 10 aa spacer corresponding to part of the MCS sequence to facilitate the configurational compatibility between bait and prey YFP fusions (Fig. 1b). All constructs were verified by DNA sequencing. Transformed Escherichia coli BL21 (DE3) cells were plated in Luria–Bertani medium with the appropriate antibiotic, and protein expression was achieved with the addition of 1 mM IPTG. Plates were incubated for 48 h at 25 °C and reconstitution of YFP fluorescence was observed in single colonies by confocal laser scanning microscopy (CLSM).
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). According to these results, we fused either the full-length or a mutated AMV CP gene lacking the C-terminal 40 aa (
40CP), to the split YFP fragments, generating constructs pNY-CPa : : CY-CPa and pNY-CPa : : CY-40CPa, respectively (Fig. 1b
). An additional pNYstop : : CYstop construct was created to confirm the lack of fluorescence by self-rearrangement of the split YFP fragments (Fig. 1b). No fluorescence signal was observed with the pNYstop : : CYstop construct, indicating that NY and CY fragments themselves did not render self-interaction (Fig. 2a, panel 1). As expected, the pNY-CPa : : CY-CPa construct showed a clear fluorescence signal, whereas the pNY-CPa : : CY-40CPa plasmid did not (Fig. 2a, panels 2 and 3, respectively). In order to confirm that the absence of fluorescent signal was due to the abolition of AMV CP–40CP interaction rather than to an altered protein expression, a Western blot analysis was carried out from total protein extracts. As shown in Fig. 2(b), fusion proteins were clearly detected using an antibody against the CP of AMV (Fig. 2b, panel AMV, lanes 2 and 3). These results demonstrate that this BiFC system can be used to analyse in vitro viral protein–protein interactions.
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). As can be observed in Fig. 2(a), there was a clear fluorescence signal in bacterial cells transformed with this construct (panel 4), indicating a strong CP–CP interaction. The involvement of the C-terminal arm of CP in dimerization has been reported for several viruses in the family Bromoviridae (Zhao et al., 1995; Choi & Loesch-Fries, 1999; Tenllado & Bol, 2000). Thus, it was reasonable to hypothesize that the C-terminal portion of the PNRSV CP might be the region involved in the CP–CP interaction. A series of CP mutants was generated to analyse the role that this region plays in dimer formation. PNRSV CP genes lacking the C-terminal 9, 18 and 27 aa were exchanged with the corresponding full-length CP gene fused to the CYFP fragment in the plasmid pNY-CPp : : CY-CPp. The resultant pNY-CPp : : CY-9CPp, pNY-CPp : : CY-18CPp and pNY-CPp : : CY-27CPp constructs (Fig. 1b) were electroporated into BL21 (DE3) cells, and the fluorescence was monitored by CLSM. In relation to the fluorescence observed using the plasmid pNY-CPp : : CY-CPp (Fig. 2a, panel 4), signal intensity was maintained in cells transformed with pNY-CPp : : CY-9CPp, whereas it decreased in pNY-CPp : : CY-18CPp, and it totally disappeared in the pNY-CPpnrsv : : CY-27CPp construct (Fig. 2a, panels 5, 6 and 7, respectively). Western blot analysis using an antiserum against the PNRSV CP showed that all mutant CPs were expressed in bacterial cells (Fig. 2b, panel PNRSV, lanes 4, 5, 6 and 7), indicating that a lack of fluorescence signal was unrelated to protein accumulation levels but that it was in fact related to loss of interaction capacity. These results suggest that (i) the C-terminal arm of PNRSV CP plays a critical role in CP–CP interaction and (ii) the domain responsible for the interaction is located between aa residues 9 and 27 from the C-end of the protein. In AMV it has been reported that, in addition to the C-terminal 21 residues, the tryptophan at position 29 (from the C terminus) is also critical for dimer stabilization by a putative intermolecular interaction mediated via aromatic residues (Choi & Loesch-Fries, 1999). Interestingly, a tyrosine at position 25 of PNRSV CP (from the C terminus; data not shown) is present in a similar position included in the domain described here for CP–CP interaction. In addition, the C-terminal portion of the PNRSV CP sequence is the highest conserved region in the protein among the different isolates sequenced so far (Aparicio et al., 1999), thus supporting the function described here for this region. Finally, and by considering the close relationship between AMV and PNRSV, we generated the pNY-CPpnrsv : : CY-CPamv construct in order to investigate putative heterologous interactions between both CPs. No reconstitution of YFP fluorescence was observed (Fig. 2a, panel 8), although a significant protein expression level of both fusion proteins was detected by Western blot analysis (Fig. 2b, panel AMV+PNRSV, lane 8). Apparently, AMV and PNRSV CPs are unable to interact with each other in spite of the fact that both proteins are interchangeable for different viral processes (Sánchez-Navarro et al., 1997). A plausible explanation for this unexpected observation could rely on the different forms of the virus particles that AMV and PNRSV adopt. Whereas AMV has bacilliform particles, PNRSV forms icosahedral virions. It would be interesting to check if this potential interaction occurs with other true ilarviruses closely related to PNRSV like Apple mosaic virus or Prune dwarf virus.
The BiCF technique offers the possibility of analysing protein interactions in living plant cells (Walter et al., 2004; Bracha-Drori et al., 2004). We decided to analyse the different PNRSV CP–CP interactions in intact plant tissue. For this purpose, the CP fusion proteins were cloned between the cauliflower mosaic virus 35S promoter and the NOS transcriptional terminator in the pMOG800 binary vector (Fig. 1c). Reconstitution of YFP fluorescence was determined by transient co-expression of the desired protein pairs. Nicotiana benthamiana leaves were infiltrated as described previously (Bendahmane et al., 2000) and examined by CLSM at 48 h following infiltration. The results obtained consistently reproduced the pattern generated in bacteria. No fluorescent signal was displayed in leaves that were co-infiltrated with p35S : NY plus p35S : CY cells, indicating the inability of the split YFP fragments to reconstitute YFP fluorescence in vivo (Fig. 3a, panel 1). Leaves co-infiltrated with p35S : NY-CPp plus p35S : CY-CPp and p35S : NY-CPp plus p35S : CY-9CPp rendered a strong YFP fluorescence signal in the cells (Fig. 3a, panels 2 and 3, respectively), whereas a lower intensity signal was detected in leaves co-expressing p35S : NY-CPp and p35S : CY-18CPp (Fig. 3, panel 4). Finally, no YFP reconstitution fluorescence was detected in leaves co-infiltrated with the pair p35S : NY-CPp plus p35S : CY-27CPp (Fig. 3a, panel 5). Analysis of the spectrum properties of the fluorescent signals derived from the PNRSV NY-CP : : CY-CP, NY-CP : : CY-9CP and NY-CP : : CY-18CP fusion protein interactions showed the typical YFP fluorescence spectrum, which reached a peak around 527 nm (Fig. 3a). Moreover, Western blot analysis against PNRSV CP showed that all fusion proteins accumulated at detectable levels in the infiltrated tissues (Fig. 3b). All these results indicate that YFP fluorescence resulted from protein–protein interactions.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aparicio, F., Sanchez Navarro, J. A., Olsthoorn, R. C., Pallás, V. & Bol, J. F. (2001). Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus. J Gen Virol 82, 947–951.
Aparicio, F., Vilar, M., Perez-Paya, E. & Pallás, V. (2003). The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology 313, 213–223.[CrossRef][Medline]
Bendahmane, A., Querci, M., Kanyuka, K. & Baulcombe, D. C. (2000). Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J 21, 73–81.[CrossRef][Medline]
Bol, J. F. (2005). Replication of alfamo- and ilarviruses: role of the coat protein. Annu Rev Phytopathol 43, 39–62.[Medline]
Bracha-Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R., Yalovsky, S. & Ohad, N. (2004). Detection of protein–protein interactions in plants using bimolecular fluorescence complementation. Plant J 40, 419–427.[CrossRef][Medline]
Choi, J. W. & Loesch-Fries, L. S. (1999). Effect of C-terminal mutations of alfalfa mosaic virus coat protein on dimer formation and assembly in vitro. Virology 260, 182–189.[CrossRef][Medline]
Choi, J., Kim, B. S., Zhao, X. & Loesch-Fries, S. (2003). The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication. Virology 305, 44–49.[CrossRef][Medline]
Codoñer, F. M. & Elena, S. F. (2006). Evolutionary relationships among members of the Bromoviridae deduced from whole proteome analysis. Arch Virol 151, 299–307.[CrossRef][Medline]
Codoñer, F. M., Cuevas, J. M., Sánchez-Navarro, J. A., Pallás, V. & Elena, S. F. (2005). Molecular evolution of the plant virus family Bromoviridae based on RNA3-encoded proteins. J Mol Evol 61, 697–705.[CrossRef][Medline]
Herranz, M. C. & Pallas, V. (2004). RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus. J Gen Virol 85, 761–768.
Herranz, M. C., Sanchez-Navarro, J. A., Sauri, A., Mingarro, I. & Pallas, V. (2005). Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement. Virology 339, 31–41.[CrossRef][Medline]
Hoff, B. & Kück, U. (2005). Use of bimolecular fluorescence complementation to demonstrate transcription factor interaction in nuclei of living cells from the filamentous fungus Acremonium chrysogenum. Curr Genet 47, 132–138.[CrossRef][Medline]
Hu, C. D., Chinenov, Y. & Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9, 789–798.[CrossRef][Medline]
Hwang, M. S., Kim, S. H., Lee, J. H., Bae, J. M., Paek, K. H. & Park, Y. I. (2005). Evidence for interaction between the 2a polymerase protein and the 3a movement protein of Cucumber mosaic virus. J Gen Virol 86, 3171–3177.
Hynes, T. R., Tang, L., Mervine, S. M., Sabo, J. L., Yost, E. A., Devreotes, P. N. & Berlot, C. H. (2004). Visualization of G protein 
dimers using bimolecular fluorescence complementation demonstrates roles for both and in subcellular targeting. J Biol Chem 279, 30279–30286.
Kruseman, J., Kraal, B., Jaspars, E. M. J., Bol, J. F., Brederode, F. T. & Veldstra, H. (1971). Molecular weight of the coat protein of alfalfa mosaic virus. Biochemistry 10, 447–455.[CrossRef][Medline]
Liu, H., Boulton, M. I., Oparka, K. J. & Davies, J. W. (2001). Interaction of the movement and coat proteins of Maize streak virus: implications for the transport of viral DNA. J Gen Virol 82, 35–44.
Neeleman, L., Linthorst, H. J. M. & Bol, J. F. (2004). Efficient translation of alfamovirus RNAs requires the binding of coat protein dimers to the 3' termini of the viral RNAs. J Gen Virol 85, 231–240.
O'Reilly, E. K., Tang, N. J., Ahlquist, P. & Kao, C. C. (1995). Biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus. Virology 214, 59–71.[CrossRef][Medline]
Pallás, V., Sanchez-Navarro, J. A. & Diez, J. (1999). In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses. Arch Virol 144, 797–803.[CrossRef][Medline]
Pouwels, J., van der Velden, T., Willemse, J., Borst, J. W., van Lent, J., Bisseling, T. & Wellink, J. (2004). Studies on the origin and structure of tubules made by the movement protein of Cowpea mosaic virus. J Gen Virol 85, 3787–3796.
Roudet-Tavert, G., German-Retana, S., Delaunay, T., Delecolle, B., Candresse, T. & Le Gall, O. (2002). Interaction between potyvirus helper component-proteinase and capsid protein in infected plants. J Gen Virol 83, 1765–1770.
Sánchez-Navarro, J. A. & Pallás, V. (1997). Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3. Arch Virol 142, 749–763.[CrossRef][Medline]
Sánchez-Navarro, J. A., Reusken, C. B., Bol, J. F. & Pallás, V. (1997). Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus. J Gen Virol 78, 3171–3176.[Abstract]
Sánchez-Navarro, J. A., Herranz, M. C. & Pallás, V. (2006). Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses, and does not require virion formation. Virology 346, 66–73.[CrossRef][Medline]
Tenllado, F. & Bol, J. F. (2000). Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement. Virology 268, 29–40.[CrossRef][Medline]
Van Der Heijden, M. W., Carette, J. E., Reinhoud, P. J., Haegi, A. & Bol, J. F. (2001). Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane. J Virol 75, 1879–1887.
Walter, M., Chaban, C., Schutze, K. & 9 other authors (2004). Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40, 428–438.[CrossRef][Medline]
Wilson, C. G., Magliery, T. J. & Regan, L. (2004). Detecting protein-protein interactions with GFP-fragment reassembly. Nat Methods 1, 255–262.[CrossRef][Medline]
Zhao, X., Fox, J. M., Olson, N. H., Baker, T. S. & Young, M. J. (1995). In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA. Virology 207, 486–494.[CrossRef][Medline]
Received 17 November 2005;
accepted 13 February 2006.
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