Instituto de Biología Molecular y Celular de Plantas (IBMCP), UPV-CSIC, Avda 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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INTRODUCTION |
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MPs of plant viruses are involved in viral nucleic acid transit through plasmodesmata. At least five different types of MP have been described: the products of the triple gene block of potexviruses and related viruses, the tymovirus MPs, a series of small polypeptides (less than 10 kDa) encoded by carmo-like viruses and some geminivirus, the hsp70-like proteins of closterovirus and the well characterized 30K superfamily, related to the 30 kDa Tobacco mosaic virus (TMV) MP. Comparative sequence analyses have assigned PNRSV MP to the 30K superfamily (Mushegian & Koonin, 1993), a meaningful grouping of proteins related in sequence and structure (Melcher, 2000
) whose members are characterized by presenting a motif of 30 highly conserved amino acids, which may comprise a hydrophobic interaction domain (Mushegian & Koonin, 1993
). Previous sequence comparison analysis (Sánchez-Navarro & Pallás, 1997
) has revealed that AMV and ilarvirus MPs have a basic region preceding the 30K motif, with a high surface probability, which makes them good candidates for RNA-binding interactions, and a transmembrane domain within the 30K motif, which could be involved in targeting of the MP to the cell wall (Berna, 1995
; van der Wel et al., 1998
). Molecular variability studies among different PNRSV isolates has revealed an absence of genetic diversity within the proposed RNA-binding domain (Aparicio & Pallás, 2002
).
In this study, we have demonstrated the RNA-binding properties of the PNRSV MP, which have not been previously described for any member of the Ilarvirus genus. In addition, the RNA-binding domain of this protein was mapped to aa 5688, located at the N terminus of the MP. Interestingly, the RNA-binding domains of Alfamo- and Ilarvirus are located at the N terminus, whereas those of Bromo- and Cucumovirus are at the C terminus of the MP. Although no significant sequence homology was found among these RNA-binding domains, they share several important features that could reflect a common origin.
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METHODS |
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Nucleic acid binding assay.
ProteinRNA binding studies were performed by EMSA. Plus-strand PNRSV RNA 4 was generated by in vitro transcription of a full-length cDNA copy (Sánchez-Navarro & Pallás, 1994). The plasmid was linearized with SacI and transcribed with T3 RNA polymerase as previously described (van der Kuyl et al., 1991
). For the EMSA, 5 ng of plus-strand PNRSV RNA 4 transcript (mixed with either equal or with tenfold mass excess of competitors, as specified) were heated for 5 min at 85 °C and cooled at room temperature for 15 min. Various amounts of purified PNRSV MP (600 ng of MP for salt and competition experiments) were added and incubated for 30 min at room temperature in a 10 µl final volume of binding buffer (BB; 10 mM Tris/HCl, pH 8·0, 100 mM NaCl, 50 % glycerol, 2 units HPRI RNase inhibitor). When indicated, the samples were incubated with 6 M urea at 60 °C for 15 min after the binding reaction.
Following incubations, 2 µl of tracking dye was added and the samples were separated by electrophoresis through a 1 % agarose gel at 60 V in TAE (40 mM Tris/acetate, 1 mM EDTA, pH 8·0). RNAs were transferred to positively charged nylon membranes (Roche Diagnostics) by electrotransference at 350 mA in TAE for 1 h at 4 °C. RNAs were fixed to the membranes with a UV cross-linker (700x100 µJ/cm2). Hybridization and detection of DIG-RNA probes was conducted as previously described (Pallás et al., 1998).
Synthesis of DIG-labelled riboprobe.
Synthesis of the PNRSV RNA 4 DIG-labelled riboprobe was carried out by in vitro transcription of a PNRSV RNA 4 full-length cDNA copy (Sánchez-Navarro & Pallás, 1994). The plasmid was linearized with KpnI and transcribed with T7 RNA polymerase as previously described (van der Kuyl et al., 1991
) in the presence of DIG-UTP.
Cloning of the PNRSV MP and mutated forms in an expression vector.
PNRSV MP was amplified from PNRSV PV32 isolate (Sánchez-Navarro & Pallás, 1997) by PCR using VP200 sense primer (5'-P-ATGGCCGGTGTCAGTAAAAAC-3') and VP104 antisense primer (5'-ACATAAGCTTCAAGCACTCCCAGAAC-3', containing a HindIII site, underlined) The MP start and stop codons are included in the sense and antisense primers, respectively. After HindIII digestion, the PCR-amplified fragment was ligated into the bacterial expression vector pMal-c2x digested with Asp700 and HindIII, to generate the recombinant plasmid pMal-200/104. The mutant forms were amplified from PNRSV PV32 RNA 3 plasmid by PCR using appropriate primers and cloned into the vector pMal-c2x digested with Asp700 and HindIII (see Fig. 4A
). pMal-246/104, in which the first 52 amino acids of the MP were deleted, was obtained using VP246 sense primer (5'-P-AACCTCCCGAAATCCAATGTACTAAGA-3') and VP104 antisense primer. In pMal-239/104, the first 86 amino acids of the protein were deleted using VP239 sense primer (5'-P-GGCCGTGTATTCCTCGTTTATGTA-3') and VP104 antisense primer. To delete the first 111 amino acids of the MP, the PCR amplification was carried out using VP234 (5'-P-AAGTTGCAGAACTCCGATACAG-3') sense primer and VP104 antisense primer to obtain pMAL-234/104. pMal-200/201 was obtained by cloning the first 125 amino acids after amplifying the fragment of the protein resulting from using VP200 sense primer and VP201 antisense primer (5'-ACATAAGCTTCACCATCTGTCCATAATAAC-3', containing a HindIII site). To obtain pMal-200
104, which had the hypothetical RNA-binding domain deleted, two PCR reactions were carried out. The N-terminal region, preceding the domain, was amplified using VP200 sense primer and VP276 antisense primer (5'-GGGGAGGTTAACCAAATG-3') and the C-terminal region, which follows the domain, by using VP277 sense primer (5'-TTGGTTAACCTCCCCGGGCGTGTATTCCTC-3', containing a SmaI site) and VP104 antisense primer. The PCR products obtained were then mixed and used as templates in a third amplification using VP200 sense primer and VP104 antisense primers.
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Northwestern assays.
Purified proteins were electrophoresed through 12 % SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Northwestern blot assays were performed as described previously (Pallás et al., 1999). Membranes were incubated four times in RN buffer (10 mM Tris/HCl, pH 7·5, 1 mM EDTA, 100 mM NaCl, 0·05 % Triton X-100, 1x Denhardt's reagent) followed by a 3 h incubation in RN buffer in the presence of the DIG-labelled riboprobe and three 15 min washes in washing buffer (10 mM Tris/HCl, pH 7·5, 1 mM EDTA, 100 mM NaCl). Finally, the DIG-labelled riboprobe was detected as describe above except that the Tween-20 was omitted from the washing solutions and a colorimetric method was used.
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RESULTS |
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The PNRSV MP binding specificity was examined by competition experiments (Fig. 2). BSA did not produce electrophoretic retardation of the RNA probe (Fig. 2
, lane 3). The ssRNA from a heterologous origin (Lettuce big vein virus; LBVV) only competed at 10-fold excess (Fig. 2
, lanes 4 and 5), whereas tRNA did not compete (lanes 6 and 7), as expected for a highly structured RNA (Li & Palukaitis, 1996
; Marcos et al., 1999
). The dsRNA, either containing probe sequences (Fig. 2
, lanes 8 and 9) or not (lanes 10 and 11), partially displaced the binding only at a 10-fold ratio (lanes 9 and 11), whereas ss- and dsDNAs did not produce any effect on the electrophoretic retardation (lanes 12 and 15). These results indicate that the PNRSV MP has a preference for ssRNA binding, although it is able to bind with a lower affinity to dsRNAs.
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Equivalent amounts of different deletion mutants were used to delimit the RNA-binding domain of the MBPMP fusion protein. The proteins were separated by SDS-PAGE (Fig. 4B), electroblotted on to a nitrocellulose membrane, renatured and incubated with the DIG-labelled riboprobe as described previously (Aparicio et al., 2003
). Analysis of mutants 200/201 and 234/104, retaining the C-terminal half or the N-terminal half of the protein, respectively, showed that only mutant 200/201 retained the RNA-binding activity (Fig. 4C
, compare lanes 1 and 2). Analysis of two N-terminal deletion mutants (239/104 and 246/104) mapped the RNA-binding domain between aa 52 and 86 (Fig. 4C
, lanes 3 and 4), which fits well with the region (aa 5688) previously predicted (Sánchez-Navarro & Pallás, 1997
). To define this region further, a mutant was generated in which the region between aa 56 and 88 was removed (mutant 200
104). As shown in Fig. 4(C, lane 5), this mutant lacked RNA-binding activity. Therefore, the analysis defined the nucleic acid binding domain between aa 56 and 88, since only constructs containing this region were able to bind efficiently to RNA (Fig. 4C
, lanes 1, 4 and 6). Interestingly, the RNA-binding domain described here and that described for AMV are located at the N terminus of the MP, whereas similar domains previously characterized in viruses of the genera Bromovirus and Cucumovirus are present at the C terminus, reflecting their phylogenetic relationships.
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DISCUSSION |
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In this work, in vitro RNA-binding properties of PNRSV MP were demonstrated and the RNA-binding domain mapped. In gel retardation experiments, PNRSV MP bound preferentially to ssRNA in a non-sequence-specific manner. A cooperative binding of PNRSV MP to RNA was demonstrated with a quantitative approach, which was supported by the absence of detectable intermediate binding complexes. Thus, the first MP molecule binds and boosts subsequent MP binding, most likely as a result of proteinprotein interactions. Interestingly, in vitro binding assays revealed the existence of two different complexes between the PNRSV MP and RNA. One of the complexes was able to enter the electrophoresis gel and was formed at a 400 : 1 protein : RNA ratio, whereas at 120 : 1 the complex formed was fully retarded without entering the gel. The use of a protein-denaturing agent strongly suggests that these two complexes adopt very different forms. That formed at the lower protein : RNA ratio could adopt a rod-like structure, whereas that formed at the higher protein : RNA ratio could adopt a globular structure. The observation that the urea treatment mainly affected the MPRNA complexes that were unable to enter the gel, and the other complexes to a much lesser extent, can be explained by the fact that that urea interacts directly with polar residues and the peptide backbone, thereby stabilizing non-native conformations (Bennion & Daggett, 2003). It is reasonable to assume that in the complexes formed at low protein : RNA ratio, where all the protein molecules must be in direct contact with the RNA, modification of protein conformation by urea would result in the lost of the capability to bind RNA, since it has been shown than binding of MPs to RNA is structure-dependent (Li & Palukaitis, 1996
). In the faster-migrating complexes, formed at a higher protein : RNA ratio, only the more external protein molecules would be affected, whereas those directly involved in the RNA binding would not. Interestingly, two different complexes have recently been reported for the TMV-encoded MP with TMV RNA (Kiselyova et al., 2001
). The MP distribution along the chain of RNA depended on the molar MP : RNA ratios at which the complexes were formed. The structure of TMV-specific complexes was visualized by atomic force microscopy. A rise in the amount of protein relative to the RNA led to a structural change of the complexes from RNase-sensitive beads-on-a-string into a thick string form that was partly resistant to RNase treatment. These complexes may be considered equivalent to the rod-like and globular complexes, respectively, suggested here for the PNRSV MPRNA interaction.
The dissociation constant (Kd) value for the PNRSV MPRNA complex was estimated to be 1·4 µM, which is within the range of values reported for other sequence-non-specific RNA binding, such as the major hnRNP proteins (Kd=0·11 µM) (Burd & Dreyfuss, 1994), the poliovirus 3D polymerase (Kd=3 µM) (Pata et al., 1995
), the NIa from TEV (Kd=1·11·3 µM) (Daròs & Carrington, 1997
) and the p7 movement protein of Carnation mottle carmovirus (Kd=0·7 µM) (Marcos et al., 1999
; Vilar et al., 2001
). This lack of specificity supports the idea that many MPs have the ability to bind different nucleic acids. In this case, heterologous ssRNA and dsRNA partially competed for PNRSV MP homologous ssRNA binding.
As previously mentioned, a sequence comparison analysis revealed that AMV and ilarvirus MPs have a basic motif preceding the 30K motif, with a high surface probability, which makes them good candidates for RNAprotein interactions (Sánchez-Navarro & Pallás, 1997). The results presented here confirmed this prediction and located this basic domain to aa 5688 at the N terminus of the PNRSV MP. Further evidence demonstrating the functionality of this characterized domain was obtained using a synthetic peptide, which behaved like the PNRSV MP in in vitro RNA-binding experiments (M. C. Herranz, A. Sauri, I. Mingarro & V. Pallás, unpublished data). The biological significance of the identified RNA-binding domain is also supported by a high degree of sequence conservation in this region among the MPs of the PNRSV isolates characterized so far (Aparicio & Pallás, 2002
).
The MP RNA-binding domain of PNRSV was compared with the other three previously characterized domains of members of the family Bromoviridae. Interestingly, a first visual inspection revealed that in AMV and PNRSV MPs, the RNA-binding domains are located at the N terminus, whereas those of Cucumber mosaic virus (CMV) and Brome mosaic virus (BMV) are at the C terminus of the MP (Fig. 5). Although the alignment of sequences did not reveal any obvious similarities, several common features can be drawn from this analysis: (i) a high proportion of basic amino acids (Arg or Lys) is present at the C terminus of the four domains; within or preceding this motif there is an unusually high number of aliphatic amino acids (Ile, Leu); (ii) secondary structure predictions revealed the presence of two
-helices; the presence of
-helices in a basic environment has been described for other proteins that bind RNA (Tau et al., 1993
; Tau & Frankel, 1995
; Marcos et al., 1999
; Vilar et al., 2001
; Gómez & Pallás, 2001
); and (iii) similar to the RNA-binding domain B of TMV (Citovsky et al., 1992
), the four RNA-binding domains compared here have a high surface probability.
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Emerging evidence suggests that many plant proteins are made up of spatially defined domains that are associated with assigned functions (Lam & Blumward, 2002). In many cases, the homology within a protein domain family could be low and might only be conserved in some crucial functional residues. On the other hand, it has been reported that some viral MPs show structural and functional similarities to some phloem proteins (Xoconostle-Cázares et al., 1999
; Vilar et al., 2001
; Gómez & Pallás, 2001
). This has led to the hypothesis that genes encoding these viral MPs have been acquired from the plant genome, providing a connection between the evolution of the virus and its plant host (Lucas & Wolf, 1999
). It is tempting to speculate that the four RNA-binding domains compared here have been acquired by a common ancestor from the plant host or that this capture could have happened in two separate events, one for AMV and the genus Ilarvirus and the other for the genera Cucumovirus and Bromovirus. The differences among them could be explained as an adaptation of these viruses to the special requirements of their different hosts, without modifying the critical amino acid residues required for protein function.
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ACKNOWLEDGEMENTS |
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Received 29 July 2003;
accepted 29 October 2003.