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1 School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seongbuk-gu, Seoul 136-713, Korea
2 Department of Molecular Biology, Sejong University, Gunja-dong, Gwangjin-gu, Seoul 143-747, Korea
Correspondence
Young In Park
yipark{at}korea.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Plant Pathology, One Shields Avenue, University of California, Davis, CA 95616, USA. 
The sequence of the primers used in this study is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). A great deal of progress has been made in understanding the MP-mediated cell-to-cell movement of plant viruses (Canto & Palukaitis, 2005; Deom et al., 1992; Li & Palukaitis, 1996; Lucas, 2006), and recent work suggests that for some plant viruses multi-protein ribonucleoprotein complexes are moving from cell to cell . For example, in tobacco mosaic virus (TMV), viral proteins including the MP, RNA polymerase and coat protein (CP) assemble to make ribonucleoprotein complexes. These complexes traffic in and between cells and contain the components capable of rapid replication after entry in adjacent cells, conferring a benefit for rapid virus spread (Kawakami et al., 2004). The interactions among some of the cucumber mosaic virus (CMV)-encoded proteins have been well characterized (Kim et al., 2002; Hwang et al., 2005). Yeast two-hybrid and co-immunoprecipitation assays have demonstrated that the CMV-encoded MP interacts with the 2a polymerase protein, and that MP also interacts indirectly with 1a protein, via 2a polymerase protein. However, the specific regions of MP and 2a polymerase proteins that are important for the interaction are not known.
CMV infects more than 1000 species of plants, and has a single-stranded, positive-sense and functionally divided genome that consists of three genomic RNAs. The CMV MP has been shown to be necessary for cell-to-cell movement of CMV in plant hosts (Kaplan et al., 1995). The CMV MP binds single-stranded nucleic acid cooperatively in vitro and forms ribonucleoprotein complexes with viral RNA (Andreev et al., 2004; Li & Palukaitis, 1996; Vaquero et al., 1997). CMV MP-mediated trafficking from cell to cell also requires the CMV capsid protein (Kaplan et al., 1998; Nagano et al., 1997, 1999, 2001)
CMV RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are involved in viral RNA replication (Buck, 1996; Palukaitis et al., 1992). These two proteins interact with each other to form the replicase complex (Kim et al., 2002; O'Reilly et al., 1998) and are regulated in part by phosphorylation (Kim et al., 2002). RNA 2 also encodes a small protein called 2b, which affects virulence of the virus and is known to suppress the initiation of RNA silencing and play a role in promoting cell-to-cell movement (Lucy et al., 2000). The RNA 3-encoded MP binds the viral genomic RNAs and interacts with host factors involved in the intercellular transport system to facilitate viral transport through the plasmodesmata into adjacent cells (Cooper & Dodds, 1995; Palukaitis et al., 1992). RNA 4 is a subgenomic mRNA derived from the 3' half of RNA 3, and encodes the CMV CP (reviewed by Palukaitis & García-Arenal, 2003). All three CMV genomic RNAs are essential for systemic plant infection and all five CMV-encoded proteins directly or indirectly affect the movement of CMV within the plant host (Palukaitis & García-Arenal, 2003; Palukaitis et al., 1992; Rao & Francki, 1982).
In this paper, we report identification of the regions of MP and 2a polymerase proteins that are important for formation of the MP-2a polymerase complex.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and products were ligated to the pAS2-1 vector. pKG2a was digested with SmaI/SalI (for 2a, position 1–21), NcoI (for 2a, position 126–588), NdeI/NcoI (for 2a, position 334–588), NdeI/PstI (for 2a, position 334–988), and EcoRI/PstI (for 2a, position 682–988) (Fig. 1a). Fragments containing 2a ORF sequences were transferred to the same sites of pAS2-1, respectively. For 2a (1–126), pKG2a (1–126) (Kim et al., 2002) was digested with SmaI/PstI and transferred to pAS2-1. For 2a (1–21/588–682), the region encoding amino acids 588–682 was amplified with specific primers as listed in Supplementary Table S1, available with the online version of this paper, containing a SalI sequence in the forward primer and PstI sequence in the reverse primer. The amplified fragment was digested with SalI/PstI, and transferred to previously prepared pAS (1–21).
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), derivatives of the CMV MP open reading frames (ORFs) were amplified by PCR using specific C-terminal reverse primers and one forward initiation primer sets (Supplementary Table S1), and digested with SmaI and SacI as previously described (Hwang et al., 2005). To create N-terminal mutants of the MP ORF (Fig. 1c), a previously described termination primer (Hwang et al., 2005) was used with the primers described in Supplementary Table S1. The nucleotide positions of the primers are indicated (CMV-As, , GenBank accession nos AF013291 and AF033667 for RNA 3 and RNA 2, respectively), and restriction sites are underlined (Supplementary Table S1). Amplified products were digested by SmaI/SacI and cloned into SmaI/SacI-digested pACT2, which harbours the GAL4 activation domain (AD) (Clontech). A further set of clones was built in order to investigate the role of each of the first 20 amino acids of MP through a series of deletion mutants, from MP(1–T6) to MP(1–S19), using an initiation forward primer (Hwang et al., 2005) and specific primers (Supplementary Table S1).
Site-directed mutagenesis using fusion PCR.
Mutant plasmids harbouring sequences encoding aspartate instead of serine at the 14th amino acid residue from the N terminus of MP were created by fusion PCR (Kuwayama et al., 2002). Briefly, two primary PCR reactions were performed with primers rna3-1, s14d-2, s14d-3 and rna3-4 (Supplementary Table S1). Primers s14d-2 and s14d-3 were designed to give the mutated amino acid sequence in the change to aspartate instead of serine in the 14th amino acid and were complementary to each other. The first PCR reaction was done with rna 3-1 and s14d-2 for the 5' region of RNA 3, and s14d-3 and rna 3-4 for the 3' region of RNA 3, respectively. The two PCR fragments were denatured, mixed to anneal and then subsequently PCR-amplified with primers rna 3-1 and rna 3-4 to generate the entire RNA 3 genomic DNA encoding the mutated amino acid residue. This mutated RNA 3 cDNA was cloned into pGEM-T and confirmed by DNA sequencing and restriction enzyme digestions. The mutated MP ORF was amplified by PCR using a specific primer set (Hwang et al., 2005; Kim et al., 2002) and then cloned into pACT2 (Clontech) for yeast two-hybrid assays.
Yeast two-hybrid assay and determination of 
-galactosidase activity.
The yeast two-hybrid assay and measurement of -galactosidase specific activity were performed as described previously (Hwang et al., 2005). Yeast strain Y190 (Saccharomyces cerevisiae, baker's yeast) and the two-hybrid vectors pAS2-1 and pACT2 were included in the MATCHMAKER GAL4 Two-Hybrid System 2 (Clontech). Recombinant bait and prey vectors were transformed into Saccharomyces cerevisiae strain Y190 by the lithium acetate method as described previously (Kim et al., 2002). One unit of -galactosidase was defined as the amount that hydrolyses 1 µmol ONPG to o-nitrophenol and D-galactose in 1 minute.
Co-immunoprecipitation of interacting proteins.
For immunoprecipitation, the cDNAs were purified, in vitro transcribed by T7 RNA polymerase and translated with a TNT T7 transcription/translation kit (Promega) according to the manufacturer's manual. In vitro translation reactions were combined with each other for 1 h at room temperature after resuspension in IP buffer [50 mM HEPES, pH 7.5, 50 mM NaCl, 10 mM EDTA, 5 mM DTT, 0.1 % Triton X-100 and 1x Complete Protease inhibitor (Roche)]. The solutions were then centrifuged at 12 000 g for 30 min at 4 °C. Following centrifugation, the supernatant was incubated with anti-2a antiserum (1 : 1000 dilutions) or anti-MP antiserum for each immunoprecipitation, respectively, for 2–4 h at 4 °C. The resulting immunocomplexes were collected on prewashed protein A Sepharose beads (Bio-Rad) by incubation for 1–2 h at 4 °C. The immunoprecipitates were recovered and separated on 8 % SDS-PAGE and transferred to nitrocellulose membranes. Proteins were analysed by immunoblotting with appropriate anti-MP antiserum (1 : 1000) and anti-2a antiserum, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), we characterized the interactions between the CMV-As MP and other CMV-encoded proteins, and showed that MP interacts with the 2a polymerase protein. Here, nine 2a polymerase protein deletion constructs of various sequence and size were created in attempts to identify the 2a polymerase protein region(s) important for interaction with MP (Fig. 1). When these constructs were used in yeast two-hybrid assays, we found two regions of the 2a protein that strongly and specifically interacted with the MP. A deletion mutant only encoding the 2a polymerase protein N-terminal 21 amino acids developed a strong blue colour in the filter-lift assay within 2–3 h, indicating interaction with MP (Table 1). Similarly, the construct encoding amino acids for the region between residues 588 and 682, which also contains the GDD motif, also interacted strongly with the MP. These results may suggest that both the N-terminal 21 amino acids and the central GDD motif of 2a polymerase play a role in interaction with the MP. None of the other constructs showed positive interactions. We next created a fusion construct containing only the two interacting regions (Fig. 1) and found that this interacted more strongly with MP than did each region alone (Table 1). The enhanced binding affinity likely indicates that the two regions act coordinately in associating with MP.
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). The latter two constructs both encode proteins containing the GDD motif, so the presence of the GDD motif alone is not sufficient for the interaction. It seems more likely that the binding activity of the 21 N-terminal amino acids or the GDD motif may be masked by the neighbouring amino acid sequences in some of the deletion mutants, but not in the complete protein, or in the small constructs where we saw specific interactions.
In order to gain confidence that the interacting regions identified here are significant, we compared them by aligning the corresponding regions from other CMV isolates. Among them, amino acids of 2a polymerase showed overall 68.2 % sequence identities (data not shown). The interacting region amino acid sequences are highly conserved among the 2a polymerase proteins present in other CMV isolates, including those of both subgroup I and subgroup II isolates (Fig. 2). In fact, the 21 amino acid sequence of the 2a polymerase of As strain perfectly matches with the sequences of another subgroup I CMV, the Fny strain, and it differs by only 5 aa from the sequence of the subgroup II, Q strain, at the N terminus. Furthermore, the central GDD motif of the 2a polymerase also has highly conserved amino acid sequences among the subgroups (Fig. 2).
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). In contrast, all MP mutants lacking these N-terminal 20 amino acids did not interact with 2a polymerase protein (Table 2). Also in contrast to what was observed for the 2a constructs, any of our MP mutants that encoded the N-terminal amino acids interacted with the 2a proteins. There were no negative effects due to additional amino acids.
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). Yeast two-hybrid assays showed that all deletion mutants encoding at least the 14 N-terminal amino acids maintained the ability to interact with 2a polymerase protein. However, mutants encoding 13 or fewer amino acids without the 14th serine did not show any interaction. These results suggest that the first 14 amino acids of MP were essential for the interaction with 2a polymerase. However, it could not be excluded that only the 13 N-terminal MP amino acids might yield a protein too small to exhibit the necessary conformation to interact with 2a polymerase protein.
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, this substitution mutant failed to interact with the 2a polymerase protein in the yeast two-hybrid system, indicating that the serine residue at the MP 14th position plays a critical role in the interaction with the 2a polymerase protein.
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, we observed that 2a polymerase–MP and 2a polymerase–S14D MP showed different co-immunoprecipitation patterns. As we showed previously (Hwang et al., 2005), 2a polymerase protein–MP interactions were detected by co-immunoprecipitation analysis, and revealed a reciprocal co-immunoprecipitation with anti-2a and anti-MP specific antibodies, respectively (Fig. 4, upper two panels). In contrast, the 2a polymerase and S14D MP did not result in reciprocal co-immunoprecipitation of either protein (Fig. 4, lower two panels). The only amino acid residue that differed between these two full-length MPs was the S14D, further demonstrating the importance of the serine at this position and confirming our yeast two-hybrid results. Furthermore, the serine at position 14 is conserved among all the CMV MPs examined by us, including those from subgroups I and II (Fig. 2), compared with 78.5 % sequence identities of the overall protein (data not shown), further suggesting its importance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kim et al., 2002), and we previously used it to search for viral proteins that associate with the MP of CMV-As. We showed that CMV MP interacts with the 2a polymerase protein (Hwang et al., 2005), and we now have further dissected the specific determinants of this interaction.
The GDD motif region is well known to be one of the most conserved regions in the RNA-dependent RNA polymerase (Kamer & Argos, 1984; Poch et al., 1989). In addition to its role in RNA amplification, the CMV 2a protein has been shown to have important roles in host defence and systemic infection. In cowpea, CMV cannot escape initially infected cells and hypersensitive response (HR) results. The HR requires two amino acid residues located close to the GDD motif in the 2a polymerase protein (amino acids 631 and 641), which are conserved among CMV RNA-dependent RNA polymerase proteins. Changes of either site to other amino acid residues allowed systemic infection to occur without HR, and increased the accumulation of both the 2a polymerase protein and viral RNA in the protoplasts (Kim & Palukaitis, 1997). Only three mutations in this region can eliminate the HR in cowpea (Kim & Palukaitis, 1997). Our data show that the 2a protein GDD motif region is also very important for interactions with the CMV MP (Table 1).
The CMV MP requires its cognate CP to mediate efficient cell-to-cell movement during infection (Canto et al., 1997; Nagano et al., 1999). An MP mutant lacking the C-terminal 33 amino acids was still able to mediate the movement of a chimeric BMV genome in the absence of CMV CP (Nagano et al., 1997), but MP deletion mutants missing more than 36 amino acids from the C-terminal end could not promote CMV movement in the absence of CP (Nagano et al., 2001). According to our results (Table 2), it appears that the MP region responsible for the interaction with the 2a polymerase protein differs from the region which is involved in the interaction with CP. When the serine at position 14 of the MP was changed to aspartate, the MP–2a interaction was not detected in the yeast two-hybrid assay or co-immunoprecipitation assays (Figs 3 and 4). Serine was changed to aspartate rather than glycine or alanine, which have smaller non-polar functional groups. Aspartate contains a negatively charged hydrophilic functional group. This ensures more likely exposure to the surface without changing protein conformation due to its size. Furthermore, this amino acid residue is a putative phosphorylation site. Therefore, we speculate that the phosphorylation status of the MP may play a critical role in determining interaction with the 2a polymerase protein. Phosphorylation of the CMV MP has been reported in MP-transgenic tobacco plants (Matsushita et al., 2002), and phosphoserine was detected in these plants. No evidence for phosphorylation of the serine at position 14 in the MP has yet emerged, but its phosphorylation in vivo cannot be excluded, and MP phosphorylation has been reported in a few other plant viruses. For example, the TMV MP is phosphorylated at its C terminus in vivo in transgenic tobacco plants (Waigmann et al., 2000). Mimicking the phosphorylation of MP by substituting with negatively charged amino acids disrupted the MP's ability to interact with plasmodesmata and to promote viral cell-to-cell movement, suggesting that the phosphorylation might have a negative effect on the plasmodesmatal transport. For the tobamovirus tomato mosaic virus, the serine 37 of the MP is phosphorylated and its phosphorylation is essential for intracellular localization and stability of the MP, which is necessary for the protein to function (Kawakami et al., 1999).
Viral protein interactions are complex and play important roles in infection of the plant host. For TMV, colocalization of the TMV MP and replicase was detected (Asurmendi et al., 2004). Furthermore, the TMV replicase, MP and CP assembled with each other to facilitate cell-to-cell movement of virus replication complexes (VRCs) as large, complex structures through plasmodesmata (Kawakami et al., 2004). They suggested that these complexes contain the components that are necessary to initiate rapid spread of infection. It is not known whether similar interactions occur for CMV in vivo. However, our data show that CMV-encoded proteins, including 1a (Hwang et al., 2005), 2a and MP, can interact and form complexes in vitro. This is the first report to dissect the regions of both 2a polymerase and MP that are important for their interaction. Future in vivo studies will further elucidate functions and specificities of these interactions.
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ACKNOWLEDGEMENTS |
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Received 1 June 2007;
accepted 7 August 2007.
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