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Short Communication |
1 Department of Agricultural Biotechnology and Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul 151-742, Korea
2 National Institute of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, Korea
Correspondence
Kook-Hyung Kim
kookkim{at}snu.ac.kr
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ABSTRACT |
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Supplementary material is available in JGV Online.
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MAIN TEXT |
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; Hill, 1999). Despite the economic importance of SMV, relatively little is understood about the detailed molecular interactions between viral proteins and the host proteins that occur during viral infections. The viral RNA-dependent RNA polymerase (nuclear inclusion protein b) of Tobacco vein mottling virus reportedly interacts with its own genome-linked viral protein (VPg) and coat protein (CP) (Hong et al., 1995; Li et al., 1997). An interaction between the CP and the helper-component protease (HC-Pro) of Zucchini yellow mosaic virus has also been reported (Peng et al., 1998). The nuclear inclusion protein a (NIa) and cylindrical inclusion proteins have also been reported to interact with each other (Guo et al., 2001). Such interactions may be important for virus replication, virus movement and insect transmission (Blanc et al., 1997; Rojas et al., 1997; Carrington et al., 1998). Recently, we applied the yeast two-hybrid system (YTHS) to identify protein interactions among ten viral proteins generated from the SMV polyprotein (Kang et al., 2004). Here, we used the YTHS to identify key amino acid sequences or domains responsible for the self-interaction of SMV CP.
Four deletion mutations were introduced into SMV-G7H CP (Fig. 1a) to define regions required for CP self-interaction. Truncated clones of the CP were fused downstream of both GAL4-DBD (pAS2-1) and GAL4-AD (pACT2; Clontech). The yeast two-hybrid plasmids pAS2-1 and pACT2, host strain AH109 (MATa, trp1-901, leu2-3, ura3-52, his3-200, gal4
, gal80), all media, buffers and methods for the yeast two-hybrid assay were adapted from the Matchmaker System 2 (Clontech). Proteins expressed by yeast cells were extracted (Yeast Protocol Handbook, Clontech) and verified by using SDS-PAGE followed by immunoblot with GAL4 antibodies (1 : 250 dilution; Santa Cruz Biotechnology) using an ECL kit (Amersham Biosciences). All truncated CP mutants were detected in yeast cells (Fig. 1b) with the exception of the CP mutant from the pAS2-1 F1 clone. The truncated mutant F1 could not be detected on this blot. Extra bands in the expected positions were seen that were not detectable with the non-transformed wild-type (wt) AH109 cells. Immunoblotting using the GAL4-TA antibody produced a considerable number of background bands as well as the target protein bands, but most background bands also existed with the non-transformed wt yeast (Fig. 1b, right). This result seems to relate to the different specificity of the two antibodies, because few background bands were detected in the immunoblot of pAS2-1-transformed yeast cells with the GAL4-DBD antibody.
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F3 AD formed no colonies on the SD media; however, the same SMV-G7H CP clone co-transformed with CP F3 AD and CP F1 AD did form colonies (Fig. 1c
; Supplementary Table S1, available in JGV Online). The clone lacking the F1 region from the full-length CP (CP F1 BD) formed colonies when co-transformed with the pACT2 clone missing the F1 region (CP F1 AD) or the pACT2 clone possessing only the F3 region (CP F3 AD). These results suggest that the F3 region was necessary for CP–CP self-interaction, but the F1 region was not. An interaction between the C-terminal regions of SMV-G7H CP (CP F3 BD and CP F3 AD) was also detected on the SD agar media and with respect to galactosidase activity. In contrast, the C-terminal deletion mutants of CP (CP F3 BD and CP F3 AD) lost their ability to self-interact; the co-transformed yeast with C-terminal deletion mutant plasmids did not grow on SD media and showed no 
-galactosidase activity (see Supplementary Table S1, available in JGV Online). These results suggest that the F3 region was not only necessary, but also sufficient, for CP–CP self-interaction. The C-terminal region (amino acid residues 170–256) of SMV-G7H CP contained crucial amino acid(s) or domain(s) responsible for CP self-interaction. Potentially imperfectly folded SMV-G7H CP fragments translated from truncated cDNA seemed to provide active structures that were sufficient for full or partial CP self-interaction.
The CP-deletion analysis allowed us to focus on the C-terminal region of the SMV CP. Alanine-substitution mutations were introduced in the C-terminal region of the SMV CP to verify the specific amino acid domain(s) required for CP–CP self-interaction. Both positively and negatively charged amino acids on the C-terminal region of SMV-G7H CP were substituted with alanine (Fig. 2a; Supplementary Table S2, available in JGV Online). Mutagenesis was performed by using PCR with mutagenic megaprimers (Picard et al., 1994). Alanine-substituted mutants were fused to the yeast two-hybrid vector pAS2-1 that encodes the GAL4 DNA-binding domain. To confirm the interactions, colonies from 12 pairs of alanine-substituted mutant (ASM) CPs and wt CPs were restreaked on SD agar medium supplemented with X--Gal reagent and liquid-cultured in YPDA broth for quantitative analysis of enzyme activity (Yeast Protocol Handbook, Clontech). Chromatic reactions produced by -galactosidase activity from co-transformed yeast cells corresponded exactly to the results of the SD agar-medium assay (Fig. 2b). Of the 12 ASM CPs, six mutants retained the ability to interact with wt CP, but the other mutants lost that ability. Alanine substitution at the amino acid positions R190, E191, E212, R245, H246 and R249 disrupted CP self-interaction, whereas substitutions at R188, D189, D198, K205, K218 and D250 did not. Mutants with underlined t-test values were considered to have mutational effects from the substitution of each amino acid residue relative to the wt CP at a significance level of P=0·05; these mutants were also grouped separately from the non-underlined mutants by Duncan's multiple-range test (Fig. 2d). Expression of ASM CPs in yeast cells was also verified. The CPs from all ASMs were the same size as the wt CPs and were detected successfully (Fig. 2e).
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a). The estimated mass of the in vitro-translated SMV CP (33 kDa) band was the greatest on the gel; smaller bands at about 30 and 25 kDa were considered to be CP derivatives. Additional proteins smaller than the expected size were also detected in the final elution products (Fig. 3a). These CP derivatives seemed to maintain the self-interaction ability despite being incomplete, possibly because the derivatives were not much smaller than the wt CP and therefore did not lose a significant degree of their function. Translation products from 13 potential initiation sites in the SMV CP were expected to be approximately 29, 25, 17, 15, 10 and 3 kDa, corresponding exactly to the blotted protein bands. With the exception of the 3 kDa protein, these derivatives still contained the six identified amino acid sites and probably retained the ability to bind in vitro. A column that was not loaded with the expressed pET25b CP was used as a negative control. Almost all in vitro-translated CP was detected in the final eluted solution, which contained the initially bound His-tagged SMV CP (Fig. 3b, lane 1). Subsequently, four CP ASMs (R189A, K218A, E191A and R245A) were used for in vitro binding with His-tagged SMV CP. Two mutated CPs (R189A and K218A) that showed active interaction in vivo also interacted with His-tagged SMV CP (Fig. 3b, lanes 2 and 4, respectively). In contrast, two additional mutated CPs (E191A and R245A) that showed negative interaction in vivo were not bound to the His-tagged SMV CP (Fig. 3b, lanes 3 and 5, respectively). To ensure that the SMV CP–CP self-interaction was specific, an unrelated Potato virus X (PVX) CP expressed similarly in Escherichia coli (Kwon et al., 2005) was also used for in vitro binding. The His-tagged PVX CP was initially bound, and in vitro-translated SMV CP was loaded. In vitro-translated SMV CP did not bind to the His-tagged PVX CP (Fig. 3b, lane 6), suggesting that the SMV CP–CP self-interaction was specific.
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; Flasinski & Cassidy, 1998; Peng et al., 1998). When virus particles were dispersed by aphids through interaction with the HC-Pro dimer (Guo et al., 1999), the HC-Pro dimer connected to the CP through the DAG motif; the surface-exposed N-terminal region is thought to be necessary to facilitate this interaction (López-Moya et al., 1999). If the N-terminal region is provisionally considered as an element for transmission only, the possibility of C-terminal involvement in CP self-interaction for virus assembly increases.
More research is needed to distinctly divide the domains or key amino acids required for assembly and to examine the other exposed regions facing inward that facilitate genomic RNA binding and/or other functions. In addition, assembled homodimeric CP units have been shown to require further assembly to form their final flexuous-rod morphology (Riechmann et al., 1992). This finding suggests that at least one of the three domains interacts in a different manner with preformed CP self-interacting units. When the SMV-G7H CP was aligned with those of other SMV strains as well as those of other selected potyviruses, the amino acid composition of the three helices and the one extended-strand region was almost identical (data not shown). In the present study, alanine substitutions that disrupted CP self-interaction were found on six residues and were roughly located in two regions (aa 190–212 and 245–249) of the SMV CP. These regions include previously reported key amino acid residues mentioned above and may also be potential regions containing additional key residues. Further research is required to reveal these residues and to elucidate the role(s) of each C-terminal-region helix in self-interaction and in the assembly of SMV CP.
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ACKNOWLEDGEMENTS |
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Received 6 September 2005;
accepted 10 October 2005.
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-galactosidase activity assay. The underlined mutants showed significant mutational effects on each amino acid residue at the critical significance level of P=0·05. (e) Expression of all substitution mutants in yeast was verified by immunoblotting using SMV antibodies. The molecular mass of the protein size markers is denoted at the top of the panel; the calculated mass of the SMV CP was approximately 57 kDa.