Báo cáo khoa học: The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus doc

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The F13 residue is critical for interaction among the coatprotein subunits of papaya mosaic virusM. E. Laliberte´Gagne´1, K. Lecours2, S. Gagne´2and D. Leclerc11 Infectious Disease Research Centre, Laval University, Que´bec, Canada2 Department of Biochemistry, Laval University, Que´bec, CanadaPapaya mosaic virus (PapMV) is a member of thepotexvirus family. Its virion is a flexuous rod that is500 nm long and 13 nm in diameter. A PapMV parti-cle is composed of 1400 subunits of the coat protein(CP) [1] assembled around a 6656 nucleotide plusstrand of genomic RNA [2]. The CP is composed of215 amino acids and has an estimated molecular massof 23 kDa. Until now, most of the informationobtained regarding assembly of potexvirus familymembers has been obtained from studying partiallydenatured CPs extracted from purified plant virus bythe acetic acid method [3]. Even though in vitro assem-bly using this method has been studied extensively [3–8], the nature of the interaction among CP subunitsand genomic RNA remains unknown.Recently, we have shown that CP expression inEscherichia coli leads to formation of nucleocapsid-likeparticles (NLPs) that are very similar to wild-typevirus purified from plants [9]. Therefore, this system isideal for investigating virus assembly as well as formapping domains of CPs involved in this process. Therecombinant NLPs, with an average length of 50 nm,represent 20–30% of the total purified proteins. Theremaining protein is essentially found as a 450 kDamultimer that forms a 20 subunit disk. Recombinantdisks self-assemble in vitro in the presence of RNA [9].We also showed that the affinity of disks for RNAwas important for protein self-assembly into NLPs.Mutated K97A disks, which cannot bind RNA, areincapable of self-assembly. Conversely, the E128Amutant, which shows improved affinity for RNA,makes longer NLPs than the wild-type protein [9]. Inanother study, we have shown that deletion of 26amino acids at the N-terminus of the CP leads to aKeywordsNucleocapsid-like particles (NLPs); PapMV;papaya mosaic virus; potexvirus; virusself-assemblyCorrespondenceD. Leclerc, Infectious Disease ResearchCentre, Laval University, QC, CanadaFax: +1 418 654 2715Tel: +1 418 654 2705E-mail: denis.leclerc@crchul.ulava(Received 31 August 2007, revised 6December 2007, accepted 21 January 2008)doi:10.1111/j.1742-4658.2008.06306.xPapaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli waspreviously showed to self-assemble in nucleocapsid-like particles (NLPs)that were similar in shape and appearance to the native virus. We have alsoshown that a truncated CP missing the N-terminal 26 amino acids is mono-meric and loses its ability to bind RNA. It is likely that the N-terminus ofthe CP is important for the interaction between the subunits in self-assem-bly into NLPs. In this work, through deletion and mutation analysis, wehave shown that the deletion of 13 amino acids is sufficient to generate themonomeric form of the CP. Furthermore, we have shown that residue F13is critical for self-assembly of the CP subunits into NLPs. The replacementof F13 with hydrophobic residues (L or Y) generated mutated forms of theCP that were able to self-assemble into NLPs. However, the replacementof F13 by A, G, R, E or S was detrimental to the self-assembly of the pro-tein into NLPs. We concluded that a hydrophobic interaction at the N-ter-minus is important to ensure self-assembly of the protein into NLPs. Wealso discuss the importance of F13 for assembly of other members of thepotexvirus family.AbbreviationsCP, coat protein; EMSA, electrophoretic mobility shift assay; NLP, nucleocapsid-like particle; PapMV, papaya mosaic virus; PVBV, peppervain banding virus; PVX, potato virus X.1474 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBSmonomeric form of the protein [10]. This protein failedto assemble, form disks or interact with RNA in vitro[10]. On the basis of this result, we hypothesized thatthe N-terminus of the CP is involved in contact amongNLP subunits.In this study, we have established precisely which ofthe N-terminal 26 amino acids are important in Pap-MV CP multimerization. We found that deletion ofonly 13 amino acids was sufficient to inhibit interac-tion among CP subunits, thus leading to a monomericform. We provide evidence that the F13 residue playsa crucial role in CP subunit interaction and assembly.ResultsExpression and purification of truncated andmutated forms of PapMV CPOur reference recombinant proteins are CP6–215 [9]and CP27–215 [10], which will be compared with allmutated forms described in this article. The expressionand purification of CP6–215 and CP27–215 have beendescribed elsewhere [9,10]. However, here we employeda French press instead of sonication for bacterial lysis.We generated two truncated versions of CP13–215 andCP14–215 (Fig. 1A), and then expressed and purifiedthe recombinant proteins as reported previously usinga His6 tag [9]. As expected, we observed differences inmolecular mass among CP6–215, CP13–215, CP14–215and CP27–215 as a consequence of deletion of a fewamino acids (Fig. 1B). In addition, we introducedsingle amino acid changes at F13, made substitutionswith amino acids of increasing hydrophobicity, andgenerated CP6–215 F13G, F13A, F13L, and F13Ymutants (Fig. 1A), with charged residues and gener-ated the F13R and F13E mutants (Fig. 1C), andfinally with a polar residue and generated the F13Smutant (Fig. 1C).Some of the mutations and deletions appear to havean impact on the stability of the resulting recombinantproteins. Indeed, only 24 h after purification, recombi-nant CP13–215, CP14–215, F13A, F13G, F13R, F13Eand F13S showed signs of degradation, and bands oflower molecular mass proteins appeared in westernblots (Fig. 1B,C).Characterization of recombinant NLPsAs shown before, purified CP6–215 can self-assemblein E. coli [9], and CP27–215 was found as a mono-meric form [10]. To monitor the capacity of the differ-ent mutated and truncated forms to produce NLPs, weexamined purified proteins by electron microscopy(Fig. 2A–D). Three mutated forms, CP13–215, F13Lmutant, and F13Y mutant, could form NLPs. CP13–215 NLPs were similar in shape and length to CP6–21(Fig. 2B). Interestingly, the F13L and F13Y mutantsA B C Fig. 1. PapMV CP mutants. (A) Schematic representation of Pap-MV CP mutant constructs expressed in E. coli. All constructs pos-sess a His6 tag. The dark rectangle in the schemata and theunderlined amino acids represent a small helix of six amino acidsthat is predicted to occur between Q18 and S23 [10]. Amino acidsthat are mutated in some constructs are in italics. (B, C) Expressionand purification of recombinant coat proteins on an SDS/PAGE gel.The left panels represent Coomassie staining profiles and the rightpanels represent western blots of purified proteins revealed withIgG directed against PapMV CP.M. E. Laliberte´Gagne´et al. F13 critical for interaction among the CP subunitsFEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1475formed NLPs that appeared to be longer than CP6–215 and CP13–215 (Fig. 2C,D).We determined the length of 250 NLPs for eachrecombinant protein, and the average lengths are givenin Fig. 2E. As expected, CP6–215 and CP13–215 NLPswere similar in length, measuring 50 nm. However,NLPs comprising the F13L and F13Y mutants werelonger than CP6–215 NLPs. Indeed, F13L NLPsappeared to be 2.5 times longer than CP6–215 NLPs,whereas F13Y NLPs were four times longer.Gel filtration analysis of recombinant proteinsPreviously, we showed that when expressed in E. coli,the CP6–215 protein occurred 80% of the time as a450 kDa multimer (disks), and the remaining 20% wasin NLPs [9]. To measure the ability of our recombi-nant CPs to form NLPs, we subjected purified proteinsto gel filtration (Figs 3 and 4). The Superdex 200 andSuperdex 75 FPLC profiles of recombinant CP6–215and CP27–215 were compared with those of otherrecombinant CPs. As shown before [9], the FPLCSuperdex 200 profile of CP6–215 first presents a peakeluting at 42.7 mL, which corresponds to molecules(larger than 670 kDa) that are excluded by the column(Fig. 3A) where NLPs are found. A second peak elutesat 50.5 mL; this corresponds to a multimer of450 kDa, which corresponds to CP6–215 disks.Finally, a third peak eluting at 78.8 mL correspondsto low molecular mass molecules composed ofdegraded forms of the CP that remain monomeric [9].The respective percentages of the total proteins0.2 µm 0.2 µm 0.2 µm 0.2 µm 0.2 µm 0.2 µm 0.2 µm 0.2 µm ABC ED350300250150Length of the NLPs (nm)500200100CP6–215 CP13–215 F13L F13YFig. 2. Characterization of recombinantNLPs self-assembled in E. coli. Electronmicroscopy of (A) CP6–215, (B) CP13–215,(C) F13L mutant and (D) F13Y high-speedpellet. Bars are 200 nm. (E) Average lengthof recombinant NLPs: CP6–215, CP13–215,F13L, and F13Y (n = 250).F13 critical for interaction among the CP subunits M. E. Laliberte´Gagne´et al.1476 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBSrepresented by the three forms were as follows: NLP,33%; disks, 36%; and monomers, 31%. This currentprofile differs slightly from the first one that we pub-lished [9]. This is probably because the methods usedfor bacterial lysis were different. Here, use of a Frenchpress permitted recovery of more proteins that werenot previously detected when sonication was employedto lyse the cells. It is likely that the heat generated bysonication affected the protein and influenced therecovery. CP27–215 was applied to a Superdex 75 26/60 column (Fig. 3B), and eluted as a single peak at164.61 mL, as previously reported [10]. The elutionABCDEF0.2 µm0.2 µm0.2 µm0.2 µmFig. 3. Gel filtration analysis of the truncated recombinant proteins and mutants F13A, F13G, F13L and F13Y mutants. (A) Black line,CP13–215; gray line, CP6–215; 2 mg of the purified proteins was loaded onto an FPLC Superdex 200 16/60 column. (B) Black line, CP14–215;gray line, CP27–215(21.2 kDa); 2 mg of the purified proteins was loaded onto an FPLC Superdex 75 26/60 column. (C) Gray line, F13Lmutant; dark line, F13Y mutant; dotted line, CP6–215; 2 mg of the recombinant proteins was loaded onto an FPLC Superdex 200 16/60 col-umn. (D) Gray line, F13A mutant; black line, F13G mutant; dotted line, CP6–215; 2 mg of the recombinant proteins was loaded onto an FPLCSuperdex 200 16/60 column. Molecular markers are shown in the right (A, C, D) or left (B) corners. HMWF, high molecular weight forms(> 670 000); disks, 20 subunits of the CP (450 000); LMWF: low molecular weight forms (< 230 000). Electron microscopy of the HMWFfractions of (E) the F13A mutant and (F) the F13G mutant.M. E. Laliberte´Gagne´et al. F13 critical for interaction among the CP subunitsFEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1477profile of CP13–215 was very similar to that of CP6–215 (Fig. 3A), but showed a lower ratio of NLPs(16%), a similar amount of disks (33%), and anincrease in the monomeric form of the protein (51%).This might indicate lower stability of the protein,which consequently impacts on the quantity of NLPsproduced. This result suggests that deletion of 12amino acids at the N-terminus of PapMV CP does notabolish its capacity to self-assemble and form NLPs.Deletion of 13 amino acids in recombinant CP14–215 led to a monomeric form, as shown by a singlepeak at 158.31 mL obtained using the Superdex 75 26/60 column. As expected, the recombinant CP14–215eluted before the truncated CP27–215, as it is 13amino acids longer. Both proteins were detected with100% frequency as monomers.Superdex 200 profiles of F13L and F13Y were alsocompared with that of CP6–215 (Fig. 3C). The twomutated forms were eluted in only two peaks, in con-trast with three peaks for CP6–215. In both cases,most of the protein was eluted in the first peak, whichoccurred at 42.6 mL for the F13L mutant and at41.7 mL for the F13Y mutant (Fig. 3C). These peakscorrespond to 80% and 90% of the total purified pro-tein respectively. These fractions contain NLPs. Inter-estingly, disks that normally elute at 50.5 mL were notdetected with these two mutants (Fig. 3C). Finally, apeak eluted at 86.1 mL for the F13L mutant and82.6 mL for the F13Y mutant. This peak is associatedwith monomeric forms that probably represent adegraded protein. These results suggest that the twomutants are highly efficient at forming NLPs.The F13A and F13G mutants were also subjected toSuperdex 200 (Fig. 3D) elution. The F13A and F13Gmutants eluted in two peaks (Fig. 3D). The first oneappeared at 42.1 mL for the F13A mutant and at40.3 mL for the F13G mutant. The top of each peakwas collected and examined by electron microscopy.Few NLPs were observed with the F13A mutant, asmost of the protein appeared as nonspecific aggregates(Fig. 3E). For the F13G mutant, NLPs were not foundon the electron microscopy grids. Only nonspecificaggregates were visible (Fig. 3F). In both cases, diskswere not found in the sample. A peak that eluted at81.4 mL for the F13A mutant and at 81.5 mL for theF13G mutant corresponds to a monomeric form(Fig. 3D). In fact, most of the purified F13A (65%)mutant was found to be monomeric. In contrast, only20% of the F13G mutant eluted as a monomer. Itseems that the F13A mutation affects the capacity ofthe recombinant CP to form NLPs, because a largeproportion of the recombinant purified protein isfound in low molecular mass forms. Also, even if 35%of the protein eluted as a large molecular mass multi-mer, the electron microscopy observation revealed thatthe proteins form nonspecific aggregates that are ineffi-cient in making NLPs. For the F13G mutant, themutation probably greatly affects its capacity to multi-merize into disks and NLPs.The F13R, F13E and F13S mutants were also sub-jected to Superdex 200 (Fig. 4A) gel filtration. In thisexperiment, we loaded smaller amount (150 lg) ofCP6–215 protein to separate the NLPs from the disksinto two distinct peaks. The F13R and F13S mutantsA B 0.2 µmFig. 4. Gel filtration of the F13R, F13E and F13S mutants. (A) Gel filtration analysis of recombinant proteins. Black dotted line, CP6–215;bright gray line, F13E mutant; dark gray line, F13R mutant; black line, F13S mutant; 500 lg of the purified F13E, F13R and F13S mutant pro-teins and 150 lg of the purified CP6–215 protein were loaded onto an FPLC Superdex 200 10/300 column. Molecular markers are shown inthe left corner. HMWF, high molecular weight forms (> 670 000); disks, 20 subunits of the CP (450 000); LMWF, low molecular weightforms (< 230 000). (B) Electron microscopy of the HMWF fraction of the F13E mutant.F13 critical for interaction among the CP subunits M. E. Laliberte´Gagne´et al.1478 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBSwere found entirely in the low molecular mass frac-tions and were unable to self-assemble into NLPs (datanot shown). Most of the protein of the F13E mutantwas found as low molecular mass forms, but a smallfraction was found in the exclusion fraction with CP6–215 NLPs (Fig. 4A). However, NLPs were absent, andonly nonspecific aggregates could be observed by elec-tron microscopy in this fraction (Fig. 4B). Therefore,we concluded that the F13E mutant was unable toself-assemble into an NLP.1H-15N HSQC spectrum analysisTo confirm that the CP14–215 monomer can be usedfor NMR analysis, we uniformly labeled the proteinwith15N and acquired preliminary NMR data that wesuperimposed on similar spectra obtained previouslywith the monomeric form of CP27–215 [10]. Conditionsdetermined previously to be optimal for NMR wereused [10]. In order to improve solubility and stabilityfor NMR sample analysis, a pH of 6.2 was selected. A2D1H-15N HSQC spectrum of CP14–215 was acquiredat 600 MHz at 25 °C (Fig. 5). Good spectral dispersion(3.5 p.p.m.) of backbone amide1H resonances indicatesthat PapMV CP is well folded under the conditionsused. Furthermore, the peak line width and signalintensity under the conditions used suggest that themutant CP14–215 is monomeric in solution, as expectedfrom the chromatography results. Superimposition ofspectra revealed that all peaks corresponding to struc-tured regions of CP27–215 are present in the CP14–215spectrum. This suggests that the structure of both trun-cated forms is very similar. Moreover, the presence ofseveral peaks in the middle of the spectrum (corre-sponding to unstructured regions) suggests that aminoacids 14–26 are not structured.Gel shift assaysTo evaluate whether the ability to form NLPs wasrelated to affinity for RNA, as we have shown previ-ously with the E128A and K97A mutants [9], we mea-sured the affinity of the mutant by electrophoreticmobility shift assay (EMSA) (Fig. 6). The high-speedsupernatant (disks) of the purified proteins was incu-bated with 165 fmol of an RNA probe labeled withc-32P made from an 80 nucleotide RNA transcriptfrom the 5¢-end of PapMV. The disks of CP6-215andCP13–215 interacted with the probe in a cooperativemanner and induced a shift when as little as 100 ng ofproteins was added (Fig. 6A,B). This result suggeststhat differences between the ability of the two proteinsto form NLPs, as shown in Fig. 3A, are not related totheir affinity for RNA.A similar experiment was performed with CP14–215and CP27–215, two proteins known to form mono-mers. As expected, both CP14–215 and CP27–215failed to interact with the first 80 nucleotides of viralRNA in vitro (Fig. 6C,D). We performed an EMSAwith the high-speed supernatant of F13A, and showedthat it failed to induce formation of a protein–RNAcomplex (Fig. 6E). This is consistent with our electronmicroscopy observations, which highlighted the inabil-ity of this protein to self-assemble into NLPs.As the F13L and F13Y mutants form only NLPs inE. coli, we needed to disrupt NLPs using the widelyemployed acetic acid treatment to isolate the disks aspreviously described [3], to test their ability to bindRNA. The same treatment was done with CP6–215NLPs as a control. Previously, we proposed that puri-fied protein NLP length was related directly to itsRNA-binding capacity [9]. Surprisingly, isolated disksof these two proteins showed a lower affinity forRNA than CP6–215 disks (Fig. 7A–C), even thoughextracted disks looked normal at the electron micros-copy level (supplementary Fig. S1). We did not test theF13G, F13E, F13R and F13S mutants, because theywere unable to form NLPs and therefore did not bindRNA.Measurement of RNA content by spectroscopyIn addition to EMSA, we evaluated the differenceobserved between the F13L and F13Y mutants andFig. 5. Superimposition of the1H-15N HSQC spectra of CP14–215and CP27–215; 0.1 mM each protein was diluted in 10 mM dithiothrei-tol, 10% D2O, 1· complete protease inhibitor cocktail, 0.1 mMNaN3and 60 lM DSS at pH 6.2.M. E. Laliberte´Gagne´et al. F13 critical for interaction among the CP subunitsFEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1479CP6–215 by spectroscopy using the A280/260 nmratio ofdifferent recombinant proteins. Measurement of theA280/260 nmratio, which was performed three times,was very consistent, and the average is presented inTable 1. Surprisingly, A280/260 nmratios obtained forthe two recombinant proteins were closer to the oneobtained for PapMV than for CP6–215 NLPs. Theseresults suggest that F13L and F13Y NLPs are compe-tent at binding RNA in spite of the lower affinity mea-sured by EMSA.The A280/260 nmratio was also calculated for disks.Results for PapMV disks and CP6–215 disks differedfrom those for F13L and F13Y disks, and suggest thatthere is still some RNA associated with recombinantF13L and F13Y disks. This could partially explain thedecreased affinity of F13L and F13Y disks in EMSA.DiscussionPrevious studies on the PapMV CP indicated that anessential domain for CP multimerization is located on26 amino acids of the N-terminus [9,10]. In this work,we investigated this region in detail, introducing dele-tions and point mutations. All mutations incorporatedin the PapMV CP gene did not affect the secondarystructure prediction of the CPs (supplementaryFig. S2). We have shown clearly that the N-terminal12 amino acids are not important for self-assembly ofthe PapMV CP. This result is consistent with the find-ings of Zhang et al. [1], who showed that cleavage ofthe N-terminus with trypsin did not affect virus parti-cles. This region probably plays a role in protein sta-bilization, rather than in NLP formation, as we foundmore degraded monomers with CP13–215 than withCP6–215 in the FPLC profiles (Fig. 3A).Deletion of 13 amino acids, mutation of residue F13for the less hydrophobic residues A or G, or replace-ment with the charged residues R or E, or the polarresidue S, had a major detrimental impact on NLPformation. This suggests that F13 is involved in ahydrophobic interaction that is crucial for interplayamong the protein subunits and formation of the disksABCEDFig. 6. EMSA with high-speed supernatantof recombinant CPs. (A) CP6–215;(B) CP13–215; (C) CP27–215; (D) CP14–215;(E) F13A mutant. Increasing proteinamounts were incubated at 22 ° C for 1 hwith 165 fmol of an RNA probe labeled withc-32P. The probe was made from an80 nucleotide RNA transcript from the5¢-end of the PapMV noncoding region. Thefree probe and the RNA–protein complexare indicated by arrows.F13 critical for interaction among the CP subunits M. E. Laliberte´Gagne´et al.1480 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBSthat are the building blocks with the RNA of theNLPs. Interestingly, F13L and F13Y substitutionsincreased NLP formation, probably through improve-ment of the RNA-binding capacity of the proteins, asshown by the A280/260 nmratio (Table 1). EMSA analy-sis of F13L and F13Y extracted disks did not showimproved affinity for RNA as compared with CP6–215, probably because they were still bound tightly toRNA, which interfered with RNA probe binding.It appears that F13 plays an important role in theaggregation state of the protein, as mutation of thisresidue led to formation of either NLPs (F13Y andF13L) or monomeric forms of the protein (F13G,F13A, F13R, F13E, F13S), which were alwaysdetrimental to accumulation of disks in bacteria. It ispossible that this regulation is important in PapMV-infected plants to ensure that only viral RNA, and notplant cellular RNA, gets encapsulated by the viral CP.It is tempting to draw a parallel with tobacco mosaicvirus CP, even if this protein is not related to the Pap-MV CP, where a hydrophobic interaction between theCP subunits was shown to be important for self-assem-bly of the virus into a rigid rod structure [11].Comparison of 2D1H-15N HSQC spectra from twomonomeric forms, CP14–215 and CP27–215, indicatesthat amino acids 14–26 are unstructured. This resultsuggests that the small helix that was predicted by bio-informatics to occur between residues 18 and 24 [10] isprobably unstable. We propose that the entire N-ter-minus from residues 1 to 36 forms an unstructured coilregion.A recent report showed that the CP of potato vir-us X (PVX) can be truncated by 22 amino acids at itsN-terminus without affecting either virus infectivity orformation of virus particles in plants [12]. The authorstook advantage of this mutant by fusing foreign pep-tides to the surface of the virus. Alignment of theN-terminus of the PVX CP with the PapMV CPrevealed that the PVX CP harbors an extension of20 amino acids in the N-terminus as compared withPapMV (Fig. 8). At position 33 of the PVX CP, wefind an F residue that aligns perfectly with the PapMVABCFig. 7. EMSA with high-speed supernatantof recombinant disks obtained from the dis-ruption of the NLPs by use of the aceticacid method [3]. (A) CP6–215; (B) F13Lmutant; (C) F13Y mutant. Increasingamounts of proteins were incubated at22 °C for 1 h with 165 fmol of an RNAprobe labeled with c-32P. The probe wasmade from an 80 nucleotide RNA transcriptfrom the 5¢-end of the PapMV noncodingregion. The free probe and the RNA–proteincomplex are indicated by arrows.Table 1. Protein A280/260 nmratio. Spectrophotometer absorbance measurements were taken three times with different protein preparations.Results were consistent among measurements. Recombinant CP6–215 NLPs were isolated from the high-speed pellet. The absorbancemeasurement was taken directly from the purified PapMV and purified F13L and F13Y recombinant proteins. The four proteins were treatedby acetic acid methodology [3] to generate disks that were used to calculate the A280/260 nmratio.Virus and NLPs Extracted disksPapMV CP6–215 F13L F13Y PapMV CP6–215 F13L F13YA280/260 nmratio0.75 1.1 0.8 0.75 1.5 1.55 0.95 0.9M. E. Laliberte´Gagne´et al. F13 critical for interaction among the CP subunitsFEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1481CP F13. Therefore, on the basis of our results, it islikely that a deletion of 32 amino acids will be toler-ated by PVX without disturbing the assembly process.Alignment of this F residue is also shared with severalother potexviral CP sequences, as seven out of the18 N-terminal sequences of the potexviruses showedconsensus for an F in the position that corresponds toF13 of PapMV CP (Fig. 8). Also, an F is present inthe same area in the CP of bamboo mosaic virus. TheCP of mint virus X presents an L in this position,which corresponds to a hydrophobic residue that couldsubstitute for an F in the PapMV CP. Therefore, onthe basis of the alignment, we propose that a hydro-phobic residue at the position that corresponds to Pap-MV CP F13 is preferred in half of the potexvirus CP.It is likely that this residue also plays an importantrole in the interactions between the subunits in thepotexviruses family.Finally, our results agree with the assembly modelrecently proposed for a potyvirus member of the Poty-viridea family: the pepper vain banding virus (PVBV)[13]. These authors proposed that the N-terminalextension of a CP subunit interacts with the C-terminalextension of an adjacent CP subunit in a head-to-tailmanner, thereby permitting formation of both thering-like intermediate and the NLPs into helix-likestructures. We propose that this model is applicablefor PapMV and probably all potexviruses. However, amajor difference between PapMV and PVBV is thatPapMV CP subunit assembly into disk structures isbased on a hydrophobic interaction, whereas PVBVCP assembly into ring-like structures (disks) was pro-posed to be driven by electrostatic interactions [13].Experimental proceduresCloning and expression of recombinant proteinsThe PapMV CP gene CP6–215 has been described previously[9], as has the truncated version of PapMV CP, CP27–215[10]. The other truncated versions of PapMV, CP13–215 andCP14–215, were amplified by PCR from the clone CP6–215inserted into a pET-3d vector. The forward primers used forthese PCR reactions were CP13–215 forward, 5¢-ACGTCATATGTTCCCCGCCATCACCCAG-3¢, and CP14–215 for-ward, 5¢-ACGTCATATGCCCGCCATCACCCAGGAA-3¢.A reverse primer, 3¢-GAAATTCTTCCTCTATATGTATACTGCA-5¢, was used for both constructs. The PCR prod-ucts were digested with NdeI, to generate the two truncatedCPs inserted into a pET-3d vector.The F13A, F13E, F13G, F13L, F13R, F13S and F13Ymutations were introduced by PCR into the CP6–215 cloneusing the following oligonucleotides: forward (F13A),5¢-GCGCCCGCCATCACCCAGGAACAA-3¢; forward(F13E), 5¢-GAACCCGCCATCACCCAGGAACAA-3¢; for-ward (F13G), 5¢-GGCCCCGCCATCACCCAGGAACAA-3¢; forward (F13L), 5¢-CTGCCCGCCATCACCCAGGAACAA-3¢; forward (F13R), 5¢-CGCCCCGCCATCACCCFig. 8. Alignment of a consensus sequence derived from 18 known potexvirus coat proteins and the PapMV CP in the N-terminalregion 1–27 of PapMV CP. Conserved hydrophobic residues that aligned with amino acid 13 of the PapMV CP are highlighted in bold. Align-ment was done using the CP sequences of: bamboo mosaic virus (BaMV); cactus virus X (CVX); clover yellow mosaic virus (ClYMV); cas-sava common mosaic virus (CsCMV); Cymbidium mosaic virus (CymMV); foxtail mosaic virus (FoMV); Hosta virus X (HVX); lily virus X (LVX);mint virus X (MVX); narcissus mosaic virus (NMV); PapMV; potato aucuba mosaic virus (PAMV); pepino mosaic virus (PepMV); plantago asi-atica mosaic virus (PlAMV); PVX; scallion virus X (ScaVX); strawberry mild yellow edge virus (SMYEV); tulip virus X (TVX); white clovermosaic virus (WClMV).F13 critical for interaction among the CP subunits M. E. Laliberte´Gagne´et al.1482 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBSAGGAACAA-3¢; forward (F13S), 5¢-AGCCCCGCCATCACCCAGGAACAA-3¢; forward (F13Y), 5¢-TATCCCGCCATCACCCAGGAACAA-3¢; and reverse (F13), 3¢-CGTAGGTGTGGGTTGTATCGG-5¢. PCR products withblunt ends were circularized to form the fourth mutated CPinserted into a pET-3d vector.Expression and purification of recombinantproteins from E. coliExpression and induction of proteins was conducted asdescribed previously [9]. Bacteria were harvested by centri-fugation for 30 min at 9000 g. The pellet was resuspendedin ice-cold lysis buffer (50 mm NaH2PO4, pH 8.0, 300 mmNaCl, 10 mm imidazole, 40 lm phenylmethanesulfonyl fluo-ride and 0.2 mgÆmL)1lysosyme), and bacteria were lysedby one passage through a French press. The lysate wasincubated with agitation for 15 min with 9000 units ofDNase and 1.5 mm MgCl2, and this was followed by twocentrifugations for 30 min at 10 000 g to eliminate cellulardebris. The supernatant was incubated with 3 mL of Ni–ni-trilotriacetic acid (Qiagen, Turnberry Lane, Valencia, CA,USA) under gentle agitation overnight at 4 °C. Proteinswere purified as described elsewhere [9], except that theywere incubated for 4 h with 2 mL of the elution buffer(10 mm Tris/HCl, pH 8.0, supplemented with 1 m imidaz-ole) before elution. Imidazole was eliminated by dialysis for24 h. Protein purity was determined by SDS/PAGE andconfirmed by western immunoblot analysis using rabbitpolyclonal antibodies generated against purified PapMVvirus.Separation of disks and NLPsTo separate the disks from NLPs, 1 mL of purified proteinswas subjected to a high-speed centrifugation for 2 h at100 000 g in a Beckman SW60Ti rotor. The pellet thatcomprised the NLPs was resuspended in 300 lLof10mmTris/HCl at pH 8.0. The supernatant with the disks and thelow molecular mass forms was retained for gel shift assays.SDS/PAGE and electroblottingProteins were mixed with one-third of the final volume ofloading buffer containing 5% SDS, 30% glycerol, and0.01% bromophenol blue. SDS/PAGE was performed asdescribed elsewhere [14].Electron microscopyNucleocapsid-like particles or viruses were diluted in10 mm Tris/HCl (pH 8.0) to a concentration of 50 ngÆlL)1,and were absorbed for 6 min on carbon-coated formvargrids. Grids were washed twice with 8 lL of water. Finally,grids were incubated in darkness for 6 min with 8 lLof2% uranyl acetate.Acetic acid degradationIsolation of disks from CP6–215, F13L and F13Y NLPswas performed by acetic acid degradation as described pre-viously [3]. Two volumes of glacial acetic acid were addedto the NLPs and incubated at 4 °C for 1 h. Centrifugationat 10 000 g for 15 min removed insoluble RNA. The super-natant was removed and subjected to high-speed centrifuga-tion at 100 000 g for 2 h in a Beckman 50.2Ti rotor toremove any residual NLPs. Proteins were dialyzed exten-sively against 10 mm Tris/HCl (pH 8.0).Gel filtrationProteins were purified by gel filtration. Columns were firstcalibrated with molecular weight markers (GE Healthcare,Baie d’Urfe´, Canada). Superdex 75 26/60 (GE Healthcare),Superdex 200 16/60 (GE Healthcare) and Superdex 200 10/300 (GE Healthcare), pre-equilibrated with gel filtration buf-fer (10 mm Tris/HCl, pH 8.0, supplemented with 150 mmNaCl), were used. The volume of protein loaded into thesample loop was 1.5 mL for Superdex 75 26/60, 1 mL forSuperdex 200 16/60, and 0.1 mL for Superdex 200 10/300.NMR spectroscopyThe 600 lL sample used for NMR spectroscopy was0.1 mm CP14–215 or CP27–215 in 90% H2O/10% D2O,10 mm dithiothreitol (pH 6.2), 1· complete protease inhibi-tor cocktail (Roche), with 0.1 mm NaN3and 60 lm 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) as the NMRchemical shift reference. The1H-15N HSQ spectra wereobtained at 25 °C on a Varian Unity 600 MHz spectrome-ter equipped with a triple-resonance cryoprobe and Z-axispulsed-weld gradient. The acquired data consisted of 768complex data points in the acquisition domain and 128complex data points in the indirectly detected domain. Thespectral width was 10 000 Hz in the1H dimension and1680 Hz in the15N dimension. NMR spectra were pro-cessed using NMRPipe [15]. Processing involved doublingof the15N time domain by linear prediction, zero-filling to2048 and 512 complex points in1H and15N, respectively, a45° shifted sine-bell apodization in the1H dimension, and a72° shifted sine-bell apodization in the15N dimension.RNA transcripts and EMSAThe probe was generated as described before [9]. LabeledRNA probe was incubated with various amounts of recom-binant proteins at room temperature for 60 min. We used165 fmol of RNA for each reaction in the in vitro assemblyM. E. Laliberte´Gagne´et al. F13 critical for interaction among the CP subunitsFEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1483[...]... M & Bancroft JB (1978) The initiation of papaya mosaic virus assembly Virology 90, 54–59 5 Abouhaidar MG & Bancroft JB (1980) The polarity of assembly of papaya mosaic- virus and tobacco mosaicvirus RNAs with PMV -protein under conditions of nonspecificity Virology 107, 202–207 6 Erickson JW & Bancroft JB (1978) The self-assembly of papaya mosaic virus Virology 90, 36–46 7 Erickson JW, Bancroft JB & Stillman... assembly of papaya mosaic potexvirus coat protein FEBS J 273, 14–25 ´ ´ Lecours K, Tremblay MH, Gagne ME, Gagne SM & Leclerc D (2006) Purification and biochemical characterization of a monomeric form of papaya mosaic potexvirus coat protein Protein Expr Purif 47, 273–280 Bendahmane M, Fitchen JH, Zhang G & Beachy RN (1997) Studies of coat protein- mediated resistance to tobacco mosaic tobamovirus: correlation... critical reading of our manuscript 12 13 References 1 Zhang H, Todderud E & Stubbs G (1993) Crystallization and preliminary X-ray analysis of papaya mosaic virus coat protein J Mol Biol 234, 885–887 2 Sit TL, Abouhaidar MG & Holy S (1989) Nucleotide sequence of papaya mosaic virus RNA J Gen Virol 70 (Pt 9), 2325–2331 3 Erickson JW, Bancroft JB & Horne RW (1976) The assembly of papaya mosaic virus protein Virology... al F13 critical for interaction among the CP subunits buffer (10 mm Tris/HCl, 4% glycerol, 1 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm EDTA, 20 mm NaCl), which contained 7.5 U of RNase inhibitor (27-0816-01; GE Healthcare) The final reaction volume was 10 lL Two microliters of loading dye was added to the sample before loading onto a 5% native polyacrylamide gel Electrophoresis was performed in 0.5· Tris/borate/EDTA... Stillman MJ (1981) Circular dichroism studies of papaya mosaic virus coat protein and its polymers J Mol Biol 147, 337–349 8 Erickson JW, Hallett FR & Bancroft JB (1983) Subassembly aggregates of papaya mosaic- virus protein Virology 129, 207–211 ´ 9 Tremblay MH, Majeau N, Gagne ME, Lecours K, Morin H, Duvignaud JB, Bolduc M, Chouinard N, ´ ´ Pare C, Gagne S et al (2006) Effect of mutations K97A 1484 14 15... The following supplementary material is available online: Fig S1 Electron microscopy of disks extracted by acetic acid methodology [3] of: (A) CP6–215; (B) F13L mutant; and (C) F13Y mutant Fig S2 Predicted secondary structure of recombinant PapMV CPs This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for. .. buffer for 90 min at 10 mA The gel was dried and subjected to autoradiography for 16 h on Kodak Bio-Max MS film (V8326886; GE Healthcare) and developed 10 11 Acknowledgements We thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the ‘Fond de Recherche sur la Nature et les Technologies’ (FQRNT) for funding our research program on papaya mosaic virus, and Dr Paul Khan for critical. .. carboxy-terminal residues are crucial for the initiation of assembly in Pepper vein banding virus: a flexuous rod-shaped virus Virology 316, 325–336 Schagger H & von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166, 368–379 Delaglio F, Grzsiek S, Vuister VW, Zhu G, Pfeifer J & Bax A (1995) NMRPipe:... assembly of mutant coat proteins and resistance J Virol 71, 7942–7950 Donini M, Lico C, Baschieri S, Conti S, Magliani W, Polonelli L & Benvenuto E (2005) Production of an engineered killer peptide in Nicotiana benthamiana by using a potato virus X expression system Appl Environ Microbiol 71, 6360–6367 Anindya R & Savithri HS (2003) Surface-exposed amino- and carboxy-terminal residues are crucial for the. .. http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS . of the protein, as mutation of this residue led to formation of either NLPs (F13Y andF13L) or monomeric forms of the protein (F13G,F13A, F13R, F13E, F13S),. The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus M. E. Laliberte´Gagne´1,
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