The F13 residue is critical for interaction among the coat protein subunits of papaya mosaic virus ´ ´ ´ M E Laliberte Gagne1, K Lecours2, S Gagne2 and D Leclerc1 ´ Infectious Disease Research Centre, Laval University, Quebec, Canada ´ Department of Biochemistry, Laval University, Quebec, Canada Keywords Nucleocapsid-like particles (NLPs); PapMV; papaya mosaic virus; potexvirus; virus self-assembly Correspondence D Leclerc, Infectious Disease Research Centre, Laval University, QC, Canada Fax: +1 418 654 2715 Tel: +1 418 654 2705 E-mail: denis.leclerc@crchul.ulava (Received 31 August 2007, revised December 2007, accepted 21 January 2008) doi:10.1111/j.1742-4658.2008.06306.x Papaya mosaic virus (PapMV) coat protein (CP) in Escherichia coli was previously showed to self-assemble in nucleocapsid-like particles (NLPs) that were similar in shape and appearance to the native virus We have also shown that a truncated CP missing the N-terminal 26 amino acids is monomeric and loses its ability to bind RNA It is likely that the N-terminus of the CP is important for the interaction between the subunits in self-assembly into NLPs In this work, through deletion and mutation analysis, we have shown that the deletion of 13 amino acids is sufficient to generate the monomeric form of the CP Furthermore, we have shown that residue F13 is critical for self-assembly of the CP subunits into NLPs The replacement of F13 with hydrophobic residues (L or Y) generated mutated forms of the CP that were able to self-assemble into NLPs However, the replacement of F13 by A, G, R, E or S was detrimental to the self-assembly of the protein into NLPs We concluded that a hydrophobic interaction at the N-terminus is important to ensure self-assembly of the protein into NLPs We also discuss the importance of F13 for assembly of other members of the potexvirus family Papaya mosaic virus (PapMV) is a member of the potexvirus family Its virion is a flexuous rod that is 500 nm long and 13 nm in diameter A PapMV particle is composed of 1400 subunits of the coat protein (CP) [1] assembled around a 6656 nucleotide plus strand of genomic RNA [2] The CP is composed of 215 amino acids and has an estimated molecular mass of 23 kDa Until now, most of the information obtained regarding assembly of potexvirus family members has been obtained from studying partially denatured CPs extracted from purified plant virus by the acetic acid method [3] Even though in vitro assembly using this method has been studied extensively [3– 8], the nature of the interaction among CP subunits and genomic RNA remains unknown Recently, we have shown that CP expression in Escherichia coli leads to formation of nucleocapsid-like particles (NLPs) that are very similar to wild-type virus purified from plants [9] Therefore, this system is ideal for investigating virus assembly as well as for mapping domains of CPs involved in this process The recombinant NLPs, with an average length of 50 nm, represent 20–30% of the total purified proteins The remaining protein is essentially found as a 450 kDa multimer that forms a 20 subunit disk Recombinant disks self-assemble in vitro in the presence of RNA [9] We also showed that the affinity of disks for RNA was important for protein self-assembly into NLPs Mutated K97A disks, which cannot bind RNA, are incapable of self-assembly Conversely, the E128A mutant, which shows improved affinity for RNA, makes longer NLPs than the wild-type protein [9] In another study, we have shown that deletion of 26 amino acids at the N-terminus of the CP leads to a Abbreviations CP, coat protein; EMSA, electrophoretic mobility shift assay; NLP, nucleocapsid-like particle; PapMV, papaya mosaic virus; PVBV, pepper vain banding virus; PVX, potato virus X 1474 FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits monomeric form of the protein [10] This protein failed to assemble, form disks or interact with RNA in vitro [10] On the basis of this result, we hypothesized that the N-terminus of the CP is involved in contact among NLP subunits In this study, we have established precisely which of the N-terminal 26 amino acids are important in PapMV CP multimerization We found that deletion of only 13 amino acids was sufficient to inhibit interaction among CP subunits, thus leading to a monomeric form We provide evidence that the F13 residue plays a crucial role in CP subunit interaction and assembly A Results Expression and purification of truncated and mutated forms of PapMV CP Our reference recombinant proteins are CP6–215 [9] and CP27–215 [10], which will be compared with all mutated forms described in this article The expression and purification of CP6–215 and CP27–215 have been described elsewhere [9,10] However, here we employed a French press instead of sonication for bacterial lysis We generated two truncated versions of CP13–215 and CP14–215 (Fig 1A), and then expressed and purified the recombinant proteins as reported previously using a His6 tag [9] As expected, we observed differences in molecular mass among CP6–215, CP13–215, CP14–215 and CP27–215 as a consequence of deletion of a few amino acids (Fig 1B) In addition, we introduced single amino acid changes at F13, made substitutions with amino acids of increasing hydrophobicity, and generated CP6–215 F13G, F13A, F13L, and F13Y mutants (Fig 1A), with charged residues and generated the F13R and F13E mutants (Fig 1C), and finally with a polar residue and generated the F13S mutant (Fig 1C) Some of the mutations and deletions appear to have an impact on the stability of the resulting recombinant proteins Indeed, only 24 h after purification, recombinant CP13–215, CP14–215, F13A, F13G, F13R, F13E and F13S showed signs of degradation, and bands of lower molecular mass proteins appeared in western blots (Fig 1B,C) Characterization of recombinant NLPs As shown before, purified CP6–215 can self-assemble in E coli [9], and CP27–215 was found as a monomeric form [10] To monitor the capacity of the different mutated and truncated forms to produce NLPs, we examined purified proteins by electron microscopy B C Fig PapMV CP mutants (A) Schematic representation of PapMV CP mutant constructs expressed in E coli All constructs possess a His6 tag The dark rectangle in the schemata and the underlined amino acids represent a small helix of six amino acids that is predicted to occur between Q18 and S23 [10] Amino acids that are mutated in some constructs are in italics (B, C) Expression and purification of recombinant coat proteins on an SDS/PAGE gel The left panels represent Coomassie staining profiles and the right panels represent western blots of purified proteins revealed with IgG directed against PapMV CP (Fig 2A–D) Three mutated forms, CP13–215, F13L mutant, 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 mutants FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1475 ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits A B 0.2 µm 0.2 µm D C 0.2 µm E 350 Length of the NLPs (nm) 0.2 µm 300 250 200 150 100 50 CP6–215 CP13–215 F13L F13Y formed NLPs that appeared to be longer than CP6– 215 and CP13–215 (Fig 2C,D) We determined the length of 250 NLPs for each recombinant protein, and the average lengths are given in Fig 2E As expected, CP6–215 and CP13–215 NLPs were similar in length, measuring 50 nm However, NLPs comprising the F13L and F13Y mutants were longer than CP6–215 NLPs Indeed, F13L NLPs appeared to be 2.5 times longer than CP6–215 NLPs, whereas F13Y NLPs were four times longer Gel filtration analysis of recombinant proteins Previously, we showed that when expressed in E coli, the CP6–215 protein occurred 80% of the time as a 450 kDa multimer (disks), and the remaining 20% was 1476 Fig Characterization of recombinant NLPs self-assembled in E coli Electron microscopy of (A) CP6–215, (B) CP13–215, (C) F13L mutant and (D) F13Y high-speed pellet Bars are 200 nm (E) Average length of recombinant NLPs: CP6–215, CP13–215, F13L, and F13Y (n = 250) in NLPs [9] To measure the ability of our recombinant CPs to form NLPs, we subjected purified proteins to gel filtration (Figs and 4) The Superdex 200 and Superdex 75 FPLC profiles of recombinant CP6–215 and CP27–215 were compared with those of other recombinant CPs As shown before [9], the FPLC Superdex 200 profile of CP6–215 first presents a peak eluting 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 elutes at 50.5 mL; this corresponds to a multimer of 450 kDa, which corresponds to CP6–215 disks Finally, a third peak eluting at 78.8 mL corresponds to low molecular mass molecules composed of degraded forms of the CP that remain monomeric [9] The respective percentages of the total proteins FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits A B C D E F 0.2 µm 0.2 µm Fig 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; 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); mg of the purified proteins was loaded onto an FPLC Superdex 75 26/60 column (C) Gray line, F13L mutant; dark line, F13Y mutant; dotted line, CP6–215; mg of the recombinant proteins was loaded onto an FPLC Superdex 200 16/60 column (D) Gray line, F13A mutant; black line, F13G mutant; dotted line, CP6–215; mg of the recombinant proteins was loaded onto an FPLC Superdex 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 HMWF fractions of (E) the F13A mutant and (F) the F13G mutant represented by the three forms were as follows: NLP, 33%; disks, 36%; and monomers, 31% This current profile differs slightly from the first one that we published [9] This is probably because the methods used for bacterial lysis were different Here, use of a French press permitted recovery of more proteins that were not previously detected when sonication was employed to lyse the cells It is likely that the heat generated by sonication affected the protein and influenced the recovery CP27–215 was applied to a Superdex 75 26/ 60 column (Fig 3B), and eluted as a single peak at 164.61 mL, as previously reported [10] The elution FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1477 ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits A B 0.2 µm Fig 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 proteins and 150 lg of the purified CP6–215 protein were loaded onto an FPLC Superdex 200 10/300 column Molecular markers are shown in the left corner HMWF, high molecular weight forms (> 670 000); disks, 20 subunits of the CP (450 000); LMWF, low molecular weight forms (< 230 000) (B) Electron microscopy of the HMWF fraction of the F13E mutant profile 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 an increase in the monomeric form of the protein (51%) This might indicate lower stability of the protein, which consequently impacts on the quantity of NLPs produced This result suggests that deletion of 12 amino acids at the N-terminus of PapMV CP does not abolish 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 single peak at 158.31 mL obtained using the Superdex 75 26/ 60 column As expected, the recombinant CP14–215 eluted before the truncated CP27–215, as it is 13 amino acids longer Both proteins were detected with 100% frequency as monomers Superdex 200 profiles of F13L and F13Y were also compared with that of CP6–215 (Fig 3C) The two mutated forms were eluted in only two peaks, in contrast with three peaks for CP6–215 In both cases, most of the protein was eluted in the first peak, which occurred at 42.6 mL for the F13L mutant and at 41.7 mL for the F13Y mutant (Fig 3C) These peaks correspond to 80% and 90% of the total purified protein respectively These fractions contain NLPs Interestingly, disks that normally elute at 50.5 mL were not detected with these two mutants (Fig 3C) Finally, a peak eluted at 86.1 mL for the F13L mutant and 82.6 mL for the F13Y mutant This peak is associated with monomeric forms that probably represent a degraded protein These results suggest that the two mutants are highly efficient at forming NLPs 1478 The F13A and F13G mutants were also subjected to Superdex 200 (Fig 3D) elution The F13A and F13G mutants eluted in two peaks (Fig 3D) The first one appeared at 42.1 mL for the F13A mutant and at 40.3 mL for the F13G mutant The top of each peak was collected and examined by electron microscopy Few NLPs were observed with the F13A mutant, as most of the protein appeared as nonspecific aggregates (Fig 3E) For the F13G mutant, NLPs were not found on the electron microscopy grids Only nonspecific aggregates were visible (Fig 3F) In both cases, disks were not found in the sample A peak that eluted at 81.4 mL for the F13A mutant and at 81.5 mL for the F13G mutant corresponds to a monomeric form (Fig 3D) In fact, most of the purified F13A (65%) mutant was found to be monomeric In contrast, only 20% of the F13G mutant eluted as a monomer It seems that the F13A mutation affects the capacity of the recombinant CP to form NLPs, because a large proportion of the recombinant purified protein is found in low molecular mass forms Also, even if 35% of the protein eluted as a large molecular mass multimer, the electron microscopy observation revealed that the proteins form nonspecific aggregates that are inefficient in making NLPs For the F13G mutant, the mutation probably greatly affects its capacity to multimerize into disks and NLPs The F13R, F13E and F13S mutants were also subjected to Superdex 200 (Fig 4A) gel filtration In this experiment, we loaded smaller amount (150 lg) of CP6–215 protein to separate the NLPs from the disks into two distinct peaks The F13R and F13S mutants FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS ´ ´ M E Laliberte Gagne et al were found entirely in the low molecular mass fractions and were unable to self-assemble into NLPs (data not shown) Most of the protein of the F13E mutant was found as low molecular mass forms, but a small fraction was found in the exclusion fraction with CP6– 215 NLPs (Fig 4A) However, NLPs were absent, and only nonspecific aggregates could be observed by electron microscopy in this fraction (Fig 4B) Therefore, we concluded that the F13E mutant was unable to self-assemble into an NLP H-15N HSQC spectrum analysis To confirm that the CP14–215 monomer can be used for NMR analysis, we uniformly labeled the protein with 15N and acquired preliminary NMR data that we superimposed on similar spectra obtained previously with the monomeric form of CP27–215 [10] Conditions determined previously to be optimal for NMR were used [10] In order to improve solubility and stability for NMR sample analysis, a pH of 6.2 was selected A 2D 1H-15N HSQC spectrum of CP14–215 was acquired at 600 MHz at 25 °C (Fig 5) Good spectral dispersion (3.5 p.p.m.) of backbone amide 1H resonances indicates that PapMV CP is well folded under the conditions used Furthermore, the peak line width and signal intensity under the conditions used suggest that the mutant CP14–215 is monomeric in solution, as expected from the chromatography results Superimposition of spectra revealed that all peaks corresponding to structured regions of CP27–215 are present in the CP14–215 Fig Superimposition of the 1H-15N HSQC spectra of CP14–215 and CP27–215; 0.1 mM each protein was diluted in 10 mM dithiothreitol, 10% D2O, 1· complete protease inhibitor cocktail, 0.1 mM NaN3 and 60 lM DSS at pH 6.2 F13 critical for interaction among the CP subunits spectrum This suggests that the structure of both truncated forms is very similar Moreover, the presence of several peaks in the middle of the spectrum (corresponding to unstructured regions) suggests that amino acids 14–26 are not structured Gel shift assays To evaluate whether the ability to form NLPs was related to affinity for RNA, as we have shown previously with the E128A and K97A mutants [9], we measured the affinity of the mutant by electrophoretic mobility shift assay (EMSA) (Fig 6) The high-speed supernatant (disks) of the purified proteins was incubated with 165 fmol of an RNA probe labeled with c-32P made from an 80 nucleotide RNA transcript from the 5¢-end of PapMV The disks of CP6-215 and CP13–215 interacted with the probe in a cooperative manner and induced a shift when as little as 100 ng of proteins was added (Fig 6A,B) This result suggests that differences between the ability of the two proteins to form NLPs, as shown in Fig 3A, are not related to their affinity for RNA A similar experiment was performed with CP14–215 and CP27–215, two proteins known to form monomers As expected, both CP14–215 and CP27–215 failed to interact with the first 80 nucleotides of viral RNA in vitro (Fig 6C,D) We performed an EMSA with the high-speed supernatant of F13A, and showed that it failed to induce formation of a protein–RNA complex (Fig 6E) This is consistent with our electron microscopy observations, which highlighted the inability of this protein to self-assemble into NLPs As the F13L and F13Y mutants form only NLPs in E coli, we needed to disrupt NLPs using the widely employed acetic acid treatment to isolate the disks as previously described [3], to test their ability to bind RNA The same treatment was done with CP6–215 NLPs as a control Previously, we proposed that purified protein NLP length was related directly to its RNA-binding capacity [9] Surprisingly, isolated disks of these two proteins showed a lower affinity for RNA than CP6–215 disks (Fig 7A–C), even though extracted disks looked normal at the electron microscopy level (supplementary Fig S1) We did not test the F13G, F13E, F13R and F13S mutants, because they were unable to form NLPs and therefore did not bind RNA Measurement of RNA content by spectroscopy In addition to EMSA, we evaluated the difference observed between the F13L and F13Y mutants and FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1479 ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits A B C D Fig EMSA with high-speed supernatant of recombinant CPs (A) CP6–215; (B) CP13–215; (C) CP27–215; (D) CP14–215; (E) F13A mutant Increasing protein amounts were incubated at 22 °C for h with 165 fmol of an RNA probe labeled with c-32P The probe was made from an 80 nucleotide RNA transcript from the 5¢-end of the PapMV noncoding region The free probe and the RNA–protein complex are indicated by arrows E CP6–215 by spectroscopy using the A280/260 nm ratio of different recombinant proteins Measurement of the A280/260 nm ratio, which was performed three times, was very consistent, and the average is presented in Table Surprisingly, A280/260 nm ratios obtained for the two recombinant proteins were closer to the one obtained for PapMV than for CP6–215 NLPs These results suggest that F13L and F13Y NLPs are competent at binding RNA in spite of the lower affinity measured by EMSA The A280/260 nm ratio was also calculated for disks Results for PapMV disks and CP6–215 disks differed from those for F13L and F13Y disks, and suggest that there is still some RNA associated with recombinant F13L and F13Y disks This could partially explain the decreased affinity of F13L and F13Y disks in EMSA Discussion Previous studies on the PapMV CP indicated that an essential domain for CP multimerization is located on 1480 26 amino acids of the N-terminus [9,10] In this work, we investigated this region in detail, introducing deletions and point mutations All mutations incorporated in the PapMV CP gene did not affect the secondary structure prediction of the CPs (supplementary Fig S2) We have shown clearly that the N-terminal 12 amino acids are not important for self-assembly of the PapMV CP This result is consistent with the findings of Zhang et al [1], who showed that cleavage of the N-terminus with trypsin did not affect virus particles This region probably plays a role in protein stabilization, rather than in NLP formation, as we found more degraded monomers with CP13–215 than with CP6–215 in the FPLC profiles (Fig 3A) Deletion of 13 amino acids, mutation of residue F13 for the less hydrophobic residues A or G, or replacement with the charged residues R or E, or the polar residue S, had a major detrimental impact on NLP formation This suggests that F13 is involved in a hydrophobic interaction that is crucial for interplay among the protein subunits and formation of the disks FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits A Fig EMSA with high-speed supernatant of recombinant disks obtained from the disruption of the NLPs by use of the acetic acid method [3] (A) CP6–215; (B) F13L mutant; (C) F13Y mutant Increasing amounts of proteins were incubated at 22 °C for h with 165 fmol of an RNA probe labeled with c-32P The probe was made from an 80 nucleotide RNA transcript from the 5¢-end of the PapMV noncoding region The free probe and the RNA–protein complex are indicated by arrows B C Table Protein A280/260 nm ratio 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 absorbance measurement was taken directly from the purified PapMV and purified F13L and F13Y recombinant proteins The four proteins were treated by acetic acid methodology [3] to generate disks that were used to calculate the A280/260 nm ratio Virus and NLPs Extracted disks PapMV A280/260 nm ratio CP6–215 F13L F13Y PapMV CP6–215 F13L F13Y 0.75 1.1 0.8 0.75 1.5 1.55 0.95 0.9 that are the building blocks with the RNA of the NLPs Interestingly, F13L and F13Y substitutions increased NLP formation, probably through improvement of the RNA-binding capacity of the proteins, as shown by the A280/260 nm ratio (Table 1) EMSA analysis of F13L and F13Y extracted disks did not show improved affinity for RNA as compared with CP6– 215, probably because they were still bound tightly to RNA, which interfered with RNA probe binding It appears that F13 plays an important role in the aggregation state of the protein, as mutation of this residue led to formation of either NLPs (F13Y and F13L) or monomeric forms of the protein (F13G, F13A, F13R, F13E, F13S), which were always detrimental to accumulation of disks in bacteria It is possible that this regulation is important in PapMVinfected plants to ensure that only viral RNA, and not plant cellular RNA, gets encapsulated by the viral CP It is tempting to draw a parallel with tobacco mosaic virus CP, even if this protein is not related to the PapMV CP, where a hydrophobic interaction between the CP subunits was shown to be important for self-assembly of the virus into a rigid rod structure [11] Comparison of 2D 1H-15N HSQC spectra from two monomeric forms, CP14–215 and CP27–215, indicates that amino acids 14–26 are unstructured This result suggests that the small helix that was predicted by bioinformatics to occur between residues 18 and 24 [10] is probably unstable We propose that the entire N-terminus from residues to 36 forms an unstructured coil region A recent report showed that the CP of potato virus X (PVX) can be truncated by 22 amino acids at its N-terminus without affecting either virus infectivity or formation of virus particles in plants [12] The authors took advantage of this mutant by fusing foreign peptides to the surface of the virus Alignment of the N-terminus of the PVX CP with the PapMV CP revealed that the PVX CP harbors an extension of 20 amino acids in the N-terminus as compared with PapMV (Fig 8) At position 33 of the PVX CP, we find an F residue that aligns perfectly with the PapMV FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1481 ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits Fig Alignment of a consensus sequence derived from 18 known potexvirus coat proteins and the PapMV CP in the N-terminal region 1–27 of PapMV CP Conserved hydrophobic residues that aligned with amino acid 13 of the PapMV CP are highlighted in bold Alignment was done using the CP sequences of: bamboo mosaic virus (BaMV); cactus virus X (CVX); clover yellow mosaic virus (ClYMV); cassava 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 asiatica mosaic virus (PlAMV); PVX; scallion virus X (ScaVX); strawberry mild yellow edge virus (SMYEV); tulip virus X (TVX); white clover mosaic virus (WClMV) CP F13 Therefore, on the basis of our results, it is likely that a deletion of 32 amino acids will be tolerated by PVX without disturbing the assembly process Alignment of this F residue is also shared with several other potexviral CP sequences, as seven out of the 18 N-terminal sequences of the potexviruses showed consensus for an F in the position that corresponds to F13 of PapMV CP (Fig 8) Also, an F is present in the same area in the CP of bamboo mosaic virus The CP of mint virus X presents an L in this position, which corresponds to a hydrophobic residue that could substitute for an F in the PapMV CP Therefore, on the basis of the alignment, we propose that a hydrophobic residue at the position that corresponds to PapMV CP F13 is preferred in half of the potexvirus CP It is likely that this residue also plays an important role in the interactions between the subunits in the potexviruses family Finally, our results agree with the assembly model recently proposed for a potyvirus member of the Potyviridea family: the pepper vain banding virus (PVBV) [13] These authors proposed that the N-terminal extension of a CP subunit interacts with the C-terminal extension of an adjacent CP subunit in a head-to-tail manner, thereby permitting formation of both the ring-like intermediate and the NLPs into helix-like structures We propose that this model is applicable for PapMV and probably all potexviruses However, a major difference between PapMV and PVBV is that 1482 PapMV CP subunit assembly into disk structures is based on a hydrophobic interaction, whereas PVBV CP assembly into ring-like structures (disks) was proposed to be driven by electrostatic interactions [13] Experimental procedures Cloning and expression of recombinant proteins The 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 and CP14–215, were amplified by PCR from the clone CP6–215 inserted into a pET-3d vector The forward primers used for these PCR reactions were CP13–215 forward, 5¢-ACGTCA TATGTTCCCCGCCATCACCCAG-3¢, and CP14–215 forward, 5¢-ACGTCATATGCCCGCCATCACCCAGGAA-3¢ A reverse primer, 3¢-GAAATTCTTCCTCTATATGTA TACTGCA-5¢, was used for both constructs The PCR products were digested with NdeI, to generate the two truncated CPs inserted into a pET-3d vector The F13A, F13E, F13G, F13L, F13R, F13S and F13Y mutations were introduced by PCR into the CP6–215 clone using the following oligonucleotides: forward (F13A), forward 5¢-GCGCCCGCCATCACCCAGGAACAA-3¢; (F13E), 5¢-GAACCCGCCATCACCCAGGAACAA-3¢; forward (F13G), 5¢-GGCCCCGCCATCACCCAGGAACAA3¢; forward (F13L), 5¢-CTGCCCGCCATCACCCAGGA ACAA-3¢; forward (F13R), 5Â-CGCCCCGCCATCACCC FEBS Journal 275 (2008) 14741484 ê 2008 The Authors Journal compilation ª 2008 FEBS ´ ´ M E Laliberte Gagne et al AGGAACAA-3¢; forward (F13S), 5¢-AGCCCCGCCAT CACCCAGGAACAA-3¢; forward (F13Y), 5¢-TATCCCG CCATCACCCAGGAACAA-3¢; and reverse (F13), 3¢-CG TAGGTGTGGGTTGTATCGG-5¢ PCR products with blunt ends were circularized to form the fourth mutated CP inserted into a pET-3d vector Expression and purification of recombinant proteins from E coli Expression and induction of proteins was conducted as described previously [9] Bacteria were harvested by centrifugation for 30 at 9000 g The pellet was resuspended in ice-cold lysis buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 10 mm imidazole, 40 lm phenylmethanesulfonyl fluoride and 0.2 mgỈmL)1 lysosyme), and bacteria were lysed by one passage through a French press The lysate was incubated with agitation for 15 with 9000 units of DNase and 1.5 mm MgCl2, and this was followed by two centrifugations for 30 at 10 000 g to eliminate cellular debris The supernatant was incubated with mL of Ni–nitrilotriacetic acid (Qiagen, Turnberry Lane, Valencia, CA, USA) under gentle agitation overnight at °C Proteins were purified as described elsewhere [9], except that they were incubated for h with mL of the elution buffer (10 mm Tris/HCl, pH 8.0, supplemented with m imidazole) before elution Imidazole was eliminated by dialysis for 24 h Protein purity was determined by SDS/PAGE and confirmed by western immunoblot analysis using rabbit polyclonal antibodies generated against purified PapMV virus F13 critical for interaction among the CP subunits grids were incubated in darkness for with lL of 2% uranyl acetate Acetic acid degradation Isolation of disks from CP6–215, F13L and F13Y NLPs was performed by acetic acid degradation as described previously [3] Two volumes of glacial acetic acid were added to the NLPs and incubated at °C for h Centrifugation at 10 000 g for 15 removed insoluble RNA The supernatant was removed and subjected to high-speed centrifugation at 100 000 g for h in a Beckman 50.2Ti rotor to remove any residual NLPs Proteins were dialyzed extensively against 10 mm Tris/HCl (pH 8.0) Gel filtration Proteins were purified by gel filtration Columns were first calibrated 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 buffer (10 mm Tris/HCl, pH 8.0, supplemented with 150 mm NaCl), were used The volume of protein loaded into the sample loop was 1.5 mL for Superdex 75 26/60, mL for Superdex 200 16/60, and 0.1 mL for Superdex 200 10/300 NMR spectroscopy Proteins were mixed with one-third of the final volume of loading buffer containing 5% SDS, 30% glycerol, and 0.01% bromophenol blue SDS/PAGE was performed as described elsewhere [14] The 600 lL sample used for NMR spectroscopy was 0.1 mm CP14–215 or CP27–215 in 90% H2O/10% D2O, 10 mm dithiothreitol (pH 6.2), 1· complete protease inhibitor cocktail (Roche), with 0.1 mm NaN3 and 60 lm 2,2dimethyl-2-silapentane-5-sulfonic acid (DSS) as the NMR chemical shift reference The 1H-15N HSQ spectra were obtained at 25 °C on a Varian Unity 600 MHz spectrometer equipped with a triple-resonance cryoprobe and Z-axis pulsed-weld gradient The acquired data consisted of 768 complex data points in the acquisition domain and 128 complex data points in the indirectly detected domain The spectral width was 10 000 Hz in the 1H dimension and 1680 Hz in the 15N dimension NMR spectra were processed using NMRPipe [15] Processing involved doubling of the 15N time domain by linear prediction, zero-filling to 2048 and 512 complex points in 1H and 15N, respectively, a 45° shifted sine-bell apodization in the 1H dimension, and a 72° shifted sine-bell apodization in the 15N dimension Electron microscopy RNA transcripts and EMSA Nucleocapsid-like particles or viruses were diluted in 10 mm Tris/HCl (pH 8.0) to a concentration of 50 ngỈlL)1, and were absorbed for on carbon-coated formvar grids Grids were washed twice with lL of water Finally, The probe was generated as described before [9] Labeled RNA probe was incubated with various amounts of recombinant proteins at room temperature for 60 We used 165 fmol of RNA for each reaction in the in vitro assembly Separation of disks and NLPs To separate the disks from NLPs, mL of purified proteins was subjected to a high-speed centrifugation for h at 100 000 g in a Beckman SW60Ti rotor The pellet that comprised the NLPs was resuspended in 300 lL of 10 mm Tris/HCl at pH 8.0 The supernatant with the disks and the low molecular mass forms was retained for gel shift assays SDS/PAGE and electroblotting FEBS Journal 275 (2008) 1474–1484 ª 2008 The Authors Journal compilation ª 2008 FEBS 1483 ´ ´ M E Laliberte Gagne et al F13 critical for interaction among the CP subunits buffer (10 mm Tris/HCl, 4% glycerol, 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 buffer for 90 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 reading of our manuscript 12 13 References 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 Sit TL, Abouhaidar MG & Holy S (1989) Nucleotide sequence of papaya mosaic virus RNA J Gen Virol 70 (Pt 9), 2325–2331 Erickson JW, Bancroft JB & Horne RW (1976) The assembly of papaya mosaic virus protein Virology 72, 514–517 Abouhaidar M & Bancroft JB (1978) The initiation of papaya mosaic virus assembly Virology 90, 54–59 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 Erickson JW & Bancroft JB (1978) The self-assembly of papaya mosaic virus Virology 90, 36–46 Erickson JW, Bancroft JB & Stillman MJ (1981) Circular dichroism studies of papaya mosaic virus coat protein and its polymers J Mol Biol 147, 337–349 Erickson JW, Hallett FR & Bancroft JB (1983) Subassembly aggregates of papaya mosaic-virus protein Virology 129, 207–211 ´ 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 and E128A on RNA binding and self 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 between 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 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 to 100 kDa Anal Biochem 166, 368–379 Delaglio F, Grzsiek S, Vuister VW, Zhu G, Pfeifer J & Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes J Biomol NMR 6, 277–293 Supplementary material 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 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 ... the aggregation state of the protein, as mutation of this residue led to formation of either NLPs (F13Y and F13L) or monomeric forms of the protein (F13G, F13A, F13R, F13E, F13S), which were always... et al F13 critical for interaction among the CP subunits monomeric form of the protein [10] This protein failed to assemble, form disks or interact with RNA in vitro [10] On the basis of this result,... F13 critical for interaction among the CP subunits spectrum This suggests that the structure of both truncated forms is very similar Moreover, the presence of several peaks in the middle of the