In another study, we have shown that deletion of 26 amino acids at the N-terminus of the CP leads to a Keywords Nucleocapsid-like particles NLPs; PapMV; papaya mosaic virus; potexvirus;
Trang 1Papaya 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
parti-cle 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
assem-bly 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 colileads 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
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 6
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 mono-meric 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-assem-bly 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 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 We also discuss the importance of F13 for assembly of other members of the potexvirus family
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.
Trang 2monomeric 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
Pap-MV CP multimerization We found that deletion of
only 13 amino acids was sufficient to inhibit
interac-tion 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
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
gener-ated 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,
recombi-nant 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
mono-meric form [10] To monitor the capacity of the
differ-ent mutated and truncated forms to produce NLPs, we
examined purified proteins by electron microscopy
(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
A
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 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.
Trang 3formed 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
in NLPs [9] To measure the ability of our recombi-nant CPs to form NLPs, we subjected purified proteins
to gel filtration (Figs 3 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
C
E
D
350
300
250
150
Length of the NLPs (nm) 50
0
200
100
CP6–215 CP13–215 F13L F13Y
Fig 2 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).
Trang 4represented 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
pub-lished [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
Fig 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, F13L mutant; 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 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.
Trang 5profile 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
con-trast 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
pro-tein respectively These fractions contain NLPs
Inter-estingly, 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
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 multi-mer, the electron microscopy observation revealed that the proteins form nonspecific aggregates that are ineffi-cient in making NLPs For the F13G mutant, the mutation 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 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
0.2 µm
Fig 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 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.
Trang 6were found entirely in the low molecular mass
frac-tions 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
elec-tron microscopy in this fraction (Fig 4B) Therefore,
we concluded that the F13E mutant was unable to
self-assemble into an NLP
1H-15N HSQC spectrum analysis
To confirm that the CP14–215 monomer can be used
for NMR analysis, we uniformly labeled the protein
with15N 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
2D1H-15N HSQC spectrum of CP14–215 was acquired
at 600 MHz at 25C (Fig 5) Good spectral dispersion
(3.5 p.p.m.) of backbone amide1H 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
struc-tured regions of CP27–215 are present in the CP14–215
spectrum This suggests that the structure of both trun-cated forms is very similar Moreover, the presence of several peaks in the middle of the spectrum (corre-sponding 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 previ-ously with the E128A and K97A mutants [9], we mea-sured the affinity of the mutant by electrophoretic mobility shift assay (EMSA) (Fig 6) The high-speed supernatant (disks) of the purified proteins was incu-bated 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 mono-mers 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 inabil-ity 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 puri-fied 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 micros-copy 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
Fig 5 Superimposition of the 1 H- 15 N HSQC spectra of CP14–215
and CP 27–215 ; 0.1 m M each protein was diluted in 10 m M
dithiothrei-tol, 10% D2O, 1· complete protease inhibitor cocktail, 0.1 m M
NaN3and 60 l M DSS at pH 6.2.
Trang 7CP6–215 by spectroscopy using the A280/260 nmratio 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 1 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
compe-tent at binding RNA in spite of the lower affinity
mea-sured 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
26 amino acids of the N-terminus [9,10] In this work,
we investigated this region in detail, introducing dele-tions and point mutadele-tions All mutadele-tions 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 find-ings of Zhang et al [1], who showed that cleavage of the 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 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 replace-ment 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
C
E
D
Fig 6 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 1 h with 165 fmol of an RNA probe labeled with c- 32 P 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.
Trang 8that are the building blocks with the RNA of the
NLPs Interestingly, F13L and F13Y substitutions
increased NLP formation, probably through
improve-ment of the RNA-binding capacity of the proteins, as
shown by the A280/260 nmratio (Table 1) EMSA
analy-sis 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
PapMV-infected 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
Pap-MV CP, where a hydrophobic interaction between the
CP 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 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 bio-informatics to occur between residues 18 and 24 [10] is probably unstable We propose that the entire N-ter-minus from residues 1 to 36 forms an unstructured coil region
A recent report showed that the CP of potato
vir-us 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 pep-tides 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
A
Fig 7 EMSA with high-speed supernatant
of recombinant disks obtained from the
dis-ruption 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 1 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.
Table 1 Protein A 280/260 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 A 280/260 nm ratio.
A280/260 nm
ratio
Trang 9CP F13 Therefore, on the basis of our results, it is
likely 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 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
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 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
Poty-viridea 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
PapMV CP subunit assembly into disk structures is based on a hydrophobic interaction, whereas PVBV
CP assembly into ring-like structures (disks) was pro-posed 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 for-ward, 5¢-ACGTCATATGCCCGCCATCACCCAGGAA-3¢
TACTGCA-5¢, was used for both constructs The PCR prod-ucts 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),
(F13E), 5¢-GAACCCGCCATCACCCAGGAACAA-3¢; for-ward (F13G), 5¢-GGCCCCGCCATCACCCAGGAACAA-3¢; forward (F13L), 5¢-CTGCCCGCCATCACCCAGGA ACAA-3¢; forward (F13R), 5¢-CGCCCCGCCATCACCC
Fig 8 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 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 clover mosaic virus (WClMV).
Trang 10AGGAACAA-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
centri-fugation for 30 min 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
fluo-ride and 0.2 mgÆmL)1 lysosyme), and bacteria were lysed
by one passage through a French press The lysate was
incubated with agitation for 15 min with 9000 units of
DNase and 1.5 mm MgCl2, and this was followed by two
centrifugations for 30 min at 10 000 g to eliminate cellular
debris The supernatant was incubated with 3 mL of
Ni–ni-trilotriacetic acid (Qiagen, Turnberry Lane, Valencia, CA,
USA) under gentle agitation overnight at 4C Proteins
were purified as described elsewhere [9], except that they
were 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 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
Separation of disks and NLPs
To separate the disks from NLPs, 1 mL of purified proteins
was subjected to a high-speed centrifugation for 2 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
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]
Electron microscopy
Nucleocapsid-like particles or viruses were diluted in
10 mm Tris/HCl (pH 8.0) to a concentration of 50 ngÆlL)1,
and were absorbed for 6 min on carbon-coated formvar
grids Grids were washed twice with 8 lL of water Finally,
grids were incubated in darkness for 6 min with 8 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 pre-viously [3] Two volumes of glacial acetic acid were added
to the NLPs and incubated at 4C for 1 h Centrifugation
at 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 to remove any residual NLPs Proteins were dialyzed exten-sively 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 buf-fer (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, 1 mL for Superdex 200 16/60, and 0.1 mL for Superdex 200 10/300
NMR spectroscopy 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 inhibi-tor cocktail (Roche), with 0.1 mm NaN3 and 60 lm 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) as the NMR chemical shift reference The 1H-15N HSQ spectra were obtained at 25C on a Varian Unity 600 MHz spectrome-ter 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 pro-cessed using NMRPipe [15] Processing involved doubling
of the 15N time domain by linear prediction, zero-filling to
2048 and 512 complex points in1H and15N, respectively, a 45 shifted sine-bell apodization in the1
H dimension, and a 72 shifted sine-bell apodization in the15N dimension
RNA transcripts and EMSA The probe was generated as described before [9] Labeled RNA probe was incubated with various amounts of recom-binant proteins at room temperature for 60 min We used
165 fmol of RNA for each reaction in the in vitro assembly