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Dissectingtheroleofprotein–proteinand protein–nucleic
acid interactionsinMS2bacteriophage stability
Sheila M. B. Lima
1
, Ana Carolina Q. Vaz
1
, Theo L. F. Souza
1
, David S. Peabody
2
, Jerson L. Silva
1
and Andre
´
a C. Oliveira
1
1 Programa de Biologia Estrutural and Centro Nacional de Ressona
ˆ
ncia Magne
´
tica Nuclear de Macromole
´
culas, Instituto de Bioquı
´
mica
Me
´
dica, Universidade Federal do Rio de Janeiro, Brazil
2 Department of Molecular Genetics and Microbiology and Cancer Research and Treatment Center, University of New Mexico School of
Medicine, Albuquerque, NM, USA
Specific protein–proteinandprotein–nucleicacid inter-
actions are required for successful assembly of a large
variety of biologically important macromolecular com-
plexes, including viruses. We used the bacteriophage
MS2 as a model for the study of such interactions.
MS2 is a member of a large group of small RNA
phages that infect Escherichia coli [1]. Its icosahedral
shell consists of 180 copies of coat protein (M
r
13 728)
arranged in a T ¼ 3 quasi-equivalent surface lattice
surrounding the ssRNA genome. Each virion also con-
tains one copy ofthe maturase (or A) protein, respon-
sible for attachment ofthe virus to E. coli through the
F-pilus. Coat protein folds as a dimer of identical sub-
units and consists of a 10-stranded antiparallel b-sheet
facing the interior ofthe phage particle, with antiparal-
lel, interdigitating a-helical segments on the virus’
Keywords
coat protein interactions; fluorescence;
hydrostatic pressure; MS2 bacteriophage;
virus-like particles
Correspondence
J. L. Silva and A. C. Oliveira, Avenida
Bauhinia, 400 – CCS ⁄ ICB ⁄ Bl E, sl. 08,
Cidade Universita
´
ria, CEP, Rio de Janeiro,
RJ 21941-590, Brazil
Fax: +55 21 3881 4155
Tel: +55 21 2562 6756
E-mail: jerson@bioqmed.ufrj.br
E-mail: cheble@bioqmed.ufrj.br
(Received 2 August 2005, revised 26 January
2006, accepted 6 February 2006)
doi:10.1111/j.1742-4658.2006.05167.x
To investigate theroleofprotein–proteinandprotein–nucleicacid interac-
tions in virus assembly, we compared the stabilities of native bacteriophage
MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assem-
bly-defective coat protein mutant (dlFG) and its single-chain variant
(sc-dlFG). Physical (high pressure) and chemical (urea and guanidine
hydrochloride) agents were used to promote virus disassembly and protein
denaturation, andthe changes in virus and protein structure were moni-
tored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluor-
escence, and light scattering. We found that VLPs dissociate into capsid
proteins that remain folded and more stable than the proteins dissociated
from authentic particles. The proposed model is that the capsid disassem-
bles but the protein remains bound to the heterologous RNA encased by
VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids,
because it lacks the 15-amino acid FG loop involved in inter-dimer inter-
actions at the viral fivefold and quasi-sixfold axes. This protein was very
unstable and, when compared with the dissociation ⁄ denaturation of the
VLPs andthe wild-type virus, it was much more susceptible to chemical
and physical perturbation. Genetic fusion ofthe two subunits ofthe dimer
in the single-chain dimer sc-dlFG stabilized the protein, as did the presence
of 34-bp poly(GC) DNA. These studies reveal mechanisms by which inter-
actions inthe capsid lattice can be sufficiently stable and specific to ensure
assembly, and they shed light on the processes that lead to the formation
of infectious viral particles.
Abbreviations
ANS, 8-anilinonaphthalene-1-sulfonate; GdnHCl, guanidine hydrochloride; VLP, virus-like particle.
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1463
outer surface. The 15-residue FG loop extends
from both ends ofthe dimer and is responsible for
many ofthe interdimer contacts essential for virus
assembly [2].
Coat protein acts both as a translational repressor
and as the principal viral structural protein, interacting
specifically with a 19-nucleotide RNA hairpin to
repress translation ofthe viral replicase and to nucle-
ate assembly ofthe viral shell on genome RNA [3–7].
It has been extensively studied using genetic approa-
ches, and a large number of mutational variants
affected in their protein–RNA and protein–protein
interactions have been produced [8,9]. We recently des-
cribed a comparative study with three ts mutants of
bacteriophage MS2, in which in each mutant only one
coat protein amino acid was substituted. Each substi-
tution was sufficient to change thestabilityof viral
capsid inthe presence of denaturing agents [10]. Here
we describe studies of additional coat protein variants.
As part of a study to define which interactions are
important for virus stability, we compared authentic
MS2 virions with virus-like particles (VLPs) produced
when coat protein is overexpressed in E. coli from a
plasmid [11]. To examine thestabilityofthe coat pro-
tein dimer inthe absence ofthe intersubunit interac-
tions found inthe capsid, we used a mutant, named
dlFG, in which the FG loop, a 15-residue sequence
connecting the F and G b-strands, is replaced with
two amino acids sufficient to make a b-turn. Although
unable to form capsids, the dlFG dimer retains its abil-
ity to bind RNA [3,5,12–17]. We also determined the
effects on stabilityof genetic fusion ofthe two sub-
units ofthe dlFG dimer using the variant named
2CTdlFG, which takes advantage ofthe physical prox-
imity ofthe N-terminus and C-termius ofthe two
monomers to covalently link them. To facilitate
purification, both dlFG and a single-chain dimer,
sc-dlFG, contain an N-terminal six-histidine nickel-
affinity tag.
Using intrinsic fluorescence of Trp residues, extrinsic
fluorescence ofthe probe bis-8-anilinonaphthalene-1-
sulfonate (bis-ANS), light scattering, and CD, we
monitored conformational changes promoted by high
pressure and high concentrations of urea and guani-
dine. Concerning the tertiary structure, the relative
stabilities ofthe different forms are as follows:
dlFG < sc-dlFG < MS2 < VLP. The higher stability
of the capsid protein bound to heterologous nucleic acid
may serve as a ‘biological sieve’. In contrast, authentic
MS2 particles dissociate and unfold co-operatively,
which would guarantee that any particle without the
authentic RNA would be locked in a state lacking the
ability to release the RNA during infection.
Results
Dissociation and denaturation of wild-type
virus and VLPs induced by urea and guanidine
hydrochloride (GdnHCl)
Intrinsic fluorescence provides a convenient means to
monitor changes in protein conformation inthe pres-
ence of denaturing agents. The tryptophan residues
buried inthe hydrophobic interior ofthe protein
emit fluorescence when excited at 280 nm. When the
protein unfolds inthe presence of denaturing agents,
the exposure of buried residues reflects the conform-
ational changes inthe protein. The coat protein of
MS2 has two tryptophan residues, Trp32 and Trp82.
Trp32 clearly resides within the hydrophobic core of
the protein. The other residue, Trp82, is partially
solvent-exposed [17]. Its environment is determined
primarily by interactions within the dimer, not by
interactions between dimers. Thus, tryptophan fluo-
rescence should predominantly monitor dimer dena-
turation rather than capsid dissociation. Meanwhile,
light-scattering measurements are sensitive to the size
of the particle and can be used to monitor capsid
dissociation. The use ofthe two methods permitted
us to discriminate between the two phenomena inves-
tigated here, dissociation ofthe capsids and denatur-
ation ofthe coat protein.
The wild-type coat protein, when expressed in E. coli
from a plasmid spontaneously associates into capsids
and packages host RNAs, thus forming VLPs. We
treated VLPs and native virus with increasing concen-
trations of urea to compare theroleof native RNA
with heterologous RNA inthestabilityofthe capsid
protein.
Surprisingly, as measured by tryptophan fluores-
cence, VLPs appear to be substantially more stable
than virus particles themselves. The spectral center of
mass of VLPs did not change significantly until 5 m
urea, indicating that the capsid protein does not dena-
ture until this urea concentration is achieved (Fig. 1A).
MS2, however, begins to significantly unfold at around
3.5–4 m. This result is all the more surprising when
compared with the VLPs of other RNA viruses, which
are generally less stable than the authentic virus in the
presence of nonviral RNA [18]. Similar behavior was
observed inthe presence of a different denaturant,
GdnHCl, as the VLP starts to denature only after
3.5 m GdnHCl (Fig. 1B). In fact, during both treat-
ments (5 m urea and 3.5 m GdnHCl), there were
minimal changes on solvent exposure of tryptophan
residues on VLP coat protein, compared with the other
forms.
Bacteriophage MS2stability S. M. B. Lima et al.
1464 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
To check whether thestability was ofthe entire par-
ticle or ofthe capsid protein, light-scattering measure-
ments were performed for MS2 (Fig. 2A) and VLPs
(Fig. 2B). When we plotted the spectral center of mass
and light scattering for MS2inthe same plot, we
observed that the curves were superimposable, indica-
ting that virus disassembly was coincident with subunit
unfolding (Fig. 2A). For the VLPs, however, the
curves were different, suggesting that protein unfolding
occurs only after capsid disassembly (Fig. 2B).
Overall, the results indicate that authentic MS2
particles dissociate and denature co-operatively. In
contrast, VLPs disassemble at lower denaturant
concentration but denature at much higher urea con-
centrations than theMS2 capsid protein. We propose
that the VLP capsid disassembles but the protein
remains bound to the heterologous RNA encased by
the VLPs. The VLPs have been previously character-
ized [11] showing that VLP capsids contain a hetero-
geneous population of RNAs, with two predominant
species corresponding to about 1800 and 200 nucleo-
tides. Northern-blot analysis shows that the coat
protein encapsidates host RNAs, including species
derived from rRNA precursors. The average size of the
Fig. 1. Effects of urea and guanidine on thestabilityof bacterio-
phage MS2 wild-type, VLPs, dlFG and sc-dlFG. (A) Effects of
increasing urea concentrations on the spectral center of mass of
(d) MS2 WT, (n) VLPs, (m) dlFG, and (r) sc-dlFG. Incubation was
overnight at each concentration. (B) Effect of guanidine was meas-
ured by the spectral center of mass of tryptophan fluorescence
emission. The sample was excited at 280 nm andthe emission
was measured at 300–420 nm. The buffer used was 10 m
M
Tris ⁄ HCl ⁄ 100 mM NaCl ⁄ 0.01 mM EDTA, pH 7.5.
Fig. 2. Comparison between spectral center of mass and light-scat-
tering values ofbacteriophageMS2 (A) and VLPs (B) during urea-
induced dissociation ⁄ denaturation. To verify the dissociation and
denaturation processes, we compared inthe same plot the spectral
center of mass and light-scattering measurements inthe presence
of increasing concentrations of urea. (A) (d) Spectral center of
mass; (s) light scattering for MS2. (B) (n) Spectral center of mass;
(h) light scattering for VLPs. For the light-scattering measurements,
the sample was excited at 320 nm andthe emission was meas-
ured at 315–325 nm. For the spectral center of mass, the sample
was excited at 280 nm and measured at 300–420 nm.
S. M. B. Lima et al. BacteriophageMS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1465
heterologous RNA is much smaller than that of the
MS2 RNA. It is noteworthy that the two particles
have similar hydrodynamic behavior, and electron
microscopy shows that they are very similar (data not
shown).
To verify the urea-induced and GdnHCl-induced
changes inthe secondary structure ofthe proteins, we
analyzed the UV CD spectra ofMS2and VLPs in the
absence and presence of 5.0 m and 9.0 m urea and
3.5 m and 5.0 m GdnHCl, the concentrations at which
the greatest difference between the samples was
observed. CD spectra were measured inthe range 300–
190 nm and showed that the loss of secondary structure
probably accompanied the loss of tertiary structure.
The CD data reveal one negative peak at 216 nm,
corresponding to a high b-sheet content, and one pos-
itive peak around 260–270 nm contributed by RNA
[19]. Inthe presence of 5.0 m urea there was a decrease
of 70% of ellipticity at 216 nm for MS2, indicating
a substantial loss of secondary structure, and at 9 m
urea no residual secondary structure was present
(Fig. 3A). A similar result was obtained with GdnHCl
treatment (Fig. 3B). However, the VLPs showed few
changes in secondary structure inthe presence of 5.0 m
urea, and no change inthe presence of 3.5 m GdnHCl,
confirming its higher stability (Fig. 3C,D).
Denaturation ofthe assembly-defective coat
protein mutants (dlFG and sc-dlFG) induced by
urea and GdnHCl
The loop between the F and G b-strands (FG loop) of
the bacteriophageMS2 coat protein subunit forms
contacts between dimers important for capsid assem-
bly. Because it lacks this loop, the dlFG mutant can-
not form capsids, but instead is encountered in the
form of dimers [5,14]. To understand the importance
of capsid protein–protein contacts for thestability of
coat protein, we determined thestabilityof dlFG and
compared it with thestabilityofthe genetically fused
coat protein dimer, sc-dlFG. We induced dissociation
of dlFG and sc-dlFG with increasing concentrations of
urea between 1 and 8 m (Fig. 1A). The data showed
significant differences between thestabilityof dlFG,
which begins to unfold at 1 m urea and unfolds
completely by 4 m, and authentic virions, whose
unfolding only begins at around 4 m and is not com-
plete until 6 m. The sc-dlFG single-chain dimer was
about as stable as the virus particle. The shift in the
spectral center of mass 1500 cm
)1
at 4 m urea sug-
gested complete denaturation of dlFG at this urea con-
centration. On the other hand, complete denaturation
of virus and sc-dlFG was observed only inthe pres-
ence of 6 m urea, and even more urea was needed to
promote complete denaturation ofthe VLPs.
A similar instability profile was observed for dlFG
in the presence of GdnHCl, where 1.5 m was enough
for complete denaturation, but the virus was denatured
only with 3.5 m. Although the urea denaturation of
sc-dlFG was very similar to the virus, in GdnHCl it
denatured more easily (Fig. 1B). It should be noted
that the FG loop deletion removes Trp82, accounting
for the differences observed between the initial spectral
center ofthe mass of both dlFG and sc-dlFG com-
pared with wild-type virus and VLPs, which both
retain Trps.
CD studies were also performed with the assembly-
defective mutants. With dlFG the presence of 2.0 m
urea promoted 50% decrease in ellipticity at 216 nm,
and no secondary structure was detected inthe pres-
ence of 5.0 m. The same behavior was observed in the
presence of 1.0 m and 3.5 m GdnHCl. The data
showed that the loss of secondary structure accompan-
ied the loss ofthe tertiary structure (Fig. 3E,F). Again,
sc-dlFG was substantially more stable to these denatu-
rants than dlFG (Fig. 3G,H).
Pressure-induced dissociation and denaturation
of wild-type virus, VLPs, dlFG and sc-dlFG
Pressure effects are governed by Le Chatelier’s princi-
ple, where an increase in pressure favors reduction of
the volume of a system, leading to dissociated forms.
A key advantage of hydrostatic pressure is that it does
not perturb the chemical composition ofthe solvents,
or the internal energy ofthe protein [2,20,21].
The samples ofMS2and VLPs were diluted to a
final concentration of 50 lgÆmL
)1
and incubated for
20 min under increasing pressures up to 3.4 kbar.
Hydrostatic pressure effects were monitored by
Fig. 3. UV CD spectra ofMS2 wild-type, VLPs, dlFG and sc-dlFG. Conformational changes inthe secondary structure ofMS2 bacteriophage
and mutant coat protein. (A,C) Treated with 4.5
M urea (dashed line), 9 M urea (dotted line) and control (solid line). (B,D) Treated with 3 M
GdnHCl (dashed line), 5 M GdnHCl (dotted line) and control (solid line). (E,G) Treated with 2 M urea (dashed line) and 5 M urea (dotted line)
and control (solid line). (F,H) Treated with 1
M GdnHCl (dashed line), 3.5 M GdnHCl (dotted line) and control (solid line). The spectra were
obtained in 10 m
M Tris ⁄ HCl buffer, pH 7.0, using a 0.1-cm path length quartz cuvette. The samples were diluted to a final concentration of
100 lgÆmL
)1
. The spectropolarimeter used was a Jasco J-715 1505 model. Wavelength range 300–200 nm. The data are representative of
three experiments.
Bacteriophage MS2stability S. M. B. Lima et al.
1466 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
S. M. B. Lima et al. BacteriophageMS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1467
changes inthe spectral center of mass and by light
scattering. In contrast with the effects of chemical
agents, we found that high pressure was able to pro-
mote only small changes inthe spectral center of mass
of theMS2 virus (Fig. 4). VLPs again seemed more
stable than the virus, as we observed no significant
changes at all inthe spectral center of mass (Fig. 4).
There were also no significant changes in light scatter-
ing for VLPs andMS2 particles.
To confirm that high pressure did not affect the
virus and VLPs, we analyzed the treated samples by
HPLC. The samples treated by pressure showed the
same behavior as the untreated ones, confirming that
pressure did not dissociate the viral particles (data not
shown).
We also investigated the denaturation of dlFG and
sc-dlFG under pressure. Structural changes in dlFG
were followed by a significant shift inthe Trp emission
spectrum, indicating increasing exposure to the polar
solvent. The coat protein mutant dlFG was more sus-
ceptible to pressure than was the virus, VLPs, or
sc-dlFG, 1.8 kbar being sufficient to promote complete
denaturation (Fig. 4). For sc-dlFG, complete denatura-
tion was observed only after 2.5 kbar.
bis-ANS binding assay
During dissociation and denaturation processes, pro-
teins expose hydrophobic segments and acquire the
ability to bind certain hydrophobic probes. As part
of our characterization of chemical-induced and pres-
sure-induced denaturation, we used the fluorophore
bis-ANS. This probe binds noncovalently to non-
polar segments in proteins, especially in proximity to
positive charges [22]. Because its binding is accom-
panied by a large increase in its fluorescence quan-
tum yield, it is useful in following protein structural
changes, and has been used to monitor conforma-
tional changes in capsid proteins during virus disas-
sembly.
At atmospheric pressure andinthe absence of urea
and GdnHCl, theMS2bacteriophageand VLPs did
not bind bis-ANS, showing that these particles do not
present exposed hydrophobic segments. High urea and
GdnHCl concentrations did not promote significant
binding to any ofthe particles, suggesting that the
denaturation ofthe protein was essentially complete
and prevented probe binding (data not shown). How-
ever, for dlFG and sc-dlFG, the probe bound the pro-
tein inthe absence of either denaturant, and at low
urea concentrations an increase occurred in bis-ANS
fluorescence emission, but when the urea concentration
was further increased, the probe became unbound. The
binding of bis-ANS to dlFG and sc-dlFG promoted a
shift inthe urea denaturation curves, suggesting a sta-
bilizing effect ofthe probe (Fig. 5A). The same effect
was not observed for GdnHCl-induced denaturation,
and sc-dlFG was actually less stable inthe presence of
the probe (Fig. 5B).
When wild-type MS2and VLPs were submitted to
high pressure inthe presence of bis-ANS, again no sig-
nificant binding was observed (data not shown). How-
ever, when dlFG was pressurized inthe presence of the
probe, there was a sevenfold increase in its emission,
suggesting that under pressure the protein exposes
hydrophobic segments and retains some degree of sec-
ondary structure. Furthermore, the conformation of
the pressure-denatured state is different from that of
the urea-denatured state (Fig. 6A).
The denaturation of dlFG promoted by pressure
was measured inthe presence and absence of bis-ANS,
and the probe showed the same protective effect
observed on urea-induced denaturation (Fig. 6B). It
should be noted, however, that in all assays conducted
in the presence of bis-ANS, the spectral center of mass
did not return to its initial value after pressure release,
and an increase inthe light-scattering value was
observed (data not shown). This result suggests that
the coat protein dimer may be undergoing aggregation.
For sc-dlFG, again we observed that the binding of
the probe destabilized the protein, as it did inthe pres-
ence of GdnHCl (Fig. 6B).
Fig. 4. Pressure stabilityofMS2bacteriophage wild-type, VLPs,
dlFG and sc-dlFG. The effects of pressure on MS2, VLPs, dlFG and
sc-dlFG at room temperature were analyzed. The effect was meas-
ured by spectral center of mass of tryptophan fluorescence emis-
sion. (d) MS2 WT; (n) VLPs; (m) dlFG; (r) sc-dlFG. The samples
were excited at 280 nm, andthe emission was measured at
300–420 nm. The buffer used was 10 m
M Tris ⁄ HCl ⁄ 100 mM
NaCl ⁄ 0.01 mM EDTA, pH 7.5. Incubation at each pressure was for
20 min.
Bacteriophage MS2stability S. M. B. Lima et al.
1468 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
Role ofthe nucleic acid–protein complex in the
stability of dlFG
As shown before, pressure was able to completely
denature dlFG above 1.8 kbar. To compare the stabil-
ity ofthe protein inthe presence of different nucleic
acids, we incubated the coat protein samples with
dsDNA [a nonspecific poly(GC) DNA], tRNA and the
RNA hairpin sequence ofMS2 (ratio 2 : 1, pro-
tein ⁄ DNA or tRNA or RNA), andthe pressure stabil-
ity was checked. DNA poly(GC) stabilized the coat
protein dimer. p
1 ⁄ 2
, the pressure necessary to promote
half denaturation, shifted to higher pressures when the
protein was mixed with DNA. On the other hand,
when we incubated the coat protein with tRNA or the
hairpin sequence, neither was able to protect the pro-
tein against denaturation (Fig. 7A). After pressure
release, the value ofthe spectral center of mass of
dlFG emission inthe presence of DNA returned
almost completely to the initial value, but again we
observed an increase in light scattering, suggesting
Fig. 5. Urea and guanidine treatment of dlFG and sc-dlFG in the
presence of bis-ANS. (A) Effects of increasing urea concentrations
on the spectral center of mass of (m) dlFG and (r) sc-dlFG in the
absence of bis-ANS, andinthe presence of 1 m
M bis-ANS (n) dlFG
and (e) sc-dlFG. Incubation was overnight at each concentration.
(B) Effects of increasing GdnHCl concentrations on the spectral
center of mass of (m) dlFG and (r) sc-dlFG inthe absence of bis-
ANS andinthe presence of 1 m
M bis-ANS for (n) dlFG and (e)
sc-dlFG. Incubation was overnight at each concentration. The effect
was measured by spectral center of mass of tryptophan fluores-
cence emission. The sample was excited at 280 nm, andthe emis-
sion was measured at 300–420 nm.
Fig. 6. bis-ANS binding to coat protein dimer (dlFG) under pressure
and pressure stabilityof dlFG and sc-dlFG inthe absence and pres-
ence of bis-ANS. (A) Structural changes in dlFG were analyzed by
fluorescence ofthe probe bis-ANS at a final concentration 1 l
M.
Excitation wavelength was 360 nm and emission wavelength range
400–600 nm. Inset: fluorescence emission spectra of bis-ANS dur-
ing the pressurization process. (B) Effect of pressure on the coat
protein inthe presence of bis-ANS for (n) dlFG and (e) sc-dlFG
and inthe absence ofthe probe for (m) dlFG and (r) sc-dlFG, was
analyzed at room temperature. The effect was measured by spec-
tral center of mass of tryptophan fluorescence emission. The sam-
ple was excited at 280 nm, andthe emission was measured at
300–420 nm.
S. M. B. Lima et al. BacteriophageMS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1469
that, like bis-ANS, this DNA promoted an aggregation
process. The assay inthe presence of DNA and bis-
ANS inthe same concentration (1 : 1 : 1) pro-
tein ⁄ DNA ⁄ bis-ANS, showed that the protein bound
bis-ANS, because there was an increase inthe fluores-
cence emission ofthe probe when the protein was sub-
jected to high pressure, but the DNA could not
protect the protein dimer against dissociation. The
presence of both DNA and bis-ANS decreased the sta-
bility ofthe protein (Fig. 7B).
Effects of high temperature on the secondary
and tertiary structure of dlFG and sc-dlFG in the
absence and presence of bis-ANS
We confirmed the results observed when we submitted
sc-dlFG to high pressure and GdnHCl inthe presence
of bis-ANS using high temperature. We subjected
dlFG and sc-dlFG to elevated temperatures in the
presence and absence of bis-ANS and analyzed the
changes by fluorescence emission (Fig. 8A) and far-UV
CD (Fig. 8B). The results confirmed that the probe
decreased thestabilityof sc-dlFG, as the temperature
necessary to start the denaturing process decreased by
almost 20 °C and for dlFG the presence ofthe probe
did not change thestability (data not shown).
Discussion
Assembly of infectious bacteriophageMS2 requires the
proper folding of coat protein dimers and their self-
assembly into an icosahedral capsid, while selectively
encapsulating a single copy of viral plus-strand RNA
by the coat, and ignoring viral minus strands and host
nucleic acids. Moreover, the particle must be stable
enough to survive outside the host, but not so stable
as to prevent the release ofthe viral genome when a
new susceptible host is encountered. The complex
formed between coat protein and RNA has been inves-
tigated [5,9,23,24], and amino acid–nucleotide inter-
actions contributing to its stability have been
characterized [13,25], but theroleof protein–protein
and protein–RNA interactionsin virus stability is not
completely understood, and we have investigated these
questions in this work. We sought to determine the
role of protein–RNA andprotein–protein interactions
in virus stability, measuring the effects of urea,
GdnHCl and high pressure on the structure and stabil-
ity of whole particles (bacteriophage MS2and VLPs)
and assembly-defective coat protein species [coat pro-
tein dimers (dlFG) and single-chain dimers (sc-dlFG)].
The coat protein ofbacteriophageMS2 expressed in
E. coli forms intracellular VLPs that package a precur-
sor form of 16S rRNA, which happens to contain a
translational operator-like sequence near its 5¢ end
[11]. The results reported here show that VLPs and
MS2 behave differently when perturbed with denatur-
ing agents. Whereas authentic MS2 particles dissociate
and denature co-operatively, VLPs undergo disassem-
bly at lower denaturant concentration and denatura-
tion at much higher concentration than the MS2
Fig. 7. Comparison ofstabilityof dlFG under pressure inthe pres-
ence and absence of nucleic acids and bis-ANS binding assay on
dlFG inthe presence and absence of DNA. (A) Effect of pressure
on the dlFG coat protein at room temperature was analyzed in the
absence of nucleic acids (m) andinthe presence of 34-bp DNA
poly(GC) (.) and yeast tRNA (,). The effect was measured by
spectral center of mass of tryptophan fluorescence emission. The
sample concentration was 1 l
M as well as the DNA and tRNA. (B)
Effect of pressure on the dlFG coat protein was analyzed in the
absence of nucleic acids (m), inthe presence of 34-bp DNA
poly(GC) (.), bis-ANS (n) andinthe presence of both bis-ANS
(1 l
M) and DNA (1 lM)(
~
). The effect was measured by spectral
center of mass of tryptophan fluorescence emission. The sample
concentration was 1 l
M.
Bacteriophage MS2stability S. M. B. Lima et al.
1470 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
capsid protein. The only feasible explanation is the for-
mation of complexes of folded capsid protein with the
small RNAs that compose the VLPs [11]. The presence
of maturase protein in authentic particles may play a
role inthe different behavior. Inthe natural virion, a
part ofthe maturase protein is present on the inner
surface ofthe capsid shell where it interacts with viral
RNA. Another part must be present on the outside of
the shell where it is responsible for adsorption of the
virus to the cellular receptor, the F-pilus. During infec-
tion, maturase enters the cell together with the RNA
genome, leaving behind an intact capsid.
The dlFG version of coat protein is dimeric and
therefore lacks both theprotein–proteinand protein–
nucleic acidinteractions that normally occur within
the capsid. Our results show that dlFG is very unstable
to denaturants when compared with MS2, VLP and
sc-dlFG (Fig. 1A,B). Further investigation is necessary
to understand theroleofthe FG loop in dimer stabil-
ity, by comparing the wild-type and dlFG mutant.
Here we demonstrate that the dimeric protein is more
susceptible to denaturation. We think this is probably
mostly due to the absence ofthe intersubunit contacts
normally present inthe capsid. Although we cannot
rule out a contribution ofthe FG-loop itself to dimer
stability, previous studies with several viruses show
that, when the wild-type protein is isolated from the
capsid, it becomes more unstable to chemical and
physical denaturing agents [26–28].
Bis-ANS-binding assays during pressurization of
dlFG and sc-dlFG suggested the exposure of hydro-
phobic residues, as the fluorescence intensity of the
probe increased about sevenfold. However, inthe pres-
ence of urea or GdnHCl, there is nonexpressive bis-
ANS binding, suggesting that the pressure-denatured
state is different from the chemical-denatured state.
Furthermore, the probe partially protected both forms
against urea (but not guanidine) denaturation. In the
presence ofthe probe, sc-dlFG was more susceptible
to denaturation by GdnHCl, pressure and heat.
Because its monomers are tethered to one another, we
believe that they maintain additional interactions
within the dimer, and that these interactions are affec-
ted negatively by the presence ofthe probe during
denaturation by guanidine, pressure and high tempera-
ture, and positively inthe presence of urea. Although
the molecular basis ofthe effect of urea and GdnHCl
on polypeptide chains is still not well understood, it is
generally thought that urea mainly affects hydrogen
bonding. The binding ofthe probe presumably pro-
tects these interactions against urea.
Bis-ANS binding seems to be different from dlFG
and sc-dlFG, as the probe protected the dimer against-
most treatments and destabilized the fused form against
all treatments except urea-induced denaturation.
High hydrostatic pressure has been a very useful
tool inthe study of folding intermediates, DNA recog-
nition, and virus assembly. Pressure studies have
revealed that there is a large step before total phage
assembly. The protein–DNA complex formed between
dlFG and poly(GC) DNA stabilizes the protein and
promotes the complete reversibility ofthe pressure
Fig. 8. Conformational changes in dlFG and sc-dlFG promoted by
high temperature. (A) The sample was treated at 25–80 °C, with an
incubation time of 5 min at each temperature. The structural chang-
es were analyzed by variation of spectral center of mass of trypto-
phan emission inthe absence of bis-ANS for (m) dlFG, (r ) sc-dlFG.
and inthe presence of bis-ANS for (e) sc-dlFG andthe return at
room temperature for both inthe absence and presence of bis-ANS
(d). The bis-ANS concentration was 1 l
M. (B) High-temperature-
induced denaturation of dlFG and sc-dlFG proteins analyzed by far-
UV CD spectroscopy. Conformational changes in (m) dlFG and (r)
sc-dlFG (in the absence of bis-ANS and (e) sc-dlFG inthe presence
of 1 l
M bis-ANS) at increasing temperatures (15–85 °C). Ellipticity
values at 216 nm are plotted as a function of increasing tempera-
ture.
S. M. B. Lima et al. BacteriophageMS2 stability
FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS 1471
denaturation process. We are unsure ofthe mechanism
of DNA-induced stabilization ofthe protein, and whe-
ther it is related to the normal RNA-binding function
of coat protein, as RNA binding had no effect.
The increased light scattering of samples after pres-
sure release inthe presence of bis-ANS or DNA
suggests that the dimer is probably undergoing an
aggregation process. This suggests that, when the pro-
tein was submitted to high pressure, hydrophobic resi-
dues were exposed, allowing probe binding and
inhibiting the aggregation, but after pressure release
the probe bound to the protein may play a nucleation
role, inducing an aggregated state. A possible mechan-
ism is the formation of a limited associated state, for
example a pentameric unit, as observed for other ani-
mal viruses [29]. The binding of bis-ANS to the coat
protein dimer under pressure andthe absence of pro-
tection from dissociation by DNA suggest that DNA
and bis-ANS may bind inthe same or nearby sites of
the protein.
Genetically fusing the subunits ofthe dlFG dimer
greatly stabilized it against all the forms of denatura-
tion we tested. This is consistent with the observation
of increased stabilityofthe single-chain version of the
wild-type dimer [30] and is reminiscent ofthe similar
stabilization engendered by subunit fusion in other
proteins [30–34]. This increased stability most likely
reflects the increased local concentration of one chain
with respect to the other when the two are covalently
tethered to one another.
Overall, our studies provide information on the
mechanisms by which theinteractionsinthe capsid
lattice are made sufficiently stable and specific to allow
the formation of a correctly assembled particle, while
maintaining sufficient instability to allow release of the
viral genome during initiation of infection. Zlotnick
[35] discussed the importance of understanding virus
stability and assembly, pointing out that the virus must
assemble at the right time andinthe right place so
as to package the correct nucleic acid. Moreover, it
must be able to undergo conformational transitions
to release its nucleic acid. The higher stabilityof the
capsid protein bound to heterologous nucleic acid may
serve as a ‘biological sieve’. Whereas authentic MS2
particles dissociate and unfold co-operatively, any
particle without the authentic RNA would be locked
in a state lacking the ability to release the RNA during
infection.
Our studies shed light on the processes that lead to
packaging ofthe correct RNA resulting in a mature
infectious viral particle. The efficient coupling of fold-
ing and assembly ofthe authentic virus particle
revealed by the co-operative processes of dissociation
and denaturation is very likely to play an important
role. The lack of this co-operativity for the VLP indi-
cates that, inthe host, the complexes of capsid protein
bound to a small nonspecific RNA may lock it in the
disassembled state. There have been few studies that
addressed the topic of specific packaging of nucleic
acids by viruses. Annamalai et al. [36] described how
an arginine-rich RNA-binding motif, situated at the
N-proximal region of cowpea chlorotic mottle virus
(CCMV) capsid protein (CP), recognizes and packages
specific RNA. Inthe case ofMS2 coat protein, it binds
to its cognate RNA hairpin 1000-fold tighter than the
corresponding DNA and any modification of one of
the 21 different RNA–protein contacts leads to a stri-
king change inthe specificity ofthe RNA–capsid pro-
tein interaction [37]. It is the delicate balance between
proper protein–RNA affinity and thermodynamic sta-
bility ofthe resulting RNA packaged particle that
drives the formation ofthe infection virus.
Experimental procedures
Chemicals
All reagents were of analytical grade. Distilled water was
filtered and deionized through a Millipore water purifica-
tion system. The probe bis-ANS was purchased from
Molecular Probes (Eugene, OR, USA). The experiments
were performed at 20 °C using the standard buffer (10 mm
Tris ⁄ HCl, 100 mm NaCl, 0.01 mm EDTA, pH 7.5).
Phage propagation
E. coli cell strain C3000 was grown in Luria–Bertani med-
ium to A
600
¼ 1.2 when they were infected with MS2. After
5 h the culture was treated with lysozyme, and bacterial
debris was removed by centrifugation at 9800 g for 10 min
at 4 °C (RPR 9.2 rotor; Hitachi, Tokyo, Japan). The super-
natant was precipitated with ammonium sulfate (330 gÆL
)1
),
and the phage pellet was collected by centrifugation at
11 800 g for 45 min at 4 °C (RPR 12.2 rotor; Hitachi). The
precipitate was dissolved in standard buffer and purified by
high-speed centrifugation (155 000 g for 14 h; SW41 rotor;
Beckman, Fullerton, CA, USA) in a sucrose gradient (10–
50%). The phage was collected, and its purity determined
by SDS ⁄ PAGE (12.5% gel) and visualized by staining with
Coomassie Blue. Sample concentrations were determined by
Lowry’s method [38].
Expression and purification of VLPs
After growing overnight in Luria–Bertani medium, E. coli
cells strain BL21 (DE3) were transformed with a plasmid
Bacteriophage MS2stability S. M. B. Lima et al.
1472 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
[...]... effect ofthe presence of nucleic acids on thestabilityof dlFG was analyzed during pressurization The protein was incubated with poly(GC) DNA, yeast tRNA andthe translational operator sequence ofbacteriophageMS2 (5¢-ACAUGAGCAUUACCCAUGU-3¢) on ratio 1 : 2 (nucleic acid ⁄ protein) The sample concentration used was 2 lm, and it was diluted in 0.01 mm Tris ⁄ HCl, pH 7.5, containing 0.1 m NaCl and 0.01... dlFG and sc-dlFG treated with urea and guanidine were analyzed TheMS2bacteriophageandthe coat protein mutant samples were diluted to a final concentration 100 lgÆmL)1, andthe spectra were obtained in 10 mm Tris ⁄ HCl ⁄ 30 mm NaCl, pH 7.5, using a 0.1-cm path length quartz cuvette The spectropolarimeter used was a Jasco J-715 1505 model (wavelength range 300–210 nm) Nucleic acid- binding assays The. .. Oliveira AC (2004) Mutations inthe hydrophobic core andinthe protein–RNA interface affect the packing andstabilityof icosahedral viruses Eur J Biochem 271, 135–145 11 Pickett GG & Peabody DS (1993) Encapsidation of heterologous RNAs by bacteriophageMS2 coat protein Nucleic Acids Res 21, 4621–4626 12 Axblom C, Tars K, Fridborg K, Orna L, Bandule M & Liljas L (1998) Structure of phage fr capsids with... 24 tion inthe FG loop: implications for viral assembly Virology 249, 80–88 Lago H, Parrott AM, Moss T, Stonehouse NJ & Stockley PG (2001) Probing the kinetics of formation oftheBacteriophageMS2 translational operator complex: identification of a protein conformer unable to bind RNA J Mol Biol 305, 1131–1144 Peabody DS & Ely KR (1992) Control of translational repression by protein–protein interactions. .. Trimble S, Fermi G, et al (1992) A human recombinant haemoglobin designed for use as a blood substitute Nature 356, 258–260 White FL & Kenneth WO (1987) Effects of crosslinking on the thermal stabilityof hemoglobin The use of bis-(3,5-dibromosalicyl)fumarate Arch Biochem Biophys 258, 51–57 Yang T & Kenneth WO (1991) Thermal stabilityof hemoglobin crosslinked inthe T-state by bis-(3,5-dibromosalicyl)fumarate... Viruses andthe physics of soft condensed matter Proc Natl Acad Sci USA 101, 15549– 15550 Annamalai P, Apte S, Wilkens S & Rao AL (2005) Deletion of highly conserved arginine-rich RNA binding motif in cowpea chlorotic mottle virus capsid protein results in virion structural alterations and RNA packaging constraints J Virol 79, 3277–3288 Dertinger D, Dale T & Uhlenbeck OC (2001) Modifying the specificity of. .. et al BacteriophageMS2stability pETCT containing the sequence of wild-type coat protein The cells were diluted 20 times, and, after 2 h at 37 °C, protein expression was induced with 1 mm isopropyl b-dthiogalactopyranoside After 2 h the cells were pelleted by centrifugation (5500 g for 20 min; RPR 9.2 rotor) at 4 °C The pellet was resuspended in standard buffer, treated with lysozyme, sonicated and. .. incubated with increasing urea concentrations (1–9 m) and increasing GdnHCl concentrations (0.5– 6 m) and allowed to equilibrate overnight before measurements were recorded The measurements were made inthe absence and presence of urea and GdnHCl The assembly-defective mutants dlFG and sc-dlFG were propagated to mid-exponential phase (A600 ¼ 0.8) in strain BL21(DE3) at 37 °C Protein expression was induced... Fridborg K, Beigelman L, Matulic-Adamic J, Warriner SL, Stockley PG & Liljas L (2000) Delection of a single hydrogen bonding atom from theMS2 RNA operator leads to dramatic rearrangements at the RNA–coat protein interface Nucleic Acids Res 28, 4611–4616 9 Peabody DS & Lim F (1996) Complementation of RNA binding site mutations inMS2 coat protein heterodimers Nucleic Acids Res 24, 2352–2359 10 Lima SMB, Peabody... that depends on a single aspartic acid residue J Biol Chem 269, 13680–13684 19 Gray DM (1996) Circular dichroism ofprotein–nucleicacidinteractionsIn Circular Dichroism andthe Conformational Analysis of Biomolecules (Fasman GD, ed.), pp 469–500 Plenum Press, New York and London Weber G (1987) Dissociation of oligomeric proteins by hydrostatic pressure In High Pressure Chemistry and Biochemistry NATO-ASI . Dissecting the role of protein–protein and protein–nucleic acid interactions in MS2 bacteriophage stability Sheila M. B. Lima 1 , Ana Carolina Q. Vaz 1 , Theo L. F. Souza 1 ,. understood, and we have investigated these questions in this work. We sought to determine the role of protein–RNA and protein–protein interactions in virus stability, measuring the effects of urea, GdnHCl. enters the cell together with the RNA genome, leaving behind an intact capsid. The dlFG version of coat protein is dimeric and therefore lacks both the protein–protein and protein– nucleic acid interactions