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
, 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–protein and protein–nucleic acid inter-

E-mail:
E-mail:
(Received 2 August 2005, revised 26 January
2006, accepted 6 February 2006)
doi:10.1111/j.1742-4658.2006.05167.x
To investigate the role of protein–protein and protein–nucleic acid 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, and the 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 and the wild-type virus, it was much more susceptible to chemical
and physical perturbation. Genetic fusion of the two subunits of the 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 in the 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.

unable to form capsids, the dlFG dimer retains its abil-
ity to bind RNA [3,5,12–17]. We also determined the
effects on stability of genetic fusion of the two sub-
units of the dlFG dimer using the variant named
2CTdlFG, which takes advantage of the physical prox-
imity of the N-terminus and C-termius of the 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 of the 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 of the 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 in the pres-

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 in the 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 MS2 stability 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 the stability was of the entire par-
ticle or of the 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 MS2 in the 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

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. Bacteriophage MS2 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 in the secondary structure of the proteins, we
analyzed the UV CD spectra of MS2 and 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 in the 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]. In the 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 in the presence of 5.0 m
urea, and no change in the presence of 3.5 m GdnHCl,
confirming its higher stability (Fig. 3C,D).

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 of the 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 in the 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 of the 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 of the solvents,
or the internal energy of the protein [2,20,21].
The samples of MS2 and VLPs were diluted to a
final concentration of 50 lgÆmL

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 in the 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 and in the absence of urea
and GdnHCl, the MS2 bacteriophage and VLPs did

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 in the 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 in the pres-
ence of GdnHCl (Fig. 6B).
Fig. 4. Pressure stability of MS2 bacteriophage 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, and the 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 MS2 stability S. M. B. Lima et al.
1468 FEBS Journal 273 (2006) 1463–1475 ª 2006 The Authors Journal compilation ª 2006 FEBS
Role of the 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 of the protein in the presence of different nucleic
acids, we incubated the coat protein samples with
dsDNA [a nonspecific poly(GC) DNA], tRNA and the
RNA hairpin sequence of MS2 (ratio 2 : 1, pro-

ence of bis-ANS. (A) Structural changes in dlFG were analyzed by
fluorescence of the 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 in the presence of bis-ANS for (n) dlFG and (e) sc-dlFG
and in the absence of the 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, and the emission was measured at
300–420 nm.
S. M. B. Lima et al. Bacteriophage MS2 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 in the presence of DNA and bis-
ANS in the same concentration (1 : 1 : 1) pro-
tein ⁄ DNA ⁄ bis-ANS, showed that the protein bound
bis-ANS, because there was an increase in the fluores-
cence emission of the 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 of the 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 in the presence
of bis-ANS using high temperature. We subjected

tein dimers (dlFG) and single-chain dimers (sc-dlFG)].
The coat protein of bacteriophage MS2 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 of stability of dlFG under pressure in the pres-
ence and absence of nucleic acids and bis-ANS binding assay on
dlFG in the 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) and in the 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), in the presence of 34-bp DNA
poly(GC) (.), bis-ANS (n) and in the 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.

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, in the 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 of the 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 of the probe during
denaturation by guanidine, pressure and high tempera-
ture, and positively in the presence of urea. Although
the molecular basis of the 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 of the 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 in the study of folding intermediates, DNA recog-
nition, and virus assembly. Pressure studies have

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 and the absence of pro-
tection from dissociation by DNA suggest that DNA
and bis-ANS may bind in the same or nearby sites of
the protein.
Genetically fusing the subunits of the dlFG dimer
greatly stabilized it against all the forms of denatura-
tion we tested. This is consistent with the observation
of increased stability of the single-chain version of the
wild-type dimer [30] and is reminiscent of the 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 the interactions in the 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

tein interaction [37]. It is the delicate balance between
proper protein–RNA affinity and thermodynamic sta-
bility of the resulting RNA packaged particle that
drives the formation of the 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

600
¼ 0.8) in strain
BL21(DE3) at 37 °C. Protein expression was induced with
2mm isopropyl b-d-thiogalactopyranoside. Three hours
after induction, the cells were centrifuged (5500 g for
20 min) at 4 °C and frozen at )20 °C overnight. After
being thawed, the cells were resuspended in lysis buffer
(0.02 m Na
2
HPO
4
, pH 7.4, 0.5 m NaCl, 0.001 m phenyl-
methanesulfonyl fluoride, 0.02 m 2-mercaptoethanol, 0.01 m
imidazole) and sonicated. The cell debris was pelleted by
centrifugation (13 500 g for 20 min). The supernatant was
added to Chelating Sepharose Fast Flow charged with
nickel ions and mixed gently for 30 min. The mixture
applied to a column, washed with increasing concentrations
of imidazole (20 mm,50mm, 100 mm, 250 mm), and the
protein was eluted with 500 mm imidazole. After purifica-
tion, the samples were dialyzed in buffer (0.01 m Tris ⁄ HCl,
pH 7.5, 0.1 m NaCl, 0.01 m EDTA).
Spectroscopic measurements and high pressure
experiments
Fluorescence spectra were recorded on an ISSK2 spectroflu-
orimeter (ISS Inc., Champaign, IL, USA). Tryptophan resi-
dues were excited at 280 nm, and emission was observed at
300–420 nm. Changes in fluorescence spectra were quanti-
tated by the spectral center of mass, <m>:
< m >¼ Rm

CD
Conformational changes in MS2 bacteriophage, VLPs,
dlFG and sc-dlFG treated with urea and guanidine were
analyzed. The MS2 bacteriophage and the coat protein
mutant samples were diluted to a final concentration
100 lgÆmL
)1
, and the 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 effect of the presence of nucleic acids on the stability
of dlFG was analyzed during pressurization. The protein
was incubated with poly(GC) DNA, yeast tRNA and the
translational operator sequence of bacteriophage MS2
(5¢-AC AUGAGCA UUACCCA UGU-3¢) on r atio 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 m EDTA.
Acknowledgements
We gratefully acknowledge Emerson Gonc¸ alves for
competent technical assistance, Cristiane Dinis Ano
Bom and Professors Fa
´
bio Almeida and Ana Paula
Valente from CNRMN ⁄ UFRJ for helpful comments
and suggestions. This work was supported in part by
an international grant from the International Centre
for Genetic Engineering and Biotechnology (ICGEB)

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