From the Programa de Biologia Estrutural,
Departamento de Bioquímica Médica, Instituto de
Ciências Biomédicas, Centro Nacional de Ressonância
Magnética Nuclear de Macromoléculas and ¶ Departamento
de Virologia, Instituto de Microbiologia Professor Paulo Góes,
Universidade Federal do Rio de Janeiro,
21941-590, RJ, Brazil
Received for publication, November 3, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Enveloped viruses fuse their membranes with
cellular membranes to transfer their genomes into cells at the
beginning of infection. What is not clear, however, is the role of the
envelope (lipid bilayer and glycoproteins) in the stability of the
viral particle. To address this question, we compared the stability
between enveloped and nucleocapsid particles of the
alphavirus Mayaro using hydrostatic pressure and
urea. The effects were monitored by intrinsic fluorescence, light
scattering, and binding of fluorescent dyes, including
bis(8-anilinonaphthalene-1-sulfonate) and ethidium bromide. Pressure
caused a drastic dissociation of the nucleocapsids as determined by
tryptophan fluorescence, light scattering, and gel filtration
chromatography. Pressure-induced dissociation of the nucleocapsids was
poorly reversible. In contrast, when the envelope was present, pressure
effects were much less marked and were highly reversible. Binding of
ethidium bromide occurred when nucleocapsids were dissociated under
pressure, indicating exposure of the nucleic acid, whereas enveloped
particles underwent no changes. Overall, our results demonstrate
that removal of the envelope with the glycoproteins leads the particle
to a metastable state and, during infection, may serve as the trigger
for disassembly and delivery of the genome. The envelope acts as a
"Trojan horse," gaining entry into the host cell to allow
release of a metastable nucleocapsid prone to disassembly.
The properties that stabilize a virus particle have to fulfill two
apparently opposite requirements. On one hand, the stability must be
great enough to keep the particle intact while it is outside the host
cell. On the other hand, the virus must be able to disassemble inside
the infected cell. Among several steps in virus infection, disassembly
of the capsid and unpacking of the nucleic acid is the least understood
(1, 2). Enveloped viruses enter cells by protein-mediated fusion of the
membrane enclosing the virus particle with the cellular membrane, which
ultimately results in release of the nucleocapsid into the cell for
processing (3). In nonenveloped viruses, biophysical studies have been
carried out to understand how the overall stability depends on
protein-protein interactions (4-7), protein-nucleic acid interactions
(8, 9), and proteolysis maturation (10, 11). Here, the alphavirus Mayaro was used as a simple model of enveloped viruses to compare the
properties of the whole and nucleocapsid particles deprived of the
lipid bilayer and transmembrane glycoproteins.
Mayaro virus is a member of the Alphavirus genus of the
Togaviridae family. The single-stranded positive-sense RNA of
alphaviruses is enclosed within a icosahedral core made up of 240 identical copies of the nucleocapsid protein (C) (12). A lipid-bilayer membrane derived from the infected host cell surrounds the nucleocapsid core (NC).1 Projecting from
the bilayer and embedded in it are the viral-encoded glycoproteins
designated E1 and E2 (also designated P1 and P2 in Mayaro virus) (13).
E2 is involved in cellular recognition and attachment, and E1 is
involved in membrane fusion. A domain of the E2 protein also interacts
with the NC inside of the lipid bilayer (13-15). RNA-protein
interactions that stabilize the nucleocapsid were also described (16).
A three-dimensional structure of the Sindbis virion to a resolution of
28 Å based on cryoelectron microscopy shows a very tight arrangement
of glycoprotein spikes distributed as 80 trimers on its outer surface
(12). Each trimer contains three E1-E2 heterodimers that appear
intertwined but bulge in a triangular shape from the membrane. The high
resolution x-ray structure of the purified capsid protein has shown
that residues 114-264 have a structural fold similar to that of
chymotrypsin (17, 18). Cryoelectron microscopy and image reconstruction carried out on Ross River virus show that the alphavirus
nucleocapsid exhibits T = 4 icosahedral symmetry
(15).
Pressure is a valuable tool to perturb the structure of macromolecular
assemblages and viruses (5, 7, 19-23). The application of pressure
shifts equilibrium toward the state that occupies a smaller volume and
accelerates processes for which the transition state has a smaller
volume than the ground state (24-26). Pressure perturbation can yield
novel information about stability, volume, and packing in a wide
variety of biological phenomena, and it has been particularly useful in
the investigation of conformational transitions in proteins (27-29).
In general, pressure maintains the secondary structure network but is
unfavorable for hydrophobic interactions, which are predominantly
responsible for maintaining the tertiary structure of a protein (20,
25). The negative volume change that occurs with protein dissociation
or unfolding arises globally from more intimate interactions between
the polypeptide chain and water. Thus pressure destabilizes hydrophobic
and electrostatic interactions in addition to eliminate cavities (20,
30, 31). A unique property of high pressure denaturation is the
formation of partially folded or molten-globule states at equilibrium
(32-35). High pressure has been used successfully to denature and
dissociate proteins, protein-DNA complexes, and virus particles (20,
25, 36-39).
Metastability has been observed for serine protease inhibitors (40,
41), heat shock transcription factors (42), viral envelope
glycoproteins (43), and proteolitically processed capsid proteins of
nonenveloped viruses (11). Here, we characterize the metastable state
of the ribonucleoprotein capsid by perturbing it with hydrostatic
pressure or urea. The nucleocapsid dissociates into partially folded
states under pressure that poorly reassemble into particles when
pressure is returned to atmospheric values. In contrast, in the whole
virus the envelope containing the lipid bilayer and the integral
membrane proteins functions as a "thermodynamic cage," preventing
irreversible dissociation of the capsid and protecting the genome.
Chemicals--
All reagents were of analytical grade. Distilled
water was filtered and deionized though a Millipore water purification
system. The experiments were performed at 22 °C in the buffer 50 mM Tris, 150 mM NaCl (pH 7.5) for Mayaro virus
and Mayaro NC. Tris was chosen as the buffer because the dependence of
its pKa on pressure is small. At 3.0 kbar, the value
of pKa increases by only 0.1 unit (44).
Bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) was purchased from
Molecular Probes (Eugene, OR), and ethidium bromide was purchased from Sigma.
Cells and Virus--
Baby hamster kidney (BHK-21) cells were
used extensively. Mayaro virus was grown on BHK-21 cells in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
3% tryptose phosphate. Mayaro virus was obtained from American Type
Culture Collection (Manassas, VA). The virus was propagated for 48 h in 2.0-liter roller bottles that were placed on a roller apparatus in
37 °C warm room. To analyze Mayaro virus, biological activity before
and after virus purification, infectivity assays were done. Virus
dilutions were added to monolayers of BHK-21 cells in 60-mm-diameter Petri dishes. After 60 min at 37 °C, virus inoculum was removed, and
the monolayers were overlaid with 2.0 ml of Dulbecco's medium supplemented with 1% of fetal bovine serum and 0.95% of agarose and
incubated in an atmosphere of CO2 at 37 °C for 48 h. The monolayers were stained with crystal violet, and plaques were
counted (45).
Virus Purification--
Mayaro virus was purified as described
previously with some modifications (46). BHK-21 cells were infected
with Mayaro virus at a low multiplicity of infection (0.1 plaque-forming unit/ml); 48 h post-infection, the supernatant was
collected and sedimented at 8,000 rpm for 20 min to remove cells
debris. After clarification, supernatant was spun in a Beckman Ti-45
rotor at 32,000 rpm for 1.5 h in a 30% sucrose cushion. The
pellet was then resuspended in 50 mM Tris, 150 mM NaCl (pH 7.5) and layered onto a discontinuous 10-50%
sucrose density gradient in 50 mM Tris, 150 mM
NaCl (pH 7.5) and spun for 1.5 h at 32,000 rpm in a Beckman SW-41
rotor. Fractions collected from the gradient were analyzed by reading the optical density at 280 and 260 nm. Purified virus was removed from
sucrose gradient by dialysis in 50 mM Tris, 150 mM NaCl (pH 7.5) and maintained at 4 °C. Fresh
preparations of whole virus were prepared for each set of experiments.
Nucleocapsids Purification--
Mayaro NC was purified as
described previously for Sindbis virus (47). Purified NC fractions were
collected and analyzed for their RNA content by measuring absorbance at
260 nm. Purified virus NC was maintained at 4 °C. Fresh preparations
of purified NC virus were prepared for each set of experiments.
High Pressure Equipment and Fluorescence Studies--
The high
pressure bomb has been described (48) and was purchased from ISS Inc.
(Champaign, IL). Fluorescence spectra were recorded on a ISS200 or on
ISSK2 computer-controlled spectrofluorometers (ISS Inc.). The spectra
at pressure p were quantified by specifying the center of
spectral mass (
The binding of bis-ANS was monitored by exciting the samples at 360 nm
and collecting the emission in the range of 400-600 nm (49). The
concentration of bis-ANS used in all experiments was 2 µM. Samples were allowed to equilibrate for 15 min before making measurements.
Light Scattering--
Light-scattering measurements were
recorded on ISS 200 or on ISSK2 spectrofluorometers (ISS Inc.).
Scattered light (320 nm) was collected at an angle of 90° of the
incident light by integrating the intensity in the 315-325-nm window.
Size Exclusion Chromatography--
High performance liquid
chromatography was carried out in a Sephacryl G-500 column. The system
was equilibrated in 50 mM Tris 0.5 M sodium
acetate buffer (pH 7.5). A flow rate of 0.3 ml/min was utilized. Sample
elution was monitored by fluorescence at 330 nm (excitation at 280 nm)
and absorbance at 260 nm.
Differences in Stability between Mayaro Virus and Mayaro
Nucleocapsid--
Although much progress has been made on the
understanding of viral structures in the last years, the thermodynamics
and the mechanism of virus assembly and disassembly are still poorly
understood. Hydrostatic pressure is an efficient tool for promoting
dissociation of oligomeric proteins and for the studies of
protein-nucleic acid interaction in virus assembly and stability (20,
25). Because interactions between nucleic acids and proteins are mainly electrostatic, they are disfavored by high pressure.
The effects of high pressure and urea on membrane-enveloped and
isolated nucleocapsids of Mayaro virus were characterized by following
the changes in intrinsic fluorescence spectra of the tryptophan (Trp)
residues and in light scattering of the samples. Although the changes
in light scattering indicate a decrease in the average size of the
particles, the changes in fluorescence spectra reveal local
modification in the environment of the Trp residues reflecting the
tertiary structure. Therefore, fluorescence measurements are efficient
means of monitoring the different states in the disassembly pathway of
a virus particle.
The changes in the Trp fluorescence were followed by the average energy
of emission (expressed in wavenumbers) for whole particles (squares) and NC (circles) (Fig.
1A). The total change in
center of spectral mass for virus particle was
The changes in light scattering as a function of pressure for the whole
virus particle (square) and for NC (circles) are
shown in Fig. 1B. There was a slight decrease in the light
scattering of the intact Mayaro virus solution, suggesting little
dissociation of the viral particle. On the other hand, isolated
nucleocapsids presented a dramatic decrease in the light scattering,
suggesting its complete disassembly induced by high pressure. The
open symbols on the left side of the panel represent the
light-scattering values after decompression. As shown, the reassembly
of nucleocapsids was not achieved after returning the samples to the
atmospheric pressure. The small change in light scattering for
enveloped particles occurred only in the first 500 bar, with
practically no changes at higher pressures, which can be explained by
the effects on a subpopulation of particles, very likely deformed or
defective ones.
To further characterize the stability induced by the envelope, urea was
used as a perturbing agent. The changes in light scattering and in Trp
fluorescence emission as a function of urea were monitored for the
whole virus (Fig. 2, squares) and for the
nucleocapsids (circles). The whole virus was more resistant
to urea than the nucleocapsids, as measured by light scattering (Fig.
2A), Trp fluorescence
intensity (Fig. 2B), and the red shift of the Trp emission
(Fig. 2C). All these results demonstrate that the intact particles presented a higher stability against urea.
Lack of Reversibility in the Dissociation of
Nucleocapsids--
The large red shift in the fluorescence emission
spectrum of the nucleocapsid incubated in 8.0 M urea and at
high pressure demonstrates the entire disassembly of these particles,
clearly visualized in the spectra of Fig.
3A. In contrast, the
structural changes promoted by pressure and urea on whole particles
were not equivalent (Fig. 3B). Thus, an end point is reached
when urea or pressure perturbs the nucleocapsids, whereas only urea
fully affects the enveloped particles.
The dramatic decrease in light scattering (Fig. 1) indicates
dissociation into subunits or small oligomers of the capsid protein. From the end point of the light-scattering curve we cannot assign the
precise size of the dissociated units. To further characterize the
products of dissociation of nucleocapsids, gel filtration chromatography was utilized (Fig. 4). It
can be noticed that the peak that corresponds to the elution of
nontreated NC particles (Fig. 4A) decreased in the sample
subjected to pressure (Fig. 4B) with the appearance of
dissociated protein at longer elution times. The elution time of the
free protein corresponds to monomeric subunits of the capsid protein. A
small peak also eluted at shorter times, indicating a fraction of
aggregates of the capsid protein. More drastic effects were produced by
treatment with 8.0 M urea (Fig. 4C) that
resulted in the elution of naked RNA (dashed line) without
the protein. We have seen in many cases that unfolded capsid proteins
become very sticky and adsorb into the column (8, 11). It should be
pointed out that the protein was in solution before injection in the
column. Our data demonstrate that NC particles are dissociated
into capsid proteins (probably monomers or small oligomers) and that
only a small fraction of the protein reassembles into ribonucleoprotein
particles after return to atmospheric pressure.
Accessibility of the RNA--
To check whether NC dissociation was
exposing the viral genome, ethidium bromide was utilized as a probe
(Fig. 5). The whole virus particle
(squares) was compared with nucleocapsid
(circles). Whole viruses and nucleocapsids were incubated
with 4 µM ethidium bromide and subjected to pressure
(Fig. 5). Whole virus presented no changes in the binding of ethidium
bromide, whereas NC showed a large increase in binding of the dye when
pressure was increased up to 2.5 kbar. These results not only confirm
that the nucleocapsid is dissociated by high pressure but also that RNA
is exposed. The isolated symbols on the left side of the
panel represent the binding of ethidium bromide values after
decompression. As shown, the RNA exposure is maintained after return to
atmospheric pressure, suggesting that only a small fraction of
ribonucleoprotein particles are formed after decompression. This result
corroborates the gel filtration chromatography of pressurized NC (Fig.
4B), where it can be observed that there is a small quantity
of protein and RNA eluting in the same position.
The changes in ethidium bromide fluorescence (Fig. 5) occur at slightly
lower pressures than the decrease in light scattering (Fig. 1). This
result may indicate that RNA becomes accessible before dissociation,
likely due to capsid expansion.
The Partially Folded State of the Coat Protein under
Pressure--
To examine the protein conformation in the nucleocapsid
and in the whole virus, we performed the pressurization of both samples in the presence of bis-ANS. The fluorescence emission of the probe incubated with the nucleocapsid at 2.5 kbar was almost twice the value
of the control at atmospheric pressure (Fig.
6A). It is noteworthy that
bis-ANS binding after decompression was even higher (four times the
control). These results indicate that protein-RNA interactions are
broken and result in binding of bis-ANS to the protein. Bis-ANS binds
to pockets in native or partially folded proteins, especially to
nucleic acid binding sites (32, 50).
When whole virus was incubated with bis-ANS, an increase in
fluorescence occurred especially because of binding to the membrane and
to the membrane viral glycoproteins (49, 51-53). The treatment of
intact particles with pressure generated a different pattern from that
found for NC particles. There was a large increase in fluorescence under pressure followed by a decrease when pressure was
returned to 1.0 bar. These results corroborate the idea that the
envelope glycoproteins suffered structural modifications induced by
high pressure (Fig. 6B); these changes resulted in loss of the ability to infect susceptible cells by the particles (49, 54). High
concentrations of urea caused complete disruption of the particles and
resulted in a remarkable decrease in bis-ANS binding for both whole
virus and nucleocapsids, suggesting complete unfolding of the protein components.
The pressure-dissociated NC protein bound bis-ANS and could bind even
more after decompression. This result indicates that the capsid protein
is in a partially folded state. In fact, the circular dichroism spectra
of pressure-dissociated NC show retention of secondary structure
(not shown). A fluctuating tertiary conformation is probably
responsible for the heterogeneous elution profile in the gel filtration
analysis with free, aggregated, and RNA-bound states.
The combination of dynamic and structural data is required to
dissect the steps of virus assembly and disassembly. Viruses propagate
in host cells by conferring unique properties to each particle that act
in a single sequential cycle: 1) assembly inside the cells and release
to the environment; 2) attachment and fusion to new host cells; 3)
disassembly and delivery of genome; 4) replication of the genome and
transcription of new viral proteins. Step 2 is different for
nonenveloped and enveloped viruses, and step 3 is not known in detail
for most viruses (2). Here we show that removal of the envelope
containing the lipid bilayer and the spike proteins (E1 and E2) results
in a metastable NC particle. Under a given perturbation of the
icosahedral structure, the "skinned" capsid dissociates into
partially folded subunits with a substantial exposure of the RNA
suitable for further processing.
Nonenveloped RNA viruses have a progressive decrease in folding
structure from assembled capsids to ribonucleoprotein particles (9,
20). Here we find that the stability of the capsid is severely weakened
when the envelope is removed. The loss of the membrane and the
transmembrane proteins is responsible for causing the particle to
become metastable. Although protein-protein interactions between the
cytoplasmic tail of E2 and the capsid protein (15) are likely perturbed
in the enveloped particle, return to the atmospheric pressure results
in reversibility of the interactions. Indeed, electron microscopy of
enveloped particles subjected to pressure does not show major changes
in the spike pattern of the envelope and its relation with the
nucleocapsid core (not shown).
RNA-protein interactions stabilize many viruses and also the
nucleocapsids of enveloped animal viruses (55). The knowledge of the
effects of high hydrostatic pressure on Mayaro virus nucleocapsids and
on the capsid protein should contribute to the understanding of the
assembly and disassembly of animal viruses. NC disassembly has been
suggested to require a conformational change in the coat protein, which
primes the core for uncoating after entry (56). It has been proposed
that residues 99 to 113 of the alphavirus NC protein are important in
binding ribosomes to facilitate NC uncoating (57, 58). Recent data
suggest that RNA encapsidation and disassembly may be related
processes, and residues involved in these events may be close in the
primary sequence of the NC protein (59). Residues 97-113 of the
Sindbis virus NC protein are important for both encapsidation and NC
assembly and disassembly (59). Residues 97-106 are clearly involved in
dictating specificity in encapsidation, and residues 108 and 110 appear
to be involved in NC assembly. Studies with Sindbis capsid protein,
expressed by Escherichia coli, showed that the
monomer-monomer interface is maintained by two pairs of hydrogen bonds
and by hydrophobic interactions. Removal of the hydrogen bonds by
single substitutions did not prevent dimer formation. However, a
mutation that reduced the hydrophobic contacts did inhibit dimer
formation (60). Our findings agree with these studies since high
pressure affects primarily hydrophobic interactions and cavities in the
interior of the protein (21, 30, 31). It is likely that mutations that
affect core formation also affect core disassembly. Further examination
of viruses containing mutations may help elucidate the contributions of
the envelope, protein-RNA, and protein-protein interactions in the
stability of alphaviruses.
Our data do indicate that the envelope restrains the intrinsic
metastability of the capsid. Removal of the envelope releases the
constraint and primes the capsid into a metastable state that dissociates in a poorly reversible way. The gel filtration studies revealed that only a small fraction (around 20%) of the capsid protein
reassembled into NC particles (Fig. 4). No empty capsids were detected,
in agreement with recent data using Sindbis capsid proteins expressed
in E. coli, which oligomerize in vitro into core-like particles in the presence of single-stranded nucleic acid
(61). Recently, we found that the maturation/cleavage of flock house
virus capsid converts the coat protein into a metastable conformation
(11), whereas cleavage-defective mutant particle coat protein
reversibly reassembles into particles. At the early stages of viral
infection, the metastable state of the cleaved coat protein would
contribute to a rapid dissociation concerted to unfolding (11). For
enveloped viruses, the membrane would eliminate the need for maturation
of the capsid coat protein. The metastability would be intrinsically
forged on the entire capsid. Although most of the proteins acquire the
most stable state, there are many examples of native proteins in a
metastable state (21, 40-42).
Fig. 7 sketches the changes in
conformation of the capsid protein as it passes through a cycle of
pressure and decompression for the NC particle and for the whole virus.
In the enveloped state, the particle undergoes completely reversible
changes, and there is no exposure of the nucleic acid (Fig. 7,
state 3a). We know that the glycoproteins from alphaviruses
change to the fusion-active state under pressure, resulting in a loss
of infectivity but without major changes in
structure.2 On the other
hand, high pressure affects protein-protein and protein-RNA
interactions in the nucleocapsid core in a way that, returning to 1 bar, there is a heterogeneous distribution of states, including free
protein and ribonucleoprotein complexes (Fig. 7, state 3b).
The high capacity to bind bis-ANS in this state suggests a partially
folded conformation of the capsid protein as responsible for this
fluctuating structure. It is noteworthy that the small fraction of
ribonucleoprotein complex formed after reassembly (Fig. 4) has the
capsid protein deprived of tertiary structure with exposure of
tryptophans to the solvent (Figs. 1 and 3). Proteins devoid of tertiary
structure were recently shown in the atomic structure of the large
ribosomal subunit (62). We propose in the scheme that the capsid
protein has different states coded by color, and the NC without
envelope is in an intermediate state, a metastable one (Fig. 7,
state 1b). Urea causes complete dissociation of the NC
particles as represented in Fig. 7, state 4b. Recent studies
reveal the importance of nucleic acid to scaffold the assembly of the
particles (8, 9). For alphaviruses, recent work demonstrates that the
initial intermediate for NC assembly is a dimer-RNA complex that acts
as a nucleation center for assembly (63).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES
p) in wavenumber
(cm
1) according to the following equation.
where Fi stands for the fluorescence emitted
at wavenumber
(Eq. 1)
i, and the summation is carried out
over the range of appreciable values of F. Tryptophan
emission was collected from 300 to 420 nm with excitation set at 280 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES
= 350 cm
1 and, for NC, was
= 500 cm
1. These results suggest a greater Trp
exposure to the polar solvent for the NC than for the whole virus
particle. The changes in Trp emission were mostly reversible for the
whole particle and irreversible for the NC. The changes in the whole
particle are not only due to changes in the capsid protein but as well
in the envelope glycoproteins (E1 and E2). The inset shows
the gel electrophoresis of purified samples of whole Mayaro virus and
of nucleocapsids (Fig. 1A), demonstrating that the NC
preparation lacks the membrane spike proteins.
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Fig. 1.
Pressure-induced effects on Mayaro virus and
Mayaro virus NC. A, the changes in the center of
spectral mass were followed as a function of pressure at 22 °C for
the native virus ( ) and virus NC (
). The center of spectral mass
values after returning to atmospheric pressure for NC (
) and whole
virus (
) is shown. The inset shows
SDS-polyacrylamide gel electrophoresis as described under
"Experimental Procedures" of purified virus and virus NC.
Lane 1 contains purified Mayaro virus (P1, 54 kDa; P2, 50 kDa; C, 34 kDa), lane 2 contains purified Mayaro NC. B, the light scattering
(LS) was followed as a function of increasing pressures at
22 °C for the native virus (
) and Mayaro NC (
). Values are
plotted as a fraction of light-scattering values at atmospheric
pressure. The isolated symbols on the left side of panel
represent the light-scattering value after returning to atmospheric
pressure of native virus (
) and virus NC (
). The spectra were
collected after 15 min at each pressure applied. The protein
concentration was 60 µg/ml.
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Fig. 2.
Urea-induced disassembly of Mayaro virus and
virus NC. A, the light-scattering change was followed
as a function of urea concentration at 22 °C, as shown on the
abscissa. Native virus ( ) and purified Mayaro NC (
)
were incubated with increasing concentrations of urea. Values are
plotted as a fraction of the light-scattering (LS) value at
no urea concentration. B, the area of the tryptophan
fluorescence emission spectrum of native virus (
) and virus NC (
)
is shown. C, the changes in center of spectral mass of
native virus (
) and virus NC (
). The spectra were collected after
a 15-min incubation at each different urea concentration. The protein
concentration was 60 µg/ml.
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Fig. 3.
Lack of reversibility in pressure- and
urea-induced disassembly of Mayaro and virus NC. A, the
fluorescence emission spectra of Mayaro virus NC at atmospheric
pressure (solid line), of NC disassembled by high pressure (2.5 kbar)
(dashed line), and after incubation with 8.0 M urea (dotted
line). B, tryptophan fluorescence emission of whole virus at
atmospheric pressure (solid line), 2.5 kbar (dashed line), and after
incubation with 8.0 M urea (dotted line). The
protein concentration was 60 µg/ml. A.U., absorbance
units.
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Fig. 4.
Pressure-induced disassembly of Mayaro virus
nucleocapsid. The virus concentration was 100 µg/ml. High
performance liquid chromatography in a size exclusion column (Sephacryl
G-500) of the native nucleocapsids (A) and pressurized
nucleocapsids after return to atmospheric pressure (B) are
shown. A gel filtration run of the sample incubated with 8.0 M urea for 60 min is shown in C. Elution was
monitored by coat protein fluorescence (excitation at 280 nm and
emission at 330 nm; continuous solid lines) and by RNA
absorbance (260 nm; dashed lines). A.U.,
absorbance units.
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Fig. 5.
Pressure-induced exposure of nucleic acid in
nucleocapsids. The binding of 4 µM ethidium bromide
on Mayaro virus and NC nucleic acid was measured by fluorescence.
Isolated NC ( ) and native virus (
) were subjected to increasing
pressures, and the spectra were collected after 15 min at each
pressure. The isolated symbols on the left represent the center of
spectral mass after returning to atmospheric pressure: NC (
) and
Mayaro virus (
). The samples were excited at 520 nm, and the
emission was measured from 550 to 700 nm. the protein concentration was
60 µ g/ml. A.U., absorbance units.
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Fig. 6.
Binding of bis-ANS to Mayaro virus and
nucleocapsids. A, nucleocapsids at atmospheric pressure
(a), at 2.5 kbar (b), after returning to
atmospheric pressure (c), and with 8.0 M urea
(d). B, Mayaro virus at atmospheric pressure
(a), at 2.5 kbar (b), after returning to
atmospheric pressure (c) and with 8.0 M urea
(d). The pressure spectra were collected after 60 min after
2.5 kbar. The samples were excited at 360 nm, and the emission was
measured from 400 to 600 nm. Protein concentration was 60 µg/ml, and
Bis-ANS concentration was 2 µM. A.U.,
absorbance units.
DISCUSSION AND CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES
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Fig. 7.
Schematic representation of pressure effects
on the whole virus particle and pressure and urea disassembly of Mayaro
NC. The degree of stability of enveloped virus and unfolding of
coat proteins of Mayaro virus NC are represented by color codes. The
top panel shows that pressure produces a noninfectious virus
particle (state 3a). The middle panel shows that
Mayaro NC disassembles irreversibly into a ribonucleoprotein complex
(state 3b) and that partially unfolded coat proteins are
formed different from the completely unfolded state obtained at high
urea concentration showed in the bottom panel (state
4b).
Cavities have been implicated in protein metastability (21, 41, 64,
65). The metastability observed here can be correlated with cavities,
which would be present in the capsids but not in the subunits. The
atomic structure of the coat protein subunit has been solved at high
resolution (17) and a comparison with the whole capsid should be
possible when its atomic structure becomes available. The high
stability and reversibility of enveloped viruses under physical
perturbation can be explained by compartmentalization induced by the
membrane. The membrane envelope acts as a Trojan horse, subverting the
host cell to allow entry and release of a metastable nucleocapsid prone
to disassembly and processing.
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ACKNOWLEDGEMENTS |
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We are grateful to Carlos F. L. Fontes for critical reading of the manuscript and to Emerson R. Gonçalves for his competent technical assistance.
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FOOTNOTES |
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* This work was supported in part by an international grant from the Howard Hughes Medical Institute (to J. L. S.) and by grants from Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico (PADCT and Pronex programs), and Financiadora de Estudos e Projetos (FINEP) of Brazil (to J. L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Scientific Instruments Co. do Brazil.
A Howard Hughes Medical Institute International Researcher. To
whom correspondence should be addressed. Tel.: 55-21-590-4548; Fax:
55-21-270-8647. E-mail: jerson@bioqmed.ufrj.br.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M010037200
2 M. S. Freitas, L. P. Gaspar, and J. L. Silva, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: NC, nucleocapsid core; bis-ANS, bis(8-anilinonaphthalene-1-sulfonate); BHK cells, baby hamster kidney cells; kbar, kilobars.
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