The Metastable State of Nucleocapsids of Enveloped Viruses as Probed by High Hydrostatic Pressure*

Luciane P. GasparDagger , Alexandre F. TerezanDagger §, Anderson S. PinheiroDagger , Débora FoguelDagger , Moacyr A. Rebello, and Jerson L. SilvaDagger ||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES

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 (upsilon p) in wavenumber (cm-1) according to the following equation.


⟨&ngr;<SUB>p</SUB>⟩=<LIM><OP>∑</OP></LIM>&ngr;<SUB>i</SUB> F<SUB>i</SUB>/<LIM><OP>∑</OP></LIM>F<SUB>i</SUB> (Eq. 1)
where Fi stands for the fluorescence emitted at wavenumber upsilon 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.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES

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 Delta  = 350 cm-1 and, for NC, was Delta  = 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 (black-square) and virus NC (). The center of spectral mass values after returning to atmospheric pressure for NC (open circle ) 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 (black-square) 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 (open circle ). The spectra were collected after 15 min at each pressure applied. The protein concentration was 60 µg/ml.

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.



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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 (black-square) 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 (black-square) and virus NC () is shown. C, the changes in center of spectral mass of native virus (black-square) and virus NC (). The spectra were collected after a 15-min incubation at each different urea concentration. The protein concentration was 60 µg/ml.

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.



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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.

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.



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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.

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.



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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 (black-square) 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 (open circle ) 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.

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).



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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.

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.


    DISCUSSION AND CONCLUSIONS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES

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).



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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.


    ACKNOWLEDGEMENTS

We are grateful to Carlos F. L. Fontes for critical reading of the manuscript and to Emerson R. Gonçalves for his competent technical assistance.


    FOOTNOTES

* 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.


    ABBREVIATIONS

The abbreviations used are: NC, nucleocapsid core; bis-ANS, bis(8-anilinonaphthalene-1-sulfonate); BHK cells, baby hamster kidney cells; kbar, kilobars.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION AND CONCLUSIONS
REFERENCES


1. Rossmann, M. G., and Johnson, J. E. (1989) Annu. Rev. Biochem. 58, 533-573[CrossRef][Medline] [Order article via Infotrieve]
2. Johnson, J. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 27-33[Abstract/Free Full Text]
3. Harrison, S., Wiley, D. C., and Skehel, J. J. (1996) Fields Virology , 3rd Ed. , pp. 59-99, Lippincott-Raven Publishers, Philadelphia
4. Silva, J. L., and Weber, G. (1988) J. Mol. Biol. 199, 149-161[Medline] [Order article via Infotrieve]
5. Prevelige, P. E., King, J., and Silva, J. L. (1994) Biophys. J. 66, 1631-1641[Abstract]
6. Reddy, V. S., Giesing, H. A., Morton, R. T., Kumar, A., Post, C. B., Brooks, C. L., and Johnson, J. E. (1998) Biophys. J. 74, 546-558[Abstract/Free Full Text]
7. Leimkuhler, M., Goldbeck, A., Lechner, M. D., and Witz, J. (2000) J. Mol. Biol. 296, 1295-1305[CrossRef][Medline] [Order article via Infotrieve]
8. Da Poian, A. T., Oliveira, A. C., and Silva, J. L. (1995) Biochemistry 34, 2672-2677[Medline] [Order article via Infotrieve]
9. Gaspar, L. P., Johnson, J. E., Silva, J. L., and Da Poian, A. T. (1997) J. Mol. Biol. 273, 456-466[CrossRef][Medline] [Order article via Infotrieve]
10. Zlotnick, A., Reddy, V. S., Dasgupta, R., Schneemann, A., Ray, W. J., Jr., Rueckert, R. R., and Johnson, J. E. (1994) J. Biol. Chem. 269, 13680-13684[Abstract/Free Full Text]
11. Oliveira, A. C., Gomes, A. M. O., Almeida, F. C. L., Mohana-Borges, R., Valente, A. P., Reddy, V. S., Johnson, J. E., and Silva, J. L. (2000) J. Biol. Chem. 275, 16037-16043[Abstract/Free Full Text]
12. Paredes, A. M., Simon, M. L., and Brown, D. T. (1993) Virology 187, 329-332[CrossRef]
13. Strauss, J. H., and Strauss, E. G. (1994) Microbiol. Rev. 58, 491-562[Abstract]
14. Helenius, A., and Kartenbeck, J. (1980) Eur. J. Biochem. 106, 613-618[Abstract]
15. Cheng, R. H., Kuhn, R. J., Olson, N. H., Rossmann, M. G., Choi, H. K., Smith, T. J., and Baker, T. S. (1995) Cell 80, 621-630[Medline] [Order article via Infotrieve]
16. Geigenmüller-Gnirke, U., Nitschko, H., and Schlesinger, S. (1993) J. Virol. 67, 1620-1626[Abstract]
17. Choi, H. K., Tong, L., Minor, W., Dumas, P., Boege, U., Rossmann, M. G., and Wengler, G. (1991) Nature 354, 37-43[CrossRef][Medline] [Order article via Infotrieve]
18. Tong, L., Wengler, G., and Rossmann, M. G. (1993) J. Mol. Biol. 230, 228-247[CrossRef][Medline] [Order article via Infotrieve]
19. Gorovits, B., Raman, C, S., and Horowitz, P. M. (1995) J. Biol. Chem. 270, 2061-2066[Abstract/Free Full Text]
20. Silva, J. L., Foguel, D., Da Poian, A. T., and Prevelige, P. E. (1996) Curr. Opin. Struct. Biol. 6, 166-175[CrossRef][Medline] [Order article via Infotrieve]
21. de Souza, P. C., Tuma, R., Prevelige, P. E., Silva, J. L., and Foguel, D. (1999) J. Mol. Biol. 287, 527-538[CrossRef][Medline] [Order article via Infotrieve]
22. Oliveira, A. C., Ishimaru, D., Gonçalves, R. B., Mason, P., Carvalho, D., Smith, T., and Silva, J. L. (1999) Biophys. J. 76, 1270-1279[Abstract/Free Full Text]
23. Tian, S. M., Ruan, K. C., Qian, J. F., Shao, G. Q., and Balny, C. (2000) Eur. J. Biochem. 267, 4486-4494[Abstract/Free Full Text]
24. Silva, J. L., and Weber, G. (1993) Annu. Rev. Phys. Chem. 44, 89-113[CrossRef][Medline] [Order article via Infotrieve]
25. Mozhaev, V. V., Heremans, K., Frank, J., Masson, P., and Balny, C. (1996) Proteins 24, 81-91[CrossRef][Medline] [Order article via Infotrieve]
26. Northrop, D. B., and Cho, Y. K. (2000) Biophys. J. 79, 1621-1628[Abstract/Free Full Text]
27. Jonas, J., and Jonas, A. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 287-318[CrossRef][Medline] [Order article via Infotrieve]
28. Heremans, K., and Smeller, L. (1998) Biochim. Biophys. Acta 1386, 353-370[Medline] [Order article via Infotrieve]
29. Desai, G., Panick, G., Zein, M., Winter, R., and Royer, C. A. (1999) J. Mol. Biol. 288, 461-475[CrossRef][Medline] [Order article via Infotrieve]
30. Frye, K. J., and Royer, C. A. (1998) Protein Sci. 10, 2217-2222
31. Hummer, G., Garde, S., Garcia, A. E., Paulaitis, M. E., and Pratt, L. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1552-1555[Abstract/Free Full Text]
32. Silva, J. L., Silveira, C. F., Correa, A., and Pontes, L. (1992) J. Mol. Biol. 223, 545-555[Medline] [Order article via Infotrieve]
33. Nash, D. P., and Jonas, J. (1997) Biochemistry 36, 14375-14383[CrossRef][Medline] [Order article via Infotrieve]
34. Ruan, K., Lange, R., Bec, N., and Balny, C. (1997) Biochem. Biophys. Res. Commun. 239, 150-154[CrossRef][Medline] [Order article via Infotrieve]
35. Ferrão-Gonzales, A. D., Souto, S. O., Silva, J. L., and Foguel, D. (2000) Proc. Natl. Acad. Sci. U. S. A. 12, 6445-6450[CrossRef]
36. Erijman, L., Lorimer, G. H., and Weber, G. (1993) Biochemistry 32, 5187-5195[Medline] [Order article via Infotrieve]
37. Foguel, D., and Silva, J. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8244-8247[Abstract]
38. Robinson, C. R., and Sligar, S. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3444-3448[Abstract]
39. Mohana-Borges, R., Pacheco, A. B. F., Sousa, F. J. R., Foguel, D., Almeida, D. F., and Silva, J. L. (2000) J. Biol. Chem. 275, 4708-4712[Abstract/Free Full Text]
40. Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966[Medline] [Order article via Infotrieve]
41. Lee, C., Park, S. H., Lee, M. Y., and Yu, M. H. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7727-7731[Abstract/Free Full Text]
42. Orosz, A., Wisniewski, J., and Wu, C. (1996) Mol. Cell. Biol. 16, 7018-7030[Abstract]
43. Carr, C. M., Chaudhry, C., and Kim, P. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14306-14313[Abstract/Free Full Text]
44. Neuman, R. C., Kauzmann, W., and Zipp, A. (1973) J. Phys. Chem. 77, 2687-2691
45. Volkmer, N., and Rebello, M. A. (1981) IRCS Med. Sci. Biochem. 9, 157-158
46. Rebello, M. C. S., Fonseca, M. E. F., Marinho, J. O., and Rebello, M. A. (1993) Acta Virol. 37, 223-231[Medline] [Order article via Infotrieve]
47. Harrison, S. C., Strong, R. K., Schlesinger, S., and Schlesinger, M. J. (1992) J. Mol. Biol. 226, 277-280[Medline] [Order article via Infotrieve]
48. Paladini, A. A., and Weber, G. (1981) Rev. Sci. Instrum. 52, 419-427
49. Silva, J. L., Peng, L., Glaser, M., Voss, E. W., and Weber, G. (1992) J. Virol. 66, 2111-2117[Abstract]
50. Rosen, C. G., and Weber, G. (1969) Biochemistry 8, 3915-3920[Medline] [Order article via Infotrieve]
51. Korte, T., and Herrmann, A. (1994) Eur. Biophys. J. 23, 105-113[Medline] [Order article via Infotrieve]
52. Korte, T., Ludwig, K., Booy, F. P., Blumenthal, R., and Herrmann, A. (1999) J. Virol. 73, 4567-4574[Abstract/Free Full Text]
53. Bonafe, C. F. S., Glaser, M., Voss, E. W., Weber, G., and Silva, J. L. (2000) Biochem. Biophys. Res. Commun. 275, 955-961[CrossRef][Medline] [Order article via Infotrieve]
54. Jurkiewicz, E., Villas-Boas, M., Silva, J. L., Weber, G., Hunsmann, G., and Clegg, R. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6935-6937[Abstract]
55. Speir, J. A., Munshi, S., Wang, G., Baker, T. S., and Johnson, J. E. (1995) Structure (Lond.) 3, 63-78[Medline] [Order article via Infotrieve]
56. Coombs, K., Brown, B., and Brown, D. T. (1984) Virus Res. 4, 297-302
57. Singh, I., and Helenius, A. (1992) J. Virol. 66, 7049-7058[Abstract]
58. Wengler, G., Würkner, D., and Wengler, G. (1992) Virology 191, 880-888[Medline] [Order article via Infotrieve]
59. Owen, K. E., and Kuhn, R. J. (1996) J. Virol. 70, 2757-2763[Abstract]
60. Choi, H. K., Lee, S., Zhang, Y. P., McKinney, B. R., Wengler, G., Rossmann, M. G., and Kuhn, R. J. (1996) J. Mol. Biol. 262, 151-167[CrossRef][Medline] [Order article via Infotrieve]
61. Tellinghuisen, T. L., Hamburger, A. E., Fisher, B. R., Ostendorp, R., and Kuhn, R. J. (1999) J. Virol. 73, 5309-5319[Abstract/Free Full Text]
62. Ban, N., Nissen, P., Hansen, J., Moore, P. B., and Steitz, T. A. (2000) Science 289, 905-920[Abstract/Free Full Text]
63. Tellinghuisen, T. L., and Kuhn, R. J. (2000) J. Virol. 74, 4302-4309[Abstract/Free Full Text]
64. Foguel, D., Teschke, C. M., Prevelige, P. E., and Silva, J. L. (1995) Biochemistry 34, 1120-1126[Medline] [Order article via Infotrieve]
65. Chen, J., Lee, K. H., Steinhauer, D. A., Stevens, D. J., Skehel, J. J., and Wiley, D. C. (1998) Cell 95, 409-417[Medline] [Order article via Infotrieve]


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