Eur J Biochem 271, 135–145 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03911.x Mutations in the hydrophobic core and in the protein–RNA interface affect the packing and stability of icosahedral viruses ´ Sheila M B Lima1, David S Peabody2, Jerson L Silva1 and Andrea C de Oliveira1 Departamento de Bioquı´mica Me´dica, Instituto de Cieˆncias Biome´dicas and Centro Nacional de Ressonaˆncia Magne´tica Nuclear de Macromole´culas, Universidade Federal Rio de Janeiro, Rio de Janeiro, Brazil; 2Department of Molecular Genetics and Microbiology and Cancer Research and Treatment Center, University of New Mexico School of Medicine, Albuquerque, NM, USA The information required for successful assembly of an icosahedral virus is encoded in the native conformation of the capsid protein and in its interaction with the nucleic acid Here we investigated how the packing and stability of virus capsids are sensitive to single amino acid substitutions in the coat protein Tryptophan fluorescence, bis-8-anilinonaphthalene-1-sulfonate fluorescence, CD and light scattering were employed to measure urea- and pressure-induced effects on MS2 bacteriophage and temperature sensitive mutants M88V and T45S particles were less stable than the wild-type forms and completely dissociated at 3.0 kbar of pressure M88V and T45S mutants also had lower stability in the presence of urea We propose that the lower stability of M88V particles is related to an increase in the cavity of the hydrophobic core Bis-8-anilinonaphthalene-1-sulfonate fluorescence increased for the pressure-dissociated mutants but not for the urea-denatured samples, indicating that the final products were different To verify reassembly of the particles, gel filtration chromatography and infectivity assays were performed The phage titer was reduced dramatically when particles were treated with a high concentration of urea In contrast, the phage titer recovered after high-pressure treatment Thus, after pressure-induced dissociation of the virus, information for correct reassembly was preserved In contrast to M88V and T45S, the D11N mutant virus particle was more stable than the wild-type virus, in spite of it also possessing a temperature sensitive growth phenotype Overall, our data show how point substitutions in the capsid protein, which affect either the packing or the interaction at the protein–RNA interface, result in changes in virus stability The protein shells of viruses generally have several key functions, including shielding of the nucleic acid, particle maturation and conferring the ability to penetrate the host cell and undergo disassembly The coat proteins are usually arranged in a shell with an icosahedral shape [1] The information required for successful assembly of a virus particle is encoded in the native conformation of a capsid protein subunit Structural and thermodynamic approaches have been employed to identify the general rules that govern virus assembly [2–7] The MS2 bacteriophage is an RNA virus of the family Leviviridae, a group of single-stranded RNA bacteriophages that infect F+ Escherichia coli cells The icosahedral shell of the MS2 virus particle has a ˚ diameter of 260 A and is made up of 180 copies of the coat protein subunit (Mr 13.7 · 103) in a T¼3 surface lattice Each virion also contains one copy of the maturase protein, which is responsible for attachment of the phage to E coli F-pili The coat protein has two functions in the viral life cycle First, it acts as a translational repressor of the replicase gene A coat protein dimer binds specifically to an RNA stem–loop structure (known as the translational operator) and prevents initiation of replicase translation [8–10] Second, coat protein serves as the major virus structural protein, forming the shell in which the RNA genome is contained [11,12] The tertiary structure and topology of the MS2 coat protein is different from those of other simple icosahedral viruses [13] The main chain of the protein subunit folds into a five stranded antiparallel b-sheet (strands bC–bG) facing the interior of the phage particle, with an N-terminal hairpin (strands bA and bB) and two a-helices (aA and aB) shielding most of the upper surface of the b-sheet from the environment Upon dimerization, extensive contacts are formed between the subunits so that the b-sheet becomes extended to form a continuous 10-stranded sheet The two polypeptide chains are so intimately intertwined that it seems clear that the dimer must be the basic unit of coat protein folding; each subunit depends on the other for acquisition of its native fold The 3D structure of the MS2 bacteriophage has ˚ been determined at 2.8 A (Fig 1A) [14] Even so, the mechanism of assembly and nucleic acid recognition are still far from completely understood Correspondence to A C de Oliveira, Avenida Bauhinia, ´ 400 - CCS/ICB/Bl E, sl 08, Cidade Universitaria, CEP 21941-590, Rio de Janeiro, RJ, Brazil Fax: + 55 21 2270 8647, Tel.: + 55 21 2562 6756, E-mail: cheble@bioqmed.ufrj.br Abbreviations: bis-ANS, bis-8-anilinonaphthalene-1-sulfonate; LB, Luria–Bertani; p.f.u., plaque-forming units; ts, temperature sensitive; WT, wild-type (Received 26 September 2003, accepted November 2003) Keywords: hydrostatic pressure; MS2 bacteriophage; temperature-sensitive mutants; urea; fluorescence Ó FEBS 2003 136 S M B Lima et al (Eur J Biochem 271) The isolated capsid and the assembly intermediates assume different partially folded states in the assembly pathway [7] Hydrostatic pressure permits controlled perturbation of the subunit interactions and is a powerful tool for using to study cavities in proteins [24,25] The coat protein of bacteriophage MS2 contains two tryptophan residues, thus permitting the use of intrinsic fluorescence as a probe of structural changes Here, we study the stability against pressure and urea of the MS2 bacteriophage and three temperature sensitive (ts) mutants (Fig 1B) Urea and hydrostatic pressure are utilized to promote capsid dissociation and denaturation, where the conformational changes are analyzed by fluorescence spectra, light scattering, CD, HPLC, infectivity assays and the bis-8-anilinonaphthalene-1-sulfonate (bis-ANS) binding assay We find that pressure promotes dissociation of the wild-type (WT) bacteriophage and ts mutants Two mutations that appear to lead to the formation of cavities, one in the hydrophobic core (M88V) and the other in the protein–RNA interaction (T45S), decrease the stability of the capsid Our findings illuminate the role of packing in the icosahedral lattice of the virus capsid Experimental procedures Chemicals All reagents were of analytical grade Distilled water was filtered and deionized through a Millipore water purification system The experiments were performed at 20 °C in standard buffer: 50 mM Tris, 150 mM NaCl, pH 7.5 MS2 bacteriophage and mutant samples Fig Structure of the whole capsid of bacteriophage MS2 and of the coat protein dimer bound to RNA (A) The MS2 bacteriophage capsid is colored according to the asymmetric units (B) The coat protein of MS2 bacteriophage bound to RNA, showing the location of the amino acids (in space fill display) substituted in the temperature sensitive (ts) mutants The two polypeptide chains of the dimer are shown as blue and green ribbons and the RNA molecule is shown in red (space fill) Met88 is represented in brown (at the dimer surface), Thr45 in yellow (interacting with RNA) and Asp11 in red, which appears to form a salt bridge to Lys113 (cyan) on the alpha-helix of the adjacent subunit of the dimer (PDB file: 2MS2) High pressure is an efficient tool for studies on the folding of proteins [15–18] and on the assembly of supramolecular structures, such as viruses [6,7,19–23] In general, it has been found that individual capsid proteins (monomers or dimers) are generally much less stable to pressure than the assembled icosahedral particles [21,22] After growth of E coli C3000 in Luria–Bertani (LB) medium to an attenuance (D), at 600 nm, of 1.2, the culture was infected with MS2 and, after a further h of incubation, was treated with lysozyme [8] The samples were processed by pelleting the bacterial debris by centrifugation (8000 r.p.m for 10 min; RPR 9.2 rotor; Beckman) at °C The supernatant was precipitated with ammonium sulfate (330 gỈL)1) The phages were precipitated by centrifugation (10000 r.p.m for 45 min; RPR 12.2 rotor; Beckman) at °C The precipitate was dissolved in standard buffer and purified by high-speed centrifugation (35 000 r.p.m for 14 h; SW41 rotor; Beckman) in a sucrose gradient (10–50%) The sample concentration utilized in all the experiments was 50 lgỈmL)1, except for CD experiments (where the sample concentration was 100 lgỈmL)1) Virus concentrations were determined by the method of Bradford [26] using lysozyme as a standard They were confirmed by measuring the absorbance at 280 nm Spectroscopic measurements under pressure Two important parts form the high pressure system: the pressure generator and the high-pressure cell [27] Fluorescence spectra and light scattering measurements were recorded on an ISSK2 spectrofluorometer (ISS Inc., Champaign, IL, USA) Fluorescence spectra were quantified by evaluating the spectral center of mass, , as follows: Ó FEBS 2003 Cavities and stability in MS2 capsid mutants (Eur J Biochem 271) 137 ẳ Rmi:F i=RF i 1ị where Fi stands for the fluorescence emitted at wavenumber mi and the summation is carried out over the range of appreciable values of F The values of center of spectral mass of tryptophan were converted into degree of denaturation/dissociation (ap), according to the following equation: ap ẳ mp mi ị=mf À mi Þ ð2Þ where mp is the value at each pressure, mi is the value at 1.0 bar, and mf is the value at 3.4 kbar The volume change DV can be calculated from the following thermodynamic relation [15,16]: lnẵan =1 p n ap ị ẳ pDV=RT ỵ lnẵkatm =n C n1ị 3ị where katm is the denaturation/dissociation constant at atmospheric pressure, p corresponds to a given pressure, R is the gas constant, T is the absolute temperature, n is the number of subunits, and C is the protein concentration Each experiment was performed at least three times with different protein preparations Light scattering Light scattering measurements were made in an ISSK2 spectrofluorometer Scattered light was collected at an angle of 90° of the incident light The samples were excited on 320 nm and collected in the same wavelength This wavelength was chosen because protein and RNA not absorb at 320 nm Chemical denaturation The samples were incubated with increasing concentrations of urea (1–9 M) and allowed to equilibrate for 30 prior to making measurements The measurements were made in the presence and absence of urea Each experiment was performed at least three times with different protein preparations Size-exclusion chromatography HPLC was carried out using a prepackaged SynChropak GPC500 column (250 · 4.6 mm inner diameter; SynChropaK Inc., Linden, IN, USA) The system was equilibrated in 50 mM Tris, 0.2 M sodium acetate buffer containing 0.5 gỈL)1 sodium azide (pH 7.0) A flow rate of 0.3 mLỈ min)1 was utilized Sample elution was monitored by the fluorescence at 330 nm (excitation at 280 nm) and the absorbance at 260 nm The equipment used was a Shimadzu model CD Conformational changes in MS2 bacteriophage and ts mutants treated with urea were analyzed The MS2 bacteriophage and ts mutant samples were diluted to a final concentration of 100 lgỈmL)1 and the spectra were obtained in 10 mM Tris, 30 mM NaCl (pH 7.5) buffer using a 0.1 cm pathlength quartz cuvette The spectropolarimeter used was a Jasco J-715 1505 model Infectivity assays An overnight culture of E coli was diluted : 20 (v/v) in LB medium and cultured at 37 °C for h in a rotary shaker Several phage dilutions, made in a standard buffer, were plated in LB semisolid medium containing E coli The plates were incubated overnight at 37 °C, after which the MS2 and ts mutants were diluted and titered by quantification of plaques resulting from phage-induced bacterial lysis The results are expressed as p.f.u (plaque-forming units) per mL Isolation of ts coat mutants The MS2 coat protein gene was randomly mutagenized by error-prone PCR [28] and introduced into pMS27 [29], a plasmid from which infectious MS2 genomic RNA can be produced by transcription from the T7 promoter Transfection into strain CSH41(pAR1219) [30], which produces T7 RNA polymerase, and plating at 32 °C led to the production of plaques, % 200 of which were picked to lawns of CSH41 on duplicate plates One plate was incubated at 32 °C and the other at 40 °C After identification of mutants exhibiting a growth defect at 40 °C, virus stocks were produced by growth in LB medium at 32 °C and the viruses were purified, by sedimentation to equilibrium, in CsCl density gradients Further characterization showed that the three mutants analysed in this study exhibited modest reductions in plating efficiencies (four to 20-fold reductions in plaque number) at elevated temperature, but in each case the plaques produced were dramatically smaller than at the permissive temperature, whereas WT plaque size was unaffected The coat genes of ts mutants were recovered by RT–PCR and cloned in pCT119 [31] and their amino acid substitutions were determined by DNA sequence analysis Results Chemical stability of ts MS2 coat mutants The coat protein of MS2 is the major structural protein of the virus and acts as a translational repressor that inhibits synthesis of the viral replicase late during infection (Fig 1) We isolated ts mutants of MS2, as described above in the Experimental procedures Each mutant particle exhibited a ts growth phenotype, producing fewer and dramatically smaller plaques at non-permissive temperatures Further characterization verified the ts character of the mutants Each exhibited a ts defect for repression of translation of a replicase–b-galactosidase fusion protein from plasmid pRZ5 [31,32] Furthermore, although WT coat protein was found almost entirely in the soluble fraction of cell lysates at either growth temperature, each of the ts mutant proteins was found predominantly in the insoluble fraction when cultured at the non-permissive temperature, but exhibited WT solubilities at the permissive temperature More than 12 ts coat mutants were produced with these characteristics (D S Peabody, unpublished results) We describe here some additional properties of three: D11N, T45S and M88V (Fig 1B) We used light scattering and intrinsic tryptophan fluorescence to monitor whole particle disassembly and subunit denaturation, respectively Intrinsic fluorescence of the coat 138 S M B Lima et al (Eur J Biochem 271) Fig Tryptophan fluorescence spectra of bacteriophage MS2 Spectra of wild-type (WT) (circles), M88V (diamonds), D11N (squares) and T45S (triangles) particles were recorded at atmospheric pressure in the absence (filled symbols), or presence of 4.5 M (unfilled symbols) or 9.0 M (lines) urea The excitation wavelength was 280 nm, and the emission wavelength range was 300–420 nm Standard buffer: 50 mM Tris/150 mM NaCl (pH 7.5) The sample concentration utilized was 50 lgỈmL)1 protein in the absence of urea is blue shifted because the tryptophan residues are buried in the hydrophobic interior of the protein As the protein unfolds, the tryptophan residues become more exposed to the solvent and their Ó FEBS 2003 fluorescence maxima shift towards the red (Fig 2) Figure shows that the WT and mutant forms have different susceptibilities at an intermediate urea concentration (4.5 M), but all fully dissociate and denature at a high urea concentration (9.0 M) Trp32 clearly resides within the hydrophobic core of the protein The other, Trp82, is only partially solvent-exposed [33] Its environment is determined primarily by interactions within the dimer, not by interactions between dimers Thus, tryptophan fluorescence should predominantly monitor dimer denaturation rather than capsid dissociation Meanwhile, light scattering measurements are sensitive to the size of the particle and can be used to monitor capsid dissociation The WT virus and each mutant were subjected to increasing concentrations of urea (1–9 M) Figure 3A shows that the curves relating to the spectral center of mass and light scattering are practically superimposable for WT MS2, indicating that the subunit dissociation and the denaturation processes are coupled The T45S and M88V mutants were both significantly less stable than the WT form (Figs and 3B,C) and denatured at rather lower urea concentrations (midpoints at % M for the mutants vs % 4.5 M for the WT form) The dissociation and denaturation processes for T45S and M88V were also coupled, as shown by the overlapping of the fluorescence and light scattering curves The large red-shift in the fluorescence emission spectra of the WT form and T45S, M88V and D11N mutants incubated with 9.0 M urea demonstrated the complete denaturation of coat protein in all cases Surprisingly however, D11N, despite its ts growth phenotype, was more stable to urea treatment than the WT capsid (Figs and 3B) Moreover, the light scattering data showed no significant change during urea treatment of the D11N mutant, suggesting that, even in the denatured state, the characteristics of a large particle are retained Apparently, the subunits remain aggregated in a particle of approximately virus-size (Fig 3C) Size-exclusion HPLC was also utilized Only for the D11N mutant was a small fraction Fig Dissociation and denaturation of wild-type (WT) and mutant MS2 particles (A) Light scattering and spectral center of mass measurements of WT MS2 as a function of urea concentration To verify the dissociation and denaturation processes, we measured the light scattering of the particles at 320 nm (d) and the spectral center of mass of the particles (s) (B) Urea-induced denaturation of MS2, as measured by tryptophan fluorescence for: (d), WT MS2; (r), M88V; (j), D11N; and (m), T45S (C) Urea-induced dissociation, as measured by light scattering for: (d), WT MS2; (r), M88V; (j), D11N; and (m), T45S For tryptophan fluorescence emission, the sample was excited at 280 nm and the emission was measured at 300– 420 nm For the light scattering measurements, the sample was excited at 320 nm and the emission measured from 315 to 325 nm Standard buffer: 50 mM Tris/150 mM NaCl (pH 7.5) Fluorescence data points are the average and standard deviation of three experiments (A and B) and light scattering curves are representative of three measurements The sample concentration utilized was 50 lgỈmL)1 Ĩ FEBS 2003 Cavities and stability in MS2 capsid mutants (Eur J Biochem 271) 139 Table Effects of pressure and urea on bacteriophage MS2 and mutants: infectivity assays (37 °C) ND, non-detected; p.f.u., plaqueforming units Titer (p.f.u.ỈmL)1) Control MS2 T45S M88V D11N 3.4 kbar of pressure 4.5 M urea 9.0 M urea 108 108 108 108 108 108 107 108 105 102 102 107 ND ND ND 102 and T45S particles with 4.5 M urea resulted in drastic reductions of phage titer (Table 1) The titer of WT MS2 was reduced by % 1000-fold M88V and T45S were the most susceptible, each showing a · 106-fold reduction in titer However, consistent with other measures of its stability, the D11N mutant was relatively unaffected and showed only a 10-fold loss of titer Pressure stability of the MS2 bacteriophage and ts mutants Fig High-performance gel filtration chromatography of bacteriophage MS2 and the temperature sensitive mutants Elution profiles of (A) M88V mutant, (B) wild-type MS2 and (C) D11N mutant The unbroken line corresponds to the samples that were incubated with 9.0 M urea, the dashed line corresponds to the samples without treatment, and the dotted line represents the samples treated with high pressure and returned to atmospheric pressure The flow rate was 0.3 mLỈmin)1 and the elution of the samples was monitored by tryptophan fluorescence (excitation at 280 nm, emission at 330 nm) of the particle eluted in the same position as untreated particles (Fig 4) In contrast, the other mutants and the WT capsid were irreversibly dissociated and denatured by a high concentration of urea (Fig 4) The monomers were expected to elute close to the total volume of the column (% 12 min) However, as previously found with other dissociated capsid proteins, the lack of elution of the proteins can be explained by non-specific binding of the denatured coat proteins to the gel [34,35] The elution of some D11N as capsids after urea treatment is probably the result of a small fraction that was not denatured by urea To explore the biological activity of the viral particles, cell infectivity assays were performed Treatment of WT, M88V The effects of high pressure on tryptophan fluorescence emission spectra of the MS2 and ts mutants were also investigated Pressure produced complete dissociation of the two mutants M88V and T45S However, up to 3.4 kbar, hydrostatic pressure was unable to promote complete dissociation of WT and D11N particles, as measured by fluorescence (Fig 5A) and by light scattering (Fig 5B) In agreement with the urea studies described above, the D11N mutant was more stable than the WT bacteriophage All the curves for pressure denaturation of WT and mutant particles seem to have more than one transition, which may indicate partially dissociated or denatured states However, because both fluorescence and light scattering reveal the average properties, we cannot fully characterize these potential intermediates The reversibility of the process was analyzed by determining the values of spectral center of mass (Fig 5A,C), which were measured after decompression and utilizing HPLC (Fig 4) Figure 5C shows that the spectra of the sample subjected to compression and decompression are similar to the non-treated sample, even in the case of the M88V mutant The elution of the samples after pressurization in the same position as the native virus showed that the particles were able to reassemble correctly, suggesting that dissociation by pressure is at least partially reversible To further investigate the recovery, infectivity assays were performed and when we used high pressure the phage titer was similar to that of the control (Table 1) Thus, in spite of the dissociation induced by pressure, the information for correct reassembly seems to be largely preserved In principle, the recovery of the titer implies that the pressure disassembly process is reversible, whereas the urea denaturation process is not The changes in stability can be clearly evaluated by the values of pressure and urea that lead to 50% denaturation (p½ and U½, respectively, shown in Table 2) Table also shows the apparent volume changes obtained by treating Ó FEBS 2003 140 S M B Lima et al (Eur J Biochem 271) Fig Pressure stability of bacteriophage MS2 and the temperature sensitive (ts) mutants The effect of pressure on the samples was analyzed at room temperature (A) The effect was measured by the tryptophan fluorescence emission of the spectral center of mass The samples were excited at 280 nm and the emission was measured from 300 to 420 nm for: (d), WT MS2; (r), M88V; j), D11N; and (m), T45S (B) Light scattering measurements of MS2 and ts mutants under pressure (d) MS2 bacteriophage and the ts mutants (r) M88V, (j) D11N, and (m) T45S The excitation wavelength was 320 nm and the emission wavelength range was 315–325 nm The incubation time at each pressure was 10 Other conditions were as described in the legend to Fig The unfilled symbols correspond to the respective values after pressure release Fluorescence data points are the average and SD of three experiments, and light scattering curves are the representative of three measurements (C) Fluorescence emission spectra of M88V mutant particles before (unbroken line), under 3.4 kbar of pressure (broken line, or after decompression (dotted lines) Table [U]½ and p½ values for wild-type (WT) bacteriophage MS2 and mutants CM, spectral center of mass; LS, light scattering p½ (kbar) Substitution Surface (S), buried (B) or RNA interaction (I) [U]½ (M) CM LS DV/na (mLỈmol)1) WT M88V T45S D11N B I S 4.6 3.2 3.0 5.8 3.0 1.6 1.8 >3.2 3.1 1.4 1.5 >3.2 4.65 19.81 14.12 – a The apparent volume change of association (DV) was determined by replotting the data in Fig according to (Eqn 3) and normalized by dividing by the number of subunits in the capsid (180) DV/n Changes in secondary structure upon dissociation and denaturation To further confirm the urea-induced changes in secondary structure, we analyzed the UV CD spectra of MS2 and the ts mutants in the presence and absence of 4.5 M urea, the concentration at which the greatest difference among the various samples was observed (Fig 6) CD spectra evidenced a great loss of secondary structure for M88V and T45S in urea In the absence of urea, the signal was smaller for the M88V mutant than for the other mutants and WT bacteriophage, although all experiments were carried out under identical conditions and virus concentrations (100 lgỈmL)1) However, the WT bacteriophage and the D11N mutant showed little change in structure, confirming their higher stabilities At higher urea concentrations, both WT bacteriophage and the D11N mutant lost the ellipticity at 218 nm, indicating complete denaturation (results not shown) the data of Fig (Eqn 3) M88V and T45S presented much larger changes in volume per unit of coat protein than the WT capsid A volume change for dissociation of D11N particles could not be determined because of the lack of change induced by pressure Bis-ANS binding assay of MS2 and ts mutants The fluorophore, bis-ANS, binds non-covalently to nonpolar segments in proteins, especially those in proximity to positive charges [36] Its binding is accompanied with a large increase in its fluorescence quantum yield and it has been Ó FEBS 2003 Cavities and stability in MS2 capsid mutants (Eur J Biochem 271) 141 Fig UV CD spectra of wild-type MS2 bacteriophage and the temperature sensitive (ts) mutants Conformational changes in the secondary structure of bacteriophage MS2 and ts mutants were analyzed in the presence of 4.5 M urea (hollow symbols) Filled symbols correspond to the samples in the absence of urea Wavelength range: 300–210 nm The samples of MS2 bacteriophage and ts mutants were diluted to a final concentration of 100 lgỈmL)1 and the spectra were measured in buffer (10 mM Tris/30 mM NaCl, pH 7.5) using a 0.1 cm pathlength quartz cuvette The data are representative of three experiments used to probe protein structural changes [37,38] At atmospheric pressure and in the absence of urea, the MS2 bacteriophage and D11N mutant did not bind bis-ANS (Fig 7B), showing that these particles not present exposed hydrophobic segments High urea concentrations did not promote significant binding of bis-ANS to any of the particles, with the exception of T45S, which bound a small amount of bis-ANS, increasing, by twofold, the emission of the probe (Fig 7B) M88V and WT particles treated with pressure did not show significant changes in bis-ANS binding However, when the T45S mutant was denatured and dissociated by pressure, a sixfold increase in the emission of the probe occurred (Fig 7A), suggesting that the conformation of the pressure-denatured state is different from that of the urea-denatured state Discussion In the last 20 years, the structure of many viruses has been solved by X-ray crystallography [1] However, despite extensive knowledge of their structures, the mechanisms of virus assembly and disassembly are still poorly understood In viruses, interactions within and between capsid subunits must be strong enough to ensure virus particle stability and protection of the genome, but weak enough to permit uncoating or release of the genome upon interaction with the cell The coat protein of the bacteriophage MS2 has two basic functions, (a) specific RNA binding for translational repression and genome encapsidation and (b) formation of the capsid structure Structural and genetic analysis permit the identification of amino acid residues involved in the protein–RNA and protein–protein interactions that mediate these functions [9,31,32,39–42] Analysis of the effects of substitutions of key amino acids should ultimately provide information about the roles they play in capsid assembly and disassembly In this work, we studied the effects of chemical (i.e urea) and physical (i.e high pressure) denaturing agents in the structure and stability of the MS2 bacteriophage, and of three ts mutants, with the aim to eventually understand the assembly and disassembly processes Our results allow us to infer how the introduction of a small cavity in the coat protein affects the whole stability of the virus particle However, local mutations also tend to affect the global conformation of the protein For the pressure sensitivity, modification in the dynamics of the protein, even with the average structure not affected, may result in changes in pressure stability [43] M88V showed a large decrease in stability when exposed to conditions of high pressure and high concentrations of urea Its diminished stability can probably be explained by the large potential of the substitution to create a cavity, as well to sterically interfere with side-chain packing in the protein’s interior Mutations that create cavities in hydrophobic environments generally cause proteins to become less stable [44,45] Using the program VMD [46] and a probe ˚ radius of 1.4 A, we analyzed the pdb coordinates of WT bacteriophage [14] and the M88V mutant (by substitution using the same program) for the existence of internal cavities in this region in the structure A significant cavity in the WT phage structure in the neighboring Met88 residue was identified After substitution of this residue with valine, the ˚ cavity volume increased to 43 A3, reflecting a reduction in ˚ the surface area of the residue (% 59 A2) and in the interactions occurring there (Fig 8) Met88 resides in the 142 S M B Lima et al (Eur J Biochem 271) Fig Binding of bis-8-anilinonaphthalene-1-sulfonate (bis-ANS) to dissociated and denatured capsids (A) bis-ANS binding to MS2 bacteriophage and mutants under pressure Inset: fluorescence emission spectra of bis-ANS during the pressurization process of the T45S mutant (B) bis-ANS binding to MS2 bacteriophage and mutants under conditions of increasing urea concentrations Structural changes were also analyzed by a fluorescent probe (bis-ANS) emission, at a final concentration of lM (d) MS2 bacteriophage and the ts mutants (m) T45S and (j) D11N The excitation wavelength was 360 nm and the emission wavelength range was 400–600 nm The data shown are representative of three experiments middle of the central b-strand of the coat protein dimer and its side-chain projects into the protein’s interior (Figs 1B and 8) The two Met88 residues are in close proximity to one another at the dimer’s twofold symmetry axis and thus interact with each other across the dimer interface Each also interacts with residues on the adjacent b-strand (F) and Ó FEBS 2003 with amino acids in the alpha-helical regions as they pass over the b-sheet Thus, the M88V mutant substitution affects the dimer interface, explaining its increased sensitivity to pressure (Dpẵ ẳ 1.4 kbar, Table 2) The increase in volume of 43 A3 corresponds to 26 mLỈmol)1 of coat subunit, which, by itself, could explain the difference (of % 15 mLỈmol)1) between the volumes measured for the M88V mutant and the WT phage (Table 2) In addition to the creation of a cavity, the substitution of Val for Met might produce steric clashes as a result of introduction of the beta branched side-chain where the unbranched Met side-chain is ordinarily packed The physical basis for the reduced stability of the T45S mutant may have a similar explanation Although residue 45 resides on the surface of the protein and makes no obvious stabilizing interactions with other amino acid sidechains, this part of the protein interacts with genomic RNA [33,41,47] Such interactions probably contribute to the stability of the virus particle (Fig 1B) The crystal structure of coat protein in complex with the translational operator shows interaction between Thr45 and RNA [41] The X-ray structure of the virus particle itself shows significant electron density in the vicinity of Thr45, indicating that many individual subunits apparently contact RNA at this position, albeit in a presumably non-specific manner [13,14] Thus, it is clear that the T45S substitution destabilizes the capsid because of a perturbation of the protein–RNA interaction, as assessed by the several criteria reported here The high sensitivity of coat protein folding/stability to Thr45 substitution was previously inferred from electrophoretic studies [41] Nineteen substitutions were introduced in position 45 and none of the amino acids was a completely acceptable replacement for threonine Every mutant showed loss of translational repression, increased insolubility and/or degradation, and failure to produce normal quantities of virus-like particles However, the T45S mutant was the most affected in the translational repressor activity [41] The T45S mutant was more sensitive to urea than to high pressure This sensitivity to urea treatment can be explained by the involvement of other forces, such as hydrogen bonds, in the interactions at the protein–RNA interface X-ray structural analysis suggests that the hydroxyl group of Thr45 makes H-bonds with N6 and N7 of A-4 [47] Moreover, the lack of binding of bis-ANS in this mutant, following treatment with urea, suggests that the dissociation and denaturation processes induced by urea and pressure occur in a distinct way More likely, urea provokes a general unfolding, not leaving any hydrophobic cleft for bis-ANS binding High pressure is well known to denature proteins to partially folded states [15,17,38,48–50] The large increase in bis-ANS binding when the T45S mutant was treated with pressure can also be explained by the presence of a cavity in the T45S mutant, which is released by dissociation, leading to binding of the dye The changes promoted by pressure were reversible for WT and all the ts mutants, whereas the effects of urea were irreversible Only the D11N mutant showed a residual titer after treatment with 4.5 M urea The UV CD spectra, under different conditions, provide further insights into the stability of the WT and mutant capsids The less stable mutants presented higher positive ellipticity peaks in the RNA region (270 nm), whereas the negative peak Ó FEBS 2003 Cavities and stability in MS2 capsid mutants (Eur J Biochem 271) 143 ˚ Fig Cavity increase occuring in the M88V mutation Using the program VMD and a probe radius of 1.4 A, we analyzed the pdb coordinates of wild-type (WT) bacteriophage and the M88V mutant for the existence of internal cavities in this region The figure shows the region on coat protein in the asymmetric unit of the capsid around residue 88 The methionine residue is shown in yellow and the valine is represented in green A significant cavity was identified in the WT phage structure (A and B) in the neighboring region of the Met88 residue After the substitution of this ˚ ˚ residue with valine (C and D), the cavity volume increased to 43 A3, reflecting a reduction in the surface area of the residue (% 59 A2) and in the interactions occurring there (corresponding to the b-sheet) is smaller An appealing interpretation for these findings is that the lower stability of the coat protein shell is counterbalanced by a more structured RNA, which results in a similar infectivity The increased stability of the D11N mutant presents a puzzle Like the others, this mutant was isolated for its ts growth phenotype, implying that it possesses decreased thermal stability Why then is the virus apparently more stable? The X-ray structure of MS2 shows that Asp11 participates in both intra- and interchain interactions It appears to form a salt bridge to Lys113 on the alpha-helix of the adjacent subunit of the dimer, and may thus help to stabilize the dimer interface (residues in red and blue colors in Fig 1B) But Asp11 makes another interaction It resides in b-strand A, just before the turn that connects it to bB Here it seems to position itself to the H-bond, through a carbonyl oxygen of its side-chain, to the main chain amide of Gly13 within the turn, thus possibly exerting a stabilizing influence on the turn The D11N substitution might be expected to have a destabilizing effect on both interactions, making it difficult to understand its stabilizing effect on the virus particle Thus, the stabilization produced by the D11N substitution is probably associated with a change in the overall conformation propagated from the local replacement One can envisage that the salt-bridge in the WT capsid protein locks the helix in a conformation leading to cavities in the interior of the hydrophobic core King and colleagues [51,52] characterized a large collection of ts mutants of the phage P22 tailspike protein Many of these are so-called temperature-sensitive for folding or tsf mutations They have the property of preventing folding at the non-permissive temperature without altering appreciably the stability of the native state, once formed A disproportionate fraction of these mutations affect residues at or near b-turns, suggesting that the proper formation of such turns represents a crucial step in the folding process Substitutions in positions near b-turns may destabilize important folding intermediates, rendering them aggregation-prone D11N is apparently in this category All the mutants were subjected to a test of their stability to assemble into virus-like particles, which is an activity sensitive to folding defects As virus-like particles have a characteristic electrophoretic behavior, this provides a simple means of assessing a mutant for acquisition of native protein structure (D S Peabody, unpublished data) Therefore, the changes in the stability observed here are not the result of defects in the assembly pathway In conclusion, our studies show the high sensitivity of single amino-acid substitutions in the coat protein of small RNA viruses Lower stability was correlated with a local increase in the cavity Interaction with RNA is also sensitive, and its perturbation (in the case of T45S replacement) also leads to lower stability On the other hand, capsid proteins cannot be highly packed otherwise they would lose the flexibility needed for virus assembly In this context, the intricate interactions between capsid protein and RNA are 144 S M B Lima et al (Eur J Biochem 271) crucial for assembling the whole particle It is also interesting that the mutants had a ts phenotype, which means a high sensitivity to both high and low temperatures The presence of cavities that confer the decreased stability to pressure is also usually related to both decreased stability to low and high temperatures [15,17,18] Acknowledgements We gratefully acknowledge Alan Witer Sousa da Silva from Labo´ ´ ratorio de Fı´ sica Biologica at Instituto de Biofı´ sica Carlos Chagas Filho/UFRJ for help with the VMD program, Emerson Goncalves for ¸ competent technical assistance, and Cristiane Dinis Ano Bom and ´ Professors Fabio 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 Howard Hughes Medical Institute to J.L.S and by grants from Programa de Nucleos de Excelencia (PRONEX), Conselho Nacional de Desenvolvi´ ˆ ´ mento Cientı´ fico e Tecnologico (CNPq), Fundacao de Amparo a ¸ ˜ Pesquisa Estado Rio de Janeiro (FAPERJ), Fundacao ¸ ˜ ´ ´ ´ Universitaria Jose Bonifacio (FUJB) of Brazil to J.L.S and A.C.O., and by a grant from the National Institutes of Health (NIH) to D.S.P 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illuminate the role of packing in the icosahedral lattice of the virus... middle of the central b-strand of the coat protein dimer and its side-chain projects into the protein’s interior (Figs 1B and 8) The two Met88 residues are in close proximity to one another at the. .. with valine (C and D), the cavity volume increased to 43 A3, reflecting a reduction in the surface area of the residue (% 59 A2) and in the interactions occurring there (corresponding to the b-sheet)