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