VIROLOGY
76,
664-669
(1977)
Self-Assembly
of Eggplant
J. P. BRIAND, Laboratoire
de Virologie,
Znstitut
Mosaic Virus Protein
J. P. BOULEY,
de Biologie Molhdaire Strasbourg-CCdex, Accepted
et Celluhire France
September
AND
J. WITZ’ du CNRS,
15, rue Descartes,
F67064
16,1976
Purified bottom component of eggplant mosaic virus (EMV) was dissociated in cold 66% acetic acid in the presence of 0.01 M dithiothreitol (DTT). EMV coat protein free from residual RNA could be obtained by dialysis against 0.02 M sodium acetate buffer, 0.001 M DTT, pH 4.0. The aggregation states of this protein were investigated after dialysis against 0.02 M sodium acetate buffers, 0.001 M DTT, pH 4.0 to 5.5. At pH 4.0, the protein reassembled essentially into threads and small aggregates sedimenting at about 8-9 S. Increasing numbers of isometric shells formed with increasing pH. At pH 5.5, they represented the totality of soluble material and sedimented at about 50 S. Once formed, these shells were stable at pH 7.0, unlike the aggregates formed at pH 4.0, which precipitated if dialyzed directly to pH 7. Conditions required for assembly and some possible implications for the assembly mechanism are discussed.
analytical ultracentrifugation in a Spinco Model E ultracentrifuge. Dissociation of G-ions. To dissociate protein subunits and RNA, 1 vol of purified bottom component at a concentration of about 25 mg/ml in 0.02 M sodium phosphate buffer, 0.03 M dithiothreitol (DTT), pH 7, was mixed with 2 vol of cold acetic acid, and the mixture was shaken for 15 min in an ice bath. Precipitated RNA was removed by low-speed centrifugation. The clear supernatant was dialyzed in the cold for 2-4 days against two changes of a lOOfold excess of 0.02 M sodium acetate buffer, pH 4.0, containing 0.001 M DTT. Residual virus was removed by sedimentation at 225,000g for 1 hr in a Beckman 60 Ti rotor. The protein solution was concentrated fiveto sevenfold by ultrafiltration through an Amicon UM-10 membrane. Optical densities at 260 and 280 nm were measured in a Zeiss PMQII spectrophotometer, and uv absorption spectra were recorded with a Beckman Acta III spectrophotometer. Approximate protein concentrations were determined by assuming E = 1.0 cm2/mg at 280 nm. Aggregation states of EMV coat protein. To study the aggregation states of EMV
INTRODUCTION
It would be of major interest to know how viruses stabilized by very strong protein-protein interactions assemble (Kaper, 1975; Jonard et al., 1976). These include tymoviruses, those related to turnip yellow mosaic virus (TYMV). However, attempts to reassemble TYMV or its empty protein shell have not yet been successful. This paper reports results obtained with another tymovirus, eggplant mosaic virus (EMV), which is more sensitive than TYMV to high concentrations of urea and to extreme pH’s (Bouley, 1975). We describe the aggregation states of EMV protein and the reassembly of isometric shells. MATERIALS
AND
METHODS
Virus. EMV was propagated and purified as described elsewhere (Bouley et al., 1976). The bottom and top components were fractionated by successive, short, high-speed centrifugations (Klug et al., 1966). Fractionated bottom component contained less than 2% top, according to ’ Author dressed.
to whom
reprint
requests
should
be ad-
664 Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
EMV
PROTEIN
protein, we dialyzed aliquots of a stock solution at a concentration of about 4-5 mg/ml in 0.02 M sodium acetate buffer, pH 4.0, containing 0.001 M DTT, overnight at room temperature against 0.02 M glycineHCl buffers (2.5 G pH G 3.7) or 0.02 M sodium acetate buffers (4.0 G pH G 5.6), all containing 0.001 M DTT. Samples were examined by analytical ultracentrifugation in a Spinco Model E equipped with schlieren optics and by electron microscopy in a Siemens Elmiskop 101 microscope working at 60 or 80 kV and a nominal magnification of 40,000 or 60,000. l?or electron microscopy, samples were diluted to about 0.1-0.2 mg/ml and negatively stained with 1% uranyl acetate. Aliquots of the samples were also fixed overnight at room temperature with 2% glutaraldehyde prior to dilution and staining. RESULTS
Properties
of EMV
Protein
at pH 4.0
The uv absorption spectrum of the protein obtained at pH 4.0 after filtration through an Amicon UM-10 membrane was characterized by a maximum at 276 nm, a minimum at 250 nm, an OD,,,/OD,i, ratio of 2.0 to 2.3, and a ratio r = OD,,,,,/ OD280nm of 0.60 to 0.70. These values suggest a very low contamination by RNA. The solubility of this protein was tested by dialysis of aliquots of a stock solution at 4-6 mg/ml against a variety of buffer and salt systems containing 0.001 M DTT. All the protein precipitated at 2.5 G pH < 4.0 in 0.02 M glycine-HCl buffers containing 0.1 M NaCl and at PI-I’S > 4.0 in 0.05 M sodium acetate buffers. Aggregation
States of EMV
Protein
Figure 1 shows analytical sedimentation patterns and electron micrographs of EMV protein after overnight dialysis against sodium acetate buffers of pH 4.0-5.6 and of reassembled shells after overnight dialysis at pH 7.0. At pH 4.0 and below, the protein sedimented at about 8-9 S (Fig. la), and many threads could be seen in the electron micrographs, together with smaller aggregates and, at pH 4.0, a few partly assembled shells (Fig. 1A). In some prepara-
AGGREGATION
665
tions, small amounts of a faster-moving component, sedimenting at about 12-14 S, were also present. From pH 4.25 to 5.25, the schlieren patterns contained two peaks, sedimenting at about 14-18 and 3540 S, respectively (Fig. lb-d). The relative amount of the latter increased noticeably with increasing pH. On the corresponding electron micrographs (Figs. 1B and D and 21, more and more empty shells, many ill shaped, could be seen in addition to threads. We noticed that, during fixation with glutaraldehyde, the threads (14 S component) aggregated and precipitated at pH’s ~4.75, whereas they remained soluble at pH 4.0. Figures 2A and B are electron micrographs obtained at pH 5.0, without and after fixation with glutaraldehyde, respectively. The schlieren pattern in Fig. lc shows that the 12-14 S component represents about 50% of the material present in that sample. Threads may be seen in Fig. 2A but are absent from Fig. 2B. At pH 5.25, shells sedimenting at about 46-50 S are the predominant aggregate (Fig. Id). At pH 5.5, no other component could be detected in the ultracentrifuge (Fig. le). In electron micrographs, these shells were similar to EMV top component (Fig. 2b in Bouley et al., 1976). The same results were also obtained when dialysis proceeded to pH 5.25 or 5.5 via a step at pH 4.75. We examined the stability of protein shells formed at pH 5.25 or 5.5 under various conditions of pH and ionic strength. Analytical ultracentrifugation showed that they dissociated again when dialyzed back against lower-pH buffers. But unlike the dissociated protein, this material only partially precipitated, about 30%, when aggregates formed at pH 5.25 or 5.5 were dialyzed against 0.02 M sodium phosphate buffer, pH 7, containing 0.001 M DTT. Figures If and g show the presence of only one fairly narrow peak sedimenting at 55 S; any material not aggregated into shells at pH 5.25 precipitated during dialysis to pH 7. Electron micrographs showed a majority of well-formed isometric shells (Fig. 1F) and only a few partly assembled (or disrupted) capsids. At pH 7, the 55 S aggre-
666
.----
BRIAND,
BOULEY
AND WITZ
.^ _--..
1. EMV protein aggregation states. Sedimentation patterns (a to g) and electron micrographs (A, B, D, and F) of EMV protein at pH 4.0, dialyzed overnight at room temperature against 0.02 M sodium acetate buffers, 0.001 M DTT. (a,A), pH 4.0; (b,B), pH 4.75; (c), pH 5.0; (d,D), pH 5.25; (e), pH 5.5; (f,F), sample d,D after further overnight dialysis against 0.02 M sodium phosphate buffer, 0.001 M DTT, pH ‘7; (g), sample e after dialysis similar to that for f. Schlieren photographs were taken about 15 to 20 min after reaching 35,600 rpm. Sedimentation, from left to right; phase angle, 55”. All dialysis bags initially (at pH 4.0) contained EMV protein at the same concentration, about 5 mg/ml. For electron microscopy, samples were diluted to about 0.1 mg/ml with the corresponding buffer. Samples A and F were fixed overnight with 2% glutaraldehyde. Negative staining was with 1% uranyl acetate. The bar corresponds to 100 nm. FIG.
gate ,remained soluble at least up to 2 M NaCl. Some Conditions Required for Reassociation of EMV Protein In systematically studying good reassociation conditions, we made several observations. Some of them are still not understood.
Uncontaminated protein could be prepared only from bottom component fractionated as described in Materials and Methods. Top component, although containing only about 2% RNA (Bouley et al., 1976), yielded presumably contaminated protein with an r = 1.0, which gave only minute amounts of 50-55 S aggregates, whereas the majority of the material pre-
EMV
PROTEIN
cipitated. In our earliest experiments, we dissociated purified EMV suspensions containing both top and bottom components (i.e., about lo-20% top). Essentially the same patterns as those described for fractionated virus were observed, aside from a higher r value for the protein. But at pH 4.0-4.5, a second aggregate, sedimenting at 28-32 S, formed in addition to the one at 12-14 S (compare Figs. la and 3a); some longer threads and a very few shells could be seen in electron micrographs (compare Figs. 1A and 3A). Furthermore the 45-50 S component obtained at pH 5.0 (Fig. 3b) seemed more heterogeneous and was present in smaller amounts than that shown in Fig. lc. The presence of some contaminated protein from dissociated top component may change the aggregation state
FIG. 2. Electron micrographs glutaraldehyde prior to staining. corresponds to 100 nm.
of sample c of Fig. All other conditions
667
AGGREGATION
and partially inhibit the reassociation of EMV protein into spherical shells. EMV bottom and top components can easily be separated by isopycnic CsCl gradient centrifugation (Bouley et al., 1976). However, virions purified in that way dissociated into presumably contaminated protein (r = 1.0) which did not aggregate but, rather, precipitated at pH ~5.0. Residual RNA could be removed from the protein by chromatography on a DEAESephadex column equilibrated with 0.02 M sodium acetate buffer, pH 4.0, containing 8 M urea and 0.001 M D’IT; but this purified protein did not aggregate either. This action of CsCl very likely reflects an effect of high salt concentrations on EMV, which is presently under investigation. Even when EMV bottom component was
1 (pH 5.0): (A), unfixed; (B), after fixation with were the same as those given in Fig. 1. The
FIG. 3. Schlieren pattern and electron microscopy of EMV protein aggregates and 5.0 (b) from protein prepared by the dissociation of a purified but unfractionated taining both top and bottom components. Arrows point to the longer threads seen photograph was taken about 15 min after the rotor speed reached 47,660 rpm. conditions were the same as those given for Fig. IA. The bar corresponds to 100
2% bar
obtained at pH 4.5 (a,A) EMV suspension conat pH 4.5. The schlieren Phase angle, 50”. Other nm.
668
BRIAND,
BOULEY
fractionated as described in Materials and Methods, a high degree of reassociation could be obtained only if the dissociation mixture in 66% acetic acid was dialyzed for at least 48 hr against 0.02 M buffer, pH 4.0. Shorter dialysis yielded protein which did not aggregate to 30-55 S components, even if the pH 4.0 buffer was changed two or three times within 36 hr. EMV protein apparently undergoes a slow conformational change at pH 4.0 before it is able to reassociate into shells. DISCUSSION
These results show that a virus related to TYMV can be dissociated into its components, and its protein can be reassociated in vitro into isometric shells in the absence of RNA. These shells are not stable at acidic pH’s, unlike EMV top component, which contains about 2% low molecular weight RNA (Bouley et al., 1976). It is not yet known if this lack of stability is due to the absence of RNA or to partially defective assembly. Reassembly conditions of EMV protein described in this work seem fairly unphysiological. All but shell-like protein aggregates are soluble only at pH’s ~5.25 and very low ionic strength. A detailed analysis of the process of shell formation will be necessary to determine the nature of ionizable groups involved in self-assembly and the contribution of “hydrophobic” intersubunit interactions, which are known to be very important in the stability of the related virus TYMV (Jonard et al., 1976). Examination of many electron micrographs showed that fewer ill-shaped shells could be seen at pH 7.0 than at pH 5.2 or 5.5. The solubility of well-assembled capsids at neutral pH and any ionic strength is a further indication that the most hydrophobic side chains of the coat protein are buried during “good” assembly. The steady increase of the sedimentation coefficient of the faster-moving peak in schlieren patterns (from about 35 to about 55 S) also indicates the better homogeneity and tighter architecture of reassembled shells at increasing pH’s. The morphology of the 12-14 S threads has not yet been determined. At pH’s
AND
WIT2
24.75 they precipitated during fixation with glutaraldehyde, whereas electron micrographs of unfixed samples (Figs. 1D and 2A) showed the presence, in addition to shells and threads, of some denatured protein which greatly reduced the contrast of the images. The presence of protein threads in reassembly experiments has already been reported for bromegrass mosaic virus protein (Pfeiffer and Hirth, 1975) and for small bacteriophage coat protein (reviewed by Hohn and Hohn, 1970). But in the first case at least they proved to be irreversible aggregates. Our results indicate that EMV protein threads may be direct precursors of isometric shells. This is suggested by: (i) comparison of the heights of the 18 and 35-50 S peaks observed at pH’s increasing from 4.0 to 5.5 and (ii) the very similar results obtained if the protein at pH 4.0 is dialyzed first against a buffer of pH 4.75 and then against a buffer of pH 5.25 or 5.5. It cannot yet be excluded that threads act as donors of smaller aggregates which would then reassemble into shells, a role which has been suggested for the double disk of tobacco mosaic virus protein in the in vitro elongation of tobacco mosaic virus (Richards and Williams, 1972). But if it is confirmed that threads are direct precursors of EMV protein shells, such a self-assembly mechanism would raise interesting problems concerning the validity of Caspar and Klug’s quasi-equivalence theory for the morphogenesis of isometric viruses and not only for their architecture (Caspar and Klug, 1962). The number of ill-shaped shells seen in electron micrographs such as Figs. 1B and D provides good evidence that the presence of threads and other structures seen at low pH’s does not automatically lead to the self-assembly of perfect isometric shells. The precipitation of the protein if a sample at pH 4.0 is dialyzed directly against a pH 7.0 buffer indicates that, whatever the mechanism of self-assembly, the formation of isometric shells is a slow process in comparison to the time required to change the pH in the dialysis bag, i.e., about 10 to 20 min.
EMV
Work is now in progress to the isometric shells described teract with EMV-RNA and if cursors of EMV top or bottom
PROTEIN
determine if here can inthey are precomponent.
ACKNOWLEDGMENTS We thank Professor L. Hirth for stimulating discussions, Dr. R. Milne for having taken some of the electron micrographs, Mr. G. de Marcillac for his skillful assistance in the analytical ultracentrifugation experiments, and Miss S. Miller for her help in preparing the manuscript. REFERENCES BOULEY, J. P. (1975). “Interactions RNA-Proteine dans le Virus de la Mosaique de l’Aubergine.” Thesis, Universitd Louis Pasteur, Strasbourg. BOULEY, J. P., BRIAND, J. P., GENEVAUX, M., PINCK, M., and WITZ, J. (1976). The structure of eggplant mosaic virus: Evidence for the presence of low molecular weight RNA in top component. Virology 69, 775-781. CASPAR, D. L. D., and KLUG, A. (1962). The physical
AGGREGATION
669
principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, l-23. HOHN, T., and HOHN, B. (1970). Structure and assembly of simple RNA bacteriophages. Aduan. Virus Res. 16, 43-78. JONARD, G., BRIAND, J. P., BOULEY, J. P., WITZ, J., and HIRTH, L. (1976). Nature and specificity of the RNA-protein interactions in the case of the tymoviruses. Phil. Trans. Roy. Sot. London B. 276, 123-129. KAPER, J. M. (197.5). “The Chemical Basis of Virus Structure, Dissociation and Reassociation,” pp. 273-320. North-Holland, Amsterdam. KLUG, A., LONGLEY, W., and LEBERMAN, R. (1966). Arrangement of protein subunits and the distribution of nucleic acid in turnip yellow mosaic virus. I. X-ray diffraction studies. J. Mol. Biol. 15, 315-343. PFEIFFER, P., and HIRTH, L. (1975). Aggregation states of bromegrass mosaic virus protein. Virology 61, 160-167. RICHARDS, K. E., and WILLIAMS, R. C. (1972). Assembly of tobacco mosaic virus in vitro: Effect of state of polymerisation of the protein component. Proc. Nat. Acad. Sci. USA 69, 1121-1124.
76,
664-669
(1977)
Self-Assembly
of Eggplant
J. P. BRIAND, Laboratoire
de Virologie,
Znstitut
Mosaic Virus Protein
J. P. BOULEY,
de Biologie Molhdaire Strasbourg-CCdex, Accepted
et Celluhire France
September
AND
J. WITZ’ du CNRS,
15, rue Descartes,
F67064
16,1976
Purified bottom component of eggplant mosaic virus (EMV) was dissociated in cold 66% acetic acid in the presence of 0.01 M dithiothreitol (DTT). EMV coat protein free from residual RNA could be obtained by dialysis against 0.02 M sodium acetate buffer, 0.001 M DTT, pH 4.0. The aggregation states of this protein were investigated after dialysis against 0.02 M sodium acetate buffers, 0.001 M DTT, pH 4.0 to 5.5. At pH 4.0, the protein reassembled essentially into threads and small aggregates sedimenting at about 8-9 S. Increasing numbers of isometric shells formed with increasing pH. At pH 5.5, they represented the totality of soluble material and sedimented at about 50 S. Once formed, these shells were stable at pH 7.0, unlike the aggregates formed at pH 4.0, which precipitated if dialyzed directly to pH 7. Conditions required for assembly and some possible implications for the assembly mechanism are discussed.
analytical ultracentrifugation in a Spinco Model E ultracentrifuge. Dissociation of G-ions. To dissociate protein subunits and RNA, 1 vol of purified bottom component at a concentration of about 25 mg/ml in 0.02 M sodium phosphate buffer, 0.03 M dithiothreitol (DTT), pH 7, was mixed with 2 vol of cold acetic acid, and the mixture was shaken for 15 min in an ice bath. Precipitated RNA was removed by low-speed centrifugation. The clear supernatant was dialyzed in the cold for 2-4 days against two changes of a lOOfold excess of 0.02 M sodium acetate buffer, pH 4.0, containing 0.001 M DTT. Residual virus was removed by sedimentation at 225,000g for 1 hr in a Beckman 60 Ti rotor. The protein solution was concentrated fiveto sevenfold by ultrafiltration through an Amicon UM-10 membrane. Optical densities at 260 and 280 nm were measured in a Zeiss PMQII spectrophotometer, and uv absorption spectra were recorded with a Beckman Acta III spectrophotometer. Approximate protein concentrations were determined by assuming E = 1.0 cm2/mg at 280 nm. Aggregation states of EMV coat protein. To study the aggregation states of EMV
INTRODUCTION
It would be of major interest to know how viruses stabilized by very strong protein-protein interactions assemble (Kaper, 1975; Jonard et al., 1976). These include tymoviruses, those related to turnip yellow mosaic virus (TYMV). However, attempts to reassemble TYMV or its empty protein shell have not yet been successful. This paper reports results obtained with another tymovirus, eggplant mosaic virus (EMV), which is more sensitive than TYMV to high concentrations of urea and to extreme pH’s (Bouley, 1975). We describe the aggregation states of EMV protein and the reassembly of isometric shells. MATERIALS
AND
METHODS
Virus. EMV was propagated and purified as described elsewhere (Bouley et al., 1976). The bottom and top components were fractionated by successive, short, high-speed centrifugations (Klug et al., 1966). Fractionated bottom component contained less than 2% top, according to ’ Author dressed.
to whom
reprint
requests
should
be ad-
664 Copyright All rights
8 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
EMV
PROTEIN
protein, we dialyzed aliquots of a stock solution at a concentration of about 4-5 mg/ml in 0.02 M sodium acetate buffer, pH 4.0, containing 0.001 M DTT, overnight at room temperature against 0.02 M glycineHCl buffers (2.5 G pH G 3.7) or 0.02 M sodium acetate buffers (4.0 G pH G 5.6), all containing 0.001 M DTT. Samples were examined by analytical ultracentrifugation in a Spinco Model E equipped with schlieren optics and by electron microscopy in a Siemens Elmiskop 101 microscope working at 60 or 80 kV and a nominal magnification of 40,000 or 60,000. l?or electron microscopy, samples were diluted to about 0.1-0.2 mg/ml and negatively stained with 1% uranyl acetate. Aliquots of the samples were also fixed overnight at room temperature with 2% glutaraldehyde prior to dilution and staining. RESULTS
Properties
of EMV
Protein
at pH 4.0
The uv absorption spectrum of the protein obtained at pH 4.0 after filtration through an Amicon UM-10 membrane was characterized by a maximum at 276 nm, a minimum at 250 nm, an OD,,,/OD,i, ratio of 2.0 to 2.3, and a ratio r = OD,,,,,/ OD280nm of 0.60 to 0.70. These values suggest a very low contamination by RNA. The solubility of this protein was tested by dialysis of aliquots of a stock solution at 4-6 mg/ml against a variety of buffer and salt systems containing 0.001 M DTT. All the protein precipitated at 2.5 G pH < 4.0 in 0.02 M glycine-HCl buffers containing 0.1 M NaCl and at PI-I’S > 4.0 in 0.05 M sodium acetate buffers. Aggregation
States of EMV
Protein
Figure 1 shows analytical sedimentation patterns and electron micrographs of EMV protein after overnight dialysis against sodium acetate buffers of pH 4.0-5.6 and of reassembled shells after overnight dialysis at pH 7.0. At pH 4.0 and below, the protein sedimented at about 8-9 S (Fig. la), and many threads could be seen in the electron micrographs, together with smaller aggregates and, at pH 4.0, a few partly assembled shells (Fig. 1A). In some prepara-
AGGREGATION
665
tions, small amounts of a faster-moving component, sedimenting at about 12-14 S, were also present. From pH 4.25 to 5.25, the schlieren patterns contained two peaks, sedimenting at about 14-18 and 3540 S, respectively (Fig. lb-d). The relative amount of the latter increased noticeably with increasing pH. On the corresponding electron micrographs (Figs. 1B and D and 21, more and more empty shells, many ill shaped, could be seen in addition to threads. We noticed that, during fixation with glutaraldehyde, the threads (14 S component) aggregated and precipitated at pH’s ~4.75, whereas they remained soluble at pH 4.0. Figures 2A and B are electron micrographs obtained at pH 5.0, without and after fixation with glutaraldehyde, respectively. The schlieren pattern in Fig. lc shows that the 12-14 S component represents about 50% of the material present in that sample. Threads may be seen in Fig. 2A but are absent from Fig. 2B. At pH 5.25, shells sedimenting at about 46-50 S are the predominant aggregate (Fig. Id). At pH 5.5, no other component could be detected in the ultracentrifuge (Fig. le). In electron micrographs, these shells were similar to EMV top component (Fig. 2b in Bouley et al., 1976). The same results were also obtained when dialysis proceeded to pH 5.25 or 5.5 via a step at pH 4.75. We examined the stability of protein shells formed at pH 5.25 or 5.5 under various conditions of pH and ionic strength. Analytical ultracentrifugation showed that they dissociated again when dialyzed back against lower-pH buffers. But unlike the dissociated protein, this material only partially precipitated, about 30%, when aggregates formed at pH 5.25 or 5.5 were dialyzed against 0.02 M sodium phosphate buffer, pH 7, containing 0.001 M DTT. Figures If and g show the presence of only one fairly narrow peak sedimenting at 55 S; any material not aggregated into shells at pH 5.25 precipitated during dialysis to pH 7. Electron micrographs showed a majority of well-formed isometric shells (Fig. 1F) and only a few partly assembled (or disrupted) capsids. At pH 7, the 55 S aggre-
666
.----
BRIAND,
BOULEY
AND WITZ
.^ _--..
1. EMV protein aggregation states. Sedimentation patterns (a to g) and electron micrographs (A, B, D, and F) of EMV protein at pH 4.0, dialyzed overnight at room temperature against 0.02 M sodium acetate buffers, 0.001 M DTT. (a,A), pH 4.0; (b,B), pH 4.75; (c), pH 5.0; (d,D), pH 5.25; (e), pH 5.5; (f,F), sample d,D after further overnight dialysis against 0.02 M sodium phosphate buffer, 0.001 M DTT, pH ‘7; (g), sample e after dialysis similar to that for f. Schlieren photographs were taken about 15 to 20 min after reaching 35,600 rpm. Sedimentation, from left to right; phase angle, 55”. All dialysis bags initially (at pH 4.0) contained EMV protein at the same concentration, about 5 mg/ml. For electron microscopy, samples were diluted to about 0.1 mg/ml with the corresponding buffer. Samples A and F were fixed overnight with 2% glutaraldehyde. Negative staining was with 1% uranyl acetate. The bar corresponds to 100 nm. FIG.
gate ,remained soluble at least up to 2 M NaCl. Some Conditions Required for Reassociation of EMV Protein In systematically studying good reassociation conditions, we made several observations. Some of them are still not understood.
Uncontaminated protein could be prepared only from bottom component fractionated as described in Materials and Methods. Top component, although containing only about 2% RNA (Bouley et al., 1976), yielded presumably contaminated protein with an r = 1.0, which gave only minute amounts of 50-55 S aggregates, whereas the majority of the material pre-
EMV
PROTEIN
cipitated. In our earliest experiments, we dissociated purified EMV suspensions containing both top and bottom components (i.e., about lo-20% top). Essentially the same patterns as those described for fractionated virus were observed, aside from a higher r value for the protein. But at pH 4.0-4.5, a second aggregate, sedimenting at 28-32 S, formed in addition to the one at 12-14 S (compare Figs. la and 3a); some longer threads and a very few shells could be seen in electron micrographs (compare Figs. 1A and 3A). Furthermore the 45-50 S component obtained at pH 5.0 (Fig. 3b) seemed more heterogeneous and was present in smaller amounts than that shown in Fig. lc. The presence of some contaminated protein from dissociated top component may change the aggregation state
FIG. 2. Electron micrographs glutaraldehyde prior to staining. corresponds to 100 nm.
of sample c of Fig. All other conditions
667
AGGREGATION
and partially inhibit the reassociation of EMV protein into spherical shells. EMV bottom and top components can easily be separated by isopycnic CsCl gradient centrifugation (Bouley et al., 1976). However, virions purified in that way dissociated into presumably contaminated protein (r = 1.0) which did not aggregate but, rather, precipitated at pH ~5.0. Residual RNA could be removed from the protein by chromatography on a DEAESephadex column equilibrated with 0.02 M sodium acetate buffer, pH 4.0, containing 8 M urea and 0.001 M D’IT; but this purified protein did not aggregate either. This action of CsCl very likely reflects an effect of high salt concentrations on EMV, which is presently under investigation. Even when EMV bottom component was
1 (pH 5.0): (A), unfixed; (B), after fixation with were the same as those given in Fig. 1. The
FIG. 3. Schlieren pattern and electron microscopy of EMV protein aggregates and 5.0 (b) from protein prepared by the dissociation of a purified but unfractionated taining both top and bottom components. Arrows point to the longer threads seen photograph was taken about 15 min after the rotor speed reached 47,660 rpm. conditions were the same as those given for Fig. IA. The bar corresponds to 100
2% bar
obtained at pH 4.5 (a,A) EMV suspension conat pH 4.5. The schlieren Phase angle, 50”. Other nm.
668
BRIAND,
BOULEY
fractionated as described in Materials and Methods, a high degree of reassociation could be obtained only if the dissociation mixture in 66% acetic acid was dialyzed for at least 48 hr against 0.02 M buffer, pH 4.0. Shorter dialysis yielded protein which did not aggregate to 30-55 S components, even if the pH 4.0 buffer was changed two or three times within 36 hr. EMV protein apparently undergoes a slow conformational change at pH 4.0 before it is able to reassociate into shells. DISCUSSION
These results show that a virus related to TYMV can be dissociated into its components, and its protein can be reassociated in vitro into isometric shells in the absence of RNA. These shells are not stable at acidic pH’s, unlike EMV top component, which contains about 2% low molecular weight RNA (Bouley et al., 1976). It is not yet known if this lack of stability is due to the absence of RNA or to partially defective assembly. Reassembly conditions of EMV protein described in this work seem fairly unphysiological. All but shell-like protein aggregates are soluble only at pH’s ~5.25 and very low ionic strength. A detailed analysis of the process of shell formation will be necessary to determine the nature of ionizable groups involved in self-assembly and the contribution of “hydrophobic” intersubunit interactions, which are known to be very important in the stability of the related virus TYMV (Jonard et al., 1976). Examination of many electron micrographs showed that fewer ill-shaped shells could be seen at pH 7.0 than at pH 5.2 or 5.5. The solubility of well-assembled capsids at neutral pH and any ionic strength is a further indication that the most hydrophobic side chains of the coat protein are buried during “good” assembly. The steady increase of the sedimentation coefficient of the faster-moving peak in schlieren patterns (from about 35 to about 55 S) also indicates the better homogeneity and tighter architecture of reassembled shells at increasing pH’s. The morphology of the 12-14 S threads has not yet been determined. At pH’s
AND
WIT2
24.75 they precipitated during fixation with glutaraldehyde, whereas electron micrographs of unfixed samples (Figs. 1D and 2A) showed the presence, in addition to shells and threads, of some denatured protein which greatly reduced the contrast of the images. The presence of protein threads in reassembly experiments has already been reported for bromegrass mosaic virus protein (Pfeiffer and Hirth, 1975) and for small bacteriophage coat protein (reviewed by Hohn and Hohn, 1970). But in the first case at least they proved to be irreversible aggregates. Our results indicate that EMV protein threads may be direct precursors of isometric shells. This is suggested by: (i) comparison of the heights of the 18 and 35-50 S peaks observed at pH’s increasing from 4.0 to 5.5 and (ii) the very similar results obtained if the protein at pH 4.0 is dialyzed first against a buffer of pH 4.75 and then against a buffer of pH 5.25 or 5.5. It cannot yet be excluded that threads act as donors of smaller aggregates which would then reassemble into shells, a role which has been suggested for the double disk of tobacco mosaic virus protein in the in vitro elongation of tobacco mosaic virus (Richards and Williams, 1972). But if it is confirmed that threads are direct precursors of EMV protein shells, such a self-assembly mechanism would raise interesting problems concerning the validity of Caspar and Klug’s quasi-equivalence theory for the morphogenesis of isometric viruses and not only for their architecture (Caspar and Klug, 1962). The number of ill-shaped shells seen in electron micrographs such as Figs. 1B and D provides good evidence that the presence of threads and other structures seen at low pH’s does not automatically lead to the self-assembly of perfect isometric shells. The precipitation of the protein if a sample at pH 4.0 is dialyzed directly against a pH 7.0 buffer indicates that, whatever the mechanism of self-assembly, the formation of isometric shells is a slow process in comparison to the time required to change the pH in the dialysis bag, i.e., about 10 to 20 min.
EMV
Work is now in progress to the isometric shells described teract with EMV-RNA and if cursors of EMV top or bottom
PROTEIN
determine if here can inthey are precomponent.
ACKNOWLEDGMENTS We thank Professor L. Hirth for stimulating discussions, Dr. R. Milne for having taken some of the electron micrographs, Mr. G. de Marcillac for his skillful assistance in the analytical ultracentrifugation experiments, and Miss S. Miller for her help in preparing the manuscript. REFERENCES BOULEY, J. P. (1975). “Interactions RNA-Proteine dans le Virus de la Mosaique de l’Aubergine.” Thesis, Universitd Louis Pasteur, Strasbourg. BOULEY, J. P., BRIAND, J. P., GENEVAUX, M., PINCK, M., and WITZ, J. (1976). The structure of eggplant mosaic virus: Evidence for the presence of low molecular weight RNA in top component. Virology 69, 775-781. CASPAR, D. L. D., and KLUG, A. (1962). The physical
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principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, l-23. HOHN, T., and HOHN, B. (1970). Structure and assembly of simple RNA bacteriophages. Aduan. Virus Res. 16, 43-78. JONARD, G., BRIAND, J. P., BOULEY, J. P., WITZ, J., and HIRTH, L. (1976). Nature and specificity of the RNA-protein interactions in the case of the tymoviruses. Phil. Trans. Roy. Sot. London B. 276, 123-129. KAPER, J. M. (197.5). “The Chemical Basis of Virus Structure, Dissociation and Reassociation,” pp. 273-320. North-Holland, Amsterdam. KLUG, A., LONGLEY, W., and LEBERMAN, R. (1966). Arrangement of protein subunits and the distribution of nucleic acid in turnip yellow mosaic virus. I. X-ray diffraction studies. J. Mol. Biol. 15, 315-343. PFEIFFER, P., and HIRTH, L. (1975). Aggregation states of bromegrass mosaic virus protein. Virology 61, 160-167. RICHARDS, K. E., and WILLIAMS, R. C. (1972). Assembly of tobacco mosaic virus in vitro: Effect of state of polymerisation of the protein component. Proc. Nat. Acad. Sci. USA 69, 1121-1124.
