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Structure of rotaviruses as studied by the freeze-drying technique
Structure of Rotaviruses as Studied by the Freeze-Drying Technique
AL~ERTO ROSETO,* JACQUES ESCAlG,f ~TIENNE DELAIN,S JEAN C~H~~,~ AND RAOUL SCHERRER§
The ~ee~~~ng technique was used to study the rno~holo~ of s~ngl~shel~ad and double-shelled particles of human, bovine, and simian rotaviruses. Analysis of the inner capsid suggests the existence of 132 capsomeres, with a skewed and icosahedral pattern characteristic of 7’ = 13. Double-shelled particles exhibit a smooth surface perforated by small holes regularly organized around five- and sixfold axes.
Rotaviruses are considered to be a major causeof gastr~nteritis in infants and young animals of varmus species (1, 2). Since the discovery of these viruses, several electron microscopic studies have been published, showing that all members share a common rno~holo~~ differing only in details from other rep~sentatiyes of the Reoviridae family (s-5), and models have been proposed for both the single-shelled and doubleshelled particles that are commonly encountered in preparations of rotaviruses (6,7). Although the negative-stainingmethod has permitted a detailed analysis of the inner stru&ure of theseparticles, some~~e~~nty r~rnai~s with respect to the surface lattice of the inner capsid and the architecture of the outer one (8, 9). In the present study we have used the freeze-drying technique (10, II) in an attempt to define more cor.rectly the architecture of rotaviruses and we propose a model based on the observation of the external appearanceof both singIeshelled and doubl~shelled vii-ions. Calf rotavirus was obtained fram gnotobiotic calves infected expe~mentally with a field isolate and from primary fetal calf kidney cells infected with the Thiverval strain (12). Human and simian (SA 11)rota-
viruses were obtained respectively from human fecal samples and from infected kidney cells (MA 104). Two types of particles were purified in isopycnic gradients. Singleshelled particles band at a density of 1.38 mg/ml, and doable-shelled particles at a density of 1.36m&ml in cesiumchloride (fS). F’urifled virus particles were placed onto colladion-coated grids for a few flutes, and then washed in ammonium acetate buffer 0.1 M, pH 6.2. The excess buffer was blatted out, and the grids were immediately dipped into slush nitrogen (-210”). The grids were then clamped in a copper specimen holder immersed in liquid nitrogen, and transfe~ed to the vacuum chamber at a Reichert Cryofract apparatus (14). Ice sublimation was performed at -80” at a pressure of about 5 x 10e8Torr, Virus particles were shadowedwith platinum at an angle of 45“, coveredwith a carbon film, and warmed up to room temperatu~ with dry nitrogen. The different thickness of platinum and carbon deposits was estimated to be respectively 2 and 4 nm using a “Kranasff quartz film thickness monitor. Examination of purified virions showed a morphological difference between singleand double-shelled particles. Single-shelled
471
0042..6822/79/140471-05$02.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reswved.
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SHORT COMMUNICATIONS
particles had a diameter of about 65 nm and exhibited a regularly spaced network of knob-like capsomers resembling golf balls. This particular aspect reflected the presence of numerous sixfold axes (Fig. la). The
distance between any capsomers was 10 nm center-to-center. The capsomers were disposed around five- and sixfold axes of symmetry (Fig. lb). At least one fivefold axis could be visualized on most of the particles
FIG. 1. Electron microscopy of freeze-dried single-shelled rotavirus ex-calf. (a) Low magnification, showing a homogeneous population of single-shelled particles obtained after cesium chloride density gradient purification (d = 1.38). Bar = 200 nm. (b) High magnification showing clearly the surface lattice and the occurrence of five and six coordinated capsomers on the inner capsid. Bar = 50 nm. Particles marked with a star present two fivefold axes, and have the same orientation. Two of these particles are enlarged (c, e), and retouched (d, f), for easier visualization of two neighboring five-coordinated capsomers. Bar = 25 nm.
FIG. 2. (a) Low magnification electron micrograph of double-shelled particles after purification in a cesium chloride density gradient (d = 1.36). Bar = 200 nm. The surface has an overall smooth surface, but small holes can be clearly distinguished in this outer layer. Inset: high magnification, showing a group of holes around a fivefold axis. Bar = 50 nm. (b) The morphological differences between single- and double-shelled particles appear clearly on this micrograph which shows also an intermediate form that has lost part of its outer capsid layer. Bar = 50 nm. (c) Double-shelled human rotavirus. Bar = 100 nm. (d) Single-shelled human rotavirus. Bar = 100 nm. (e) single-shelled simian rotavirus Bar = 100 nm. (f) Model of rotavirus particle showing the presence of 132 capsomers with a skewed and icosahedral pattern characteristic of T=13. In this model the capsomers of the outer capsid coincide with those of the inner capsid.
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which had been correctly shadowed. For about 10% of the particles, it was possible to visualize two fivefold axes (Figs. lc-f). The minimum number of sixfold axes between two vertices was always three, but it must be emphasized that these axes were not located on the edgesjoining two vertices; moreover these sixfold axes were not symmetrical with respect to the edges, but just to the center of them. This pattern correlates well with that of an icosahedral lattice with T = 13. The lattice shown on Fig. 2f corresponds to an icosadeltahedron of the skew classP=h2+hlc+kZ=13(h=1;IC=3) with f = 1 (15). Consequently the total number of capsomers is 132 [lo (T - 1) = 120 hexameres and 12 pentameres] (15). Double-shelIed particles had a different morphology (Fig. 2a). They had a diameter of 75 nm and exhibited a smooth surface perforated by holes; the latter was 3 nm in diameter and the distance between any two holes was 11 nm (center-to-center). They were regularly organized around fiveand sixfold axes. However, it was not possible to localize two fivefold axes on a single particle; thus the T value of the external surface could not be determined. In Fig. 2b, a particle clearly appears to have lost part of its external layer, the thickness of which can be estimated to be 5 nm. Extensive examination of human, calf and simian rotavirus particles revealed identical structures for both single- and double-shelled particles (Figs. 2b-e). In the study of viral structures the freezedrying technique offers several advantages over negative staining, especially because a single-sided image can be obtained which is much easier to interpret than the superimposed images obtained by negative staining; further, viral particles may be better preserved by freeze-drying. The T value determined in this study (7’ = 13) places this virus among the rare virus types that have a skewed co~o~tion (16,ZT’). Esparza and Gil (8) recently reported T = 16 for human rotavirus. This result was obtained by the negative-staining method from the observation of slightly distorted particles. However these authors counted an average of 21 units at the periphery of the virions; therefore, the total number of units should
be (21)Yrr = 140 mo~holo~cal units. This last figure is si~ificantly different from 162 (co~esponding to T = 16), but closer to 132 (corresponding to T = 13). Concerning double-shelled particles, the images given by our freeze-drying method correspond perfectly to the smooth aspect obtained by negative staining. The method used here permits the visualization of the particle surface and reveals the existance of small holes. Although it was not possible to determine the T value for the double-shelled particles, it seems highly probable that it corresponds to that found for the singleshelled particles. In the model proposed by Stannard and Schoub (9) the external layer was represented by a honeycom~like lattice with large holes. The freeze-drying technique did not enable us to discern structural units on the outer capsid; however, holes do exist, but of a smaller size than shown earlier, even if one takes into account the thickness of the metal deposit. On the basis of the data reported here, we propose the model depicted in Fig. 2f. This model suggests the existence of an inner capsid composed of 132 capsomers with a skewed pattern characteristic of 7’ = 13. The outer layer is depieted as a shell with small holes that correspond one by one with those of the inner capsid. From a technical point of view, the freezedrying method appears to be a promising tool for further comparison with the structure of members of the Reoviridae family, as well as that of other viruses. REFERENCES 1. FLEWETT, T. H., BRYDEN, A. S., and DAVIES, H., Lancet 2, 1497 (1973). 2. MEBUS, C. A., UNDERDALH, M. R., RHODES, M. B., and TWIEHAUS, M. J., Univ. Neb. Agr. Exp. Stat. Res. Bull. 233 (1969). 3. LECASTAS, G., Luncet 2, 524 (1974). 4. MUCH, D. H., and ZAJAC, I., Zfkc. Znzmun. 6, 1019-1024 (1972). 5. WOODE, G. N., BRIDGER, J. C., JONES, J. M., FLEWETT, T. H., BRMEN, A. S., DAVIES, H. A., and WHITE, G. B. B., Znfec. Z~~~n.
14, 804-810 (1976). 6. BRIDOER, J. C., and WOODE, G. N., J. Gem. Viral. 31, 245-250 (1976). 7. MARTIN, M. L., PALMER, E. L., and MXDDLIXON, P. J., Virology
68, 146-153 (1976).
SHORT
COMMUNICATIONS
8. ESPARZA, J., and GIL, F., Virology 91, 141-150 (1978). 9. STANNARD, L. M., and SCHOUB, B. D., J. Gen. viroz. 37, 435-439 (1977). IO. NERMUT, M. Y., In “Freeze-Etching Techniques and Applications” (L. Benedetti and P. Favard, eds.). Sot. Franc. Micros. Electr., Paris, 1973. 11. KISTLER,J., and KELLENBERGER, stmct. Res. 59, 70-75 (1977).
E., J. Ultm-
475
12. L’HARIDON, R., and SCHERRER, R., Ann. Rech. Vet. 7, 373-381 (1975). 13. COHEN, J. J. Gen. Viral. 36, 395-402 (1977). 14. ESCAIG, J., and NICOLAS, G., C. R. Acad. Sci. Paris 233D, 1245- 1248. 15. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, 1-24 (1962). 16. KLUG, A. J., J. Mol. Biol. 11, 424-432 (1965). 17. KLUG, A., and FINCH, J. T., J. Mol. Biol. 11, 403-412 (1965).
AL~ERTO ROSETO,* JACQUES ESCAlG,f ~TIENNE DELAIN,S JEAN C~H~~,~ AND RAOUL SCHERRER§
The ~ee~~~ng technique was used to study the rno~holo~ of s~ngl~shel~ad and double-shelled particles of human, bovine, and simian rotaviruses. Analysis of the inner capsid suggests the existence of 132 capsomeres, with a skewed and icosahedral pattern characteristic of 7’ = 13. Double-shelled particles exhibit a smooth surface perforated by small holes regularly organized around five- and sixfold axes.
Rotaviruses are considered to be a major causeof gastr~nteritis in infants and young animals of varmus species (1, 2). Since the discovery of these viruses, several electron microscopic studies have been published, showing that all members share a common rno~holo~~ differing only in details from other rep~sentatiyes of the Reoviridae family (s-5), and models have been proposed for both the single-shelled and doubleshelled particles that are commonly encountered in preparations of rotaviruses (6,7). Although the negative-stainingmethod has permitted a detailed analysis of the inner stru&ure of theseparticles, some~~e~~nty r~rnai~s with respect to the surface lattice of the inner capsid and the architecture of the outer one (8, 9). In the present study we have used the freeze-drying technique (10, II) in an attempt to define more cor.rectly the architecture of rotaviruses and we propose a model based on the observation of the external appearanceof both singIeshelled and doubl~shelled vii-ions. Calf rotavirus was obtained fram gnotobiotic calves infected expe~mentally with a field isolate and from primary fetal calf kidney cells infected with the Thiverval strain (12). Human and simian (SA 11)rota-
viruses were obtained respectively from human fecal samples and from infected kidney cells (MA 104). Two types of particles were purified in isopycnic gradients. Singleshelled particles band at a density of 1.38 mg/ml, and doable-shelled particles at a density of 1.36m&ml in cesiumchloride (fS). F’urifled virus particles were placed onto colladion-coated grids for a few flutes, and then washed in ammonium acetate buffer 0.1 M, pH 6.2. The excess buffer was blatted out, and the grids were immediately dipped into slush nitrogen (-210”). The grids were then clamped in a copper specimen holder immersed in liquid nitrogen, and transfe~ed to the vacuum chamber at a Reichert Cryofract apparatus (14). Ice sublimation was performed at -80” at a pressure of about 5 x 10e8Torr, Virus particles were shadowedwith platinum at an angle of 45“, coveredwith a carbon film, and warmed up to room temperatu~ with dry nitrogen. The different thickness of platinum and carbon deposits was estimated to be respectively 2 and 4 nm using a “Kranasff quartz film thickness monitor. Examination of purified virions showed a morphological difference between singleand double-shelled particles. Single-shelled
471
0042..6822/79/140471-05$02.00/O Copyright 6 1979 by Academic Press, Inc. All rights of reproduction in any form reswved.
472
SHORT COMMUNICATIONS
particles had a diameter of about 65 nm and exhibited a regularly spaced network of knob-like capsomers resembling golf balls. This particular aspect reflected the presence of numerous sixfold axes (Fig. la). The
distance between any capsomers was 10 nm center-to-center. The capsomers were disposed around five- and sixfold axes of symmetry (Fig. lb). At least one fivefold axis could be visualized on most of the particles
FIG. 1. Electron microscopy of freeze-dried single-shelled rotavirus ex-calf. (a) Low magnification, showing a homogeneous population of single-shelled particles obtained after cesium chloride density gradient purification (d = 1.38). Bar = 200 nm. (b) High magnification showing clearly the surface lattice and the occurrence of five and six coordinated capsomers on the inner capsid. Bar = 50 nm. Particles marked with a star present two fivefold axes, and have the same orientation. Two of these particles are enlarged (c, e), and retouched (d, f), for easier visualization of two neighboring five-coordinated capsomers. Bar = 25 nm.
FIG. 2. (a) Low magnification electron micrograph of double-shelled particles after purification in a cesium chloride density gradient (d = 1.36). Bar = 200 nm. The surface has an overall smooth surface, but small holes can be clearly distinguished in this outer layer. Inset: high magnification, showing a group of holes around a fivefold axis. Bar = 50 nm. (b) The morphological differences between single- and double-shelled particles appear clearly on this micrograph which shows also an intermediate form that has lost part of its outer capsid layer. Bar = 50 nm. (c) Double-shelled human rotavirus. Bar = 100 nm. (d) Single-shelled human rotavirus. Bar = 100 nm. (e) single-shelled simian rotavirus Bar = 100 nm. (f) Model of rotavirus particle showing the presence of 132 capsomers with a skewed and icosahedral pattern characteristic of T=13. In this model the capsomers of the outer capsid coincide with those of the inner capsid.
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COMMUNICATIONS
which had been correctly shadowed. For about 10% of the particles, it was possible to visualize two fivefold axes (Figs. lc-f). The minimum number of sixfold axes between two vertices was always three, but it must be emphasized that these axes were not located on the edgesjoining two vertices; moreover these sixfold axes were not symmetrical with respect to the edges, but just to the center of them. This pattern correlates well with that of an icosahedral lattice with T = 13. The lattice shown on Fig. 2f corresponds to an icosadeltahedron of the skew classP=h2+hlc+kZ=13(h=1;IC=3) with f = 1 (15). Consequently the total number of capsomers is 132 [lo (T - 1) = 120 hexameres and 12 pentameres] (15). Double-shelIed particles had a different morphology (Fig. 2a). They had a diameter of 75 nm and exhibited a smooth surface perforated by holes; the latter was 3 nm in diameter and the distance between any two holes was 11 nm (center-to-center). They were regularly organized around fiveand sixfold axes. However, it was not possible to localize two fivefold axes on a single particle; thus the T value of the external surface could not be determined. In Fig. 2b, a particle clearly appears to have lost part of its external layer, the thickness of which can be estimated to be 5 nm. Extensive examination of human, calf and simian rotavirus particles revealed identical structures for both single- and double-shelled particles (Figs. 2b-e). In the study of viral structures the freezedrying technique offers several advantages over negative staining, especially because a single-sided image can be obtained which is much easier to interpret than the superimposed images obtained by negative staining; further, viral particles may be better preserved by freeze-drying. The T value determined in this study (7’ = 13) places this virus among the rare virus types that have a skewed co~o~tion (16,ZT’). Esparza and Gil (8) recently reported T = 16 for human rotavirus. This result was obtained by the negative-staining method from the observation of slightly distorted particles. However these authors counted an average of 21 units at the periphery of the virions; therefore, the total number of units should
be (21)Yrr = 140 mo~holo~cal units. This last figure is si~ificantly different from 162 (co~esponding to T = 16), but closer to 132 (corresponding to T = 13). Concerning double-shelled particles, the images given by our freeze-drying method correspond perfectly to the smooth aspect obtained by negative staining. The method used here permits the visualization of the particle surface and reveals the existance of small holes. Although it was not possible to determine the T value for the double-shelled particles, it seems highly probable that it corresponds to that found for the singleshelled particles. In the model proposed by Stannard and Schoub (9) the external layer was represented by a honeycom~like lattice with large holes. The freeze-drying technique did not enable us to discern structural units on the outer capsid; however, holes do exist, but of a smaller size than shown earlier, even if one takes into account the thickness of the metal deposit. On the basis of the data reported here, we propose the model depicted in Fig. 2f. This model suggests the existence of an inner capsid composed of 132 capsomers with a skewed pattern characteristic of 7’ = 13. The outer layer is depieted as a shell with small holes that correspond one by one with those of the inner capsid. From a technical point of view, the freezedrying method appears to be a promising tool for further comparison with the structure of members of the Reoviridae family, as well as that of other viruses. REFERENCES 1. FLEWETT, T. H., BRYDEN, A. S., and DAVIES, H., Lancet 2, 1497 (1973). 2. MEBUS, C. A., UNDERDALH, M. R., RHODES, M. B., and TWIEHAUS, M. J., Univ. Neb. Agr. Exp. Stat. Res. Bull. 233 (1969). 3. LECASTAS, G., Luncet 2, 524 (1974). 4. MUCH, D. H., and ZAJAC, I., Zfkc. Znzmun. 6, 1019-1024 (1972). 5. WOODE, G. N., BRIDGER, J. C., JONES, J. M., FLEWETT, T. H., BRMEN, A. S., DAVIES, H. A., and WHITE, G. B. B., Znfec. Z~~~n.
14, 804-810 (1976). 6. BRIDOER, J. C., and WOODE, G. N., J. Gem. Viral. 31, 245-250 (1976). 7. MARTIN, M. L., PALMER, E. L., and MXDDLIXON, P. J., Virology
68, 146-153 (1976).
SHORT
COMMUNICATIONS
8. ESPARZA, J., and GIL, F., Virology 91, 141-150 (1978). 9. STANNARD, L. M., and SCHOUB, B. D., J. Gen. viroz. 37, 435-439 (1977). IO. NERMUT, M. Y., In “Freeze-Etching Techniques and Applications” (L. Benedetti and P. Favard, eds.). Sot. Franc. Micros. Electr., Paris, 1973. 11. KISTLER,J., and KELLENBERGER, stmct. Res. 59, 70-75 (1977).
E., J. Ultm-
475
12. L’HARIDON, R., and SCHERRER, R., Ann. Rech. Vet. 7, 373-381 (1975). 13. COHEN, J. J. Gen. Viral. 36, 395-402 (1977). 14. ESCAIG, J., and NICOLAS, G., C. R. Acad. Sci. Paris 233D, 1245- 1248. 15. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, 1-24 (1962). 16. KLUG, A. J., J. Mol. Biol. 11, 424-432 (1965). 17. KLUG, A., and FINCH, J. T., J. Mol. Biol. 11, 403-412 (1965).