1977; Esparza & Gil, 1978: Kogasaka et al., 1979; Roseto et al., 1979). .... effect on complex protein,,;, in which their interaction with the aqueous solvent is ...
J. gen. Virol. (1986), 67,
1721
1721-1725. Printed in Great Britain
Key words: rotavirus/capsid structure~chemical degradation
The Structure of the Rotavirus Inner Capsid Studied by Electron Microscopy of Chemically Disrupted Particles By J. E. L U D E R T , F. G I L , F. L I P R A N D I *
AND J. E S P A R Z A *
Instituto Venezolano de lnvestigaciones Cientificas, Apdo. 1827, Caracas IOIOA, Venezuela (Accepted 23 April 1986) SUMMARY
The inner capsid structure of the OSU strain of porcine rotavirus was studied by electron microscopy of freeze-dried preparations and of negatively stained chemically disrupted virus particles. The analysis of the particles by the freeze-drying technique revealed a T : 13 1 (laevo) symmetry for the organization of the inner capsid. Treatment of single-capsid rotavirus particles with 30~ formamide or 5 M-urea resulted in their degradation, giving rise to very similar products, corresponding to isolated vertices, edges and faces of the virus icosahedron. An analysis of such structures confirmed the triangulation number and handedness of the rotavirus inner capsid, and provided evidence for the open-mesh model, in which the five- and six-coordinated axes are represented by 'holes" formed by smaller trimeric morphological subunits.
The structure of the rotavirus particle has been the subject of numerous investigations, resulting in a number of conflicting interpretations (Martin et al., 1975; Stannard & Schoub, 1977; Esparza & Gil, 1978: Kogasaka et al., 1979; Roseto et al., 1979). One of the earlier interpretations proposed that the inner capsid of human rotavirus is composed of large ring-like morphological subunits (Martin et al., 1975; Palmer et al., 1977). That interpretation was challenged by several authors (Stannard & Schoub, 1977; Esparza & Gil, 1978) who proposed that the ring-shaped capsomeres result from moir6 pattern artefacts caused by the superimposition of two-sided images, and suggested models based on an open lattice arrangement of small morphological subunits. For the human rotavirus, we proposed the existence of an inner capsid composed of morphological subunits arranged as an open mesh, showing a regular pattern of cavities or 'holes', constituting the five- and six-coordinated units of the virion (Esparza & Gil, 1978). Closer examination of the morphological subunits has revealed that they are trimeric structures (Martin et al., 1975; Esparza & Gil, 1978), an observation that has found recent biochemical support (Gorziglia et al., 1985). In our model we suggested the existence of 162 holes formed by the icosahedrat arrangement of 320 subunits, with a pattern characteristic of T : 16. However, Roseto et al. (t979) studied the structure of rotaviruses using the freeze-drying technique and found evidence suggesting the existence of 132 capsomeres, arranged in a skew symmetry with T : 13. The photographs presented by the authors could also be interpreted as suggesting the existence of 132 holes, which in turn, according to our previous model, should be surrounded by 260 trimeric morphological subunits, giving a total of 780 protomeric molecules per virion (Novo & Esparza, 1981 ; Esparza et al., 1984). A useful approach to the study of virus morphology is the controlled degradation of virus particles using chemical or physical treatments. This approach has recently been used with rotaviruses (Almeida et al., 1979; Bican et al., 1982; Palmer & Martin, 1982; Gorziglia et al., 1985), and has been extensively used in the past to dissect adenoviruses (Neurath et al., 1968; Stasny et al., 1968) and herpesviruses (McCombs & Williams, 1973; Palmer et al., 1975). In this tPresent address: Microbiology and Immunology Unit, World Health Organization, 1211 Geneva 27, Switzerland. 0000-7049 ~ 1986 SGM
1722
Short communication .,
(d)
v
Fig. 1. (a, b) Electron microscopy of freeze-dried single-shelled porcine rotavirus particles. The micrograph in (b) was retouched to indicate the pentonal and peripentonal holes on the surface of the virions. (c) Virus cores and inner capsid subunits released after treatment with 1-5 M-CaC12. The arrow shows a large ring-shaped morphological unit composed of six smaller wedge-shaped subunits. (d) Amorphous structure produced after treatment with 0.1 ~ SDS. (e) Low magnification of subviral structures produced after treatment with 3 0 ~ formamide. (f) Low magnification of subviral structures produced after treatment with 5 M-urea. For (a) to (d), bar marker in (b) represents 50 nm ; for (e) and (f), bar marker in (e) represents 100 nm.
communication we present an electron microscopic study of single-shelled rotavirus particles degraded by urea, formamide and other chemical agents, leading to the cleavage of intersubunit bonds with the formation of subviral structures, which provides further details on the subunit structure of the rotavirus inner capsid. All experiments were done with the OSU strain of porcine rotavirus (Theil et al., 1977), which was grown and purified as previously described (Gorziglia et al., 1985). Purified rotavirus was studied by the freeze-drying technique, essentially as described by Roseto et al. (1979), using a JEOL JEE-4C vacuum evaporator. The solutions used for degradation of the virus were prepared in 10 mM-Tris-HCl buffer pH 7-4, as follows: 5 M-urea, 30~ formamide, 1-5 M-CaCI2,
1723
Short communication
(j)
~ ~e, ~
Fig. 2. Subviral structures formed after treatment of single-shelled porcine rotavirus with 30~o formamide (a, e, .]) or 5 M-urea (b, c, g to j). (a) Peripentonal subunits (viewed through a fivefold rotational axis) generated after treatment with formamide. (b) Peripentonal subunits generated after treatment with urea. (c) Rotational enhancement (n = 5) of (b). (d) A model for the arrangement of trimeric morphological subunits around a 5-coordinated hole. (e to h) Subunits corresponding to edges of the virus icosahedron. (i,j) Subunits corresponding to isolated faces. The structures shown in (]) and (h) were retouched to indicate pentonal and peripentonal 'holes' in (e) and (g), respectively. Likewise, the structure shown in (j) was retouched to indicate pentonal 'holes'. Bar marker represents 50 nm. All photographs are at the same total magnification.
0" 1 ~ SDS, 10 ~ pyridine, 5 ~ lithium chloride and 80 ~ acetone. The degradation of rotaviruses was visualized with the electron microscope by first attaching the virus to ionized collodioncarbon coated grids (by floating them on drops of purified virus for 3 rain), followed by three washes with distilled water and floating on drops of the appropriate solution. Five min after incubation at room temperature, the grids were rinsed with distilled water and stained for 30 s with 1 ~ uranyl acetate. The samples were examined at 60 kV with a J E O L 100B electron microscope. Special precautions were taken when inserting the grids in the electron microscope, as well as during the photographic enlargement of the plates, to ensure correct orientation of the specimens (Klug & Finch, 1965). The analysis of the rotavirus particles by the freeze-drying technique confirmed the earlier report of Roseto et al. (1979) of a T : l 3 skewed symmetry for the organization of the inner capsid (Fig. 1 a, b). Two or even three 5-coordinated axes of symmetry could be located in a single particle, showing in all cases a T : 13 l (laevo) structure. However, definition of the 5-coordinated
1724
Short communication
axes either as 'solid' capsomeric structures or 'holes' of an open mesh arrangement, could not be easily obtained using such a technique. To obtain this information we examined the chemical degradation of the rotavirus particle. Of the reagents tested, pyridine, lithium chloride and acetone did not appear to affect the particles under the conditions used. Treatment with 1.5 M-CaCI2 resulted in the generation of smooth cores (Bican et al., 1982), showing little or no ultrastructural detail (Fig. 1 c). However, in some cases disrupted cores and inner capsid subunits were also present; on some occasions, as shown in Fig. 1 (c, arrow), large ring-shaped morphological units, composed of six smaller wedge-shaped subunits, were observed (Palmer & Martin, 1982). Particles treated with 0-1 SDS were completely distorted, showing no discernible morphological characteristics (Fig. 1d). On the other hand, treatment with 30~ formamide (Fig. 1e) or 5 M-urea (Fig. l f ) resulted in the degradation of the particle,;, giving rise to very similar definite degradation products, namely, structures corresponding to vertices, edges and faces of the virus icosahedron. One of the most commonly observed structures corresponded to isolated vertices (Fig. 2a to c). Careful examination of these structures allowed us to construct a model for the arrangement of the trimeric morphological subunits, in which the 5-coordinated axis in fact corresponds to a 'hole' on the virion surface (Fig. I d). Similar conclusions could be drawn from the analysis of structures corresponding to edges (Fig. 2 e to h) and faces (Fig. 2 i and j) of the virus icosahedron. In all cases, the 5- and 6-coordinated axes were 'holes' formed by the smaller wedge-shaped trimeric subunits, arranged with a pattern characteristic of T : 13. It is interesting to note that the handedness of the subviral structures appears in the photographs as dextro, probably because they represent internal views of the disrupted virus capsids. The results obtained witlh formamide and urea were basically similar, possibly due to their effect on complex protein,,;, in which their interaction with the aqueous solvent is altered, resulting in structural changes in the virus proteins (Neurath et al., 1968). Our results suggest that the inner capsid of rotavirus is stabilized by ionic bonds between the core proteins. Neither formamide nor urea appear to affect the association between the core proteins and the major inner capsid polypeptide (p45K or VP6), which is broken only by chaotropic agents (Bican et al., 1982). On the other hand, hydrophobic bonds may also play a role, especially in stabilizing the trimeric form of p45K (Gorziglia et al., 1985). We believe that our re,;ults present definitive proof of an open mesh structure for the arrangement of the trimeric morphological subunits of the inner capsid of rotaviruses with a T : 13 1. A similar conclusion was reached by Metcalf (1982) for the outer capsid of reovirus type 3, and also advanced, in a fc~tnote of his paper, for the NCDV and SA-11 rotaviruses. However, it should be borne in mind that other rotaviruses may show a different handedness in their capsid structure, as has been recently proposed for some members of the Birnaviridae family (0zel & Gelderblom, 1985). T h a n k s are due to T. A v e n d a n o and J. Halpin for typing the manuscript, and to M. R o m a n o for the electron microscopy. This work was supported in part by G r a n t S1-1311 from the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICIT), Caracas, Venezuela.
REFERENCES ALMEIDA, J., BRADBURNE, A. F. & WREGHrl-f, T. G. (1979). The effect of sodium thiocyanate on virus structure. Journal of Medical Virology 4, 269-277. BICAN, P., COHEN, J., CHARPlLIENNE,A. & SCHERRER,R. (1982). Purification and characterization of bovine rotavirus cores. Journal of Virology 43, 1113-1117. ESPARZA, J. & GIL, F. (1978). A study on the ultrastructure of h u m a n rotavirus. Virology 91, 141-150. ESPARZA, L, GORZIGLIA, M. & LIPRANDI, F. (1984). Studies on the molecular biology of rotaviruses: a review. In Proceedings of the Fourth International Symposium on Neonatal Diarrhoea, pp. 1-26. Edited by S. D. Acres. Saskatoon: Veterinary Infectious Diseases Organization. GORZIGLIA, M., LARREA,C., LIPRANDI, F. & ESPARZA,J. (1985). Biochemical evidence for the oligomeric (possibly trimeric) structure of the major inner capsid polypeptide (45K) of rotaviruses. Journalof General Virology66, 1889 1900. KLUG, A. & FINCH, J. T. (1965). Structure of viruses of the papilloma-polyoma type. I. H u m a n wart virus. Journalof Molecular Biology 11, 403-423.
Short communication
1725
KOG~AKA,R., ~KIHARA,M., HORINO,g., CHINA,S. a NAKAO,T. (1979). A morphological study of human rotavirus. Archives of Virology 61, 41-48. McCOMBS,R. M. & WILLIAMS,G. A. (1973). Disruption of herpes virus nucleocapsids using lithium iodide, guanidine and mercaptoethanol. Journal of General Virology 20, 395-400. MARTIN,M. L., PALMER, E. L. & MIDDLETON,P. J'. (1975). Ultrastructure of infantile gastroenteritis virus.
Virology68,
146-153. METCALF, P. (1982). The symmetry of the reovirus outer shell. Journal of Ultrastructure Research 78, 292-301. NEURATH, A. R., RUBIN, B. A. a STASNY,J. T. (1968). Cleavage by formamide of intercapsomer bonds in adenovirus types 4 and 7 virions and hemagglutinins. Journal of Virology 2, 1086-1095. NOVO,E. & ESPARZA,J. (1981). Composition and topography of structural polypeptides of bovine rotavirus. Journal of General Virology 56, 325-335. OZEL, M. & GELDERBLOM,H. (1985). Capsid symmetry of viruses of the proposed Birnavirus group. Archives of Virology 84, 149-161. PALMER,E. L. & MARTIN,M. L. (1982). Further observations on the ultrastructure of human rotavirus. Journal of General Virology 62, 105-111. PALMER,E. L., I~t~,TIN,M. L. & GARY,W. (1975). The ultrastructure of disrupted herpesvirus nucleocapsids. Virology 65, 260-265. PALMER, E. L., MARTIN, M. L. & MURPHY, F. A. (1977). Morphology and stability of infantile gastroenteritis virus:
comparison with reovirus and bluetongue virus. Journal of General Virology 35, 403-414. ROSETO,A., ESCAIG,J., DELAIN,E., COHEN,J. & SCHERRER,R. (1979). Structure of rotaviruses as studied by the freeze-
drying technique. Virology 98, 471-475. STAtCNARD,L. M. & SCHOUI3,B. D. (1977). Observations on the morphology of two rotaviruses. Journal of General Virology 37, 435-439. STASNY,J. T., N~URATH,A. R. & RUBIN, B. A. (1968). Effect of formamide on the capsid morphology of adenovirus types 4 and 7. Journal of Virology 2, 1429-1442. TrmIL, K. w., BOUL,E. n. & AGNES,A. G. (1977). Cell culture propagation of porcine rotavirus (reovirus-like agent). American Journal of Veterinary Research 38, 1765-1768.
(Received 20 January 1986)
