No. 45~6 March 10, 1956 NATURE 473 STRUCTURE OF SMALL VIRUSES I T is a striking fact that almost all small viruses are either rods or spheres. The purpose of this m-tiole IJ to explain this observation by means of the fol- lowing simple hypothesis : a small virus contains identical subunits, packed together in a regular manner. It has been suggested before' that viruses are constructed from sub-units ; but the idea has not previously been described in precise terms or put forward as a general feature of all smaU viruses. We believe that there is conclusive evidence for this hypothesis in two cases and suggestive evidence in a number of others. As most of the present evidence comes from the plant viruses, we shall restrict our discussion to these, except for a few remarks on animal viruses at the end of the article. Plant Viruses Notice first that all plant viruses which have been studied carefully are extremely regular in their shape and sizea. In electron micrographs their dimensi~ are constant. One particle of turnip yellow mosarc virus, for example, is the same size as another, t6 within the errors of measurement. Momwer, the `spherical' viruses have shapes very close to that of a sphere-there seem to be no ellipsoidal plant viruses. All cases where they have appeared as flattened spheres have been shown to be due to the surface tension caused by drying prior to eleotron microscope examination. In good photographs there are sometimes suggestions that the `spheres' are more nearly regular polyhedra, which, as we shall see, is. what one might expect. The great regularity of plant viruses is shown even more strikingly by t,heir ability to form cry&& (or paracrystals) which give good X-ray photographs~, often with reflexions extending to small spacings. From this we can infer that a very high degree of order exists within such viruses, and that, to 8 resolution almost at the atomic level, one .virns particle appears identical, or at least very similar, to all its sister virus particles. A plant virus can thus be considered a `molecule' in the sense used by protein crystallographers-an entity, the major part I. of which bee its atoms arranged in definite (relativo) positions in space. All known plant viruses consist of two chemical components only : protein and ribonucleic acid. It seems likely that there is a general plan for their relative positions and that the majority of the protein lies on the outside of the virus, surrounding a central core composed largely, if not entirely, of ribonucleic acid. This arrangement is well established for only two vir uses-the spherically shaped turnip yellow mosaic virus (by Markham~) and the rod-shaped tobacco mosaia virus (by both the Tubing& and Berkeley groups*)-but we believe that it is likely to apply to all simple viruses. That is, the protein component of 8 round virus is a spherical shell, and of a rod-shaped virus, a cylindrical shell. Our hypo- thesis, is that in both asses these shells are con- structed from a Iarge~number of identical protein molecules, of small or moderate size, packed together in 8 regular manner. Our hypothesis may apply, though in 8 slightly different form, to the ribonucleic acid component. This is discussed in more d&ail later. Tobacco Mosaic Virus This rod-shaped virus is the best studied and we shall therefore consider ita structure in detail. - Tobacco mosaia virus contains 24 per cent protein and 6 per oent r&nucleic aoid'. The characteristic particle, which is closely connected with the infect- ivity, has a `molecular weight' of about 46 million, a length close to 3000 A. end a diameter of about 170 A. The early X-ray works showed clearly that this pa&ale is made up of sub-units of some sort. More reesntly it was realized that the basic feature of the structure is its helical natu@. The protein part of the virus is constructed from a large number of struaturally equivalent sub-units (smalI globular prot.eins) set in helical array about the central axis. The pitoh of the helix is 23 A. The number of sub- 1mit.e per turn is more difliault to establish-the most probable value (Franklin, R. E., and Holmes, K. C., personal communication) gives a molecular weight for the sub-unit of about 20,000. A very similar value is suggested by the chemical evidence. Harris and Knights 6rst examined the carboxyl end-groups of the polypeptide chains, and found that the virus particle had about 2,600 terminal groups, all threonine. This suggested that the virus contains 2,500 identical polypeptide chains, an idea whiah hris been further strengthened by the recent work of both the Tubingen and Berkeley" groups, who have identif%ed the terminal three residues at the carboryl end of this polypeptide chain. Some additional feature is obviously needed to determine the length of the protein shell, and we would guess that in the intact virus-this is controlled by the length of the ribonucleia acid core. This would explain why rods of indefinite length are pro- duced when undonatured protein sub-units are re- aggregated in the absence of ribonucleia aoidlZ. Moreover, when the w-aggregation. occurs in the presence of ribonucleic acid, it is reported by Fraenkel-Conrat and Wllliamsl~ that rods of 3000 A. in length occur very frequently. The stnmturs of tobacco mosaia virus, then, is bseed On 8 Mix, or, in other words, it has a screw fiXis-in thi0 afma 8 iron-integer screw axis. This agmmetrg axis implies that all the protein sub-units in the body of the virus have the same environ- ment. The samoco+ot points between neighbouring 474 NATURE March 10, 1956 VOL. 177 sub-unite are used over and over @in as we move along the helix: This feature is the clue to the general principle which we can apply whenever, on the' molecular level, a structure of a definite size and shape bee to be built tip from,smaller units ;' namely, that the packing arragementa are likely to be repeated again and again-d hence that the sub- units are likely to b6 related by symmetry elements. So far we have been mainly concerned with the protein, and have neglected the ribonucleic acid component of the virus. Is that, too, made up of sub-unite ? The ribonucleic acid content of tobacco mosaic virus is rather low, and not more than four nucleotides can be associated with a given prot.e& sub-unit. Now if all these gTOUp8 were identical, the analytical composition of the ribonucleic acid Gould be based on the number 4, which it certainly is notI'. Moreover, the ribonucleic acid is probably connected with the genetic properties of the virus, and so -its fundamental unit must contain a much larger number of nucleotides. This does not mean, however, that ribonucleic acid sub-unite do not exist, since it is possible that the ribonucleic acid core contains 8 number of identical strands systematically interacting with the protein shell. The important consideration is that the packing arrangement should be repeated over and over again ; and thii can be done if the symmetry of the ribo- nucleic acid is the same as the symmetry of the protein and if the symmetry applies only to the sugar-phosphate backbone and not to the sequence of bases. It remains to be seen whether this type of arrangement ca6 be established experimentally. Spherical Plant Viruses We have seen that the rod-shaped helical form of tobacco mosaic virus repreeenta a patural way of constructing a large container from identical much smaller building blocks. The question we must now ask is whether the protein shell of the spherical viruses is likewise constructed by a regular aggre- gation of one type Of small protein mole&e, a&i, if so. how this is done. Unfortunatelv. there has been. to,our knowledg& no ~tematic c&mica1 search for the presence of sub-units in spherical viruma and so we must rely almost completely on crystallographic evidence. It has been shown in two cases-bushy stunt virus" and turnip yellow mosaic v&t+@-that spherical viruses crystallize in a unit cell which has the shape of a cube ; but unfortunately the X-ray photographs did not establish whether the symmetry also was cubic. This is important because, as has been pointed out by Dr. Dorothy Hodgkin' and Dr. Barbara Low', if the lattice possemes true cubic symmetry so must the ti particle, since there is only one particle in the primitive unit cell. It has now been clearly established by Caspar (see following communication) that the unit cell of bushy stunt virus has cubic symmetry, and that, in this particular case, the virus has an even higher sym- metry than the unit cell. Though this evidence applies to only one virus, we expect that further investigation will show- that many small spherical viruses have cubic symmetry, for the reasons given below. Now a virus possessing cubic symmetry must necessarily be built up by the regular aggregation of smaller asymmetrical building bricks, and this - be done only in a very limited number of ways. Since viruses are made of protein and ribonucleic acid, both Toblel. TEETE~EE POSSXBLS CFBIC RUST GROUPS FOR ASSPEERICAI I- Crystallo~aphic demription l- Th 3 e-fold 4 3-fold 6 e-fold 4 )-fold 3 4-fold 15 S-fold 10 S-fold 6 &fold . -. us - -- 7 60 Dodecahedron Icosahedron ~numwr Of BUtI-lmlca WI11 bE the %Une aa, or 0 multiple Of, the number of asymmetric units 23 452 552 VIR No. and type of rotation *xes present x0. of Platonic solid asymmetric with these sym- units metry elements 12 Tetrahedron I 24 Cube Octahedron : of which contain asymmebric carbon atoms of one particular hand only, those symmetry e1ement.s (mirror planes and centres of symmetry) which turn a right hand into e left hand are impossible. Thus we can only have rotation axes, and for cubic sym- ._ metry this limits us to only three different, com- binations of symmetry elements. Each of these three classes must contain at least. four three-fold axea and three two-fold axes, arranged as for a tetrahedron. The first class contains no additional type of axis, while the second and third have four- and five-fold axes, respectively. Such an arrangement of symmetry elements is known as a `point group', in contrast to a space group which applies to a regular arrangement extending t.o infinity. In Table 1 are listed t,he three cubic point groups possible for virus part.icles and also the regular polyhedra which have these symmetry elementa (among others). Notice that in all these point groups the minimum number of asymmetric units must be a multiple of 12. Three further points must be made to prevent misunderstanding. First, it is possible to arrange sub-units in other ways to produce a spherical shell, but the symmetry will not be cubic, and as they are less likely we shall not discuss them further here. Second, the asymmetric unit, upon which the sym- metry elements act to build up the spherical shell. may consist of several identical sub-units joinctl together in some unsvmmetrical fashion. This occurs quite often in pro& crystals and would not be unexpected. Nor need the sub-unit be a single protein molecule in the chemist's sense of a unit joined together by chemical bonds. Several different. protein molecules may aggregate to form the asym- metric unit. Third, our predictions concern the symmetry elements present in a virus particle, not, ifs exact shape. However, this is likely to be approximately spherical, and may, under high resolution, appear polyhedral or perhaps with bumps on, like a rather symmetrical mulberry. Both these forms have been seen in electron micrographs. It is not easy to explain in a short space why there are so few ways of building a spherical shell, but t.he reader can soon convince himself that it is difficult by trying to draw identical shapes which completel! cover the surface of a tennis ball. It is impossible. for example, to do this entirely with hexagons, even if their shape is irregular. The point is very well stewed in D'Arcy Thompson's "On Growth and Form"ll, in which we find "the broad, general prin- ciple' that we cannot group as we please any number and sort of polygons into a polyhedron, but that the number and kind of facets in t,he latter is strict,ly limited to a narrow range of possibilities". The reason is essentially a topological one. NO. 4506. March 10; `1956 NATURE 4iB From the present X-ray evidence we 8re unable to distinguish the respective contributions of the protein end the ribonucleic scid, so we cannot be syre whether the cubic symmetry is perfect and 8pplies strictly to both of them. We cannot tell whether the protein sub-units contain identical sequences of amino-acids, or whether the ribonucleic acid sub-units (if they exist) h8ve identical sequences of nucleotides. It should not be very di&ult, by end-group analysis, to decide whether the protein components are all 8pproxim8tely equ8l. By analogy with tobacco moseic virus we would guess that this will be found to be the c8se. With the ribonucleic acid component, however, the problem is more dii%cult than it was in the c8se of tobacco mosaic virus, as the number of nucleotides per sub-unit is certainly much larger. (This follows from the higher percent8ge of ribo- nucleic acid' and the much smaller number of protein sub-units.) Only with 8 more detailed understanding of the ribonucleic acid core is the problem likely to be settled. . Animal and Other Viruses For animal viruses we are handicapped bicause there is no X-ray evidence avsilable so f8r. However, it is now becoming cleer* that meny of the smaller animal viruses, such as poliomyelitis and the various encephalitic viruses, are morphologically very similer to the spheric81 plant viruses. Not only are they of similar size (approximately 300 A. diameter) ; buf it hes recently been shown10 that poliomyelitis virus 81SO contains ribonucleic acid and can form crystals which appe8r as rc3gd8r as those produced by the plant viruses. We thus think it very probable thet cubic symmetry also extends to these animal viruses, 8nd thet the soluble entiger@ (of &out 120 A. di8meter) frequently observ?d in infected cells &e related to the sub-units nOITII8lly used in the aARembly of the tinal infective virus. We else see no reason why our hypothesis should not be valid for viruses containing deoxyribonucleic acid rather than ribonucleic acid. Although the structure of bacteriophsges is USU8lly more complex t,han the smaller viruses discussed here, the fact that, their heeds appear polyhedral &ggests that idea8 of this general type may Spply to them, too. On the other hand, it is less likely thst they wiI1 be relevant to t,he structure of the larger viruses like vaccinia. Conclusion We can now describe our hypothesis in a more general manner. We assume that, the basic structural requirement, for a small virus is the provision of a shell of protein to protect its highly specific packet of ribonucleic acid. This shell is necessarily rat.her large, and the virus, when in the cell, finds it easier to control the production of a large number of identical small protein molecules rather than t.hat of one or two very large molecules to act as its shell. These small protein molecules then aggregate around the ribonucleic acid in 8 regular manner, which they can only do in 8 limited number of W8yS if they are to use the same packing arrangement repeatedly. Hence Small viruses are either rods or spheres. The number of sub-units in a rod-shaped virus is probably unrestricted. but for 8 spherical virus the number is likely`t,o be a multiple of 12. Every small virus will contain symmetry elements and .in favourable cases these c8n be discovered experimentally. We believe that this hypotheeis is likely to apply (in this form or 8 simple variant of it) t.0 811 small viruses which have a fixed size and shape. F. H. C. CRICK J. D. WATSON* &diC81 Besesrch Council Unit for the Studv of the Molecular Structure of Biologic81 Systems, Cevendish Laboratory, . Cambridge Jan. 23. o On leave from the Blolosy Department. Earvard University, and supparted by a grant from the National Science Foundation, U.S.A. a Among tbe more important references are Eodgkln, D. C., Cold Sprinq Hrwbr S~~JIP.. 14.65 (1050). Low. B.. in "The Proteins". 1. 235 (Academic Prem. New York, 1053). Schramm, Q.. 2. Natwforuh., 2b, 112, 240 (1047). * `tVilllEUUs. R. C.. Cold &ring H&or Symp.. 13, 185 (1053) ; "Ad- vances in Vlrua Research", 2, 184 (Academic Press, New York, 1054). `Bernal, J. D., and Far&when, I., J. Gen. Phpciol.. !?& 111, 147 (1041). a Ma&ham. R., Dire. Farad. Sot.. 11. 221 (1051). See alao Bernal, J. D.. and Carlisle. C. H.. Dim. Farad. SOL, 11. 227 (105lj. and Schmidt. P.. Eaeaberg, P.. and Beeman. W. W., Biochtm. rl Biophpr. AC& 14, 1 (1054). ' Schramm. G., Schumacher, G.. and Zillig, W.: Nature. 175. 549 (1055). ' Hart. R.. PTOC. U.S. Nlrt. dead. Sci., U. 201 (1055). z Knight, C. A., "Advances in Virus Research". 2. 153 (beadernie Press, Sew York, 1064). a Watson, J. D., Bimhim. cl B&whys. A&. 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