The fine structure of polyoma virus

VIROLOGY

11,

444-457

The P. WILDY,

(1969)

Fine

Structure

M. G. P. STOKER,

The Medical Research of Virology,

Council Glasgow

I. A. MACPHERSON,

Unit for University, Cambridye Accepted

Preparations of polyoma using the negative contrast were found to have a mean shell (capsid) of elongated these surrounded a central Though there is no evidence showed 5:3:2 axial symmetry. 42 capsomeres, each aligned

of Polyoma

Experimental Virus and the Cavendish Unioersity March

Virus AND R. W. HORNE Research, Laboratory,

Department

22, 1960

virus were examined by electron microscopy, method. Spherical particles identified as viruses diameter of 453 A. All the particles showed a hollow subunits (capsomeres). In the majority, core; but some particles appeared to be empty. of icosahedral shape, the capsomere arrangement It has been deduced that the particle contains radially along an axis of symmetry. INTRODUCTION

The negative contrast method developed by Brenner and Horne (1959) has now been applied to a number of viruses of widely different character. Because of the great increase in resolution and preservation provided by this method, our understanding of the fine structure of these viruses has been greatly extended. So far, no work has been reported on the fine structure of any virus known to be oncogenic, and for this reason we have studied the structure of a strain of polyoma virus. This virus was chosen because it gives high yields in cultured cells, it is easily enumerated by the plaque technique and by hemagglutination, and it readily gives tumors in laboratory animals. For reference to virus morphology, we shall use the terminology recently proposed by Lwoff et al. (1959). The infective virus particle is referred to as the vision; the shell which enclosesthe genetic material is referred to as the capsid; and the subunits which go to build this structure, as the capsomeres. Because we do not know whether the genome occupies all the central part of the particle, this will be referred to as the core. 444

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STRUCTURE

MA’IXRIALS

OF

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MEX’HOI

VIRVS

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Virus &rain The strain of polyoma virus used was originally isolated in Toronto by McCulloch et al. (1959) and was kindly supplied by Dr. I,. Simonovitch. It is untigenically related to the SE polyoma virus of Stewart et al. (1957) (Howatson et al., 1960) and to t’he >lill Hill virus isolat,ed by Kegroni et al. (1959) (M. Cr. I’. Stoker and 1. A. Macpherson, unpuhlished). Virus for seed was propagated in mouse rmbryo cells and st,ored at 1” or -70” until required.

Primary or secondary monolayer cultures of mouse embryo cells were used hot)h for producing and Gtrating virus. l’rimary cultures were made by mincing young embryos and separat,ing t)he cells by t)reatment, wit,h 0.25 % trypsin (Difco 1:250). After washing, suitable numbers of cells were added to petri dishes (GO-mm diameter) with 5.0 ml medium, or t)o bott)les (600-ml capacity) with 40 ml of medium. Secondary cultures were prepared from confluent primary cultures after suspension wit,11 0.25 % trypsin. A modified Eagle’s medium which contains t)wice the standard concentrat,ion of amino acids and vitamins has been used t>hroughout. For growth of cells before virus inoculabion, 10 %I calf serum was added. All cultures in petri dishes were placed in illc*uhat.ors at 37” which wc’crc flushrd wit)h CO:! to maint;ain t,he pH at 7.2.

1. Infect,ivit~y t,itrations were made by a modification of the plaque assay methods of Dulhecco and Freeman (1959) and Winocour and Sachs (1959). Suitable dilutions of virus in 0.2-ml volumes were inoculat,ed into monolayer cultures in GO-mm petri dishes. Aft’er 2 hours had elapsed, the cultures were overlaid with 6 ml medium containing 2.5% horse serum and 0.9 % agar (Difco Bacto). At the fourth and eighth day, respect)ively, after inoculation, a further 3 ml of agar medium was added ; t,he final layer also contained 0.008 %, neutral red. Plaques became visible on subsequent days and were counted when the number had reached a maximum. I;our petri dishes were counted for each dilution and the t,iter was expressed as the number of plaque-forming units (PFU) per millilitclr. 2. Htmagglutinin was titrated in Perspex hemagglutinatjion t,rays at

446

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HORNE

4’. Using 0.2-ml volumes, twofold dilutions of virus were made in buffered saline (Dulbecco and Vogt, 1954). One volume of 0.5% guinea pig erythrocytes in buffered saline made up with 0.01% horse serum, was added to each cup. The trays were put into the refrigerator and the sedimentation patterns were examined 4-24 hours later. One hemngglutinating unit (HAU) was taken as the minimal amount required to give partial agglutination. Production

of Virus for Electron Microscopy

The medium was removed, then cultures in petri dishes were inoculated with lo* to lo5 PFU virus in 0.2 ml; those in bottles received 10’ to lo* PFU in l-10 ml. After l-2 hours, medium containing 5 % horse serum was added, 5 ml to each petri dish and 40 ml to each bottle. The cultures were replaced at 37” and 1 week later, the medium was changed; after incubating for a further 3-7 days, the virus was harvested. At this time the medium was found to contain only a small proportion of the virus and was accordingly discarded. The cells were taken up in distilled water (1 ml per petri dish or 10 ml per bottle) and extracted at 0” for 5 minutes by blending at top speed in a microblender (Measuring and Scientific Equipment Ltd.). The brei was centrifuged at 1700 g for 10 minutes and the supernatant fluid retained. The deposit was then re-extracted in the same way with fresh distilled water and the cell debris resedimented. The two supernatants were pooled and generally contained 10’ to lo8 PFU and lo4 to lo5 HAU per milliliter. Concentration

and Purification

of Virus

Some preparations of virus were examined after concentration by sedimentation at 60,000 g for 1 hour. The pellets were taken up in one-fiftieth to one-tenth the original volume and sprayed at once. Other preparations were examined after they had been partially purified by one of the following methods: (1) extraction with fluorocarbon (Girardi, 1959) ; (2) extraction with fluorocarbon and adsorption and elution from guinea pig red blood cells (Kahler et al., 1959); (3) chromatography on brushite columns (Taverne et al., 1958), followed by dialysis overnight against distilled water at 4”. Electron Microscopy Crude suspensions of virus were mixed with equal volumes of 2 % phosphotungstic acid which had been brought to pH 7.0 with N KOH. Concentrated or partially purified suspensions were mixed with half

FINE

STRUCTURE:

OF

POLYOM.4

VIHlY4

-I17

their volume of phosphotungstic acid. The mixtures were immediately sprayed onto carbon grids, using special precautions to avoid dissemination of infected material (Horne and Nagington, 1959). The grids were subsequently examined with a Siemens Elmiskop electron microscope, using double condenser illumination at instrumental magnifications of 8000 and 40,000.

identity

of the Particles

Studied

Characteristic particles were seen in all preparations from infected cells. In unpurified material debris was also present, but this was almost, completely removed in suspensions purified by any of the methods described. Three separttte pieces of evidence have enabled us to identify t,htb particles seen as polyoma virions or empty capsids. First, they were invariably found in preparations made from infected material and, despite exhaustive searching, were never found in material from uninfected t,issue cultures. Moreover, although we are not yet able to make quantitatJive determinations with the negative contrast method, the concentration of particles seen, bore a rough relationship with the infectivity and hemagglutinin titers. Secondly, the particles were found in preparations that had retained infectivity and hemagglutinat!ion titers after sub stantial purification (by chromatography, by treatment with fluorocarbon, or by elution from red cells). k’inally, the sizes of the particles correspond with some of the published data, as will be shown in late1 sections. In \.iew of these considerations, we arc satisfied t’hat, thrb part,iclrs that, we describe are correctly identified. General Features of the Particles I;igurr I is a low magnification micrograph which clearly illustrates the general appearance of the particles. It’ will be noted that they have nearly circular profiles from which a number of processes (the capsomeres) project. It is possible to distinguish three categories of particle: (I) Jlost of the particles seen have been uniformly less dense t,han the phosphot,ungstate in which they were embedded and have been int,crpreted as int’act virions. (2) In many preparations, a proportion of particles found appeared to be filled with phosphotungst,at,e, and these are believed to be empty capsids. This circumstance has euabled us to ohserve the space normally occupied by the core, and it’ will be seen that this space also possesses a nearly circular profile (Figs. I and 2). (3) In

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AND

HORNE

FIG. 1. Low magnification of polyoma virions embedded in electron-dense phosphotungstate; note the central region of some of the particles, filled with phosphotungstate. The particles appear to be spherical with surface projections. Magnification: X 360,000.

some preparations

a number

of particles

have

been seen whose

profiles

are distorted and whose capsomeres appear disarranged to a variable extent (Fig. 3). These particles are taken to be partially disrupted virions. In no preparation have we seen bodies suggestive of separated cores, nor has there been any evidence of an outer envelope like that noted with herpes virus (Wildy et al., 1960). Particle Size The over-all diameters of 189 randomly selected particles were measured to a limit of 5A and gave a mean of 4538. The distribution of sizes among these particles is shown in Fig. 4. It will be noted that the range

FINE

STRUCTURE

OF

l’0LY01M.k

VIRT‘S

43-9

FIG. 2. Higher magnification electron micrograph of polyoma virions, showing capsomeres arranged on the surface. One of the particles (insert,) appears to rontain phosphotrlngstrtte in the center, suggesting an empty shrll. illagnificntion: x 500,ooo.

450

WILDY,

STOKER,

FIG. 3. Two partially disrupted X 500,000. Magnification:

MACPHERSON,

AND

HORNE

virions showing evidence of hollow capsomeres.

from 410 to 520A and that the distribution appears to have at least three modes. These correspond with the three categories of particle described above. Forty-nine of the particles measured were empty capsids and their size distribution is shown in the figure; the extent to which these structures contribute to the mode at 420-4298 is obvious. Including those observed in other series, a total of 90 hollow capsidshave been measured, showing a mean external diameter of 4288 (range 4104508). The remaining 140 particles shown in Fig. 4 comprising intact and partially disrupted virions had a mean diameter of 460A. Ninety-four of them, judged to be intact virions, had a mean diameter of 4478 (range 42G47OA). The remaining 46 particles were partially disrupted, and these, together with 44 disrupted particles from other series, were 470 to 520A in diameter.

extends

Fine Xtructure of the Particles At high magnifications, the fine structure of the capsid becomes apparent. In Figs. 2, 3, and 5 the individual capsomeres which go to build the capsid can be resolved clearly. It will be noted that these structures are elongated cylinders or prisms which stick out like the prickles of a hedgehog. Direct measurement suggeststhat they are 50A long and 45-508 wide (50 capsomeres measured), but it is believed that the former dimension is too short be-

FISE

STRUCTURE

420

440

OF

POLYOMA

Ijli

460 480 Diameter A

151

VIRUS

500

520

cause it is difficult to ensure that a capsomere is lying with its axis parallel to the plate. The length of the capsomeres c&n also be deduced from measurements on hollow capsids. The ext’ernal diameter of these was 328A and the internal diameter was 275280.4, giving a capsomcw length of about 75A. In some particles, especially in those that> are partially disrupted (cf. Fig. :<) each rapsomere is seen t,o possess an axial hole whose diameter has been estimated to be 20A (15 cupsomeres measured), though mensurement.s at this lcvrl of resolution are difficult owing t.o phase contrasteffects. There arc serious difficulties in int,erpreting the precise disposition of elongated capsomeres in a part,icle, and this question will he dealt with in the discussion, but8 the following features require attention. First,

magnification electron micrographs of pol.yoma virions showi F‘IG. 5 a. High of the capsomercs. The surface projections seen at the periphe the arrangement (see arrow) do not appear to be in contact, thus allowing t of t ,he particles to penetrate between the radially displaced capsomeres; nc pho sphotungstatc particle having a central capsomere surrounded by five neighbors equa; the This particle should be compared to the model shown spat ?ed (see arrows). X 560,000. Fig . 6b. Magnification: to 5~3, but the partieln indicated has a central capsomere surround bI. Similar !ed equally spaced (see arrows), and should be compared to Fig. 6a by 2Gz neighbors she, wing the model i II lhe same orient,ation. Magnification: X 500,000. 452

FINE

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15:3

many particles have been seen orientated so that’ one cent’ral capsomere appeared to be surrounded by five equally spaced neighbors (Fig. 5a). Other particles were found in which one capsomere was seen at the center of a ring of six others (Fig. 5b). These appearances strongly suggest that t’he capsomeres are arranged in accordance wit’h 5::2: 2 symmet,ry. Secondly, no particles have been seen in which t,he triangular facet,s of the icosahedron were apparent; this is in marked contrast to the appearance of adenovirus, for example (Horne et al., 1959). Thirdly, the capsomeres do not seem to be closely packed; indeed, they have invariably appeared to be widely separated by furrows into which t)hc phosphotungst,ate has penetrated deeply. Finally, all the capsomeres appear to be tilted wit)11 respect, to each other, and, by tilting the object, in t#he microscope, it has I)een found t,hut they lie with their long axes on t)he radii of t,he parti&s. From t’hese dat,a, it, has been possible t,o construc*t a model (Fig. 6, a and b) from 30 hexagonal and 12 pentagonal prisms; though t)hc holes are not, shown this closely resembles t’he part,iclt:s in appcaral~cr (iis colwtruct,ioll is described in t’he Discussion).

There is considerable divergence among the sizes recorded for the particles seen in polyoma-induced tumors, infected tissue cultures, and purified preparations. Exuminat~ion of t,he published results shows that the range of particle sizes found with the t’hree antJigenically rclnt~cd strains extends from 270 to 1100X in diameter (Banfield ct al., 195!); Bernhard et al., 1959; Dmochowski ct al., l!W; Dourmashkiu and Scgroni, 1959; Kahler et al., 1959; Segroni ct al., 1!)59; Howatson et ul., l!KO). However, all investigations in which thin s&ions ha\~ been examined have revealed small particles of uniform density, 270-400A in diumet>er, which are mostly seen in the nuclei of cells (wit)h both in rCo and irl vitra material). Xone of the larger part,icles that have been seen was intranuclrar and all were different,iuted into an inlrer denser part, which was surrounded by a “membrane.” The SE virus was also examined after purificnt,ion by shadow-cast electron micrography (Kahler ct ul., 1959), and t,he particles, which were evidently flattened, were foul~i to have an average diameter of UOA. It is inter&iug that, our e&mate of 45XA diameter agrees with t’hat, of Kahler’s on purified virus. Howatson et al. (1960) and Banfield et al. (I!L59) have suggested t)hat the smallest particles seen in thin section may be identical with those in the purified virus and that differences in techniques rnay account for the different sizes. The appearance of the small particles iI1 the scct,ions, however,

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HORNE

suggests that they could be viral cores, for they are uniformly dense, have no apparent “membrane” or capsid, and in fact agree well with our estimate of 2788 for core size. If this is so, it is tempting to equate our capsid with the “membrane” of the cytoplasmic particles seen, for example, by Dourmashkin and Negroni (1959). Before discussing the structures observed in the present studies and their interpretation, it is necessary to refer to the structure of Tipula iridescent virus, adenovirus, and herpes simplex virus (Williams and Smith, 1958; Smith and Hills, 1959; Valentine and Hopper, 1957; Horne et al., 1959; Wildy et al., 1960). These viruses have been reported to be in the form of icosahedra having a central core, probably containing nucleic acid, surrounded by a shell or capsid of regularly arranged subunits or capsomeres.In the case of adenovirus, the particle is approximately 700A in diameter and the capsid is composed of 252 closely packed capsomeres which have a center-to-center distance of about 70A. The particle clearly shows icosahedral symmetry, and facets are seen as equilateral triangles the edgesof which comprise six capsomeres which are shared between the facets. Using the negative contrast method, a particle in the form of an icosahedron having spherical subunits will show the packing arrangement clearly when viewed in favorable orientations in the electron microscope. In Pig. 6c, a model made from 42 balls is shown viewed along one of the thirty axes of twofold symmetry, a position in which the balls forming the nearest facets will be aligned with those of the opposite side. The same model when viewed along a fivefold axis will have only the subunits at the apices in alignment owing to the axial rotation through 180” of the facets on opposite sides. Particles with elongated subunits (such as herpes virus or polyoma) will not show the icosahedral symmetry at all clearly with the negative contrast method for two reasons, which will readily be appreciated by examining the simple model shown in Fig: 6, a and b. First, it will beseen that subunits arranged along theaxes of symmetry become tilted relative to each other, and secondly, largely because of this fact, most of the subunits overlap one another, obscuring what, with spherical subunits, would be a simple pattern. For the reasons outlined above, we have not been able to solve the structures of herpes virus, polyoma virus, or a fowl orphan virus, which also has elongated capsomeres (to be published), in the straightforward way that was successful with adenovirus. With herpes and the avian virus, the particles seen had markedly angulated profiles, as did the inner margins of the capsids, and with both viruses particles were found which showed triangular facets. Moreover, with herpes a number of

Frc;. A. a anti h. TN-O views of model conxt,rtlctc4 of elongated units in t hck form of 30 hex:Lgorral and 12 peni,:Lgonal prisms arranged on the sgmmetr?; :ISW of au iros:thedron and showing radial displacement. ‘I’he appearance of the synmrtry planes is clear when the model is viewed in t hc orientation sho\vn ill a. c. A model of an iconahedron constructed from -12 Mls. ‘lb facets :II’P in t Iw form of flat equilateral triangle8 wit.h three units forming the facet, edges. d. A model of an icosahedron constructed to show- thr radial displa.c.emrnt of the symmetry xceti.

pentagonal capsomeres were seen among the hexagonal members. With polyoma virus none of these features has been seen and the disposition of the capsomeres has been detected by determining their positions relative to each ot,her on the particle (see Fig. 5, a and I,). ITig. 6d shows the

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HORNE

5 : 3 : 2 axes of symmetry projected from the center of an icosahedron, and it is clear that if hollow prisms are slipped over each of these spokes they must be tilted relative to one another. The model shown in Fig. 6, a and b was constructed on these lines, using 12 pentagonal members located on the axes of fivefold symmetry and 30 hexagonal members arranged on the twofold axes. When viewed along the twofold axes, one central hexagonal prism is seen surrounded by six neighbors and the symmetry planes are obvious. When viewed along the fivefold axes a pentagonal prism is found surrounded by five hexagonal members, but the symmetry planes are not clearly seen. The importance of close packing of the capsomeres in determining the shape of the capsid is well illustrated by this method of constructing a model, for if the prisms be closely packed at their bases we construct a body whose periphery is nearly spherical, but whose internal shape is a truncated icosahedron. If, on the other hand the prisms are not closely packed, they may be slipped over the spokes at constant radii and all vestige of the icosahedral shape disappears, though the symmetry axes remain well marked. We are not able to decide whether the capsomeres of the polyoma virion are situated at constant radii or not, though the absence of close packing would allow this. It might be argued that the capsomeres must be closely packed if a stable structure is to result, but it must be remembered that substances may exist which stain positively with phosphotungstate under the conditions used, and the capsomeres may be embedded in a matrix of this sort. What does emerge is that even though the particle may be spherical it nevertheless still shows evidence of the 5 : 3 : 2 axial symmetries predicted by Crick and Watson (1956). REFERENCES BANFIELD, W. G., DAIVE, C. J., and BRINDLEY, D. C. (1959). Intracellular and extracellular particles in tissue cultures inoculat.ed with parotid tumor agent (polyoma virus). J. IVutZ. Cancer Inst. 23, 1123-1136. BERNHARU, W., FEBVRE, H. L., and CRAMER, R. (1959). Mise en Evidence au microscopic 6lectronique d’un virus dans des cellules infectkes in vitro par l’agent du polyome. Compt. rend. acad. sci. 249, 483-485. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochim. et Riophys. Acta 34, 103-110. CRICK, F. H. C., and WATSON, J. D. (1956). Structure of small viruses. Xatwe 177, 473-475. DMOCHOWSKI, L., GREY, C. E., and MAGEE, L. A. (1959). Studies of polyoma virus. Electron microscope observations on tissue culture cells and animals infected with the virus. J. App. Physics 30, 2038.

It. It., and ?;EGRONI, (+. (1959). Identification with the electron microscope of particles associated with polyoma virus in indrlretl parotid gland tumows of C3H mice. Exptl. Cell Research 18, 573-576. 1 )I~Iz3wc’o, Ii.. and FREEMAN, (;. (1959). I’iaqur prodlic*tiofl t)?- t hc po~~om:t virus. Viroloyy 8, 396-397. I)I~I,BECCO, R., and VOOT, M. (1954). Plaque formation arltl isolation of prlrc’ lines with poliomyelitis viruses. J. Erp/l. Merl. 99, 167-182. (;IRARDI, .4. ,J. (1959). The use of fluororarhon for “rlnmasking” polyom:~ virrw hcmaggliitinin. l’iroloqy 9, 488-189. HORNE, It. W., and SAGTNGTOS, J. (1959). Elect ran microscope studies of thca tlcvclopmcrrt and structure of poliom)-elitis virus. J. Nol. Hio(. 1, X33-338. I~ORNE, I<. W., BRENSER, S., WATERSON, A. P., :tlld WII.I)Y. I’. (1959). The icw sahcdral form of an adenovirus. J. Mol. Hiol. 1, 81-86. HO~.~TSOS, .;2. F., ~I(‘CI’l.l,O(‘H, I<:. A., ~4IXEIIL4, J. l)., SI\IIN(IVIT(YI, l,., AsEI.H.\I), A. A., and H.4nr, .4. W. (1!)60). Zn vilro, in uivo and electron micbrosrope studies of a virus recovered from a C3H mmlse mammaq~ t tImor: I~rlationshil~ IO polyon~a virlls. .I. .Vat(. Pacer Zrkst. in press. IC.~III,E:R, H., Itow~, W. l’., LLOYD, B. J., and HARTLEY, .I. W. (1959). I~:lrc~~ ran microscopy of mouse parot id 1 imior (pol~~om:i) viriis. J. .Vtrf/. C’nftwr Ins/. I)OURMASHKIN,

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J,wow, .4., ANDERSON, T. F., and SACOB, P. (1959). Itrmarq~w sur lew c:tr:wtPristiqiles de la particiile viral infertiruse. .-lnu. irrsf. I’usfeu). 97, 281&2X!). M~C~I,I.O~~~I, 1~:. A., HO~ATSON, A. F., SIMINOVI~~~H, I,., AXELRAD, .4. A., :rntl FI.~M, A. W. (1959). A cytopathogenic agent from a mammary tumor in a CXH ~O\LHP that produws tllmors in Saiss mice and hwmstc~rs. .Vn/uw 188, 1535-1536. ~E(:ROSI, (:., l)OI~RI\IASHKlN, It., and (:HESTERM.IS, F‘. (‘. (1950). A “~,Ol~Olrl:l” virus derived from a mouse leukaemia. Hrit. Ned. J. II, 135+1360. HMITII, K. hl., and HILLS, G. J. (1959). Further .studies on t hc tlevrlopmrnt:~l stages of the tipula iridescent virus. d. Gen. Microbial. 21, is. STE!VVART,

S. I(;.,

Ifouu,

H.

I,:.,

(;. 15., (1!)57). The induction tumors t)y tissue cult,ure.

~:OC’HEKOI-R,

of neoplasms I’irolog!g

A.

Al.,

with

~)IOR(;HESE,

a substance

N.

(;.,

relcascd

anti

from

(~RI’BRS.

mo,w

3, 38lHOO.

J. H., nud FI.LTON, F. f 1958). The purification :tnd of viruses and virus solill)le antigens 011 c~alcirim l)hosl’h:ttcx. .I. Ge/r dlicrobiol. 19, 451-461. V.II,ENTIXE, IL C., arid HOPPER, 1’. Ii. (1957). Polyhedral shapc~ of aclenovirus particles as shown t)y electron microwop>-. .Yatrrrcx, 180, !I‘%. Wrmu, I’., I~VSSELI+ W. C., and HORSE, It. W. (1960). To he pul)lishetl. WII,LI.~M~, It. (2. and SMITH, K. RI. (1958). The polyhedral form of thcl tipIll: iridescent virus. Biochin~. el Riophgs. .I& 28, -161~-tS!). WINUCOCR, li;., and SACHS, I,. (1959). .4 plquc nssa?for the polyom:~ virus. Virology 8, 397-400.

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ration

MARSHALL,