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A study of tubes produced in plants infected with a strain of turnip yellow mosaic virus
VIROLOGY
36, 50-70
(1968)
A Study
of Tubes Strain
Produced
of Turnip
in Yellow
J. H. HITCHBORN Agricultural
Research
Council,
Vims
AND
Research
Accepted
Unit,
February
Plants
Infected
Mosaic
with
a
Virus
G. J. HILLS Huntingdon
Road,
Cambridge,
England
14, 1968
Electron microscopic examination of crude specimens made from plants infected with a necrotic strain of turnip yellow mosaic virus showed that infection led to the production of tubes as well as virus particles. These tubes, which usually appeared to occur in coaxial groups, were between 110 and 230 rnp wide and up to several microns long. With the aid of magnesium bentonit,e and differential centrifugation, almost pure preparations of the tubes could be made from the sap of infected plants. Such preparations contained mainly protein and were serologically related to the virus. Optical diffractometry and photographic integration techniques applied to electron micrographs of the tubes, revealed that they were made up of hexagonally packed, hexagonal rosettes, comparable in size to the hexamer morphological subunits of the virus. Strong evidence of a clockwise skew on the rosettes was obtained. The orientation of the hexagonal lattice was not the same on all tubes, but the variation was limited, the short axes of the hexagons comprising the lattice always lying within 15 degrees of the tube axis. Moreover, optical diffractometry of shadowed carbon replicas of the tubes revealed that in all but a small minority, the longrunning rows of hexagons of the lattice formed right-handed helices. Cones, structurally related to the tubes, were occasionally found. There was no evidence that tubes were produced in plants infected with three other strains of turnip yellow mosaic virus, but a small number of tubes about 5,5 rnp in width were seen in specimens made from pumpkins infected with wild cucumber mosaic virus.
of negatively stained t,ubes initially suggested that the fine detail was in disorder. However, the clear optical transforms which could be obt’ained from many tubes demonstrated the presence of a high degree of structural order and supplied basic information on the nature of this structure. Data obtained from optical diffraction studies also enabled photographic integration techniques (Markham et al., 1963, 1964) to be applied to electron micrographs of tubes to help in the elucidation of their structure. Some of the work described in the present paper has been briefly reported elsewhere (Hitchborn and Hills, 1967).
INTRODUCTION
During electron microscopic examination of a number of plant viruses in crude specimens (Hitchborn and Hills, 1965b), those made from plants infected with a necrotic strain of turnip yellow mosaic virus (TYRIV) were found to contain some apparently tubular bodies. They occurred very rarely and were not investigated closely at that time. Later, however, a necrotic strain was isolated which produced tubes in relatively large amounts and offered an opportunity of studying them in some detail. It is the purpose of this paper to present an account of such a study. A method was found which enabled almost pure preparations of the tubes to be obtained, and much of the work was carried out on such preparations. Direct inspection of electron micrographs
MATERIALS
AND
METHODS
The strain of virus used originated as a single necrotic local lesion on a leaf of Chinese cabbage (B~assica chine&s L.) 50
TUBES
ASSOCIATED
inoculated wit,h necrotic T‘llhIV (Dunn and Hitchborn, 196.5, 196(i), and was shown to be serologically related to Denmark TTXV. The host plant used throughout) was Chinese cabbage. Purification of t)he tubes was effected as follows. Infected leaves, from \\-hich the midribs had been removed, were ground using a pestle and mortar or were homogenized in a blender after the addition of 500 ml of 0.01 X l\lgSO, to every 100 g of tissue, and the extract was expressed through muslin. To the extract one-tnentiet,h of its volume of a suspension of magnesium bentonite (Dunn and Hitchborn, 1965) at I concentrat,ion of 10 mg/ml in 0.01 3f :\IgSOA, uxs slo\vly added wit,h rapid st,irring. The mixture was then centrifuged in a Servall RC2 centrifuge operating at about 5”, using 50.ml t,ubes in an HB swinging-bucket rot,or run for ,j minutes at 1250 rpm. The green supernntant suspension \vas removed from the centrifuge tubes by sucking, and the loose green pellets were discarded. The cycle of bentonite addition and low speed centrifugntion was repeated until the suspension \vns no longer green; t’his usually required 2-4 cycles. It was found import’ant, not to add more bentonite than was needed to bring about removal of the green material, as such addition led to partial or complete loss of the tubes. Residual bent)onite was removed by centrifuging t’he suspeIlsioIl at 7000 rpm for 20 minutes in SO-ml tubes in the SS-34 rotor of the Servall centrifuge. In a t’est, experiment, when suspensions of magnesium bentonite (0.3 mg/ml) in 0.01 111 -\lgS04 were subjected to this centrifugat’ion, optical densit,y measurement made on the supernatant indicated that all the bent’onit#e had been pelleted. Sext, the suspension, xvhich was straw-coloured and turbid, was centrifuged in 1.5.ml tubes in t,he SS-X4 rotor at 15,000 rpm for 30 minutes. The supernnt,ant) was discarded, and the colourless, gelatinous pellets \verc resuspended in 0.01 d/ phosphate buffer, pH 7.4, or in water. Finally, the centrifugation and resuspension I\-cre repeated twice more; the opalescent suspension was stored at S”. Elect,ron microscopic examination of such preparations showed t)hat they consisted mainly of tubes, contaminat,ion \vit’h virus particles being relnt,ively low (I’&. 2). Yields were of t,he
WITH
TYMV
in L’ivo
51
order of 10 mg per 100 g of tissue. In the Spinco Model E analytical ultracentrifuge, suspensions at a concent’ration of about 1 mg/ml produced a single, rapidly flattening schlieren peak representing material sedimeriting at’ a mean S of about 300 S. No peaks due to virus or its top component were observed. Virus was prepared from infected plants, using bentonit,e and differential centrifugation as described by Dunn and Hit,chborn (196.5). Estimation of amounts was based on ultraviolet, spectra traced in a Cary Model 14 recording spect)rophotometer. Serological tests were of the precipitation type and were made in tubes at room temperature. Two elect,ron microscopes were used, a Siemens Elmiskop IA and an A.E.I. E.M.GB. They a-ere calibrated by phot’ographing and measuring the (20 T) lat’ticeplanes of crystals of platinum phthalocyanine, as described by Markham et al. (1964). The normal practice in this laboratory has been t,o calibrate the microscope every few weeks by t’his means. However, during the present work there was evidence t’hat a relatively large variation in the magnification given by the Siemens Elmiskop IA could occur over a short period of time. For instance, with the microscope set, to give a nominal magnification of 60,000 times, measurement of six crystals phot)ographed on separate plates indicated t,hat t,he actual magnification was bet]ween 63,600 and 64,200 times. When, after 48 hours, another six crystals were photographed and measured,the microscopemagnified between 3,100 and 61,,500times. Therefore, whenever it was appropriate during the present work, immediately prior to phot,ographing TYMV t,ubes, platinum phthalocyanine crystals were photographed for the purpose of recalibrat,ing the microscope. Crude negatively stained specimens were made as described by Hitchborn and Hills (1965b), and purified preparations were examined stained with a 2% solution of potassium phosphotungstate at pH 6.4, or saturated solutions of uranyl acet)ate or uranyl formate at’ about pH 4. Carbon replicas were prepared by drying down a suspensionof t)ubes onto a Formvar-coated grid and shadowing with gold-palladium at a low angle. The shadowed specimenswere
52
HITCHBORK
then coated with carbon from an evaporating source immediately above them, and the Formvar substrate was subsequentfly dissolved away with chloroform. Finally, the specimens were soaked for 10 minutes in a concentrated aqueous solut’ion of sodium hydroxide to digest the tubes and then washed well in water.
FIG. 1. Electron micrographs of groups uranyl acetate. “Full” (VF) and “empty” associated with tubes being “empty.”
AND
HILLS
Tissue and pelleted t,ubes were prepared for sectioning by fixation in a 2 %’ solution of glutaraldehyde in 0.05 M cacodylate buffer, pH '7.2 (Gordon et al., 1963) and leaving for 2% hours in a saturated solution of uranyl acetate in a water-et’hanol mixture (1: 1, v/v). The material was then transferred to solut,ions containing progressively higher con-
of TYMV tubes in crude specimens negatively stained with (VE) virus particles can also be seen, most of the particles
TUBES
FIG. 2. Electron
ASSOCIATED
WITH
micrograph
of a purified
centrations of ethanol and left overnight in a saturated solution of uranyl acetjatjc in absolute ethanol. It, \vas then washed in 1-Z epoxypropane (Luft, 1961) and embedded in resin (Fin&, 1960). *An LIMB microtome was used to cut, sections which were stained with lead acetat,e (~Iillonig, 1961) before electron microscopic examination. The optical diffractometer used was t)he one described by Bancroft et al. (1967) except t,hat it incorporated an alternative light source consisting of a Spectra-Physics Model 130B gas laser. Electron micrographs of platinum phthalocyanine crystals were used to calibrate the diffractomet,er. One member of each family of spots in all transforms presemed in figures has hccn numbered so that equivalent spots in different transforms cm be identified. The basic apparatus and procedure used in linear integration \vere t,hose employed by l\larkham et al. (1964). For the present, work two modifications t)o the original apparatus \vere made. I;irst,ly;, the photograph under investigation was
TYMV
in
preparat,ion
Vim
of tubes.
illuminated from underneath and VW in the form of a transparency on thick film. Secondly, this film was attached to a rigid transparent sheet lying flat on the sliding table, t)he sheet and attached photograph being capable of rotation through a measurable angle. Rotational integration was carried out as described by JIarkham of al. (19K~). RESULTS
In crude electron microscope specimens made from plants infected with the necrotic strain of TYJIV, the tubes, which were presumably flattened, appeared as shown in I’ig. 1. Two aspects of their gross morphology were consistent with the view t)hat t’hey lvere, in fact, tubular in construction. l~irstly, they had parallcl sides, and secondly, t,hesc sides oft)en showed in relatively high conk&, suggesting that there was a greater deijth of material here than elsewhere. These features sho\ved pnrticu-
54
micrographs I PIGS. 3 and 4. Electron purified tubes. Fig. 4. Leaf 3. 1 ‘elleted ing tubes mainly in groups (2’).
HITCHBORN
AND
HILLS
of sections. Stains used: tissue of Chinese cabbage
uranyl acetate infected with
and lead acetate. Fig. necrotic TYMV, sl
TUBES
ASSOCIATED
larly clearly in purified preparations (Fig. 2). Firm evidence that the structures were tubular was obtained by electron microscopic examination of sect’ioned pellets of the pure, fixed material. Such sections appeared to have been made through randomly orientat#ed tubes (Fig. 3). Two properties of the tubes in crude electron microscope specimens are of interest. Firstly, many were present in characteristic aggregates. These probably originated in the plant’ and not during specimen preparation, for sectioned infected tissue contained what appeared to be coaxial groups of tubes (Fig. 4). Secondly, t#hey showed a preferent’ial association with “empty” virus particles (Fig. I). Of about 400 particles lying under, over, or in, 40 randomly chosen groups of tubes, 54 % were “empt,y” and the rest “full.” In cont’mst, among the particles examined which were not associated with t’uhes, only 15% were “empt’y,” a figure corresponding roughly with that arrived at from measurement of the areas under the schlieren peaks produced by the two components during analytical ult,racentrifugation of sap of infected plants. Measurement of a large number of t#ubes in negatively stained electron microscope specimens made from sap which had been part,ially clarified with the aid of bentonit,e
55
II0 TUBE
WCMV
TUBES
TYMV
TUBES
165 WIDTH
220
2%
(my)
FIG. 5. Histogram showing the frequency distribution of wild cucumber mosaic virus (WCMT’) and TYMT’ tubes of different, widths in negatively stained crude electron microscope specimens.
WITH
TYMV
in
V&J
3.5
FIG. 6. Schlieren pattern produced during centrifugat,ion of sap from a plant infected with necrotic TYMT-. Sedimentation was from left to right, and the photograph was taken approsimately 16 minutes after a speed of 24,630 rpm had been reached. P, plant protein; VT, “top” component of virus; K, ribosomes; VB, “bottom” component, of virus; T, tubes.
showed t,hat they varied in width between 110 and 250 rnp (l;ig. 5) and were up to 3 p long. A variation in width of his magnitude is much too great to be accounted for by different, degrees of flattening of tubes of equal diameter and it, must he concluded, therefore, that the t,ubes vary in diameter. Assuming complete flattening, the tubes present would have had diameters of bet,ween 70 and 160 mp. In t,he analytical ult,racentrifuge sap from infect)ed plants produced, in addition to schlieren peaks representing normal plant constituent’s and “full” and “empty” particles of the virus, a flat peak due to polydisperse material \\-ith a mean sedimentation rate of about, 1.5 times that of the “full” particles of the virus, indicatJing a mean Stow of about 180 S (Fig. 6). This presumabl!
56
HITCHBORN
AND
HILLS
represented the tubes, for no such peak was produced on centrifuging sap from healthy Chinese cabbage or sap from plants infected with several other strains of TYRIV. The fact that the material in purified preparations of tubes had a higher mean S suggests that much low X tubular material had been discarded during purification. In some samples of sap, a series of small, sharp peaks could be seen superimposed on the trailing edge of the flat peak, as in Fig. 6. These may have been artifacts or each may have represent#ed sedimenting tubes of preferred size. Purified suspensions, examined in the elect’ron microscope immediately after preparation, could be seen to contain mainly well preserved tubes (Fig. 2). After storage overnight in 0.01 M phosphate buffer pH 6.2-7.5, alt,hough preparations were still opalescent and contained mainly material sedimenting at about 300 S, electron microscope specimens made from them usually cont,ained stringy, disorganized masses of material and very few identifiable tubes. It seems, therefore, that tubes stored under these conditjions remained intact but became
FIG. 8. Tubes (A) (R) present in crude, microscope specimens fected with WC&IV.
0.2
O-1
0 240
260
280
WAVELENGTH
300
320
@‘y)
FIG. 7. Ultraviolet absorption spectrum of a purified preparation of tubes in water. A, imme,diately after purification; B, after storage overnight at 8”.
and double-shelled particles negatively stained electron made from pumpkins in-
suscept’ible to disint,egrat’ion on preparation for t’he electron microscope. Suspensions stored overnight in distilled water appeared to contain largely fiuely broken down material for they were clear or nearly so, and most of the material detectable in the analytical ultracentrifuge sedimented at about 7 S. The appearance of such preparations in the elect,ron microscope was similar to that of stored buffered suspensions. The ult’raviolet absorption spectrum of a suspension of tubes in water, measured
TUBES
FIG. optiral
9. (A) Electron micrograph transform of the ringed part long arrow. The transform
ASSOCIATED
of a TYM\of the tube,
WITH
TYNV
in
Vizw
tube negatively stained with the relative direction of the
uranyl
acetate.
(B)
An
tltbe axis being indicated on hexagonal lattices and of spot,s given by the detail of the hexagotlal lattices photograph althoi&r they
is made up of two sets of spots arranged by t,he represents detail on both sides of the flatterled tribe. One spot of each family on one side has the same labelling as the equivalent spots on the transforms show:1 in Fig. 11. Several of the spots equivalent to 4tt do tlot appear in the could be seen in t,he diffractometer. 1lagnifiratioll of electrorl mic:rograph: X 150,000. immediately after preparation and aft,er storage overnight at 8” are given in l;ig. 7. The change in the spectrum xvith time is further evidence that most of t,he tubes disintegrat.e on storage in water. The spectrum of t*he suspension of tubes after storage \vas closel> similar to that of the “top” component of TT;\IV (Alarkham and Smith, 1949) and had an absorption maximum at, 273 mp and a minimum at, 2.3 rng. If t,he pH of aqueous suspensions of virus or tubes ~-as louvered by adding an equal volume of 0.15 d1 citric acid-sodium phosphate buffer at pH Z.Gci.0, both precipitatccl, but over different pH ranges. Whereas virus precipitated most rapidly at pH 3.2 and mithin 24 hours at. pH 2.4~33, the most rapid precipitation of tubes 1v-a~ at pH 4.4, and t’he range over I\-hich precipitation tool; place xvithin 24 hours \vas pH R.S-5.2. A clear serological relationship betnccn tubes and virus ~vas demonskated. Tests carried out. by mixing antiserum prepared against purified necrotic TIYIV Iyith a
fresh preparation of tubes in water, resulted in the immediate development of turbidity Tvhich n-as rapidly folio\\-ed by flocculation. When t,his test \vns carried out. on some of the same preparation of tubes stored overnight, at 8” there n-as no immediate visible reaction, a slight precipitate forming after 24 hours. This change in response to the addition of ant’iserum probably reflects the breakdo\vn of the tubes int,o small units on storage. There was no reaction \\-hen normal serum was used in&ad of ant,iserurn in these tests. Amino acid analysis of HC1-hydrol>~zatjes of purified preparations of tubes and of the protein extracted from purified necrotic strain TTAIV, 1%.a.scarried out in :I Beckman Spinco amino acid analyzer (Spackmnn et al., 1958). Results shoxved that the tubes consisted largely or entirely of a prot,ein having an amino acid composition lyhich ~vas closely similar or identical to that of the virus protein. Electron microscopic examination of crude
5s
HITCHBORN
extracts from plants infected with the Denmark, Cauliflower, and Type strains (Symons et al., 1963) of TYMV was made without tubes being found. However, pumpkins (Cucwbita pepo L.) infected with wild cucumber mosaic virus (WCXIV), which has a number of properties in common with TYJZV (Tamazaki and Kaesberg, 1961; Mncleod and Jlarkham, 1963), did occas-
AND
HILLS
ionally contain tubelike bodies. Only a limited study of these was possible because of the infrequency with which they occurred. However, the fifty or so observed had characteristic wavy outlines, were narrower and much more uniform in width than the TYMV tubes (Fig. 5), and were similar in diameter to the large shells sometimes seen enclosing empty wcnm particles (Fig. S).
FIG. 10. (A) Electron micrograph of a single sheet of hexagonally packed hexagonal rosettes present among tubes and virus particles in a negatively stained crude specimen made from a plant infected with necrotic TYMV. The direction in which short axes of the rosettes lie is indicated by the arrow in B, which is an optical transform, in equivalent orientation, of a similar sheet. The system of labelling the transform spots corresponds to that used in Figs. 9 and 11. The position of spot 4b is marked, but the family of spots it represents cannot be seen in the photograph. (C) Selected virus particles showing morphological subrlnits. Magnification of electron micrographs: X 400,000.
FIG. 11. Models representing the negatively stained fine struct,ure of a ‘l’LII\~ t,ttbe (A and C) and their optical transforms (B and I>, respectively). (A) A network of hexagons with central dots, representing staiu both between rosettes and in their centres. (C) A network representing rosettes with unstained centres. Features on the models have been numbered f-4 in order of decreasing size of repeat interval, and, as a means of identification, one of the two transform spots produced by each has the same numbering.
Firze Structure
Careful examinat’ion of electron micrographs of negatively stained TYMV tubes sho\ved the presenceof sets of evenly spaced faint’ straight lines running across and along the tubes, and suggested t,hat regularly repeating fine detail had been preserved and resolved. Proof of this came from the strikingly clear optical t,ransforms obtainable from many elect,ron micrographs (Fig. 9). Typically, such t,ransforms consisted of two nearly identical set’s of spots arranged on
hexagonal lattices superimposed on one another at an angle. A double pattern of this kind suggestedbhat t’he tubes were helical in construction and had both sides visualized in the electron microscope (Hug and Rerger, 1964; Finch et al., 1964). At least part of the confused appearance of the tubes would, therefore, appear to be at’tributable to superimposition, out’ of register, of images of the two sides. Occasionally sheets of material \vere seen which gave optical transforms consisting of single set,s of spots (Ij’ig. 10
60
HITCHBORN
A, B). These presumably consisted of singlelayered pieces of material similar to that making up the tubes and appeared to be construct,ed of hexagonally packed hexagonal rosettes resembling the hexamer morphological subunits of the virus particles (Fig. 10 C). The centre-to-centre dist’ance bet,ween adjacent rosettes was about SS A, and in Fig. 10 they can ‘be seen t’o be orientated with short axes lying in the direction indicated by the arrow. By comparing the transforms 20-
ml I IS’ ANGLE
BETWEEN
LATTICE
AND AXIS OF TUBE
FIG. 12. Histogram showing the frequency distribution of different angles between lattice and tube axis among TYRITtubes in negatively specimens. St ained
AND
HILLS
of this material wit’h that of t’he tube shown in Fig. 9, it can be deduced that the rosettes making up the tube would lie with their short, axes within a fen- degrees of the tube axis. Experimentation with models showed that an optical transform basically similar to that obtained from the single layered material was given by a stain pattern consisting of a honeycomb of hexagons, each hexagon containing a central dot (Fig. 11 A, B). The spacing labelled 2 is equal to half the centreto-cent,re distance between adjacent rosettes. The transform of a model consisting of a honeycomb of hexagons devoid of central dots has additional st’rong spots (Fig. 11 C, U). A similar transform was obtained from a model in which only about half of the hexagons were devoid of central dots, but the diffract(ion spot,s 1 and 3 were then of about equal intensity. Of the transforms given by models, this was t,he one which most nearly resembled those obtained from tubes, and it is concluded that in these structures a proportion of t,he rosettes have centres unpenetrated by the stain. From measurement of the transforms of a large number of tubes, it,
FIG. 13. (A) Ele:tron micrograph of shadowed carbon replicas of TYMTtribes. (B) A single replica of a-hich the optical transform (C) was obtained after plazing a mask around the outlined re:tangular area, which lies with its long axis parallel to that of the tube. The transform of the mask forms a cross and enables the relative orientation of the transform of the tltbe and t,he trtbe axis to be determined. The direction of the tube axis relative to the transform is indicated by the long arrow. (A) X 41,000; (B) x 80,000.
TUBES
ASSOCIATED
was calculated that the average centre-tocentre distance between roset’tes was 90 A, which is near the figure for the dist,ance between the centers of rosettes in the single sheet’ material, arrived at from measurement of electron micrographs. This figure differs slightly from that published earlier (Hitchborn and Hills, 1967), which n-as based on only a few measurements. The angle bet,\\-een the hexagonal lattices containing the two sets of spots making up the diffraction pat’terns of the upper and lower surfaces of a negatively stained tube cn11 be easily and accurately measured. This provides a convenient way of finding the angle at which each lattice is set to the axis of the tube. From measurement made OII the transforms of a large number of tubes, the histogram shown in b’ig. 12 was drawn. It show-s that the lattice angle did not exceed 15 degrees and hints at the possible existence of peaks between 0 and 7 degrees. The peak at 0 degrees may be artificial and result from the difficulty of distinguishing between pat’terns which are superimposed in register and hhose of&et by a degree or two. As there is no means of relating each of the t,\vo sets of transform spots produced by a negatively stained tube to the side of the tube lvhich produced it,, t)he sense of the slope on the lattice of such a tube cannot, be determined. This uxs investigated by diffractometry of carbon replicas, which represented only the structure on the upper surface of tubes. Dexpitc the unpromising appearance of such rc~plic:r~ many gave clear diffrnction patterns
FIG. 14. Histogram showing t,he frequency distribut.ion of different angles between lattice and tube axis among shadowed carbon replicas of T17VIV tubes. The signs + and - define the sense of the helices formed by the lollg-rllnning rows of hexagons of a 1st tice, + indicating that the helires are right-handed and - that they are left-handetl.
WITH
TYMV
in
61
T’il,o
. l
I
I
I
0
50
100 TUBE
FIG. 15. Tube tween structural
width lattice
l
I
150 WIDTH
200
I
250
hv)
plotted against angle and axis of tube.
be-
consisting of a single hexagon of spots, each analogous to that, labeled 1 in Figs. 9 B and 11 D and representing structure repeating at 78 A (Fig. 13). The angle at which the lattice lay relative to the tube axis and the sense of the slope of the lattice were assessed by enclosing an electron micrograph of a replica in a rectangular mask, positioned so that the long axis of the mask was parallel to that of the tube (Fig. IS B), and examining the masked replica in the diffractomet,er. The direction of the tube axis was determined from the transform of the mask, although as t,he placing of the mask was subject to t,he possibilit,y of slight error, the measurement of the latt’ice angle by this means would not be expected to be highly accurat’e. The result of measuring this feature on a number of replicas is given in Fig. 14. The variation in lat,tice angle was similar to that found among negatively stained tubes, and, in all but, four, the la&es were orientated so that t,he long running rows of rosettes either ran parallel to the axis or formed right-handed helices. In order to test the possibilit’y that lattice angle and width of tube were related, both measurements were made on a number of negatively stained tubes. The results obtained gave no evidence of a simple relationship between the tn-o feat’ures (Fig. 15). When electron micrographs of the aggregat’es present in crude preparations were examined in the diffractometer t’hey gave
62
HITCHBORN
transforms which appeared to arise from the superimposition of almost identical patterns, suggesting that the successive layers of material making up each aggregate were structurally related. Figure 16 shows t,he transforms of two aggregat,es which have lattices intersecting the axes of the aggregates at angles of 4.5 and 12.5 degrees, respectively. As shown in Fig. 12, an angle of intersect,ion of 12.5 degrees is unusually large and makes it highly improbable that the components of the aggregate have this feat ,ure in common by chance.
AND
HILLS
Using the information obtained from the diffractometry of electron micrographs of t’ubes as a basis, it was possible to employ the technique of linear integration t,o obtain patterns representing the ordered structure present. The procedure adopt’ed was to prepare a photograph of part of a tube at a magnification of about 2,000,OOO times (lcig. 17B) and to secure it to the integration bable with the axis of the tube lying in t’he direction of motion of the table. The t,ransparent sheet t,o which the photograph n-as &ached was then rot’ated througrh that angle which
FIG. 16. Electron micrographs of two groups of tubes and by the side of each the optical diffraction pattern given by the area marked. Only spots equivalent to that labelled 2 in Fig. 9 show. The tubes in group A have lattices set at an angle of 4.5 degrees to the long axis of the group and those in group B at an angle of 12.5 degrees. Magnification of electron micrographs: X 102,000.
TUBES
ASSOCIATED
WITH
TYMV
in
Vitlo
63
FIG. li. Linear integration of an electron micrograph of a tube. (A) Electron micrograph of a negati vely stained tube, with the area for integrat,ion outlined. (B) This area on a transparency at about a used in integration. (C) Photograph obt.ained by integrating B at 7 degrees to th lird of the magnification equivalent to 90 A on the original specimen. It represents repeating structh he tube axis and at int.ervals tu re present on one side of the tube and running at 7 degrees to the tube axis. Y indicates the directil 311 of the tube axis; and X, the direction in which integration took place. (A) X 200,000; (R and C) X 620,000.
64 an optical transform would bring rows of one side of the tube in of motion of the t’able. graph was then taken consecutive exposures,
HITCHBORN
of the Oube indicated structural rosettes on line with the direction An integrated photoby making about 25 the distance through
AND
HILLS
which the t’able was moved between one exposure and t’le next being equivalent’ to 90 A. One photograph obt,ained in this way is shown in Vig. 17 C. The supression of bot)h “noise” and the structure of one side of the tube has revealed the main repeating
FIG. 18. Integration of an electron micrograph of a TYMG tube at various angles to the tube axis. The integration interval was constant and corresponded to 90 A on the original specimen. The integrated photographs were taken after the axis of the tube had been rotated, by the anglesshown, clockwise (-) or anticlockwise (f), away from the direction of motion of the integration table. The regular patterns obtained with the axis in positions -7 and +6 degrees represent repeating structure on the two surfaces of the tube. X 455,000.
FIG. 19. Pattern obtained from an electron micrograph of a TYMV tube using a combination and rotational integration. The individual hexagonal rosettes forming the pattern are skew regions of high density at the vertices.
of linear and show
TUBES
ASSOCIATED
features on the other side. By bringing t’he equivalent rows of rosettes on the reverse side of the tube in line with the direction of motion of the table, the pattern made by t,hese could also be photographed. A series of integrat,ion photographs taken with t’he axis of :I tube rotated through 10 degrees clockwise (-) relative to the direction of motion of the t,able, 10 degrees anticlocknisc (+), and at l-degree intervals bet)t\-een these t)n-o extremes, showed that greatest’ regularity in overall pattern was produced when the axis was in positions +6 and -7 degrees. In positiow more than 1 degree on either side
WITH
TYMV
in
Viva
05
of these, no regularit,y \vas apparent. l’art of this series of photographs is she\\-n in l:ig. 1% The angle of intersection of equivalent1 rows of rosettes on the upper and loivcr sides of the tube, measured in this \vay, is l:$ degrees, and is very close to the angle (13.5 degrees) by \\-hich the diffraction pattjerns of the upper and lower surfaces of the tube jvcrc rot’at’ed with respect’ t#oone another. Some of t’he integrated pictures suggested t)hat’ substructure OII the hexagonal rosettjos \V:LG resolved, although poorly. Attempts \\-we mwdc in two \vnys to clarify this detail. One involved a second linear ‘integration of
FIG. 20. A4 and C are models representing hexagonally packed rosettes of six subunits, arranged so that, in -4, the rosettes are placed symmetrically in the rows and, in C, they are skew. The transforms of these models are shown in B and D, respectively. In the transform of the model with symmetrically arranged rosettes, spots ,$a and 4b are of equal intensity (B); in the transform of the model with skew rosettes, these spots are of unequal intensit.y (I)).
66
HITCHBORN
the pictures at 60 degrees to the direction in which the original integration t,ook place. The impression conveyed by photographs obt’ained in this way was that each hexagonal rosette was composed of six subunits, one being located at each vertex. It also appeared that t’he roset>tes were arranged slightly skew relative to the direction in which the rows ran. The second technique used t,o ext,ract’ further information from the once-integrated picture was that of rotational integration, as described by Markham
AND
HILLS
et al. (1963). The rotation was made about the cent,re of one of the rosettes, and the angle through which t,he rotation table n-as turned between exposures was 60 degrees. As n-it’h the double linear integration photographs there were areas of high density at the vertices of the rosettes, which again appeared skew (Fig. 19). In view of the foregoing evidence that the rosettes were skew it n-as relevant to compare optical transforms of two models, both constructed of hexagonal clust)ers of dots
FIG. 21. A and B are diagrammatic representations of optical diffraction patterns of the two surfaces of a TYMV tube negatively stained with uranyl acetate. The spots have the same labelling as the equivalent spots on the transform shown in Fig. 9. Patterns A and B have object-mirror image relationship and differ from each other as a result of the difference in intensity between spots 4a and 4b. Patterns C and II were made by superimposing A and B, and represent the two hypothetically possible forms of the transform of both sides of a tube having a structural lattice inclined at about 5 degrees to the long axis of the tube, the direction of which is indicated by the arrows. Among the transforms of tubes examined, only those of t,ype C were seen (e.g., Fig. 9).
TUBES
ASSOCIATED
arrayed hexagonally, but with the clusters of one arranged symmetrically relative to the rows, and the other having skew clusters (I:ig. 20). These gave broadly similar dif-
WITH
TYI\IJ’
in T’iw
6’7
fraction patt,erns but the relative intensity of the spots in t,he two was different. What, is of greatest, significance for the present purpose is that,, in the transform of the model having symmetrically arranged rosettes, the members
of t,he two
families
of spotIs rcpre-
sented by /ta and db are of equal intensity, Tvhereasin t,he model having skew rosettes t,hesespots are of unequal intensity. If, as in Fig. 20 C, t,he ros&tes have :I slight, clockwise skew, the more int’ensc spot, da, can be seen to be on t,he right of /,b. In :I transform of a model with rosettes skewed ant)iclockwise, the brighter spot was on t’he left. Careful inspection of a large number of transforms of tubes and of pieces of singlelayer material revealed the presence on some of the clearest), of spots equivalent t#o /ta and ,$b, and of unequal intensity (Figs.
9 and 10). This is substantial additional evidence that the roset,tes are skew. As the diffraction patterns produced by the two sides of a tube have an objectmirror
image
relationship,
the
element
of
asymmet,ry in t,he ring of spots labclled 4 will lead to t,he two patterns being different at, this radius, in the manner shown diagrammatically
in Fig. 21 A, B. This
meaus
t,hat t,wo dist)inct superimposition patt’erns (Fig. 21 C, D) are possible. I’ntt’ertt C is produced n-hen the senseof the slteu- on t,he rosettes is the same as t,he seuse of t’he helix formed by the long-running rows of rosettes,
and pattern D when these sensesare opposite. Of the 2:3t8ransformsin \I-hich the relevant, outer spots could be seen, all were of type C (as in Fig. 9). This indicates that in the majority of tubes, in which the structural latkice the rosett,es
Flo. 22. Structures of triangular outline present in preparations of TYMV tubes. They have apical angles of A, 30 degrees; B, 60 degrees and C, 90 degrees, and are probably flattened cones having an essential similarity to the tubes in being const,ructed on the basis of the same hexagonal lattice.
slopes to the right, is clockwise.
t#he skew on
Colzes. Most preparations of tubes cow tained a small proport’ion of triangular structures which gave diffraction pnt’t’erns of the samekind as those given by tubes. If, as seemslikely, such bodies were flat’tened cones they would be expected to exist) in a limit#ed number of well d&ted categories. This follows from the fact that a hexagonal lat8tice cart cover without discontinuity only the surfaces of cones which, when flat’t,ened, appear
as triangles
having
apical
angles
of
30, 60, 90, 120, or 150 degrees. Ittspection of
6s
HITCHBORN
electron micrographs of preparations of tubes revealed that the majority of the triangular bodies present did, indeed, fall precisely int’o several of these categories. The most’ commonly occurring were t,hose having apical angles of 30 or 60 degrees, and represent,atives of the 90 degree class were also encountered (Fig. 22). Examples of those Tvith the widest, apical angles were not identified with certainty, but this may simply reflect the improbability of such struct’ures flattening to form triangles. The diffraction patt’erns given by the cones included indication that the rosett,es of which t’hey were composed were skew. There was, however, no evidence of any limitat’ion on the angle of intersection between the hexagonal lattices of the upper and lower surfaces. Such limitation would not’ be expected, for, with a cone, the angle of intersection will depend only on where the surface folds on flattening.
AND
HILLS
are relat,ed t’o the large spheres which this virus produces. However, the tubes produced by TYMV are very variable in size, even the narrowest being much greater in diameter than the virus particles. This seemst,o preclude the possibilit? that they arise as the result of a simple mlst,ake in the construction of virus particles (Caspar and Klug, 1962). In common with T4 polyheads (Icavre et al., 1965; Kellenberger and Boy de la Tour, 1965)) TYS IV tubes are built’ on a somewhat flexible plan which allows variation in structure and with TTJIV even permits the construction of conical forms. However, there appears to be a definit’e difference bet’ween t’he polyhead tubes and those of TTMV in the degree of constructional freedom shown. From Moir6 analyses of polyhead tubes, Kellenberger and Boy de la Tour (1965) were able to show that, there was a great variation in the orientation of the hexagonal lattice in different tubes, DISCUSSION Although t’he absence of a preferential orientation could not be demonstrated beThe morphological, serological, and chemicauseof the small number of tubes amenable cal evidence given in the present paper to analysis, t’hey did encounter examples of supports the view that bot’h t’he virus parpolyheads with lattice pitches near to 30 t’icles of necrotic T‘lXV and the associated and 0 degrees, t,hat is, with the hexagons of tubes are composed of the same prot,ein the lattice having long and short axes, subunits. There is circumstant’ial evidence respectively, nearly parallel to the t’ube axis. t,hat the prot,ein of a number of other viruses In contrast’, the large number of TYRIV can assemble to form either spheres or tubes, tubes we examined only showed a variation for both types of st’ructure, sometimes in lattice angle between 0 and 15 degrees, clearly composed of morphologically similar subunits, have been found in extract’s of and about 80% had lattices set ai S degrees or lessto the axis. Moreover, in all but a few tissue infected with polyoma (Howatson of the tubes examined, the lattices were inand Almeida, 1960)) papilloma (Williams clined so that the long-running row of et aZ., 1960; E’inch and Klug, 1965), human rosettes formed right-handed helices. This wart (Noyes, 1964), and FV 1 (Darlington limitation in construction is of interest beet al., 1966). With cow-pea chlorotic mottle cause it runs counter to the expectation that’ virus the evidence is even stronger, for in the orientation of the lattice would be ranvitro conditions have been found (Bancroft dom (Kellenberger and Boy de la Tour, et al., 1967) under which t’he spherical virus particles will disrupt and their substituents 1965). As the “left-handed” tubes formed recombine to form tubes. These viruses such a small minority it, seemsworthwhile to appear to produce t’ubes of one or two sizes consider in some detail t’he possibility that only, each having a diameter comparable they were artifact,s. A “right-handed” tube with that, of an associated spherical part’icle will appear “left-handed” through incorrect and each capable of forming a hemispherical placing of either t’he specimen in the electron microscope or the electron microscope plate cap. The tubes found associated with WCMV in crude ext,racts during the present in the diffractometer. Both t,hese possible work are of a uniformity and size which explanations are ruled out by the fact, that make it reasonable to postulat’e that they the plates containing the “left-handed”
TUBES
ASSOCIATED
tubes also contained “right-handed” ones the p&es were positioned the same \va,v round in the diffractometer. The method of est’imating the tube axis would allow error in measuring t#he slope of the lattice but, Jvhcrc the replicas appeared to he prccis:cly parallel-sided, t,his error \vould he expected to he lirnikd to a degree or two. The sides of the three replicas \vith “left-handed” I:ltticcs set) at 4 degrees or more to the axis appeared to he slightlJ~ out of parallel, but this does not affect the present’ issue, for t,hey appeared “left,-handed” regttrdless of \\-hich of their sides n-as set parallel to the axis of the mask. It) is possible that these replicas are of approximately rectangular pieces of the single sheet material encountered occasionally in negatively stained prcparatjions. Ho\\-ever, the shadows along the edges of the replicas do not suggest that t,he material thcv represent was unusually thin. It seems difficult, therefore, to escape the conclusion that’ a very small proportion of the tubes are, in fact, “left-handed.” Inv&igation into the st,ructure of particlcs of broad bean mottle virus has led to the hypothesis that, the morphological subunits are skew, I\-hile parallel observations on TT1IV I\-ere consistent with the view that the subunit,s of this virus are symmetricall\ arranged on the particles (lcinch and Mug, 1967). Our results suggest that this co~xlusion may not. apply to at, least, the necrotic strain of TIT1\IV, for if the hexamer morphological subunits of the virus part’icles are identical \vith the tube roset,tes, for xvhich there is strong evidence, then, as these roset,tes appear t,o be arranged skew, it seems likely that the hexamers in the virus particles n-ould be skew also. Electron microscopic examination of both ncgat ivel) stained crude specimens and sections of infected tissue seems to indicate that in ukw man>- of t’he T\;MV t,ubes occur in \vhat appear to be coaxial groups, the diflkaction patterns given by which suggest, that each contains structurally related tubes. A possible explanation for this is that, OIW tube cm act as a former for the construction of another which thereby has imposed on it’ the structural features of t’he first. However, t,his is not a complet,ely satisfact,ory hypothcI\-1~11
WITH
TYl\l\-
in, Vito
69
sis for, if true, it seems likely that the tubes in a group u-ould be closely applied t)o one another, Tl-hereas they usually appear to be separakd by an appreciable gap. Albernatively, the structural homogeneity of the few complexes examined \2-hich gave clear transforms could be due to each being, not, a series of tubes, but a rolled-up single sheet. ACKSOWLEIXIZIESTS We wish to thank Professor It. Markham for helpful discllssion and Mr. RI. W. Rees for carrying ollt the amino acid analyses. We are also grateful for the technical assistauce of nIiss Y. 11. Carter, Miss A. l’laskitt, and 1Ir. S. Frey. Sectioning was carried out on a microtome generously donat,ed by the Wellcome Folmdation. REFERENCES B.\NCHOFT, J. B., ITILLS, G. J., and ~\LIRKH.~M, R. (1967). A study of t,he self-assembly process in a small spheriral virlls. Formation of organized structures from protein suhlurits in oilro. Virology 32, 354-379. CMP.~ I). L. I>., and KllLg, A. (1962). Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. &ant. Biol. 27, l-24. DARLINGTOIL’, R. W., GIL.\NOFF, A., and B~xzI,:, I). C. (1966). Viruses and renal carcinoma of Runa pipienx. II. Ultrastructural studies and sequential development of virus isolated from normal and tumor tissue. Virology 29, 149-156. L)CNN, 1). B., and IIITCHIWRN, J. H. (1965). The use of bentonite in the prlrification of plant virllses. ViTology 25, 171-192. DUNN, I). B., and HITCHIWRX, J. IT. (1966). The extraction of infectious, high molecular weight RNA from turnip yellow mosaic virus with ethanol. Virology 30, 598607. FAVRIC, It., Boy DI: LA TonIt, E., SEGR$:, N., and K~:LLI:NI~I.:ROI~R, E. (1963). Studies on the morphopoiesis of the head of phage Y-even. 1. Morphological, immunological, and genetic characterization of polyheads. .T. TJZtrastruct. Res. 13, 318-342. FINCH, J. T., and KLUG, A. (196.5). The structure of virllses of the papilloma-polyoma type. III. Htructllre of rabbit papilloma virus. 1. Mol. Biol. 13, I-12. FINCH, J. T., and KLVG, A. (1967). Struct,ure of broad bean mottle virus. I. Analysis of elect,ron micrographs and comparison with turnip yellow mosaic virus and its top component. J. Mol. Biol. 24,289-302. FINCH, J. T., KLUG, A., and STRETTON, A. 0. W. (1964). The structure of the “polyheads” of T4 bacteriophage. J. Mol. Biol. 10, 570~.i7A.
70
HITCHBORN
FJNCK, H. (1960).
Epoxy resins in electron microsJ. Biophys. Biochem. Cytol. 7, 27-30. GORDON, G. B., MILLEIL, L. R., and BENSCH, K. G. (1963). Fixation of tissue culture cells for ultrastructural cytochemistry. E’xptl. Cell. Rea. 31, copy.
440-443. IIITCHNRN,
J. H., and HILLS, G. J. (1965a). Electron microscopic examination of wild cucumber mosaic virus in negatively stained crltde preparations. Virology 26, 756-758. HITCHBORN, J. FT., and HILLS, G. J. (196513). The use of negative staining in the electron microscopir examination of plant viruses in crude extracts. Virology 27, 528-540. HITCHBORN, J. H., and HILLS, G. J. (1967). Tubular structures associated with t,llrnip yellow mosaic virus in viva. Science 137, 705-706. HOWATSON, A. F., and ALMEID,~, J. 1). (1960). Observations on the fine structllre of polyoma virus. J. Biophys. Biochem. Cytol. 8, 828-834. KELLENBERGI~ZR, E., and BOY DE 1,s TOCR, E. (196.5). St,ltdies on the morphopoiesis of the head of phage T-even. II. Observations on the fine structure of polyheads. J. Ultrastructure Res. 13, 343-358. KLUG, A., and BERGER, J. E. (1964). An optical method for the analysis of periodicities in elect,ron micrographs, and some observations on the mechanism of negative staining. J. Mol. Biol. 10, 565-569. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz. 9, 409-414.
BNI)
HILLS
MACLEOD, R., and M,\RKHM,
R. (1963). Experievidence of a relationship between yellow mosaic virus and wild cucumber virus. ViroZogy 19, 190-197. MARKHAM, R., and SMITH, K. 11. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. MM~ICHAM, R., FILEY, S., and HILLS, G. J. (1963). Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Vi’iroZogy 20, 8&102. mental turnip mosaic
MARKHAM, R., HITCHBORN, J. H., HILLS, G. J., and FRICY,S. (1964). The anatomy of the tobacco mosaic
virus. Vi’irology 22, 342-359. G. (1961). A modified procedure for lead staining of thin sections. J. Biophys. Biothem. Cytol. 11,736-739. NOYES, W. F. (1964). Structure of the human wart virus. Virology 23, 65-72. SPXKMAN, D. H., STEIN, W. H., and MOORE, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. SYMONS, R. II., Rms, XI. W., SHORT, PI. N., and MARKH.\M, R. (1963). Relationships between the ribonucleic acid and protein of some plant viruses. J. Mol. BioZ. 6, l-15. WILLTIMS, It. C., Kass, S. J., and KNIGHT, C. A. (1960). Structure of Shope papilloma virus particles. Virology 12, 48-58. YAMAZAKI, H., and KAESINXG, P. (1961). Biophysical and biochemical properties of wild cucumber mosaic virus and of tw-o related viruslike particles. Biochim. Biophys. Acta 51, 9-18.
MILLONIG,
36, 50-70
(1968)
A Study
of Tubes Strain
Produced
of Turnip
in Yellow
J. H. HITCHBORN Agricultural
Research
Council,
Vims
AND
Research
Accepted
Unit,
February
Plants
Infected
Mosaic
with
a
Virus
G. J. HILLS Huntingdon
Road,
Cambridge,
England
14, 1968
Electron microscopic examination of crude specimens made from plants infected with a necrotic strain of turnip yellow mosaic virus showed that infection led to the production of tubes as well as virus particles. These tubes, which usually appeared to occur in coaxial groups, were between 110 and 230 rnp wide and up to several microns long. With the aid of magnesium bentonit,e and differential centrifugation, almost pure preparations of the tubes could be made from the sap of infected plants. Such preparations contained mainly protein and were serologically related to the virus. Optical diffractometry and photographic integration techniques applied to electron micrographs of the tubes, revealed that they were made up of hexagonally packed, hexagonal rosettes, comparable in size to the hexamer morphological subunits of the virus. Strong evidence of a clockwise skew on the rosettes was obtained. The orientation of the hexagonal lattice was not the same on all tubes, but the variation was limited, the short axes of the hexagons comprising the lattice always lying within 15 degrees of the tube axis. Moreover, optical diffractometry of shadowed carbon replicas of the tubes revealed that in all but a small minority, the longrunning rows of hexagons of the lattice formed right-handed helices. Cones, structurally related to the tubes, were occasionally found. There was no evidence that tubes were produced in plants infected with three other strains of turnip yellow mosaic virus, but a small number of tubes about 5,5 rnp in width were seen in specimens made from pumpkins infected with wild cucumber mosaic virus.
of negatively stained t,ubes initially suggested that the fine detail was in disorder. However, the clear optical transforms which could be obt’ained from many tubes demonstrated the presence of a high degree of structural order and supplied basic information on the nature of this structure. Data obtained from optical diffraction studies also enabled photographic integration techniques (Markham et al., 1963, 1964) to be applied to electron micrographs of tubes to help in the elucidation of their structure. Some of the work described in the present paper has been briefly reported elsewhere (Hitchborn and Hills, 1967).
INTRODUCTION
During electron microscopic examination of a number of plant viruses in crude specimens (Hitchborn and Hills, 1965b), those made from plants infected with a necrotic strain of turnip yellow mosaic virus (TYRIV) were found to contain some apparently tubular bodies. They occurred very rarely and were not investigated closely at that time. Later, however, a necrotic strain was isolated which produced tubes in relatively large amounts and offered an opportunity of studying them in some detail. It is the purpose of this paper to present an account of such a study. A method was found which enabled almost pure preparations of the tubes to be obtained, and much of the work was carried out on such preparations. Direct inspection of electron micrographs
MATERIALS
AND
METHODS
The strain of virus used originated as a single necrotic local lesion on a leaf of Chinese cabbage (B~assica chine&s L.) 50
TUBES
ASSOCIATED
inoculated wit,h necrotic T‘llhIV (Dunn and Hitchborn, 196.5, 196(i), and was shown to be serologically related to Denmark TTXV. The host plant used throughout) was Chinese cabbage. Purification of t)he tubes was effected as follows. Infected leaves, from \\-hich the midribs had been removed, were ground using a pestle and mortar or were homogenized in a blender after the addition of 500 ml of 0.01 X l\lgSO, to every 100 g of tissue, and the extract was expressed through muslin. To the extract one-tnentiet,h of its volume of a suspension of magnesium bentonite (Dunn and Hitchborn, 1965) at I concentrat,ion of 10 mg/ml in 0.01 3f :\IgSOA, uxs slo\vly added wit,h rapid st,irring. The mixture was then centrifuged in a Servall RC2 centrifuge operating at about 5”, using 50.ml t,ubes in an HB swinging-bucket rot,or run for ,j minutes at 1250 rpm. The green supernntant suspension \vas removed from the centrifuge tubes by sucking, and the loose green pellets were discarded. The cycle of bentonite addition and low speed centrifugntion was repeated until the suspension \vns no longer green; t’his usually required 2-4 cycles. It was found import’ant, not to add more bentonite than was needed to bring about removal of the green material, as such addition led to partial or complete loss of the tubes. Residual bent)onite was removed by centrifuging t’he suspeIlsioIl at 7000 rpm for 20 minutes in SO-ml tubes in the SS-34 rotor of the Servall centrifuge. In a t’est, experiment, when suspensions of magnesium bentonite (0.3 mg/ml) in 0.01 111 -\lgS04 were subjected to this centrifugat’ion, optical densit,y measurement made on the supernatant indicated that all the bent’onit#e had been pelleted. Sext, the suspension, xvhich was straw-coloured and turbid, was centrifuged in 1.5.ml tubes in t,he SS-X4 rotor at 15,000 rpm for 30 minutes. The supernnt,ant) was discarded, and the colourless, gelatinous pellets \verc resuspended in 0.01 d/ phosphate buffer, pH 7.4, or in water. Finally, the centrifugation and resuspension I\-cre repeated twice more; the opalescent suspension was stored at S”. Elect,ron microscopic examination of such preparations showed t)hat they consisted mainly of tubes, contaminat,ion \vit’h virus particles being relnt,ively low (I’&. 2). Yields were of t,he
WITH
TYMV
in L’ivo
51
order of 10 mg per 100 g of tissue. In the Spinco Model E analytical ultracentrifuge, suspensions at a concent’ration of about 1 mg/ml produced a single, rapidly flattening schlieren peak representing material sedimeriting at’ a mean S of about 300 S. No peaks due to virus or its top component were observed. Virus was prepared from infected plants, using bentonit,e and differential centrifugation as described by Dunn and Hit,chborn (196.5). Estimation of amounts was based on ultraviolet, spectra traced in a Cary Model 14 recording spect)rophotometer. Serological tests were of the precipitation type and were made in tubes at room temperature. Two elect,ron microscopes were used, a Siemens Elmiskop IA and an A.E.I. E.M.GB. They a-ere calibrated by phot’ographing and measuring the (20 T) lat’ticeplanes of crystals of platinum phthalocyanine, as described by Markham et al. (1964). The normal practice in this laboratory has been t,o calibrate the microscope every few weeks by t’his means. However, during the present work there was evidence t’hat a relatively large variation in the magnification given by the Siemens Elmiskop IA could occur over a short period of time. For instance, with the microscope set, to give a nominal magnification of 60,000 times, measurement of six crystals phot)ographed on separate plates indicated t,hat t,he actual magnification was bet]ween 63,600 and 64,200 times. When, after 48 hours, another six crystals were photographed and measured,the microscopemagnified between 3,100 and 61,,500times. Therefore, whenever it was appropriate during the present work, immediately prior to phot,ographing TYMV t,ubes, platinum phthalocyanine crystals were photographed for the purpose of recalibrat,ing the microscope. Crude negatively stained specimens were made as described by Hitchborn and Hills (1965b), and purified preparations were examined stained with a 2% solution of potassium phosphotungstate at pH 6.4, or saturated solutions of uranyl acet)ate or uranyl formate at’ about pH 4. Carbon replicas were prepared by drying down a suspensionof t)ubes onto a Formvar-coated grid and shadowing with gold-palladium at a low angle. The shadowed specimenswere
52
HITCHBORK
then coated with carbon from an evaporating source immediately above them, and the Formvar substrate was subsequentfly dissolved away with chloroform. Finally, the specimens were soaked for 10 minutes in a concentrated aqueous solut’ion of sodium hydroxide to digest the tubes and then washed well in water.
FIG. 1. Electron micrographs of groups uranyl acetate. “Full” (VF) and “empty” associated with tubes being “empty.”
AND
HILLS
Tissue and pelleted t,ubes were prepared for sectioning by fixation in a 2 %’ solution of glutaraldehyde in 0.05 M cacodylate buffer, pH '7.2 (Gordon et al., 1963) and leaving for 2% hours in a saturated solution of uranyl acetate in a water-et’hanol mixture (1: 1, v/v). The material was then transferred to solut,ions containing progressively higher con-
of TYMV tubes in crude specimens negatively stained with (VE) virus particles can also be seen, most of the particles
TUBES
FIG. 2. Electron
ASSOCIATED
WITH
micrograph
of a purified
centrations of ethanol and left overnight in a saturated solution of uranyl acetjatjc in absolute ethanol. It, \vas then washed in 1-Z epoxypropane (Luft, 1961) and embedded in resin (Fin&, 1960). *An LIMB microtome was used to cut, sections which were stained with lead acetat,e (~Iillonig, 1961) before electron microscopic examination. The optical diffractometer used was t)he one described by Bancroft et al. (1967) except t,hat it incorporated an alternative light source consisting of a Spectra-Physics Model 130B gas laser. Electron micrographs of platinum phthalocyanine crystals were used to calibrate the diffractomet,er. One member of each family of spots in all transforms presemed in figures has hccn numbered so that equivalent spots in different transforms cm be identified. The basic apparatus and procedure used in linear integration \vere t,hose employed by l\larkham et al. (1964). For the present, work two modifications t)o the original apparatus \vere made. I;irst,ly;, the photograph under investigation was
TYMV
in
preparat,ion
Vim
of tubes.
illuminated from underneath and VW in the form of a transparency on thick film. Secondly, this film was attached to a rigid transparent sheet lying flat on the sliding table, t)he sheet and attached photograph being capable of rotation through a measurable angle. Rotational integration was carried out as described by JIarkham of al. (19K~). RESULTS
In crude electron microscope specimens made from plants infected with the necrotic strain of TYJIV, the tubes, which were presumably flattened, appeared as shown in I’ig. 1. Two aspects of their gross morphology were consistent with the view t)hat t’hey lvere, in fact, tubular in construction. l~irstly, they had parallcl sides, and secondly, t,hesc sides oft)en showed in relatively high conk&, suggesting that there was a greater deijth of material here than elsewhere. These features sho\ved pnrticu-
54
micrographs I PIGS. 3 and 4. Electron purified tubes. Fig. 4. Leaf 3. 1 ‘elleted ing tubes mainly in groups (2’).
HITCHBORN
AND
HILLS
of sections. Stains used: tissue of Chinese cabbage
uranyl acetate infected with
and lead acetate. Fig. necrotic TYMV, sl
TUBES
ASSOCIATED
larly clearly in purified preparations (Fig. 2). Firm evidence that the structures were tubular was obtained by electron microscopic examination of sect’ioned pellets of the pure, fixed material. Such sections appeared to have been made through randomly orientat#ed tubes (Fig. 3). Two properties of the tubes in crude electron microscope specimens are of interest. Firstly, many were present in characteristic aggregates. These probably originated in the plant’ and not during specimen preparation, for sectioned infected tissue contained what appeared to be coaxial groups of tubes (Fig. 4). Secondly, t#hey showed a preferent’ial association with “empty” virus particles (Fig. I). Of about 400 particles lying under, over, or in, 40 randomly chosen groups of tubes, 54 % were “empt,y” and the rest “full.” In cont’mst, among the particles examined which were not associated with t’uhes, only 15% were “empt’y,” a figure corresponding roughly with that arrived at from measurement of the areas under the schlieren peaks produced by the two components during analytical ult,racentrifugation of sap of infected plants. Measurement of a large number of t#ubes in negatively stained electron microscope specimens made from sap which had been part,ially clarified with the aid of bentonit,e
55
II0 TUBE
WCMV
TUBES
TYMV
TUBES
165 WIDTH
220
2%
(my)
FIG. 5. Histogram showing the frequency distribution of wild cucumber mosaic virus (WCMT’) and TYMT’ tubes of different, widths in negatively stained crude electron microscope specimens.
WITH
TYMV
in
V&J
3.5
FIG. 6. Schlieren pattern produced during centrifugat,ion of sap from a plant infected with necrotic TYMT-. Sedimentation was from left to right, and the photograph was taken approsimately 16 minutes after a speed of 24,630 rpm had been reached. P, plant protein; VT, “top” component of virus; K, ribosomes; VB, “bottom” component, of virus; T, tubes.
showed t,hat they varied in width between 110 and 250 rnp (l;ig. 5) and were up to 3 p long. A variation in width of his magnitude is much too great to be accounted for by different, degrees of flattening of tubes of equal diameter and it, must he concluded, therefore, that the t,ubes vary in diameter. Assuming complete flattening, the tubes present would have had diameters of bet,ween 70 and 160 mp. In t,he analytical ult,racentrifuge sap from infect)ed plants produced, in addition to schlieren peaks representing normal plant constituent’s and “full” and “empty” particles of the virus, a flat peak due to polydisperse material \\-ith a mean sedimentation rate of about, 1.5 times that of the “full” particles of the virus, indicatJing a mean Stow of about 180 S (Fig. 6). This presumabl!
56
HITCHBORN
AND
HILLS
represented the tubes, for no such peak was produced on centrifuging sap from healthy Chinese cabbage or sap from plants infected with several other strains of TYRIV. The fact that the material in purified preparations of tubes had a higher mean S suggests that much low X tubular material had been discarded during purification. In some samples of sap, a series of small, sharp peaks could be seen superimposed on the trailing edge of the flat peak, as in Fig. 6. These may have been artifacts or each may have represent#ed sedimenting tubes of preferred size. Purified suspensions, examined in the elect’ron microscope immediately after preparation, could be seen to contain mainly well preserved tubes (Fig. 2). After storage overnight in 0.01 M phosphate buffer pH 6.2-7.5, alt,hough preparations were still opalescent and contained mainly material sedimenting at about 300 S, electron microscope specimens made from them usually cont,ained stringy, disorganized masses of material and very few identifiable tubes. It seems, therefore, that tubes stored under these conditjions remained intact but became
FIG. 8. Tubes (A) (R) present in crude, microscope specimens fected with WC&IV.
0.2
O-1
0 240
260
280
WAVELENGTH
300
320
@‘y)
FIG. 7. Ultraviolet absorption spectrum of a purified preparation of tubes in water. A, imme,diately after purification; B, after storage overnight at 8”.
and double-shelled particles negatively stained electron made from pumpkins in-
suscept’ible to disint,egrat’ion on preparation for t’he electron microscope. Suspensions stored overnight in distilled water appeared to contain largely fiuely broken down material for they were clear or nearly so, and most of the material detectable in the analytical ultracentrifuge sedimented at about 7 S. The appearance of such preparations in the elect,ron microscope was similar to that of stored buffered suspensions. The ult’raviolet absorption spectrum of a suspension of tubes in water, measured
TUBES
FIG. optiral
9. (A) Electron micrograph transform of the ringed part long arrow. The transform
ASSOCIATED
of a TYM\of the tube,
WITH
TYNV
in
Vizw
tube negatively stained with the relative direction of the
uranyl
acetate.
(B)
An
tltbe axis being indicated on hexagonal lattices and of spot,s given by the detail of the hexagotlal lattices photograph althoi&r they
is made up of two sets of spots arranged by t,he represents detail on both sides of the flatterled tribe. One spot of each family on one side has the same labelling as the equivalent spots on the transforms show:1 in Fig. 11. Several of the spots equivalent to 4tt do tlot appear in the could be seen in t,he diffractometer. 1lagnifiratioll of electrorl mic:rograph: X 150,000. immediately after preparation and aft,er storage overnight at 8” are given in l;ig. 7. The change in the spectrum xvith time is further evidence that most of t,he tubes disintegrat.e on storage in water. The spectrum of t*he suspension of tubes after storage \vas closel> similar to that of the “top” component of TT;\IV (Alarkham and Smith, 1949) and had an absorption maximum at, 273 mp and a minimum at, 2.3 rng. If t,he pH of aqueous suspensions of virus or tubes ~-as louvered by adding an equal volume of 0.15 d1 citric acid-sodium phosphate buffer at pH Z.Gci.0, both precipitatccl, but over different pH ranges. Whereas virus precipitated most rapidly at pH 3.2 and mithin 24 hours at. pH 2.4~33, the most rapid precipitation of tubes 1v-a~ at pH 4.4, and t’he range over I\-hich precipitation tool; place xvithin 24 hours \vas pH R.S-5.2. A clear serological relationship betnccn tubes and virus ~vas demonskated. Tests carried out. by mixing antiserum prepared against purified necrotic TIYIV Iyith a
fresh preparation of tubes in water, resulted in the immediate development of turbidity Tvhich n-as rapidly folio\\-ed by flocculation. When t,his test \vns carried out. on some of the same preparation of tubes stored overnight, at 8” there n-as no immediate visible reaction, a slight precipitate forming after 24 hours. This change in response to the addition of ant’iserum probably reflects the breakdo\vn of the tubes int,o small units on storage. There was no reaction \\-hen normal serum was used in&ad of ant,iserurn in these tests. Amino acid analysis of HC1-hydrol>~zatjes of purified preparations of tubes and of the protein extracted from purified necrotic strain TTAIV, 1%.a.scarried out in :I Beckman Spinco amino acid analyzer (Spackmnn et al., 1958). Results shoxved that the tubes consisted largely or entirely of a prot,ein having an amino acid composition lyhich ~vas closely similar or identical to that of the virus protein. Electron microscopic examination of crude
5s
HITCHBORN
extracts from plants infected with the Denmark, Cauliflower, and Type strains (Symons et al., 1963) of TYMV was made without tubes being found. However, pumpkins (Cucwbita pepo L.) infected with wild cucumber mosaic virus (WCXIV), which has a number of properties in common with TYJZV (Tamazaki and Kaesberg, 1961; Mncleod and Jlarkham, 1963), did occas-
AND
HILLS
ionally contain tubelike bodies. Only a limited study of these was possible because of the infrequency with which they occurred. However, the fifty or so observed had characteristic wavy outlines, were narrower and much more uniform in width than the TYMV tubes (Fig. 5), and were similar in diameter to the large shells sometimes seen enclosing empty wcnm particles (Fig. S).
FIG. 10. (A) Electron micrograph of a single sheet of hexagonally packed hexagonal rosettes present among tubes and virus particles in a negatively stained crude specimen made from a plant infected with necrotic TYMV. The direction in which short axes of the rosettes lie is indicated by the arrow in B, which is an optical transform, in equivalent orientation, of a similar sheet. The system of labelling the transform spots corresponds to that used in Figs. 9 and 11. The position of spot 4b is marked, but the family of spots it represents cannot be seen in the photograph. (C) Selected virus particles showing morphological subrlnits. Magnification of electron micrographs: X 400,000.
FIG. 11. Models representing the negatively stained fine struct,ure of a ‘l’LII\~ t,ttbe (A and C) and their optical transforms (B and I>, respectively). (A) A network of hexagons with central dots, representing staiu both between rosettes and in their centres. (C) A network representing rosettes with unstained centres. Features on the models have been numbered f-4 in order of decreasing size of repeat interval, and, as a means of identification, one of the two transform spots produced by each has the same numbering.
Firze Structure
Careful examinat’ion of electron micrographs of negatively stained TYMV tubes sho\ved the presenceof sets of evenly spaced faint’ straight lines running across and along the tubes, and suggested t,hat regularly repeating fine detail had been preserved and resolved. Proof of this came from the strikingly clear optical t,ransforms obtainable from many elect,ron micrographs (Fig. 9). Typically, such t,ransforms consisted of two nearly identical set’s of spots arranged on
hexagonal lattices superimposed on one another at an angle. A double pattern of this kind suggestedbhat t’he tubes were helical in construction and had both sides visualized in the electron microscope (Hug and Rerger, 1964; Finch et al., 1964). At least part of the confused appearance of the tubes would, therefore, appear to be at’tributable to superimposition, out’ of register, of images of the two sides. Occasionally sheets of material \vere seen which gave optical transforms consisting of single set,s of spots (Ij’ig. 10
60
HITCHBORN
A, B). These presumably consisted of singlelayered pieces of material similar to that making up the tubes and appeared to be construct,ed of hexagonally packed hexagonal rosettes resembling the hexamer morphological subunits of the virus particles (Fig. 10 C). The centre-to-centre dist’ance bet,ween adjacent rosettes was about SS A, and in Fig. 10 they can ‘be seen t’o be orientated with short axes lying in the direction indicated by the arrow. By comparing the transforms 20-
ml I IS’ ANGLE
BETWEEN
LATTICE
AND AXIS OF TUBE
FIG. 12. Histogram showing the frequency distribution of different angles between lattice and tube axis among TYRITtubes in negatively specimens. St ained
AND
HILLS
of this material wit’h that of t’he tube shown in Fig. 9, it can be deduced that the rosettes making up the tube would lie with their short, axes within a fen- degrees of the tube axis. Experimentation with models showed that an optical transform basically similar to that obtained from the single layered material was given by a stain pattern consisting of a honeycomb of hexagons, each hexagon containing a central dot (Fig. 11 A, B). The spacing labelled 2 is equal to half the centreto-cent,re distance between adjacent rosettes. The transform of a model consisting of a honeycomb of hexagons devoid of central dots has additional st’rong spots (Fig. 11 C, U). A similar transform was obtained from a model in which only about half of the hexagons were devoid of central dots, but the diffract(ion spot,s 1 and 3 were then of about equal intensity. Of the transforms given by models, this was t,he one which most nearly resembled those obtained from tubes, and it is concluded that in these structures a proportion of t,he rosettes have centres unpenetrated by the stain. From measurement of the transforms of a large number of tubes, it,
FIG. 13. (A) Ele:tron micrograph of shadowed carbon replicas of TYMTtribes. (B) A single replica of a-hich the optical transform (C) was obtained after plazing a mask around the outlined re:tangular area, which lies with its long axis parallel to that of the tube. The transform of the mask forms a cross and enables the relative orientation of the transform of the tltbe and t,he trtbe axis to be determined. The direction of the tube axis relative to the transform is indicated by the long arrow. (A) X 41,000; (B) x 80,000.
TUBES
ASSOCIATED
was calculated that the average centre-tocentre distance between roset’tes was 90 A, which is near the figure for the dist,ance between the centers of rosettes in the single sheet’ material, arrived at from measurement of electron micrographs. This figure differs slightly from that published earlier (Hitchborn and Hills, 1967), which n-as based on only a few measurements. The angle bet,\\-een the hexagonal lattices containing the two sets of spots making up the diffraction pat’terns of the upper and lower surfaces of a negatively stained tube cn11 be easily and accurately measured. This provides a convenient way of finding the angle at which each lattice is set to the axis of the tube. From measurement made OII the transforms of a large number of tubes, the histogram shown in b’ig. 12 was drawn. It show-s that the lattice angle did not exceed 15 degrees and hints at the possible existence of peaks between 0 and 7 degrees. The peak at 0 degrees may be artificial and result from the difficulty of distinguishing between pat’terns which are superimposed in register and hhose of&et by a degree or two. As there is no means of relating each of the t,\vo sets of transform spots produced by a negatively stained tube to the side of the tube lvhich produced it,, t)he sense of the slope on the lattice of such a tube cannot, be determined. This uxs investigated by diffractometry of carbon replicas, which represented only the structure on the upper surface of tubes. Dexpitc the unpromising appearance of such rc~plic:r~ many gave clear diffrnction patterns
FIG. 14. Histogram showing t,he frequency distribut.ion of different angles between lattice and tube axis among shadowed carbon replicas of T17VIV tubes. The signs + and - define the sense of the helices formed by the lollg-rllnning rows of hexagons of a 1st tice, + indicating that the helires are right-handed and - that they are left-handetl.
WITH
TYMV
in
61
T’il,o
. l
I
I
I
0
50
100 TUBE
FIG. 15. Tube tween structural
width lattice
l
I
150 WIDTH
200
I
250
hv)
plotted against angle and axis of tube.
be-
consisting of a single hexagon of spots, each analogous to that, labeled 1 in Figs. 9 B and 11 D and representing structure repeating at 78 A (Fig. 13). The angle at which the lattice lay relative to the tube axis and the sense of the slope of the lattice were assessed by enclosing an electron micrograph of a replica in a rectangular mask, positioned so that the long axis of the mask was parallel to that of the tube (Fig. IS B), and examining the masked replica in the diffractomet,er. The direction of the tube axis was determined from the transform of the mask, although as t,he placing of the mask was subject to t,he possibilit,y of slight error, the measurement of the latt’ice angle by this means would not be expected to be highly accurat’e. The result of measuring this feature on a number of replicas is given in Fig. 14. The variation in lat,tice angle was similar to that found among negatively stained tubes, and, in all but, four, the la&es were orientated so that t,he long running rows of rosettes either ran parallel to the axis or formed right-handed helices. In order to test the possibilit’y that lattice angle and width of tube were related, both measurements were made on a number of negatively stained tubes. The results obtained gave no evidence of a simple relationship between the tn-o feat’ures (Fig. 15). When electron micrographs of the aggregat’es present in crude preparations were examined in the diffractometer t’hey gave
62
HITCHBORN
transforms which appeared to arise from the superimposition of almost identical patterns, suggesting that the successive layers of material making up each aggregate were structurally related. Figure 16 shows t,he transforms of two aggregat,es which have lattices intersecting the axes of the aggregates at angles of 4.5 and 12.5 degrees, respectively. As shown in Fig. 12, an angle of intersect,ion of 12.5 degrees is unusually large and makes it highly improbable that the components of the aggregate have this feat ,ure in common by chance.
AND
HILLS
Using the information obtained from the diffractometry of electron micrographs of t’ubes as a basis, it was possible to employ the technique of linear integration t,o obtain patterns representing the ordered structure present. The procedure adopt’ed was to prepare a photograph of part of a tube at a magnification of about 2,000,OOO times (lcig. 17B) and to secure it to the integration bable with the axis of the tube lying in t’he direction of motion of the table. The t,ransparent sheet t,o which the photograph n-as &ached was then rot’ated througrh that angle which
FIG. 16. Electron micrographs of two groups of tubes and by the side of each the optical diffraction pattern given by the area marked. Only spots equivalent to that labelled 2 in Fig. 9 show. The tubes in group A have lattices set at an angle of 4.5 degrees to the long axis of the group and those in group B at an angle of 12.5 degrees. Magnification of electron micrographs: X 102,000.
TUBES
ASSOCIATED
WITH
TYMV
in
Vitlo
63
FIG. li. Linear integration of an electron micrograph of a tube. (A) Electron micrograph of a negati vely stained tube, with the area for integrat,ion outlined. (B) This area on a transparency at about a used in integration. (C) Photograph obt.ained by integrating B at 7 degrees to th lird of the magnification equivalent to 90 A on the original specimen. It represents repeating structh he tube axis and at int.ervals tu re present on one side of the tube and running at 7 degrees to the tube axis. Y indicates the directil 311 of the tube axis; and X, the direction in which integration took place. (A) X 200,000; (R and C) X 620,000.
64 an optical transform would bring rows of one side of the tube in of motion of the t’able. graph was then taken consecutive exposures,
HITCHBORN
of the Oube indicated structural rosettes on line with the direction An integrated photoby making about 25 the distance through
AND
HILLS
which the t’able was moved between one exposure and t’le next being equivalent’ to 90 A. One photograph obt,ained in this way is shown in Vig. 17 C. The supression of bot)h “noise” and the structure of one side of the tube has revealed the main repeating
FIG. 18. Integration of an electron micrograph of a TYMG tube at various angles to the tube axis. The integration interval was constant and corresponded to 90 A on the original specimen. The integrated photographs were taken after the axis of the tube had been rotated, by the anglesshown, clockwise (-) or anticlockwise (f), away from the direction of motion of the integration table. The regular patterns obtained with the axis in positions -7 and +6 degrees represent repeating structure on the two surfaces of the tube. X 455,000.
FIG. 19. Pattern obtained from an electron micrograph of a TYMV tube using a combination and rotational integration. The individual hexagonal rosettes forming the pattern are skew regions of high density at the vertices.
of linear and show
TUBES
ASSOCIATED
features on the other side. By bringing t’he equivalent rows of rosettes on the reverse side of the tube in line with the direction of motion of the table, the pattern made by t,hese could also be photographed. A series of integrat,ion photographs taken with t’he axis of :I tube rotated through 10 degrees clockwise (-) relative to the direction of motion of the t,able, 10 degrees anticlocknisc (+), and at l-degree intervals bet)t\-een these t)n-o extremes, showed that greatest’ regularity in overall pattern was produced when the axis was in positions +6 and -7 degrees. In positiow more than 1 degree on either side
WITH
TYMV
in
Viva
05
of these, no regularit,y \vas apparent. l’art of this series of photographs is she\\-n in l:ig. 1% The angle of intersection of equivalent1 rows of rosettes on the upper and loivcr sides of the tube, measured in this \vay, is l:$ degrees, and is very close to the angle (13.5 degrees) by \\-hich the diffraction pattjerns of the upper and lower surfaces of the tube jvcrc rot’at’ed with respect’ t#oone another. Some of t’he integrated pictures suggested t)hat’ substructure OII the hexagonal rosettjos \V:LG resolved, although poorly. Attempts \\-we mwdc in two \vnys to clarify this detail. One involved a second linear ‘integration of
FIG. 20. A4 and C are models representing hexagonally packed rosettes of six subunits, arranged so that, in -4, the rosettes are placed symmetrically in the rows and, in C, they are skew. The transforms of these models are shown in B and D, respectively. In the transform of the model with symmetrically arranged rosettes, spots ,$a and 4b are of equal intensity (B); in the transform of the model with skew rosettes, these spots are of unequal intensit.y (I)).
66
HITCHBORN
the pictures at 60 degrees to the direction in which the original integration t,ook place. The impression conveyed by photographs obt’ained in this way was that each hexagonal rosette was composed of six subunits, one being located at each vertex. It also appeared that t’he roset>tes were arranged slightly skew relative to the direction in which the rows ran. The second technique used t,o ext,ract’ further information from the once-integrated picture was that of rotational integration, as described by Markham
AND
HILLS
et al. (1963). The rotation was made about the cent,re of one of the rosettes, and the angle through which t,he rotation table n-as turned between exposures was 60 degrees. As n-it’h the double linear integration photographs there were areas of high density at the vertices of the rosettes, which again appeared skew (Fig. 19). In view of the foregoing evidence that the rosettes were skew it n-as relevant to compare optical transforms of two models, both constructed of hexagonal clust)ers of dots
FIG. 21. A and B are diagrammatic representations of optical diffraction patterns of the two surfaces of a TYMV tube negatively stained with uranyl acetate. The spots have the same labelling as the equivalent spots on the transform shown in Fig. 9. Patterns A and B have object-mirror image relationship and differ from each other as a result of the difference in intensity between spots 4a and 4b. Patterns C and II were made by superimposing A and B, and represent the two hypothetically possible forms of the transform of both sides of a tube having a structural lattice inclined at about 5 degrees to the long axis of the tube, the direction of which is indicated by the arrows. Among the transforms of tubes examined, only those of t,ype C were seen (e.g., Fig. 9).
TUBES
ASSOCIATED
arrayed hexagonally, but with the clusters of one arranged symmetrically relative to the rows, and the other having skew clusters (I:ig. 20). These gave broadly similar dif-
WITH
TYI\IJ’
in T’iw
6’7
fraction patt,erns but the relative intensity of the spots in t,he two was different. What, is of greatest, significance for the present purpose is that,, in the transform of the model having symmetrically arranged rosettes, the members
of t,he two
families
of spotIs rcpre-
sented by /ta and db are of equal intensity, Tvhereasin t,he model having skew rosettes t,hesespots are of unequal intensity. If, as in Fig. 20 C, t,he ros&tes have :I slight, clockwise skew, the more int’ensc spot, da, can be seen to be on t,he right of /,b. In :I transform of a model with rosettes skewed ant)iclockwise, the brighter spot was on t’he left. Careful inspection of a large number of transforms of tubes and of pieces of singlelayer material revealed the presence on some of the clearest), of spots equivalent t#o /ta and ,$b, and of unequal intensity (Figs.
9 and 10). This is substantial additional evidence that the roset,tes are skew. As the diffraction patterns produced by the two sides of a tube have an objectmirror
image
relationship,
the
element
of
asymmet,ry in t,he ring of spots labclled 4 will lead to t,he two patterns being different at, this radius, in the manner shown diagrammatically
in Fig. 21 A, B. This
meaus
t,hat t,wo dist)inct superimposition patt’erns (Fig. 21 C, D) are possible. I’ntt’ertt C is produced n-hen the senseof the slteu- on t,he rosettes is the same as t,he seuse of t’he helix formed by the long-running rows of rosettes,
and pattern D when these sensesare opposite. Of the 2:3t8ransformsin \I-hich the relevant, outer spots could be seen, all were of type C (as in Fig. 9). This indicates that in the majority of tubes, in which the structural latkice the rosett,es
Flo. 22. Structures of triangular outline present in preparations of TYMV tubes. They have apical angles of A, 30 degrees; B, 60 degrees and C, 90 degrees, and are probably flattened cones having an essential similarity to the tubes in being const,ructed on the basis of the same hexagonal lattice.
slopes to the right, is clockwise.
t#he skew on
Colzes. Most preparations of tubes cow tained a small proport’ion of triangular structures which gave diffraction pnt’t’erns of the samekind as those given by tubes. If, as seemslikely, such bodies were flat’tened cones they would be expected to exist) in a limit#ed number of well d&ted categories. This follows from the fact that a hexagonal lat8tice cart cover without discontinuity only the surfaces of cones which, when flat’t,ened, appear
as triangles
having
apical
angles
of
30, 60, 90, 120, or 150 degrees. Ittspection of
6s
HITCHBORN
electron micrographs of preparations of tubes revealed that the majority of the triangular bodies present did, indeed, fall precisely int’o several of these categories. The most’ commonly occurring were t,hose having apical angles of 30 or 60 degrees, and represent,atives of the 90 degree class were also encountered (Fig. 22). Examples of those Tvith the widest, apical angles were not identified with certainty, but this may simply reflect the improbability of such struct’ures flattening to form triangles. The diffraction patt’erns given by the cones included indication that the rosett,es of which t’hey were composed were skew. There was, however, no evidence of any limitat’ion on the angle of intersection between the hexagonal lattices of the upper and lower surfaces. Such limitation would not’ be expected, for, with a cone, the angle of intersection will depend only on where the surface folds on flattening.
AND
HILLS
are relat,ed t’o the large spheres which this virus produces. However, the tubes produced by TYMV are very variable in size, even the narrowest being much greater in diameter than the virus particles. This seemst,o preclude the possibilit? that they arise as the result of a simple mlst,ake in the construction of virus particles (Caspar and Klug, 1962). In common with T4 polyheads (Icavre et al., 1965; Kellenberger and Boy de la Tour, 1965)) TYS IV tubes are built’ on a somewhat flexible plan which allows variation in structure and with TTJIV even permits the construction of conical forms. However, there appears to be a definit’e difference bet’ween t’he polyhead tubes and those of TTMV in the degree of constructional freedom shown. From Moir6 analyses of polyhead tubes, Kellenberger and Boy de la Tour (1965) were able to show that, there was a great variation in the orientation of the hexagonal lattice in different tubes, DISCUSSION Although t’he absence of a preferential orientation could not be demonstrated beThe morphological, serological, and chemicauseof the small number of tubes amenable cal evidence given in the present paper to analysis, t’hey did encounter examples of supports the view that bot’h t’he virus parpolyheads with lattice pitches near to 30 t’icles of necrotic T‘lXV and the associated and 0 degrees, t,hat is, with the hexagons of tubes are composed of the same prot,ein the lattice having long and short axes, subunits. There is circumstant’ial evidence respectively, nearly parallel to the t’ube axis. t,hat the prot,ein of a number of other viruses In contrast’, the large number of TYRIV can assemble to form either spheres or tubes, tubes we examined only showed a variation for both types of st’ructure, sometimes in lattice angle between 0 and 15 degrees, clearly composed of morphologically similar subunits, have been found in extract’s of and about 80% had lattices set ai S degrees or lessto the axis. Moreover, in all but a few tissue infected with polyoma (Howatson of the tubes examined, the lattices were inand Almeida, 1960)) papilloma (Williams clined so that the long-running row of et aZ., 1960; E’inch and Klug, 1965), human rosettes formed right-handed helices. This wart (Noyes, 1964), and FV 1 (Darlington limitation in construction is of interest beet al., 1966). With cow-pea chlorotic mottle cause it runs counter to the expectation that’ virus the evidence is even stronger, for in the orientation of the lattice would be ranvitro conditions have been found (Bancroft dom (Kellenberger and Boy de la Tour, et al., 1967) under which t’he spherical virus particles will disrupt and their substituents 1965). As the “left-handed” tubes formed recombine to form tubes. These viruses such a small minority it, seemsworthwhile to appear to produce t’ubes of one or two sizes consider in some detail t’he possibility that only, each having a diameter comparable they were artifact,s. A “right-handed” tube with that, of an associated spherical part’icle will appear “left-handed” through incorrect and each capable of forming a hemispherical placing of either t’he specimen in the electron microscope or the electron microscope plate cap. The tubes found associated with WCMV in crude ext,racts during the present in the diffractometer. Both t,hese possible work are of a uniformity and size which explanations are ruled out by the fact, that make it reasonable to postulat’e that they the plates containing the “left-handed”
TUBES
ASSOCIATED
tubes also contained “right-handed” ones the p&es were positioned the same \va,v round in the diffractometer. The method of est’imating the tube axis would allow error in measuring t#he slope of the lattice but, Jvhcrc the replicas appeared to he prccis:cly parallel-sided, t,his error \vould he expected to he lirnikd to a degree or two. The sides of the three replicas \vith “left-handed” I:ltticcs set) at 4 degrees or more to the axis appeared to he slightlJ~ out of parallel, but this does not affect the present’ issue, for t,hey appeared “left,-handed” regttrdless of \\-hich of their sides n-as set parallel to the axis of the mask. It) is possible that these replicas are of approximately rectangular pieces of the single sheet material encountered occasionally in negatively stained prcparatjions. Ho\\-ever, the shadows along the edges of the replicas do not suggest that t,he material thcv represent was unusually thin. It seems difficult, therefore, to escape the conclusion that’ a very small proportion of the tubes are, in fact, “left-handed.” Inv&igation into the st,ructure of particlcs of broad bean mottle virus has led to the hypothesis that, the morphological subunits are skew, I\-hile parallel observations on TT1IV I\-ere consistent with the view that the subunit,s of this virus are symmetricall\ arranged on the particles (lcinch and Mug, 1967). Our results suggest that this co~xlusion may not. apply to at, least, the necrotic strain of TIT1\IV, for if the hexamer morphological subunits of the virus part’icles are identical \vith the tube roset,tes, for xvhich there is strong evidence, then, as these roset,tes appear t,o be arranged skew, it seems likely that the hexamers in the virus particles n-ould be skew also. Electron microscopic examination of both ncgat ivel) stained crude specimens and sections of infected tissue seems to indicate that in ukw man>- of t’he T\;MV t,ubes occur in \vhat appear to be coaxial groups, the diflkaction patterns given by which suggest, that each contains structurally related tubes. A possible explanation for this is that, OIW tube cm act as a former for the construction of another which thereby has imposed on it’ the structural features of t’he first. However, t,his is not a complet,ely satisfact,ory hypothcI\-1~11
WITH
TYl\l\-
in, Vito
69
sis for, if true, it seems likely that the tubes in a group u-ould be closely applied t)o one another, Tl-hereas they usually appear to be separakd by an appreciable gap. Albernatively, the structural homogeneity of the few complexes examined \2-hich gave clear transforms could be due to each being, not, a series of tubes, but a rolled-up single sheet. ACKSOWLEIXIZIESTS We wish to thank Professor It. Markham for helpful discllssion and Mr. RI. W. Rees for carrying ollt the amino acid analyses. We are also grateful for the technical assistauce of nIiss Y. 11. Carter, Miss A. l’laskitt, and 1Ir. S. Frey. Sectioning was carried out on a microtome generously donat,ed by the Wellcome Folmdation. REFERENCES B.\NCHOFT, J. B., ITILLS, G. J., and ~\LIRKH.~M, R. (1967). A study of t,he self-assembly process in a small spheriral virlls. Formation of organized structures from protein suhlurits in oilro. Virology 32, 354-379. CMP.~ I). L. I>., and KllLg, A. (1962). Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. &ant. Biol. 27, l-24. DARLINGTOIL’, R. W., GIL.\NOFF, A., and B~xzI,:, I). C. (1966). Viruses and renal carcinoma of Runa pipienx. II. Ultrastructural studies and sequential development of virus isolated from normal and tumor tissue. Virology 29, 149-156. L)CNN, 1). B., and IIITCHIWRN, J. H. (1965). The use of bentonite in the prlrification of plant virllses. ViTology 25, 171-192. DUNN, I). B., and HITCHIWRX, J. IT. (1966). The extraction of infectious, high molecular weight RNA from turnip yellow mosaic virus with ethanol. Virology 30, 598607. FAVRIC, It., Boy DI: LA TonIt, E., SEGR$:, N., and K~:LLI:NI~I.:ROI~R, E. (1963). Studies on the morphopoiesis of the head of phage Y-even. 1. Morphological, immunological, and genetic characterization of polyheads. .T. TJZtrastruct. Res. 13, 318-342. FINCH, J. T., and KLUG, A. (196.5). The structure of virllses of the papilloma-polyoma type. III. Htructllre of rabbit papilloma virus. 1. Mol. Biol. 13, I-12. FINCH, J. T., and KLVG, A. (1967). Struct,ure of broad bean mottle virus. I. Analysis of elect,ron micrographs and comparison with turnip yellow mosaic virus and its top component. J. Mol. Biol. 24,289-302. FINCH, J. T., KLUG, A., and STRETTON, A. 0. W. (1964). The structure of the “polyheads” of T4 bacteriophage. J. Mol. Biol. 10, 570~.i7A.
70
HITCHBORN
FJNCK, H. (1960).
Epoxy resins in electron microsJ. Biophys. Biochem. Cytol. 7, 27-30. GORDON, G. B., MILLEIL, L. R., and BENSCH, K. G. (1963). Fixation of tissue culture cells for ultrastructural cytochemistry. E’xptl. Cell. Rea. 31, copy.
440-443. IIITCHNRN,
J. H., and HILLS, G. J. (1965a). Electron microscopic examination of wild cucumber mosaic virus in negatively stained crltde preparations. Virology 26, 756-758. HITCHBORN, J. FT., and HILLS, G. J. (196513). The use of negative staining in the electron microscopir examination of plant viruses in crude extracts. Virology 27, 528-540. HITCHBORN, J. H., and HILLS, G. J. (1967). Tubular structures associated with t,llrnip yellow mosaic virus in viva. Science 137, 705-706. HOWATSON, A. F., and ALMEID,~, J. 1). (1960). Observations on the fine structllre of polyoma virus. J. Biophys. Biochem. Cytol. 8, 828-834. KELLENBERGI~ZR, E., and BOY DE 1,s TOCR, E. (196.5). St,ltdies on the morphopoiesis of the head of phage T-even. II. Observations on the fine structure of polyheads. J. Ultrastructure Res. 13, 343-358. KLUG, A., and BERGER, J. E. (1964). An optical method for the analysis of periodicities in elect,ron micrographs, and some observations on the mechanism of negative staining. J. Mol. Biol. 10, 565-569. LUFT, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz. 9, 409-414.
BNI)
HILLS
MACLEOD, R., and M,\RKHM,
R. (1963). Experievidence of a relationship between yellow mosaic virus and wild cucumber virus. ViroZogy 19, 190-197. MARKHAM, R., and SMITH, K. 11. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. MM~ICHAM, R., FILEY, S., and HILLS, G. J. (1963). Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Vi’iroZogy 20, 8&102. mental turnip mosaic
MARKHAM, R., HITCHBORN, J. H., HILLS, G. J., and FRICY,S. (1964). The anatomy of the tobacco mosaic
virus. Vi’irology 22, 342-359. G. (1961). A modified procedure for lead staining of thin sections. J. Biophys. Biothem. Cytol. 11,736-739. NOYES, W. F. (1964). Structure of the human wart virus. Virology 23, 65-72. SPXKMAN, D. H., STEIN, W. H., and MOORE, S. (1958). Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1190-1206. SYMONS, R. II., Rms, XI. W., SHORT, PI. N., and MARKH.\M, R. (1963). Relationships between the ribonucleic acid and protein of some plant viruses. J. Mol. BioZ. 6, l-15. WILLTIMS, It. C., Kass, S. J., and KNIGHT, C. A. (1960). Structure of Shope papilloma virus particles. Virology 12, 48-58. YAMAZAKI, H., and KAESINXG, P. (1961). Biophysical and biochemical properties of wild cucumber mosaic virus and of tw-o related viruslike particles. Biochim. Biophys. Acta 51, 9-18.
MILLONIG,