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Partial purification, structure and infectivity of complete maize rough dwarf virus particles
VIROLOGY
53, 130-141 (1973)
Partial Purification, Structure and Infectivity of Complete Maize Rough Dwarf Virus Particles ROBERT G . MILNE,' MAURIZIO CONTI,
AND
VITTORIA LISA
Laboratorio di Fitovirologia applicata del C .N .R ., Via 0 . Vigliani 104-10135, Turin, Italy Accepted February 7, 1973 Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation . The product consisted mostly of complete particles, but some subviral particles were present . The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions ; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid . In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis . Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 5057 Ran subviral particle possessing 12 previously hidden spikes (B spikes) each about S nmlong, coaxial with the A spikes . Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate . Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures . Detached B spikes were composed of 5 morphological subunits surrounding a central hole . Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley . Crude and purified preparations containing the large virus particles were infectious ; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious . INTRODUCTION Maize rough dwarf virus (MRDV) causes a severe disease in maize and also multiplies in its planthopper vector . The data and literature on the virus have been summarized by Lovisolo (1971) and Harpaz (1972) . Wetter et al. (1969) purified a spherical 55 mn particle which contained doublestranded RNA (Redolfi and Pennazio, 1972) from diseased plants and from the vector ; but later it was found by Redolfi, Pennazio, and Paul (1973) to be a subviral particle I Present address : Laboratoire des Virus des Plantes, Institut de Botanique, 8 Rue Goethe, 67 Strasbourg, France .
(SVP) . The complete particle has a reolike double capsid some 70 nm in diameter . Lesemann (1972) studied further the structure of the SVP in purified preparations and that of the complete particle in crude plant extracts . He suggested that, the SVP retained at its 5-fold vertices parts of the outer capsid in the form of projecting capsomeres and found that on treatment with potassium phosphotungstate these were removed to leave a smooth SVP like that illustrated by Wetter et al . Harpaz and Klein (1969) and Conti and Lovisolo (1971) succeeded in transmitting the virus by injecting crude material from 130
Copyright © 1973 by Academic Press, Inc . All rights of reproduction in any form reserved .
MAIZE ROUGH DWARF VIILUS infected plants into the vector, but the method has not been applied systematically and the infectivity of purified preparations has not been reported . We report the partial purification of the complete virus particle and some new observations on the structure, properties, and infectivity of the virus .
MATERIALS AND METHODS Source of virus . The virus was purified mainly from naturally infected maize growing near Turin, using roots that were, split due to abnormal phloem growth (Luisoni et at ., 1968) . Roots or leaf enations of glasshouse-grown experimentally infected plants were also used with similar results . The glasshouse virus was an isolate obtained from Novara, Northern Italy, by Conti et al . (1968) and propagated by their method, which was to make hoppers viruliferous by feeding them on infected plants or by injecting them with virus preparations . These hoppers were then used to inoculate maize seedlings, which in turn were ready for harvest in 2-3 months . Planthoppers and infectivity assays . Colonies of MRDV-free Laodelphax striatellus Fallen were reared in the glasshouse according Harpaz et al . (1965) and the mature females were injected with preparations of infected and healthy plants in a cold room as described by Conti (1969) . Single hoppers were then caged on pots containing one maize and one barley plant at the coleoptile stage and were moved to fresh plants at weekly intervals . Both maize and barley were used because maize is the better indicator plant, but the hopper survives for only a few days when fed on it exclusively (Harpaz, 1961), whereas barley is a good food plant . During infectivity tests the number of surviving insects was recorded daily, arid the plants were checked for symptoms for up to 3 months, though most symptoms had appeared after I month . A minimum incubation period of 8 days was necessary to make the hoppers inoculative after the injection, so the percentage of transmitters was calculated on the number of insects which survived at least that period . Random samples of oninjected hoppers from the stock were also
131
routinely tested for infectivity to ensure that they were free of MRDV . Purification . Because the virus was unstable, all operations were performed in the cold as rapidly as possible ; usually, processing took 7-8 hr . The best method found is given below . For preliminary clarification, we used Freon or carbon tetrachloride as indicated by Kimura and Black (Black, 1970) and Kitagawa and Shikata (197 .1) for wound-tumor and rice black-streaked dwarf viruses . In each run, about 100 g of washed chilled roots were passed through the rollers of a sap press (Hosch, 1960 ; Luisoni, 1969) with dropwise addition of an equal amount (w/v) of cold extraction solution containing 0.05 ]41 Na2H1'O,, 0 .005 A! disodium EDTA and 0.0131 Na2SO,, pH 7 .8 (Wetter et al ., 1969) . Extracts were passed through nylon mesh, centrifuged at 4500 g for 5 min and then shaken for 5 min with an equal volume of Freon 113 (fluorocarbon 1,1,2-trifluoro1,2,2-trichloroethanc, from Schuchardt) . After centrifugation at 4500 g for 10 min to separate the emulsion, the upper aqueous phase was retained and 30 ml was layered in Spinco SW 25 .2 tubes on top of a minigradient consisting of 3 .5 ml each of 30, 40, 50, 60, and 70% (w/v) sucrose dissolved in extraction solution, made up 12 hr previously and equilibrated in the cold . The tubes were centrifuged for 90 min at 25,000 rpm and 5 ml of the light-scattering viruscontaining zone were collected from above by syringe . This material was dialyzed for 2 hr against the extraction solution and then layered on a second sucrose density gradient, in SW 25 .2 tubes, consisting of 10 ml each of 30, 40, 50, and 60% (w/v) sucrose prepared as before . The tubes were centrifuged at 25,000 rpm for 45 min (sometimes up to 3 hr) to give a clean light-scattering viruscontaining zone that was collected as before . The material was diluted in one volume of extraction solution and pelleted in the Spinco rotor 40 at 40,000 rpm for 30 min, to be subsequently resuspended very gently in 0.85 % NaCl for injection into hoppers or in 0 .02 M phosphate buffer, pH 7 .5, for ITV spectrophotometry . Steps in purification were checked in the electron microscope
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MILNE, CONTI, AND LISA
and the final product was scanned in a Pyc-Unicam SP 1800 UV spectrophotometer . Each level of purification (crude preparation, partially purified preparation, final product) was on several occasions checked for infectivity, and infectivity tests were also made with several other preparations, details of which are given in the Results . Electron microscopy . Virus preparations were touched to 400-mesh grids coated with Formvar and carbon . The grids were immediately washed with several drops of negative stain and drained with filter paper . Unbuffered 2 % aqueous uranyl acetate (UA) was used routinely as a negative or positive stain ; as alternatives, unbuffered 2 % aqueous uranyl formate (UF) or 2 % neutral aqueous potassium phosphotungstate (KPT), ammonium molybdate, and sodium silicotungstate were also used . Material on grids was sometimes fixed for 10 min with 2 .5% glutaraldehyde in 0.05 M phosphate buffer pH 7 before staining . Preparations were examined in a Philips EVM 100 electron microscope and studies at higher resolution were made with a Philips EM 300 instrument (at the Centro di Microscopia Elettronica dell'Universita, in Turin) for which liquid nitrogen anticontamination was sometimes available . Virus particles were measured against tobacco mosaic virus particles whose normal length was taken as 300 nm and against the lattice spacing of crystalline catalase, which was taken to be 8 .6 nm (Wrigley, 1968) . Deliberate contamination of virus particles in the electron beam . When rather hydrophobic support films carrying virus particle's were washed with UA, the background negative staining was often absent though the particles themselves were positively stained and probably also fixed . If such particles were held in the focused electron beam for 10-20 sec with the decontamination device not operating, a sometimes informative halo of contamination could be built up around the particle, as seen in Fig . 4 .
RESULTS Purification The light-scattering zone of the second density gradient (Fig . 1) contained SO-95%
Pie . 1 . Light-scattering zone of virus obtained in the second sucrose density gradient centrifugation after 3 hr . of whole virus particles, but some loss of these occurred during the last pelleting and resuspension so that the final product contained 70-80% of whole particles, 20-30 of SVPs and traces of host material (Fig . 2) . The LTV absorption spectrum of the final product is shown in Fig . 3 . The maximum and minimum absorption occurred at 260 and 245 nm and the absorption ratios 280 : 260 and 260 :245 were 0 .68 and 1 .15, respectively . Processing about 30 g of roots and resuspending the final pellet in 2 ml buffer gave 0 .5 OD,ea unit per ml. When carbon tetrachloride was used for clarification, the result as seen in the electron microscope was similar to that with Freon but the yield, as measured by optical density, was slightly less and the infectivity considerably reduced . If clarification was omitted, the yield of virus was similar but much membrane material remained, and this was only partly removable by diatomaceous earth filtration . Negative Staining UA and OF gave similar results and in them the virus appeared the same with or without prior fixation ; indeed UA itself had a fixing effect so that washing a preparation on the grid with a few drops of UA prevented subsequent breakage of the particles by KPT . Sodium silicotungstate gave negative staining results essentially similar to those of UA and UF, but was less satis-
MAIZE ROUGH DWARF VIRUS
133
FIG . 2 . A sample of the final virus product, negatively stained in UA . Arrows indicate subviral particles . X 92,000.
is
2
0 .8
04
200
240
290
320
wavelengthlnm) FIG. 3 . Uncorrected U V absorption spectrum of the purified virus . This sample contained about 80% of whole particles and 20% subviral particles with B spikes . The maxima and minima occurred at 260 and 245 nm and the ratio max :min was 1 .15 . The absorption ratio 280 :260 nm was 0 .68 .
factory, and ammonium molybdate had a stripping effect similar to that of KPT . Particle Structure A spikes . Figures 4-6 show selected but characteristic MRDV particles or parts of them . Fig . 4A shows a virion negatively stained with UA, and 4B shows a virion
fixed in glutaraldehyde and negatively stained in I{PT ; in each case, six spikes may he. seen projecting . If the images represent particles with icosahedral symmetry resting on their 3-fold axes, then the virion possesses 12 spikes, here called A spikes, one at each 5-fold vertex . The spikes appear different in UA and TUT, being globular and 11 rim long in UA but acute and 16 tan long in KPT . No trace of a knob such as adenoviruses possess was found distal to the spike . Figure 4C shows a virion positively stained in UA ; two of the spikes (arrows) appear double, indicating that 8 spikes are visible . Reference to an icosahedral model shows that this appearance would be seen with a particle resting on an "edgi .e ., a 2-fold axis, but tilted slightly . Figure 4I) shows a particle positively stained in UA and deliberately contaminated in the electron beam ; spikes are not clearly seen but the hexagonal contamination pattern on the "round" particle is evidence of their presence . In crude preparations, complete virus particles were often found enclosed in tubes (see Conti and Lovisolo, 1971 ; Lesemann, 1972) . Such particles, as well as free-lying ones, also possessed A spikes (Fig . 4E) . B Spikes . Figures 4H-Ii illustrate stages in the stripping off of A spikes and parts of the outer capsid to reveal the inner capsid and certain resistant outer eapsomeres,
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MILNE, CONTI, AND LISA
FIG . 4
MAIZE ROUGH DWARF VIRUS here called B spikes and first described for MRWV by Lesemann (1972) . Figure 4K shows loss of the B spikes induced by KPT . In Figs . 4H and I, arrows indicate A spikes in positions corresponding to the hidden B spikes, and such pictures indicate that each A spike is attached to the outer end of a B spike but falls off as the particle is degraded . Figures 4J, L, M, N, and 0 together provide evidence that there are 12 B spikes arranged with 5, 3, 2 symmetry ; thus Figs . 4J and N show differently stained particles resting on 3-fold axes, Figs . 4L and M rest on 5-fold axes, and Fig . 40 on a 2-fold axis . The B spikes were about S nm long and 11 nm wide . Brief KPT treatment of unfixed complete virus or of physically produced Bspiked SVPs resulted in stripping of all the outer capsid to give smooth particles 50-57 nm in diameter as seen in Fig . 4K . No trace of the stripped material was found . In contrast, SVPs prepared by the chloroform method of Wetter et at . (1969) were disrupted by KPT as seen in Fig . 5A : the B spikes appeared to be detached but not destroyed . In plane view they appeared as a ring of 5 subunits surrounding a hole, and in profile they were brick-shaped (Fig . 5B) . Baseplates In empty subviral particles each B spike could be seen to have a circular or possibly pentagonal baseplate (Figs . 5C-J) that was a differentiated part of the inner capsid . Ghosts
After exposure in solution to I % KPT for 30 nun at 40° or after briefly shaking
135
with n-butanol, MRJV particles were reduced to smooth though somewhat collapsed SVPs apparently devoid of nucleic acid (Fig . 6) . Attached to the capsid wall and apparently projecting inward were structures smaller than the baseplates and probably different from them . In some cases they too appeared to be arranged with icosahedral symmetry . General Appearance
In other respects the virion appeared much as in the description of Lesemann (1972) . When negatively stained in UA, the particle, excluding A spikes, was 63-67 rim in diameter and in KPT it appeared larger, measuring 66-71 Mr . Sometimes distinct "capsomeres" could be distinguished around the perimeter or half the perimeter of particles and counts indicated between 22 and 20 . Luftig et al . (1972) suggest that a count of 20 indicates a particle with about 127 capsomeres, but we also saw, in some particles, 4 "capsomeres" beneath and between two A spikes and if the A spikes mark the vertices of the particle, then there may be 4 true capsomeres along an "edge," indicating a 92-capsomere particle . Figure 4F shows a particle appearing to have 10 capsomeres around half its periphery, but also, in two places, indicating 4 capsomeres along an "edge ." This interpretation is drawn in Fig . 4G. Similarly, Fig . 41 shows 4 "capsomeres" between the arrows whereas the right-hand side of Fig . 4H shows 5 "capsomeres" in the same position .
Fie . 4 . (A-U) A spikes on the outer capsid . (A) Negatively stained with UA ; (B) negatively stained withKPT after fixation ; (C) positively stained with UA ; (D) similar, but shows the hexagonal layer that surrounds contaminated positively stained particles . In (C) arrows show where the spikes appear double (a contamination halo is also evident) . X 340,000 . (E) Virus particles in a tube, from a crude preparation . Arrows indicate two of the A spikes . X 270,000 . (F) A virus particle interpreted in (G) . There appear to he 4 "capsomeres" from A to B and from C to D, but 5 from B to C, giving 4 "capsomeres" along the "edges" AB and CD, but 10 in half the periphery . This leads to two different estimates of the total number of outer capsomeres, namely 92 and about 127 . X 210,000.
(11-K) Stages in the removal of the outer capsid . In (H) and (1), arrows indicate A spikes overlying the as yet hidden B spikes, suggesting that the A and B spikes are coaxial . For further reference to (H) and (I), see text . X 340,000 . (J, L, M, N, and 0) . B-spiked subviral particles inn various orientations ; the B spikes are arranged with 5, 3, 2 symmetry. (J) and (N) lie on 3-fold axes but are differently stained ; (L) and (M) lie on 5-fold axes, and (0) on a 2-fold axis . X 340,000 .
J FIG . 5 . (A) B-spiked subviral particles purified according to the chloroform method of Wetter et at .
(1969) (kindly provided by Dr . G . Buccardo) treated on the grid with 2% ICPT . All but one of the B spikes (arrow) have been removed but lie in the background, X 340,000. (B) Detached B spikes enlarged : upper row in face view, lower row in profile . X 500,000 . (0-I) Empty B-spiked subviral particles showing haseplates (arrows) . In (U-I), the particles lie on 5-fold axes so the upper and lower B spikes and haseplates are superimposed and their images are reinforced . (J) Diagram summarizing the structures shown in Fig . 5 . X 340,000 . 136
MAIZE ROUGH DWARF VIRUS
137
RFiG. 6 . Subviral ghosts prepared by incubating purified rus in 1% KPT for 30 min at d0° . Arrows indicate structures that project inward from the capsid wall . At lower leftt is a particle in which these structures show 5-fold symmetry . Arrows indicate one structure surrounded by 5 others . X 300,000 .
Properties in Vitro The following treatments converted whole virus to SVPs with B spikes (Figs . 4L-O, and see below) : storage at 4° for 3 weeks, heating at 50° for 10 min, one cycle of freeze-thawing, 5 min shaking with chloroform . Brief treatment of unfixed particles with 1 % neutral KPT produced SVPs without B spikes (Fig . 4K) and stronger treatment, e .g ., 30 nun at 40 ° produced empty ghosts (Fig . 6) . Shaking whole virus with n-butanol for 5 min directly produced such ghosts .
Infectivity Tests were made with several kinds of preparation, listed below. Their infectivity and appearance in the electron microscope are shown in Table 1 . 1 . CE . Crude extracts made by grinding leaf enations or roots in 10 volumes of 0 .85% NaCl and centrifuging at low speed to remove debris . 2 . PPE . Partially purified extracts made by crushing roots in extraction solution, filtering through diatomaceous earth and pelleting by high speed centrifugation (Wetter et at ., 1969) . Pellets were resuspended in approximately 10 volumes of 0 .85 % NaCl.
3 . Freon-P . Virus suspensions prepared as described in Materials and Methods, using Freon clarification . Pellets were resuspended in about 100 volumes of 0 .85% NaCl . 4 . CCI,-P . As in 3, but using carbon tetrachloride instead of Freon . 5 . P . As in 3, but not treated with organic solvents ; diluted in 10 or 100 volumes of 0.85 % NaC . 6 . P + KPT . As in 5, finally treated with an equal volume of 2 % KPT for 15 min . 7 . P + CHC1 3 . As in 5, finally shaken with ;3 volume of chloroform for 5 min and centrifuged to separate the aqueous phase for injection . S . CHCI,-P . Chloroform-clarified preparations from roots as described by Wetter et al . (1969) . 9 . P-control . As in 5 but prepared from virus-free plants . Samples from the gradients were collected from regions corresponding to the virus-containing zones of infected preparations . Table 1 summarizes the data from several separate experiments in each of which homogeneous groups of hoppers were injected with some of the different preparations, always including a crude extract as a standard .
138
MILNE, CONTI, AND LISA TABLE 1 INFECTIVITY OP MRDV PREPARATIONS
Preparation (see text)
Particle type
Hoppers Injected
Survivors
( 0/,)
Transmitters
(%)
CE Freon-P (1 :100) CCI, (1 :100) P (1 :10) P (1 :100) P + KPT (1 :20) P + CHC1, (1 :10) P-control (1 :10)
• • • • •
(A) (A) (A) (A) (A) 0 A None
179 67 67 205 68 75 179 60
72 48 43 113 34 32 84 47
(40) (72) (64) (43) (50) (43) (47) (78)
23 24 5 53 8 0 0 0
(32) (50) (12) (47) (23) (0) (0) (0)
CE PPE (1 :10)
• •
A A
236 228
98 70
(42) (31)
47 28
(46) (40)
CE CHCI,-P (1 :10)
•
A A
92 137
72 100
(78) (73)
33 2
(46) (2)
Complete particles, • ; B-spiked SVPs, A ; naked SVPs, C . Parentheses indicate that the type of particle was present in small amounts . The results show that preparations purified with Freon or carbon tetrachloride, crude extracts and partially purified preparations (PPE and P), all containing complete particles and some SVPs, were infectious . Infectivity was highest for the Freon preparations but, in contrast, very low with carbon tetrachloride preparations . The chloroform-clarified preparations made according to Wetter et al . (CHC1,-P, Table 1) appeared to contain only B-spiked SVPs and were very poorly infectious . Partially purified preparations after treatment with chloroform or TPT, were not infectious and contained only B-spiked SVPs or naked SVPs, respectively . All symptoms produced on test plants by infectious preparations included dwarfing and production of leaf enations and were typical of the disease . Injection always caused the death of some hoppers, whether these were inoculated with virus preparations or virus-free extracts .
DISCUSSION
Puri,/icallus The purification here described is a basis for further studies of the complete virus particle but there remain some problems . It is not practicable to raise large enough amounts of infected material in the glass-
house and one must rely on naturally infected field plants available only in July, August, and part of September . It has not so far been possible to store purified virus for more than 24 hours without some particle breakdown, and we were unable to free the whole virus completely from SVPs . This was because SVPs were easily formed from the whole particles and because sucrose density gradients did not effect a complete separation when centrifuged for either a short or a long period . The UV absorption spectrum of the purified virus was reproducible but cannot be interpreted exactly because there remained small amounts of impurities and because the complete virus was mixed with some 20-30% of SVPs .
Structure l-IRDV shares many properties with the reovirus group, but none of the published pictures of reoviruses or their relatives show A spikes like those of MRDV . In Fig . 3b of Lesemann (1972) the fourth virus particle from the top and perhaps others show some evidence of A spikes, though Lesemann did not have the opportunity to study purified preparations of complete particles, and in the crude extracts 1vhicb lie examined, the A
MAIZE ROUGH DWARF VIRUS spikes are not easily seen . This may explain why they were not reported by him . The spikes may be unique to MRDV but it is just possible that they are present on some of the other reo-related viruses and have been overlooked . A careful reexamination would be interesting . Recently it has been suggested by Wood (1973) that MRDV may be related to the cytoplasmic polyhedrosis virus of silkworms, as that is a double-strand RNA icosahedral virus with spikes (Asai et al ., 1972) . It is not at present clear, however, whether this spiked particle is entire or is an SVP (Lewandowski and Traynor, 1972) . We estimated between 22 and 20 "capsomeres" around the periphery of MRDV, but Lesemann (1972) estimated 18 . Luftig et al. (1972) gave convincing evidence for 5 "capsomeres" in certain selected 90 ° arcs of reovirus type 3 outer capsids and claimed that, there were therefore 20 per complete periphery, indicating a capsomere total of around 127 . This, if not exact, was at least very different from the 92 or 180 (Amano et al ., 1972) required by current reovirus models . Our counts of peripheral "capsomeres" agree with those of Luftig et al ., but we also estimated, using A spikes as markers, that there were sometimes 4 "capsomeres" along an edge, indicating 02 in all . Probably the discrepancy arises because the "capsomeres" countable around certain sectors (but never the whole) of the periphery are not in fact images of single capsomeres but interference patterns caused by superposition of two or more . This is especially likely in "spherical" particles such as MRDV or rcoviruses and a simple count is therefore probably misleading . The B spikes seen by Lesemann (1972) and by us on MRDV SVPs closely resemble those found on reovirus type 3 by Smith et al . (1969) and by Luftig et al. (1972) and further confirm the similarity of the viruses . Luftig et al . suggested that the spikes do not reach the outer surface of the virus but our measurements and those of Lesemann (1972) suggest that in MRDV they do . In our case, MRDV cores were 50-57 nm in diameter and the B spikes were about 8 nm long ; adding twice 8 to the core diameter, we obtain 6673 run, a diameter at least as large as that
139
found for the whole virion, excluding A spikes . The finding of baseplates and structures within the ghosts adds complexity to the MRDV particle and it now seems that there are at least 5 (perhaps 6) morphological components, each likely to be made of a different protein : A spikes, B spikes, the rest of the outer capsid, baseplates, the rest of the inner capsid and the structures within the ghosts . Silverstein et al. (1972), Astell et al. (1972), Luftig et al . (1972), and Smith et al . (1969) have found 7 polypeptides in the reovirus capsid and though MRDV is still some way from such analysis, our results may be of interest to reovirus workers in their attempts to assign the polypeptides to specific viral structures . Wetter et al . (1969) tested scrologically the survival of MRDV antigens (almost certainly the B-spiked SVP or its products) and found them rather stable ; they therefore suggested that MRDV could be a relatively stable virus and that difficulty in purification might be due to surface properties . This and the fact that chloroform damaged the particles has led to the suggestion that the virus might have associated lipid (Wetter et al ., 1069 ; Harpaz, 1972) . In fact, the MRDV particle as a whole is sufficiently unstable in vitro to account for difficulties in purification and it now appears from its structure that MRDV contains no lipid . Structural and chemical analyses of reoviruses (see Luftig et al ., 1972) indicate that they also do not contain lipid .
Infectivity Preparations containing complete virus particles were infectious while those containing only SVPs were not . Infectivity seems therefore to reside in the complete particle only . The preparations we made by clarifying the sap with chloroform (CHUbP, Table 1) gave 2 infective hoppers out of 100 survivors, and it is not clear whether this low figure represents the true infectivity of the B-spiked SVPs or is due to a few remaining complete particles not detected in the electron microscope . Our method of testing infectivity did not
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MILNE, CONTI, AND LISA
allow assessment of the true proportion of hoppers that became viruliferous since it only accounted for those that survived and became inoculative . Actually, 22-469% of the injected hoppers died within 8 days of injection and we do not know whether a proportion of these contained multiplying virus . It may also be that some hoppers died from the effects of MRDV multiplication in them . It is known that MRDV damages eggs and immature hoppers (Harpaz, 1972) and it may likewise affect the adults . It has also been shown for some propagative viruses that viruliferous hoppers may not be able to transmit to plants (Sinha and Reddy, 1964 ; Paliwal, 1968) . For all these reasons, our estimates of infectivity can only be interpreted qualitatively but at this stage our aim was to establish which kinds of virus particles were infectious and could reproduce the typical disease in plants. ACKNOWLEDGMENTS This work was supported by a grant from the Italian Consiglio Nazionale delle Ricerche to R . G . Milne, who would like to thank the staff of the Laboratorio di Fitovirologia applicator for their unbounded kindness and cooperation . REFERENCES AMANO, Y ., KATAGIRI, S ., ISHIcA, N ., and WATANABE, Y . (1972) . Spontaneous degradation of reovirus capsid into subunits . J . Virol . 8, 805-808 . A5AI, .1 ., KAWAMOTO, F ., and KAWASE, S . (1972) . On the structure of the cytoplasmic-polyhedrosis virus of the silkworm, Bombyx mori . J. Invertebr . Pathol . 19, 279-280 . ASTELL, C ., SILVERSTEIN, S . C ., LEVIN, D . II ., and Acs, G . (1972) . Regulation of the reovirus RNA transcriptase by a viral capsomere protein . Virology 48,648-654 . BLACK, L . M . (1970) . Wound tumor virus . CMII/ AAB descriptions of plant viruses No . 34 (A. J . Gibbs, B . D . Harrison, and A . F . Murant, eds .) . CONTI, RI . (1969) . Transmission of barley yellow striate mosaic virus by mechanically injected Laodelphax striatellus Fallen. Ric . Sri . 39, 701707 . CONTI, M ., and Lov] sc Lo, 0 . (1971) . Tubular structures associated with maize rough dwarf virus particles in crude extracts : electron microscopy study . J . Gen . Virol . 13, 173- 176 . CONTI, M ., WETTER, C ., Lrxsoor, E ., and
LovisoLO, 0 . (1968) . Puriflcazione parziale del virus del nanismo ruvido del mail (MRDV) dell'insetto vet tore Laodelphax striatellus Fallen e sua identificazione sierologica. Atli Accad . Sci . Torino 102, 563--56S. HARPAZ, 1 . (1961) . Calligypona marginate, the vector of maize rough dwarf virus . PAO Plant Protection Bull . 9, 144-147 . HARPAZ, I . (1972) . Maize rough dwarf, a planthopper disease affecting maize, rice, small grain and grasses . Israel Universities Press, Jerusalem . HARPAZ, L, and KLEIN . M . (1969) . Vector-induced modifications in a plant virus . Entomol . FJxp . Appl . 12, 99-106, HARPAZ, I ., VInANO, C ., Lovisono, 0 ., and CONTI, M. (1965) . Indagini comparative so Javesella pellucida (Fabricius) a Laodelphax striatellus (Fallen) quali vettori del virus del nanismo ruvido del mais (maize rough dwarf virus) . Atli Accad . Sri. Torino 99, 8&5-901 . Hoscn, L . (1960) . Zwei Gerato zur Arbeitserleichterung and -beschleunigung bei den Reihenuntersuchungen uber Viruskrankheiten der Pflanzenkartoffeln . Nachrichtenbl . Dent . Pftanzenschutzdienstes (Braunschweig) 12, 154 . KITAGAWA, Y ., and SHIKATA, E . (1971) . On some properties and purification of rice black streaked dwarf virus . Rev . Plant Protec . Res . 4, 113-114 . LESEMANN, D . (1972) . Electron microscopy of maize rough dwarf virus particles . J. Gen . Virol . 16, 273-284 . LEWANpowsKI, L. J ., TRAYNOR, B . L . (1972) . Comparison of the structure and polypeptide composition of three double-stranded ribonucleic acid-containing viruses (diplornaviruses) : cytoplasmic polyhedrosis virus, wound tumor virus and reovirus . J. Virol. 10, 1053-1070. Lovisono, 0 . (1971) . Maize rough dwarf virus . CMI/AAB descriptions of plant viruses No . 72 (A. J . Gibbs, B . D . Harrison, and A . F . Murant, eds .) . LUFTIO, R . B ., KILHAM, S . S ., HAY, A . J ., ZWEERINK, H . J ., and JOKLIK, W . K . (1972) . An ultrastructural study of virions and cores of reovirus type 3 . Virology 48, 170-181 . LUISONI, E . (1969) . Partial purification of barley yellow striate mosaic virus . Ric . Sci . 39, 708-713 . LursONI, E ., WETTER, C ., Lovisoia, O ., and Coo,',, M . (1968) . Purificazione e sierologia del virus del nanismo ruvido del mais (MRDV) da piante di Zea mays L . Atli Accad . Sci . Torino 102, 539-544 . PALIWAL, Y. C, (1968), Changes in relative virus concentration in Endria inimica in relation to its ability to transmit wheat striate mosaic virus . Phytopathology 58, 386-387 . REDOLFI, P ., and PENNAZIO, S . (1972) . Double-
MAIZE ROUGH DWARF VIRUS stranded ribonucleic acid from maize rough dwarf virus . Acta Virol . (Prague) 16, 369-375. REDOLEI, P ., PENNAZIO, S ., and PAUL, TI . L . (1973) . Some properties of maize rough dwarf virus subviral particles . Acta Virol. (Prague) 17, in press. SILVERSTEIN, S . C ., ASTELL, C ., LEVIN, D . H ., SCHONBERG, M . and Acs, U . (1972) . The mecha-
nisms of reovirus uncoating and gene activation in vivo. Virology 47, 797-806 . SINHA, R . C ., and REDDY, D . V . R . (1964) . Improved fluorescent smear technique and its application in detection virus antigens in an insect vector . Virology 24, 626-634 .
141
SMITH, R . E ., ZWEERINK, H . J ., and ,loKLIK, W . K .
(1969) . Polypeptide components of virions, top component and cores of reovirus type 3 . Virology 39, 791-810 . WETTER, C ., LunSONI, E ., CoNTi, M ., and LoviSOLO, 0 . (1969) . Purification and serology of maize rough dwarf virus from plant and vector . Phytopalhol . Z . 66, 197-212 . WOOD, H . A. (1973) . Double-stranded RNA viruses . J . Gen . Virol . in press . WRIGLEY, N . G . (1968) . The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy . J . Ultrastruct . Res . 24, 454-464 .
53, 130-141 (1973)
Partial Purification, Structure and Infectivity of Complete Maize Rough Dwarf Virus Particles ROBERT G . MILNE,' MAURIZIO CONTI,
AND
VITTORIA LISA
Laboratorio di Fitovirologia applicata del C .N .R ., Via 0 . Vigliani 104-10135, Turin, Italy Accepted February 7, 1973 Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation . The product consisted mostly of complete particles, but some subviral particles were present . The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions ; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid . In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis . Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 5057 Ran subviral particle possessing 12 previously hidden spikes (B spikes) each about S nmlong, coaxial with the A spikes . Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate . Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures . Detached B spikes were composed of 5 morphological subunits surrounding a central hole . Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley . Crude and purified preparations containing the large virus particles were infectious ; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious . INTRODUCTION Maize rough dwarf virus (MRDV) causes a severe disease in maize and also multiplies in its planthopper vector . The data and literature on the virus have been summarized by Lovisolo (1971) and Harpaz (1972) . Wetter et al. (1969) purified a spherical 55 mn particle which contained doublestranded RNA (Redolfi and Pennazio, 1972) from diseased plants and from the vector ; but later it was found by Redolfi, Pennazio, and Paul (1973) to be a subviral particle I Present address : Laboratoire des Virus des Plantes, Institut de Botanique, 8 Rue Goethe, 67 Strasbourg, France .
(SVP) . The complete particle has a reolike double capsid some 70 nm in diameter . Lesemann (1972) studied further the structure of the SVP in purified preparations and that of the complete particle in crude plant extracts . He suggested that, the SVP retained at its 5-fold vertices parts of the outer capsid in the form of projecting capsomeres and found that on treatment with potassium phosphotungstate these were removed to leave a smooth SVP like that illustrated by Wetter et al . Harpaz and Klein (1969) and Conti and Lovisolo (1971) succeeded in transmitting the virus by injecting crude material from 130
Copyright © 1973 by Academic Press, Inc . All rights of reproduction in any form reserved .
MAIZE ROUGH DWARF VIILUS infected plants into the vector, but the method has not been applied systematically and the infectivity of purified preparations has not been reported . We report the partial purification of the complete virus particle and some new observations on the structure, properties, and infectivity of the virus .
MATERIALS AND METHODS Source of virus . The virus was purified mainly from naturally infected maize growing near Turin, using roots that were, split due to abnormal phloem growth (Luisoni et at ., 1968) . Roots or leaf enations of glasshouse-grown experimentally infected plants were also used with similar results . The glasshouse virus was an isolate obtained from Novara, Northern Italy, by Conti et al . (1968) and propagated by their method, which was to make hoppers viruliferous by feeding them on infected plants or by injecting them with virus preparations . These hoppers were then used to inoculate maize seedlings, which in turn were ready for harvest in 2-3 months . Planthoppers and infectivity assays . Colonies of MRDV-free Laodelphax striatellus Fallen were reared in the glasshouse according Harpaz et al . (1965) and the mature females were injected with preparations of infected and healthy plants in a cold room as described by Conti (1969) . Single hoppers were then caged on pots containing one maize and one barley plant at the coleoptile stage and were moved to fresh plants at weekly intervals . Both maize and barley were used because maize is the better indicator plant, but the hopper survives for only a few days when fed on it exclusively (Harpaz, 1961), whereas barley is a good food plant . During infectivity tests the number of surviving insects was recorded daily, arid the plants were checked for symptoms for up to 3 months, though most symptoms had appeared after I month . A minimum incubation period of 8 days was necessary to make the hoppers inoculative after the injection, so the percentage of transmitters was calculated on the number of insects which survived at least that period . Random samples of oninjected hoppers from the stock were also
131
routinely tested for infectivity to ensure that they were free of MRDV . Purification . Because the virus was unstable, all operations were performed in the cold as rapidly as possible ; usually, processing took 7-8 hr . The best method found is given below . For preliminary clarification, we used Freon or carbon tetrachloride as indicated by Kimura and Black (Black, 1970) and Kitagawa and Shikata (197 .1) for wound-tumor and rice black-streaked dwarf viruses . In each run, about 100 g of washed chilled roots were passed through the rollers of a sap press (Hosch, 1960 ; Luisoni, 1969) with dropwise addition of an equal amount (w/v) of cold extraction solution containing 0.05 ]41 Na2H1'O,, 0 .005 A! disodium EDTA and 0.0131 Na2SO,, pH 7 .8 (Wetter et al ., 1969) . Extracts were passed through nylon mesh, centrifuged at 4500 g for 5 min and then shaken for 5 min with an equal volume of Freon 113 (fluorocarbon 1,1,2-trifluoro1,2,2-trichloroethanc, from Schuchardt) . After centrifugation at 4500 g for 10 min to separate the emulsion, the upper aqueous phase was retained and 30 ml was layered in Spinco SW 25 .2 tubes on top of a minigradient consisting of 3 .5 ml each of 30, 40, 50, 60, and 70% (w/v) sucrose dissolved in extraction solution, made up 12 hr previously and equilibrated in the cold . The tubes were centrifuged for 90 min at 25,000 rpm and 5 ml of the light-scattering viruscontaining zone were collected from above by syringe . This material was dialyzed for 2 hr against the extraction solution and then layered on a second sucrose density gradient, in SW 25 .2 tubes, consisting of 10 ml each of 30, 40, 50, and 60% (w/v) sucrose prepared as before . The tubes were centrifuged at 25,000 rpm for 45 min (sometimes up to 3 hr) to give a clean light-scattering viruscontaining zone that was collected as before . The material was diluted in one volume of extraction solution and pelleted in the Spinco rotor 40 at 40,000 rpm for 30 min, to be subsequently resuspended very gently in 0.85 % NaCl for injection into hoppers or in 0 .02 M phosphate buffer, pH 7 .5, for ITV spectrophotometry . Steps in purification were checked in the electron microscope
132
MILNE, CONTI, AND LISA
and the final product was scanned in a Pyc-Unicam SP 1800 UV spectrophotometer . Each level of purification (crude preparation, partially purified preparation, final product) was on several occasions checked for infectivity, and infectivity tests were also made with several other preparations, details of which are given in the Results . Electron microscopy . Virus preparations were touched to 400-mesh grids coated with Formvar and carbon . The grids were immediately washed with several drops of negative stain and drained with filter paper . Unbuffered 2 % aqueous uranyl acetate (UA) was used routinely as a negative or positive stain ; as alternatives, unbuffered 2 % aqueous uranyl formate (UF) or 2 % neutral aqueous potassium phosphotungstate (KPT), ammonium molybdate, and sodium silicotungstate were also used . Material on grids was sometimes fixed for 10 min with 2 .5% glutaraldehyde in 0.05 M phosphate buffer pH 7 before staining . Preparations were examined in a Philips EVM 100 electron microscope and studies at higher resolution were made with a Philips EM 300 instrument (at the Centro di Microscopia Elettronica dell'Universita, in Turin) for which liquid nitrogen anticontamination was sometimes available . Virus particles were measured against tobacco mosaic virus particles whose normal length was taken as 300 nm and against the lattice spacing of crystalline catalase, which was taken to be 8 .6 nm (Wrigley, 1968) . Deliberate contamination of virus particles in the electron beam . When rather hydrophobic support films carrying virus particle's were washed with UA, the background negative staining was often absent though the particles themselves were positively stained and probably also fixed . If such particles were held in the focused electron beam for 10-20 sec with the decontamination device not operating, a sometimes informative halo of contamination could be built up around the particle, as seen in Fig . 4 .
RESULTS Purification The light-scattering zone of the second density gradient (Fig . 1) contained SO-95%
Pie . 1 . Light-scattering zone of virus obtained in the second sucrose density gradient centrifugation after 3 hr . of whole virus particles, but some loss of these occurred during the last pelleting and resuspension so that the final product contained 70-80% of whole particles, 20-30 of SVPs and traces of host material (Fig . 2) . The LTV absorption spectrum of the final product is shown in Fig . 3 . The maximum and minimum absorption occurred at 260 and 245 nm and the absorption ratios 280 : 260 and 260 :245 were 0 .68 and 1 .15, respectively . Processing about 30 g of roots and resuspending the final pellet in 2 ml buffer gave 0 .5 OD,ea unit per ml. When carbon tetrachloride was used for clarification, the result as seen in the electron microscope was similar to that with Freon but the yield, as measured by optical density, was slightly less and the infectivity considerably reduced . If clarification was omitted, the yield of virus was similar but much membrane material remained, and this was only partly removable by diatomaceous earth filtration . Negative Staining UA and OF gave similar results and in them the virus appeared the same with or without prior fixation ; indeed UA itself had a fixing effect so that washing a preparation on the grid with a few drops of UA prevented subsequent breakage of the particles by KPT . Sodium silicotungstate gave negative staining results essentially similar to those of UA and UF, but was less satis-
MAIZE ROUGH DWARF VIRUS
133
FIG . 2 . A sample of the final virus product, negatively stained in UA . Arrows indicate subviral particles . X 92,000.
is
2
0 .8
04
200
240
290
320
wavelengthlnm) FIG. 3 . Uncorrected U V absorption spectrum of the purified virus . This sample contained about 80% of whole particles and 20% subviral particles with B spikes . The maxima and minima occurred at 260 and 245 nm and the ratio max :min was 1 .15 . The absorption ratio 280 :260 nm was 0 .68 .
factory, and ammonium molybdate had a stripping effect similar to that of KPT . Particle Structure A spikes . Figures 4-6 show selected but characteristic MRDV particles or parts of them . Fig . 4A shows a virion negatively stained with UA, and 4B shows a virion
fixed in glutaraldehyde and negatively stained in I{PT ; in each case, six spikes may he. seen projecting . If the images represent particles with icosahedral symmetry resting on their 3-fold axes, then the virion possesses 12 spikes, here called A spikes, one at each 5-fold vertex . The spikes appear different in UA and TUT, being globular and 11 rim long in UA but acute and 16 tan long in KPT . No trace of a knob such as adenoviruses possess was found distal to the spike . Figure 4C shows a virion positively stained in UA ; two of the spikes (arrows) appear double, indicating that 8 spikes are visible . Reference to an icosahedral model shows that this appearance would be seen with a particle resting on an "edgi .e ., a 2-fold axis, but tilted slightly . Figure 4I) shows a particle positively stained in UA and deliberately contaminated in the electron beam ; spikes are not clearly seen but the hexagonal contamination pattern on the "round" particle is evidence of their presence . In crude preparations, complete virus particles were often found enclosed in tubes (see Conti and Lovisolo, 1971 ; Lesemann, 1972) . Such particles, as well as free-lying ones, also possessed A spikes (Fig . 4E) . B Spikes . Figures 4H-Ii illustrate stages in the stripping off of A spikes and parts of the outer capsid to reveal the inner capsid and certain resistant outer eapsomeres,
134
MILNE, CONTI, AND LISA
FIG . 4
MAIZE ROUGH DWARF VIRUS here called B spikes and first described for MRWV by Lesemann (1972) . Figure 4K shows loss of the B spikes induced by KPT . In Figs . 4H and I, arrows indicate A spikes in positions corresponding to the hidden B spikes, and such pictures indicate that each A spike is attached to the outer end of a B spike but falls off as the particle is degraded . Figures 4J, L, M, N, and 0 together provide evidence that there are 12 B spikes arranged with 5, 3, 2 symmetry ; thus Figs . 4J and N show differently stained particles resting on 3-fold axes, Figs . 4L and M rest on 5-fold axes, and Fig . 40 on a 2-fold axis . The B spikes were about S nm long and 11 nm wide . Brief KPT treatment of unfixed complete virus or of physically produced Bspiked SVPs resulted in stripping of all the outer capsid to give smooth particles 50-57 nm in diameter as seen in Fig . 4K . No trace of the stripped material was found . In contrast, SVPs prepared by the chloroform method of Wetter et at . (1969) were disrupted by KPT as seen in Fig . 5A : the B spikes appeared to be detached but not destroyed . In plane view they appeared as a ring of 5 subunits surrounding a hole, and in profile they were brick-shaped (Fig . 5B) . Baseplates In empty subviral particles each B spike could be seen to have a circular or possibly pentagonal baseplate (Figs . 5C-J) that was a differentiated part of the inner capsid . Ghosts
After exposure in solution to I % KPT for 30 nun at 40° or after briefly shaking
135
with n-butanol, MRJV particles were reduced to smooth though somewhat collapsed SVPs apparently devoid of nucleic acid (Fig . 6) . Attached to the capsid wall and apparently projecting inward were structures smaller than the baseplates and probably different from them . In some cases they too appeared to be arranged with icosahedral symmetry . General Appearance
In other respects the virion appeared much as in the description of Lesemann (1972) . When negatively stained in UA, the particle, excluding A spikes, was 63-67 rim in diameter and in KPT it appeared larger, measuring 66-71 Mr . Sometimes distinct "capsomeres" could be distinguished around the perimeter or half the perimeter of particles and counts indicated between 22 and 20 . Luftig et al . (1972) suggest that a count of 20 indicates a particle with about 127 capsomeres, but we also saw, in some particles, 4 "capsomeres" beneath and between two A spikes and if the A spikes mark the vertices of the particle, then there may be 4 true capsomeres along an "edge," indicating a 92-capsomere particle . Figure 4F shows a particle appearing to have 10 capsomeres around half its periphery, but also, in two places, indicating 4 capsomeres along an "edge ." This interpretation is drawn in Fig . 4G. Similarly, Fig . 41 shows 4 "capsomeres" between the arrows whereas the right-hand side of Fig . 4H shows 5 "capsomeres" in the same position .
Fie . 4 . (A-U) A spikes on the outer capsid . (A) Negatively stained with UA ; (B) negatively stained withKPT after fixation ; (C) positively stained with UA ; (D) similar, but shows the hexagonal layer that surrounds contaminated positively stained particles . In (C) arrows show where the spikes appear double (a contamination halo is also evident) . X 340,000 . (E) Virus particles in a tube, from a crude preparation . Arrows indicate two of the A spikes . X 270,000 . (F) A virus particle interpreted in (G) . There appear to he 4 "capsomeres" from A to B and from C to D, but 5 from B to C, giving 4 "capsomeres" along the "edges" AB and CD, but 10 in half the periphery . This leads to two different estimates of the total number of outer capsomeres, namely 92 and about 127 . X 210,000.
(11-K) Stages in the removal of the outer capsid . In (H) and (1), arrows indicate A spikes overlying the as yet hidden B spikes, suggesting that the A and B spikes are coaxial . For further reference to (H) and (I), see text . X 340,000 . (J, L, M, N, and 0) . B-spiked subviral particles inn various orientations ; the B spikes are arranged with 5, 3, 2 symmetry. (J) and (N) lie on 3-fold axes but are differently stained ; (L) and (M) lie on 5-fold axes, and (0) on a 2-fold axis . X 340,000 .
J FIG . 5 . (A) B-spiked subviral particles purified according to the chloroform method of Wetter et at .
(1969) (kindly provided by Dr . G . Buccardo) treated on the grid with 2% ICPT . All but one of the B spikes (arrow) have been removed but lie in the background, X 340,000. (B) Detached B spikes enlarged : upper row in face view, lower row in profile . X 500,000 . (0-I) Empty B-spiked subviral particles showing haseplates (arrows) . In (U-I), the particles lie on 5-fold axes so the upper and lower B spikes and haseplates are superimposed and their images are reinforced . (J) Diagram summarizing the structures shown in Fig . 5 . X 340,000 . 136
MAIZE ROUGH DWARF VIRUS
137
RFiG. 6 . Subviral ghosts prepared by incubating purified rus in 1% KPT for 30 min at d0° . Arrows indicate structures that project inward from the capsid wall . At lower leftt is a particle in which these structures show 5-fold symmetry . Arrows indicate one structure surrounded by 5 others . X 300,000 .
Properties in Vitro The following treatments converted whole virus to SVPs with B spikes (Figs . 4L-O, and see below) : storage at 4° for 3 weeks, heating at 50° for 10 min, one cycle of freeze-thawing, 5 min shaking with chloroform . Brief treatment of unfixed particles with 1 % neutral KPT produced SVPs without B spikes (Fig . 4K) and stronger treatment, e .g ., 30 nun at 40 ° produced empty ghosts (Fig . 6) . Shaking whole virus with n-butanol for 5 min directly produced such ghosts .
Infectivity Tests were made with several kinds of preparation, listed below. Their infectivity and appearance in the electron microscope are shown in Table 1 . 1 . CE . Crude extracts made by grinding leaf enations or roots in 10 volumes of 0 .85% NaCl and centrifuging at low speed to remove debris . 2 . PPE . Partially purified extracts made by crushing roots in extraction solution, filtering through diatomaceous earth and pelleting by high speed centrifugation (Wetter et at ., 1969) . Pellets were resuspended in approximately 10 volumes of 0 .85 % NaCl.
3 . Freon-P . Virus suspensions prepared as described in Materials and Methods, using Freon clarification . Pellets were resuspended in about 100 volumes of 0 .85% NaCl . 4 . CCI,-P . As in 3, but using carbon tetrachloride instead of Freon . 5 . P . As in 3, but not treated with organic solvents ; diluted in 10 or 100 volumes of 0.85 % NaC . 6 . P + KPT . As in 5, finally treated with an equal volume of 2 % KPT for 15 min . 7 . P + CHC1 3 . As in 5, finally shaken with ;3 volume of chloroform for 5 min and centrifuged to separate the aqueous phase for injection . S . CHCI,-P . Chloroform-clarified preparations from roots as described by Wetter et al . (1969) . 9 . P-control . As in 5 but prepared from virus-free plants . Samples from the gradients were collected from regions corresponding to the virus-containing zones of infected preparations . Table 1 summarizes the data from several separate experiments in each of which homogeneous groups of hoppers were injected with some of the different preparations, always including a crude extract as a standard .
138
MILNE, CONTI, AND LISA TABLE 1 INFECTIVITY OP MRDV PREPARATIONS
Preparation (see text)
Particle type
Hoppers Injected
Survivors
( 0/,)
Transmitters
(%)
CE Freon-P (1 :100) CCI, (1 :100) P (1 :10) P (1 :100) P + KPT (1 :20) P + CHC1, (1 :10) P-control (1 :10)
• • • • •
(A) (A) (A) (A) (A) 0 A None
179 67 67 205 68 75 179 60
72 48 43 113 34 32 84 47
(40) (72) (64) (43) (50) (43) (47) (78)
23 24 5 53 8 0 0 0
(32) (50) (12) (47) (23) (0) (0) (0)
CE PPE (1 :10)
• •
A A
236 228
98 70
(42) (31)
47 28
(46) (40)
CE CHCI,-P (1 :10)
•
A A
92 137
72 100
(78) (73)
33 2
(46) (2)
Complete particles, • ; B-spiked SVPs, A ; naked SVPs, C . Parentheses indicate that the type of particle was present in small amounts . The results show that preparations purified with Freon or carbon tetrachloride, crude extracts and partially purified preparations (PPE and P), all containing complete particles and some SVPs, were infectious . Infectivity was highest for the Freon preparations but, in contrast, very low with carbon tetrachloride preparations . The chloroform-clarified preparations made according to Wetter et al . (CHC1,-P, Table 1) appeared to contain only B-spiked SVPs and were very poorly infectious . Partially purified preparations after treatment with chloroform or TPT, were not infectious and contained only B-spiked SVPs or naked SVPs, respectively . All symptoms produced on test plants by infectious preparations included dwarfing and production of leaf enations and were typical of the disease . Injection always caused the death of some hoppers, whether these were inoculated with virus preparations or virus-free extracts .
DISCUSSION
Puri,/icallus The purification here described is a basis for further studies of the complete virus particle but there remain some problems . It is not practicable to raise large enough amounts of infected material in the glass-
house and one must rely on naturally infected field plants available only in July, August, and part of September . It has not so far been possible to store purified virus for more than 24 hours without some particle breakdown, and we were unable to free the whole virus completely from SVPs . This was because SVPs were easily formed from the whole particles and because sucrose density gradients did not effect a complete separation when centrifuged for either a short or a long period . The UV absorption spectrum of the purified virus was reproducible but cannot be interpreted exactly because there remained small amounts of impurities and because the complete virus was mixed with some 20-30% of SVPs .
Structure l-IRDV shares many properties with the reovirus group, but none of the published pictures of reoviruses or their relatives show A spikes like those of MRDV . In Fig . 3b of Lesemann (1972) the fourth virus particle from the top and perhaps others show some evidence of A spikes, though Lesemann did not have the opportunity to study purified preparations of complete particles, and in the crude extracts 1vhicb lie examined, the A
MAIZE ROUGH DWARF VIRUS spikes are not easily seen . This may explain why they were not reported by him . The spikes may be unique to MRDV but it is just possible that they are present on some of the other reo-related viruses and have been overlooked . A careful reexamination would be interesting . Recently it has been suggested by Wood (1973) that MRDV may be related to the cytoplasmic polyhedrosis virus of silkworms, as that is a double-strand RNA icosahedral virus with spikes (Asai et al ., 1972) . It is not at present clear, however, whether this spiked particle is entire or is an SVP (Lewandowski and Traynor, 1972) . We estimated between 22 and 20 "capsomeres" around the periphery of MRDV, but Lesemann (1972) estimated 18 . Luftig et al. (1972) gave convincing evidence for 5 "capsomeres" in certain selected 90 ° arcs of reovirus type 3 outer capsids and claimed that, there were therefore 20 per complete periphery, indicating a capsomere total of around 127 . This, if not exact, was at least very different from the 92 or 180 (Amano et al ., 1972) required by current reovirus models . Our counts of peripheral "capsomeres" agree with those of Luftig et al ., but we also estimated, using A spikes as markers, that there were sometimes 4 "capsomeres" along an edge, indicating 02 in all . Probably the discrepancy arises because the "capsomeres" countable around certain sectors (but never the whole) of the periphery are not in fact images of single capsomeres but interference patterns caused by superposition of two or more . This is especially likely in "spherical" particles such as MRDV or rcoviruses and a simple count is therefore probably misleading . The B spikes seen by Lesemann (1972) and by us on MRDV SVPs closely resemble those found on reovirus type 3 by Smith et al . (1969) and by Luftig et al. (1972) and further confirm the similarity of the viruses . Luftig et al . suggested that the spikes do not reach the outer surface of the virus but our measurements and those of Lesemann (1972) suggest that in MRDV they do . In our case, MRDV cores were 50-57 nm in diameter and the B spikes were about 8 nm long ; adding twice 8 to the core diameter, we obtain 6673 run, a diameter at least as large as that
139
found for the whole virion, excluding A spikes . The finding of baseplates and structures within the ghosts adds complexity to the MRDV particle and it now seems that there are at least 5 (perhaps 6) morphological components, each likely to be made of a different protein : A spikes, B spikes, the rest of the outer capsid, baseplates, the rest of the inner capsid and the structures within the ghosts . Silverstein et al. (1972), Astell et al. (1972), Luftig et al . (1972), and Smith et al . (1969) have found 7 polypeptides in the reovirus capsid and though MRDV is still some way from such analysis, our results may be of interest to reovirus workers in their attempts to assign the polypeptides to specific viral structures . Wetter et al . (1969) tested scrologically the survival of MRDV antigens (almost certainly the B-spiked SVP or its products) and found them rather stable ; they therefore suggested that MRDV could be a relatively stable virus and that difficulty in purification might be due to surface properties . This and the fact that chloroform damaged the particles has led to the suggestion that the virus might have associated lipid (Wetter et al ., 1069 ; Harpaz, 1972) . In fact, the MRDV particle as a whole is sufficiently unstable in vitro to account for difficulties in purification and it now appears from its structure that MRDV contains no lipid . Structural and chemical analyses of reoviruses (see Luftig et al ., 1972) indicate that they also do not contain lipid .
Infectivity Preparations containing complete virus particles were infectious while those containing only SVPs were not . Infectivity seems therefore to reside in the complete particle only . The preparations we made by clarifying the sap with chloroform (CHUbP, Table 1) gave 2 infective hoppers out of 100 survivors, and it is not clear whether this low figure represents the true infectivity of the B-spiked SVPs or is due to a few remaining complete particles not detected in the electron microscope . Our method of testing infectivity did not
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allow assessment of the true proportion of hoppers that became viruliferous since it only accounted for those that survived and became inoculative . Actually, 22-469% of the injected hoppers died within 8 days of injection and we do not know whether a proportion of these contained multiplying virus . It may also be that some hoppers died from the effects of MRDV multiplication in them . It is known that MRDV damages eggs and immature hoppers (Harpaz, 1972) and it may likewise affect the adults . It has also been shown for some propagative viruses that viruliferous hoppers may not be able to transmit to plants (Sinha and Reddy, 1964 ; Paliwal, 1968) . For all these reasons, our estimates of infectivity can only be interpreted qualitatively but at this stage our aim was to establish which kinds of virus particles were infectious and could reproduce the typical disease in plants. ACKNOWLEDGMENTS This work was supported by a grant from the Italian Consiglio Nazionale delle Ricerche to R . G . Milne, who would like to thank the staff of the Laboratorio di Fitovirologia applicator for their unbounded kindness and cooperation . REFERENCES AMANO, Y ., KATAGIRI, S ., ISHIcA, N ., and WATANABE, Y . (1972) . Spontaneous degradation of reovirus capsid into subunits . J . Virol . 8, 805-808 . A5AI, .1 ., KAWAMOTO, F ., and KAWASE, S . (1972) . On the structure of the cytoplasmic-polyhedrosis virus of the silkworm, Bombyx mori . J. Invertebr . Pathol . 19, 279-280 . ASTELL, C ., SILVERSTEIN, S . C ., LEVIN, D . II ., and Acs, G . (1972) . Regulation of the reovirus RNA transcriptase by a viral capsomere protein . Virology 48,648-654 . BLACK, L . M . (1970) . Wound tumor virus . CMII/ AAB descriptions of plant viruses No . 34 (A. J . Gibbs, B . D . Harrison, and A . F . Murant, eds .) . CONTI, RI . (1969) . Transmission of barley yellow striate mosaic virus by mechanically injected Laodelphax striatellus Fallen. Ric . Sri . 39, 701707 . CONTI, M ., and Lov] sc Lo, 0 . (1971) . Tubular structures associated with maize rough dwarf virus particles in crude extracts : electron microscopy study . J . Gen . Virol . 13, 173- 176 . CONTI, M ., WETTER, C ., Lrxsoor, E ., and
LovisoLO, 0 . (1968) . Puriflcazione parziale del virus del nanismo ruvido del mail (MRDV) dell'insetto vet tore Laodelphax striatellus Fallen e sua identificazione sierologica. Atli Accad . Sci . Torino 102, 563--56S. HARPAZ, 1 . (1961) . Calligypona marginate, the vector of maize rough dwarf virus . PAO Plant Protection Bull . 9, 144-147 . HARPAZ, I . (1972) . Maize rough dwarf, a planthopper disease affecting maize, rice, small grain and grasses . Israel Universities Press, Jerusalem . HARPAZ, L, and KLEIN . M . (1969) . Vector-induced modifications in a plant virus . Entomol . FJxp . Appl . 12, 99-106, HARPAZ, I ., VInANO, C ., Lovisono, 0 ., and CONTI, M. (1965) . Indagini comparative so Javesella pellucida (Fabricius) a Laodelphax striatellus (Fallen) quali vettori del virus del nanismo ruvido del mais (maize rough dwarf virus) . Atli Accad . Sri. Torino 99, 8&5-901 . Hoscn, L . (1960) . Zwei Gerato zur Arbeitserleichterung and -beschleunigung bei den Reihenuntersuchungen uber Viruskrankheiten der Pflanzenkartoffeln . Nachrichtenbl . Dent . Pftanzenschutzdienstes (Braunschweig) 12, 154 . KITAGAWA, Y ., and SHIKATA, E . (1971) . On some properties and purification of rice black streaked dwarf virus . Rev . Plant Protec . Res . 4, 113-114 . LESEMANN, D . (1972) . Electron microscopy of maize rough dwarf virus particles . J. Gen . Virol . 16, 273-284 . LEWANpowsKI, L. J ., TRAYNOR, B . L . (1972) . Comparison of the structure and polypeptide composition of three double-stranded ribonucleic acid-containing viruses (diplornaviruses) : cytoplasmic polyhedrosis virus, wound tumor virus and reovirus . J. Virol. 10, 1053-1070. Lovisono, 0 . (1971) . Maize rough dwarf virus . CMI/AAB descriptions of plant viruses No . 72 (A. J . Gibbs, B . D . Harrison, and A . F . Murant, eds .) . LUFTIO, R . B ., KILHAM, S . S ., HAY, A . J ., ZWEERINK, H . J ., and JOKLIK, W . K . (1972) . An ultrastructural study of virions and cores of reovirus type 3 . Virology 48, 170-181 . LUISONI, E . (1969) . Partial purification of barley yellow striate mosaic virus . Ric . Sci . 39, 708-713 . LursONI, E ., WETTER, C ., Lovisoia, O ., and Coo,',, M . (1968) . Purificazione e sierologia del virus del nanismo ruvido del mais (MRDV) da piante di Zea mays L . Atli Accad . Sci . Torino 102, 539-544 . PALIWAL, Y. C, (1968), Changes in relative virus concentration in Endria inimica in relation to its ability to transmit wheat striate mosaic virus . Phytopathology 58, 386-387 . REDOLFI, P ., and PENNAZIO, S . (1972) . Double-
MAIZE ROUGH DWARF VIRUS stranded ribonucleic acid from maize rough dwarf virus . Acta Virol . (Prague) 16, 369-375. REDOLEI, P ., PENNAZIO, S ., and PAUL, TI . L . (1973) . Some properties of maize rough dwarf virus subviral particles . Acta Virol. (Prague) 17, in press. SILVERSTEIN, S . C ., ASTELL, C ., LEVIN, D . H ., SCHONBERG, M . and Acs, U . (1972) . The mecha-
nisms of reovirus uncoating and gene activation in vivo. Virology 47, 797-806 . SINHA, R . C ., and REDDY, D . V . R . (1964) . Improved fluorescent smear technique and its application in detection virus antigens in an insect vector . Virology 24, 626-634 .
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(1969) . Polypeptide components of virions, top component and cores of reovirus type 3 . Virology 39, 791-810 . WETTER, C ., LunSONI, E ., CoNTi, M ., and LoviSOLO, 0 . (1969) . Purification and serology of maize rough dwarf virus from plant and vector . Phytopalhol . Z . 66, 197-212 . WOOD, H . A. (1973) . Double-stranded RNA viruses . J . Gen . Virol . in press . WRIGLEY, N . G . (1968) . The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy . J . Ultrastruct . Res . 24, 454-464 .