Purification of potato virus X without aggregation

VIROLOGY

16,

8-15 (1961)

Purification

of

Potato

Virus

X Without

Aggregation’

M. K. CORBETT Plant

Pathology

Department,

University

Accepted

May

of Florida, 2,

Gainesville,

Florida

1961

Purification of potato virus X (PVX) by differential ultracentrifugation yielded preparations of aggregated entwmed masses of flexuous rods. Nonaggregated particles were obtained by rate zonal density-gradient centrifugation. Pigments and microsomal material were removed by preferential adsorption to activated charcoal (Merck N. F. 18351) without a reduction in PVX infectivity. The virus occurred in the gradient column in a visible zone sufficiently pure for electron microscopy. Dissociation or aggregation of flexuous rods 513 rnp in length was very slight. Infectivity tests were conducted with plants of Cassia occidentalis L. Differences in concentration of 20% or more between two PVX inocula were consistently detected, and occasionally 10% differences were detected.

ods of purifying nonaggregated infectious virus were sought. Potato virus X (PVX) was first isolated This paper describes a method of purifyby Smith (1931) from potatoes infect,ed by ing nonaggregated flexuous rods of PVX two or more viruses. The virus particles and reports Cassia occidentalis as a satisassociated with the potato mottle disease factory local-lesion host for bioassay. have been partially purified by chemical precipitation (Bawden and Pirie, 1938)) difMATERIALS AND METHODS ferential ultracentrifugation (Loring and A moderately mild strain of PVX, obWyckoff, 1937)) and chromatography tained from A. F. Ross, Cornell University, (Levin, 1958). They occur as long flexuous was used in all experiments. The virus, origrods which have a tendency to aggregate end inally very mild in several hosts (Rochow on end to form large entangled massesthat and Ross, 1955)) produced throughout this become insoluble jellies (Kleczkowski and study mild rings in inoculated leaves of Nixon, 1950). Markham (1959) concluded tobacco (Nicotiana tabacum L. var. Turkthat it is undoubtedly this property of ish), and a mild mottle in systemically inaggregation that has prevented PVX from fected leaves. The virus was maintained being extensively studied. Reichmann and increased in plants of Turkish tobacco. (1959) reported purification of nonaggre- Ten to 14 days after inoculation, systemgated PVX by dialysis against sodium cit.- ically infected leaves were ground in a food rate and differential ultracentrifugation of chopper and the juice was expressedthrough the dialyzate. Since attempts to prepare cheesecloth. Juice thus obtained was used nonaggregated PVX by Reichmann’s i 1959) for all inoculations and purification experiprocedure gave largely aggregated noninfecments. A Servall angle centrifuge (model tious rods under our conditions, other meth- SP) was used for low-speed centrifugation INTRODUCTION

(15 minutes at 3000 rpm) . Cassia occiden,talis, reported by Anderson (1958) to produce numerous local lesions when inoculated with PVX, was used for all infectivity tests. Lesions were

’ Florida Agricultural Experiment Station Journal Series, No. 1240. This investigation was supported in part by research grant E-3148 from the National Institute of Allergy and Infectious Diseases, United States Public Health Services. 8

PURIFICATION

OF

counted 4-6 days after inoculation and analyzed by Student’s t test (Snedecor, 1946). The plants were grown as described earlier by Corbett (1960). Density-gradient centrifugation similar to that described by Brakke (1951, 1953) was used. Gradient columns for rate zonai ccntrifugation were made with sucrose dissolved in buffer (0.01 M KCl, 0.005 M K-HP04, 0.0005 iM KH,POa pH 7.7) at the rate of 100,200,300, and 400 g per liter; the respective solutions were layered (4, 7, 7, and 7 ml) in nitrocellulose centrifuge tubes. All gradients were made at least 1 clay before use and stored at 10°C. A 2-1111 sample of virus suspension was layered on top of each of 2 gradients and centrifuged for 1.5 hours at. 23,000 rpm in the SW 25.1 rotor of a Spinco model L ultracentrifuge. Two tubes of each run contained material from syst,emically infected tobacco plants; the third contained material from noninoculated tobacco plants of comparable age. After centrifugation, the tubes were viewed and the visible zones removed as described by Corbett (1960). The zones thus obtained were used for infectivity studies and electron microscopy. Material to be examined was mixed with a small amount of polystyrene latex and sprayed onto collodion-covered specimen grids according to the technique of Backus and Williams (1950). The latex suspension (Dow Chemical Company, Midland, Michigan) was from run LS-057-A, with an average particle size of 264 rnp * 6 mp. Screens were shadowed at tan-l, 0.31 with chromium, and viewed with a Philips electron microscope, model 100. Fields were picked at random and the virus particles measured by comparison with replica gratings. of Cassia Occidentalis of Potato Virus X Infectizhj

Suitability

for rissay

Cassia occidentalis proved to be a suitable plant for bioassay studies with PVX. Small, discrete lesions that are easily counted appear on the inoculated leaflets 34 days after inoculation and continue to enlarge slightly for a few days. The leaflets may abscise after 4 days if the lesion count is high. Lesions appear on the older leaves

POTATO

VIRUS

9

S

l-2 days earlier than on younger leaves. Plants containing 5-10 leaves with 6 leaflets per leaf are easy to obtain and the opposite leaflets offer comparable physiological material for comparative st.udies. Lesion numbers were approximately inversely proportional to the dilution of the inoculated virus at low concentration. Unpurified virus from systemically infected tobacco plants was diluted in 0.01 M neutral phosphate buffer. Each dilution was applied to 30 leaflets in such a way that each occurred the same number of times in the same leaflet position. The average number of lesions per Icaflct in two experiments was as follows: undiluted, 115; lo-‘, 106; 10W2, 18; 10-3, 2; lo-“, 0.5; lo-“, 0.08; lo-“, 0. Two inocula, one considered the “standard” and the other the “unknown,” were prepared by dilution with 0.01 M neutral phosphate buffer from a single sample of crude juice. The “standard” was a 1:IO dilution and the “unknown” differed from this by known amounts. The two were compared on opposite leaflets of 15 evenly pinnate compound leaves in experiments replicated four times, Inocula differing in PVX content by 20% or more consistently gave lesion counts that were significantly different statistically (Table 1). Occasionally, differences of 10% were detected. The sensitivity of t’he host thus appears to be similar to that of Go?nphrena globosa L. (Rochow et al., 1955). DIFFERENTIAL

CENTRIFUGATIOX

Because of previous reports of aggregation of PVX during purification by ultraccntrifugation, the PVX strain used in the present, work was purified by this method to provide a reference for the other methods used. Crude infectious plant juice was clarified by centrifugation for 15 minutes at 7500 rpm in a No. 21 Spinco rotor. The resultant supcrnatant, which was light green, was centrifuged for 2 hours at 37,500 rpm in a h’o. 40 Spinco rotor. The resultant, dark greenish-brown pellets were resuspended in 2 ml of 0.01 Jf neutral phosphate buffer per tube and the cycle was repeated three times. The final pellets were gelatinous and had a brownish tinge. When resuspended in distilled water to give a thirteenfold concentra-

10

CORBETT TABLE

1

OF OPPOSITE LEAFLETS OF Cassia occidentalisTO DIFFERENCES IN INFECTIVITY OF Two POTATO VIRUS X INOCULA DILUTED WITH 0.01 M NEUTRAL PHOSPHATE BUFFER

SENSITIVITY

Virus content of “unknown” as percentage of “standard””

Number

of lesions

per leafletb

“Standard”

“Unknown”

46 52 59 65 59 43 137 38 62 102 72 121 104 104 140

40 43 52 54 57 24” 82c 14c 35c 48” 38” 58” 59” 79” 92c

fore and after each step in the procedures. After clarification, the material was subjected to density-gradient centrifugation, and the various zones obtained were assayed for infectivity on C. occidentalis and examined in the electron microscope for homogeneity and degree of aggregation. Low Speed Centrifugation

100 100 90 90 90 90 80 80 75 70 70 65 65 50 50

Centrifugation for 15 minutes at 3000 rpm in a Servall model SP centrifuge of otherwise untreated crude juice resulted in a light-green supernatant and a dark-green pellet containing a moderate amount of starch (Table 2). The loss of virus in the pellet was probably due to entanglement of the flexuous rods with large, heavy cellular components. The gradient columns after centrifugation were greenish. Zones were visible when a narrow beam of light was directed into the column from above in a dark room. Two zones, one 5 mm in width and 3-8 mm below the meniscus, and the 6 “Standard” wasinfectious juice diluted 1:lO. other approximately 4-5 mm in width and bEach figure is the averageof 60leaflets. occurring 1.2-1.6 cm below the meniscus, c Difference between “standard” and “un- were common to all tubes. When juice from known” was statistically significant at the 1% PVX-infected plants was used, an addilevel. tional zone 5 mm in width and located 1% 2.3 cm below the meniscus was evident. tion the preparation appeared opalescent After the zones were removed and the tubes with a characteristic sheen. had drained, the small green pellet at the When the 13 x preparation was diluted bottom of the tube was resuspended in 2 1: 10 with water and rubbed on C. occi- ml of distilled water. When infectious madentalis, the average lesion count per leafterial was used, samples from each zone, let was 128. The 13 x preparation was from areas above and below each zone, and sprayed directly onto collodion-filmed grids from resuspended pellets were assayed and examined in the electron microscope. without further dilution (Table 2). Small The particles were excessively aggregated samples of these materials, plus comparaend on end, and some parallel aggregation ble samples from tubes used for juice from occurred. Such preparations are of little use healthy plants, were examined under the in determining a normal length for the electron microscope. virus particle or in determining the effects of The zone which occurred in all tubes 3-8 various treatments upon particle morpholmm below the meniscus was not tested for ogy. In order to obtain unaggregated prepa- infectivity becauseprevious tests had shown rations of PVX for studies on the nature of that it did not contain any infectious maflexuous rod viruses (as compared to the terial. Zones from the tubes used for juice rigid rod virus particle associated with tofrom healthy plants were not tested for infectivity because the plants were indexed bacco mosaic) different techniques of purification were sought. for the presence of virus at the beginning of the experiment. PRELIMINARY CLARIFICATION Electron micrographs showed the virus to Various methods for clarifying crude juice occur as long flexuous rods, with little parwere tested. Infectivity tests were made be- ticle aggregation or dissociation. The sample

PURIFICATIO?T

OF POTATO

contained considerable amounts of microsomal and larger cellular material. Flexuous rods were not present in samples from the tubes used for juice from healthy plants.

VIRUS

11

S TABLE

2

AGGREGATION BY ITS

OF POTATO VIRUS X DISTRIBUTION AFTER RATE

TRIFUGATION OF EXTRACTS 01’s TECHNIQUES

AS

IWXCATED ZONAL CENBY VARI-

CLARIFIED

Freezing The supernatant resulting from low speed centrifugation of crude juice was frozen at Clarification -24”C, thawed, and recentrifuged at low procedure” speed. The resulting supernatant was light brown, and the pellet light green (Table 2). After rat,e zonal centrifugation of the super- ~natant, zones similar to those of the previous Low speed experiment were obtained (Table 2). The Freezing gradients were clearer than when clarificaHeat tion was by low speed centrifugation only, Detergent and the zones were more distinct. Chloroform The virus particles appeared to be broken, Charcoal and the sample contained a considerable (1 Each amount of microsomal material when eleccidentalis. tron micrographed. Helrtiny

nt 40” C

The supernatant resulting from low speed centrifugation was heated for 1 hour at 40” and recentrifuged at low speed. The resulting supernatant was greenish brown. After rate zonal density-gradient centrifugation, zones were evident in all three tubes. A 5mm zone 1.8-2.3 cm below the meniscus in the gradient,s used for juice from diseased plants differed from the comparable ones in the other treatments in that it consisted of two layers so close together that it was impossible to separate them. Samples from the middle zone, lowest zone, the area below the zone, and the bottom of the tube were assayed (Table 2). When viewed in the electron microscope the virus particles were markedly aggregated and the sample contained somemicrosomal material. The anionic synthetic detergent Igepon T-73 (active ingredient sodium N-methyl X-oleoyl taurate) was added to crude juice after low speed centrifugation; the final concentration of Igepon T-73 was 0.1% (Brakke, 1959). The mixture was heated at 40” for 1 hour and then centrifuged at low speed. The supernatant was light brown, and the pellet was light green in color. After rate zonal cent,rifugation, the 1.8-2.2 cm zone again appeared as two layers. Samples

I Lesions

Virus sus( pension

per leaflet given fraction” Rate

zonal

by indicated

centrifugationc

4hove zoned

Zonec

0.9 15 2 1 5 0.5

122 155 115 120 169 136

‘.-_ i 30” 1 96l 1 33, ! 41k I 164’ 107” is an average

15 / 82 50

-

of 28 leaflets

b See t,ext for details. c Centrifuged for 1.5 hours at SW 25.1 rotor (Ppinco model I,). d Sample removed from area mm below meniscus of tube. e Sample removed from area mm below meniscus of tube. f Sample removed from area mm below meniscus of tube. Q Pellet from drained tubes ml of water and assayed. h Low speed supernatant. The let induced 20 lesions. i Low speed supernatant. The let induced 71 lesions. i Low speed supernatant. The let induced 21 lesions. k Low speed supernatant. The let induced 32 lesions. t Aqueous phase. m Fi1trat.e.

, ;

53 144 72

75 1

36

48 13

83 12

on C:assia

23,000

rpm

oc-

in an

1.2-1.6

cm

i

1

1.8-2.3

cm

i

I

2.5-3.0

rm

f

1

in

2

resuspended resuspended

pel-

resuspended

pel-

resuspended

pel-

resuspended

pel-

from the zone at 1.2-1.6 cm, from the zone at 1.8-2.2 cm, from the area immediately below the zone, and from the resuspended material from the bottom of the tube were assayed (Table 2). When viewed under the electron microscope the preparation was seen to contain very little microsomal material, and the virus particles were fairly well dispersed. Aggregation was not as extensive as that received from heat t.reat,ment without the detergent.

12

CORBETT

Chloroform

Emulsion

Equal volumes of chloroform and of the supernatant from low speed centrifugation were shaken for 5 minutes. The emulsion was broken by low speed centrifugation, and t,he light brown aqueous phase was removed by decantation and assayed (Table 2). After rate zonal centrifugation, the first 7 mm below the meniscus was greenish yellow in color. A zone at 1.2-1.6 cm occurred in all three tubes. A zone at 1.8-2.2 cm occurred only in those gradients used for juice from diseased material. The zones, areas below the zones, and the pellets were assayed (Table 2). The virus particles were not very aggregated and the sample was relat,ively free from microsomal material when electron micrographed. Adsorption

with

Activated

Charcoal

The supernatant resulting from low speed centrifugation was mixed with activated

charcoal (Merck N. F. 18351) at the rate of 0.1 g/ml. The mixture was stirred occasionally during the next 30 minutes and then filtered through Whatman No. 1 filter paper in a Biichner funnel. The filtrate, which was clear with a slight greenish tinge, was assayed (Table 2). It induced an average of 107 lesions per leaflet compared to 30 lesions induced by the starting material. No change in pH occurred during the adsorption process, and the volume reduction (one-third) of the filtrate was not sufficient to account for the increase in lesion number. After rate zonal centrifugation, the gradients were very clear and the zones very distinct (Table 2). Figure 1 is part of a droplet pattern obtained from sprayed material from the zone at 1.8-2.3 cm. The virus particles were not extensively aggregated or dissociated. The preparation was almost completely devoid of microsomal or larger contaminating material. The particles were

Fig. 1. Particles of potato virus X from visible zone (1.S2.3 cm) obtained by rate tonal density-gradient centrifugation of infectious plant juice clarified by low speed centrifugution and adsorption by activated charcoal. Preparation shadowed with chromium at tan-’ 0.31. Magnification: X 31,000; the polystyrene latex particles have a diameter of 264 2 6 rnp. Photo by J. W. Carlisle.

PURIFICATION

OF

of uniform length and were generally distributed evenly on the specimen screen. Sixty-three per cent of 400 particles counted from fields picked at random were 513 rnp in length (Fig. 2). The zone at 1.8-2.3 cm was removed from some tubes and layered on gradients varying in sucrose concentrations from 10% to 40%. After 4 hours of centrifugation at 23,500 rpm in the SW 25.1 rotor, 2 visible zones occurred in the tubes. Both the top zone (4.1-4.3 cm below the meniscus), and t’he bottom zone (4.5-4.6 cm below the meniscus) were assayed and also used for electron microscopy. The top zone induced an average of 99 lesions per leaflet and consisted essentially of nonaggregated 513-rnp rods. The bottom zone, which induced 32 lesions per leaflet, showed excessive particle aggregation. DISCUSSION

There is much information on the structure, substructure, chemical properties, anomalous proteins, and nucleic acid infectivity of tobacco mosaic virus (TMV) This may be attributed partly to the fact that TMV was the first plant virus described, or to its stability, concentration, infectiousness, and ease of purification and assay. The uniqueness of TMV among plant viruses is readily recognized, and the need for supporting investigations with other plant viruses is imperative. Until recently (Bawden and Kleczkowski, 1959)) very little has been accomplished with the viruses which occur as flexuous rods. The lack of investigations on these viruses may be attributed in part to the difficulty of obtaining unaggregated virus preparations, to inadequate methods of assay, and to the instability of the virus. Although Gomphrena globosa has proved adequate for the assay of PVX in other areas (Rochow et al., 1955), it is not as satisfactory under Florida conditions as Cassia occidentalis. As grown in Florida, plants of C. occidentalis provide more usable genetically similar leaf material than do those of G. globosa. Plants having 6-10 leaves, each with 6 leaflets, are easily obtained, allowing for 36-60 comparisons on 1 plant. The lesions occur in less time than do those in G. globosa and are discrete and

POTATO

351

VIRUS

I rr, I I I I 0 50

13

X

NUMBER

FIG. 2. Length

! 150

100 OF

I 200

, 250

I 300

PARTICLES

distribution of potato virus particles obtained by rate zonal centrifugation juice clarified with activated charcoal.

X of

easy to count. The plant reacts with local necrosis to infection by other viruses (Anderson, 1958; Corbett, 1960) and is very useful for routine indexing and plant virus identification. The method of charcoal adsorption and density-gradient centrifugation is superior to other methods used for purification of potato virus X. The final preparation was more homogeneous than that obtained by other methods, as indicated by freedom from microsomal material and uniformity in particle size and morphology. If infect,ivity below the zone and in the pellet is taken as a measure of aggregation, then considerably less than 10% of the virus particles may have been aggregated because the dilution curve became flattened when the lesion count, was about 100. The procedure is very mild and may be compared in principle to ion exchange chromatographic purification (Shainoff and Lauffer, 1956, 1957; Levin, 1958). In the case of charcoal adsorption, the column consists of a large, flat surface formed in the Biichner funnel as compared to the tall, narrow surface formed in the chromatographic column. Also in the case of charcoal adsorption, the procedure is to adsorb the pigments, cellular fragments, and microsomal material, allowing the virus to pass through into the filtrate ; this avoids the use of acids or basesfor elution. The increase in infectivity following charcoal treatment cannot be explained on the basis of change in pH or concentration because of volume reduction. Presumably, the

14

CORBETT

charcoal adsorbs substances deleterious to the initiation of infection or contributes to the dissociation of aggregated virus. The results obtained with charcoal may depend greatly upon the type and grade of charcoal used. Experiments with other grades and sources of activated charcoal were not comparable to those obtained with the Merck product N. F. 18351. In density-gradient centrifugation, infectivity was directly correlated with the presence of flexuous rods 513 rnp in length. This particle length compares favorably with the 500-525 mp length reported for PVX strains by Bode and Paul (1955) but differs considerably from the length (540-620 rnp) reported by Reichmann (1958). Using centrifugation, dialysis, and buffers of sodium citrate or Versene, Reichmann (1959) reported purification of nonaggregated PVX particles. The degree of aggregation was determined by measurements of flow birefringence, which eliminates the drying procedure associated with specimen preparation for electron microscopy. When PVX obtained from tobacco pIants grown under Florida conditions was purified by Reichmann’s (1959) technique, it gave largely noninfectious particles that were aggregated when examined under the electron microscope. Density-gradient centrifugation without chsrcoal treat,ment will generally yield unaggregated preparations of PVX; however, contaminating microsomal material is not separated from the virus. Relatively pure preparations of active virus may be obtained after clarification of the juice by the chloroform emulsion technique. This procedure and all others except that involving charcoal adsorption, had a tendency to spread the virus through the gradient column, as indicated by the amount of infectivity found below the visible zone and in the bottom of the tube. This may be the result of aggregated particles descending farther than the unaggregated virus, or of adsorption of virus particles by normal cellular components. SCKNOWLEDGMENTS

Grateful acknowledgment is made to Dr. A. F. R.oss for supplying the potato virus X isolate, and

to J. W. Carlisle, Physics Department, of Florida, for electron microscopy.

University

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ANDERSON,

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KLECZKOWSKI, A., and NIXON, H. L. electron-microscope study of potato different stages of aggregation. J. Gen. 4,220-224. LEVIN, 0. (1958). Chromatography mosaic virus and potato virus X. them.

Biophys. 78,33-45. H. S., and WYCKOFF, R.

(1950). An virus X in Microbial.

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W. G. (1937). The ultracentrifugal isolation of latent mosaic virus protein. J. Biol. Chem. 121,225-230. MARKHAM, R. (1959). The biochemistry of plant viruses. In “The Viruses” (F. M. Burnet and W. M. St.anley, eds.), Vol. 2, pp. 33-125. Academic Press, New York. REICHMAN~, M. E. (1958). Potato X virus. I. The size and size distribution of potato virus X in tobacco sap. Can. J. Chem. 36, 1603-1611. REICHMANN, M. E. (1959). Potato X virus. II. Preparation and properties of purified, non-aggregated virus from tobacco. Can. J. Chem. 37, 4-10. ROCHOW, W. F., and Ross, .4. F. (1955). Virus LORIM,

PURIFICATION multiplication in plants doubly infected tato viruses X and Y. Vimldgy l, lO-27. ROCHOW,

W.

F.,

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A.

F., and

SIEGEL,

OF POTATO by poB. M.

(1955). Comparison of local-lesion and electronmicroscope particle-count methods for assay of potato virus X from plants doubly infected by potato viruses X and Y. Virology 1,28-39. SHAINOFF, J. R., and LAIJFFER, M. A. (1956). Chromatographic purification of southern bean mosaic virus. Arch. Biochem. Biophys. 64,315318.

VIRUS

X

15

J. R., and LAUFFER, M. A. (1957). An application of ion exchange chromatography to the identification of virus activity with characteristic particles. Viro2ogy 4, 418-434. SMITH, K. M. (1931). On the composite nature of certain potato virus diseases of the mosaic group. Proc. Roy. Sot. B109,251-267. SNEDECOR, G. W. (1946). “Statistical Methods,” 4th ed., 485 pp. Collegiate Press, Ames, Iowa.

SHAINOFF,