Some properties of apple chlorotic leaf spot virus and their relation to purification problems

VIROLOGY

46,240-251 (1971)

Some Properties

of Apple

Their Relation

Chlorotic

Leaf Spot Virus and

to Purification

R. M. LISTER

Problems’

A. F. HADIDP

AND

Department of Botany and Plant Pathology, Purdue University,

Lafayette, Indiana 47907

Accepted March 19, 197i The filamentous apple virus, chlorotic leaf spot, seems unusual among elongate plant viruses in several respects. Its ultraviolet absorption characteristics are unusual for a rod-shaped virus. It has a critical, pH-dependent, structural requirement for some divalent cations, and their removal by dialysis, exchange, or chelation causes degradation. Its RNA is accessible to ribonuclease in the intact particle. Attempts to improve purification procedures made use of some of these properties. Yields and viral stability were improved, especially by providing divalent cations during leaf extraction and dialysis of viral preparations. INTRODUCTION

Two filamentous plant viruses, apple chlorotic leaf spot (CLSV) and apple stem grooving (ASGV), both implicated in the etiology of important and widespread diseases of apple and other fruit plants, have presented special problems in purification (Lister et al., 1965; Saksena and Mink, 1969a,b; de Sequeira and Lister, 1969). Previous work established methods by which these viruses could be prepared sufficiently pure for detailed studies, but the yields obtainable (0.20-0.25 mg/lOO g of leaf) were so low as to suggest that considerable losses occurred during purification (de Sequeira and Lister, 1969). The possibility of aggregation was invoked to explain much of such loss, but it was realized that, especially with CLSV, viral instability might also be responsible (Lister et al., 1965). To clarify these possibilities, we have now investigated the properties of CLSV further, and found some possible reasons for its instability in purified preparations. Some of the results obtained were shown to be applicable in improving purification and viral stability. 1Journal paper No. 4281, Purdue Agricultural Experiment Station. 2 Postdoctoral Assista,nt.

MATERIALS

AND METHODS

Viruses used. Most work was done with the C-S isolate of CLSV (Lister et al., 1965), originally obtained by mechanical transmission to Chenopoclium spp. from a Malus source maintained in the Purdue clonal collection. This isolate was previously shown to induce chlorotic leaf spot disease when reintroduced into the appropriate woody indicator-Russian apple R-12740-7A (Lister et al., 1964). Some comparisons were made with another serologically related isolate of CLSV, designated B-38E, which was supplied in infected leaves of Chenopodium quinoa Willd. by Dr. R. M. Gilmer, Geneva, New York. This isolate was the source of the CLSV investigated by Saksena and Mink (1969b). The isolate of ASGV used was as previously described (de Sequeira and Lister, 1969), and designated C-431. Viruses were maintained, cultured, and their infectivity assayed, in C. quinoa plants. For routine transfers and batch inoculations, phosphate or Tris .HCl buffers at 0.01 M and pH 7.6-7.8 were used for extracting leaves. Buffers, leaves, and instruments were chilled to 4” before use, and inocula were kept in ice. Virus puri$cation. Virus purification was by the bentonite/polyethylene glycol pro240

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OF APPLE

CHLOROTIC

cedure already described (de Sequeira and Lister, 1969). Leaf extract,s made by blending in 0.01-0.05 ill cold phosphate buffer at pH 7 -8, at a ratio of 2: 1 (v/w), were clarified by treatment with bentonite. Further clarification, and concentration of virus, was achieved by precipitation with polyethylene glycol (AI. W. 6000 = PEG) and resuspension in buffer. Virus was separated from the resulting product by one cycle of differential ultracentrifugation followed by centrifugat,ion on sucrose density gradient’s Reducing and antifoaming agents were not used in extraction. In experiment,s carried out, over a period, tests of ultracentrifugal pellet,s from low-speed supernat,ants following t,he PE(+ treatment showed that 4% (w/v) PEG was adequate for precipitating all detectable virus from extracts made during wimer, but, up t,o X %I was required for extract,s made during summer. The latter concentrntion was t’herefore used routinely. Similarly, since for this step the addition of NaCl to 0.0% 31 (de Sequeira and Lister, 1969) did not improve virus yields, it was omitted in most experiments. Herttor~ite peparatiorr. Bentonite was usually prepared by suspension in 0.01 M pH T pot)assium phosphate buffer, but use of bemonitr suspended in Tris . HCl buffer gave similar yields. For a typical batch, 20 g of bentonite (Bentonite powder, U.S.P., Fisher Scientific Co.) was suspended in 400 ml of buffer by blending in a Waring blendor. The bentonit,e fraction which did not pellet, in 1 min at, 3000 y but which did pellet in 10 min at 6000 !, n-as t,hen obtained by centrifugation. This fraction was finally suspended in 100 ml of buffer by blending, making a suspension for use containing about 40 mg/ml of brntonitr, which did not settle on storage. Density grarlietlt cefltrifugation. Den&y gradient centrifugation (Brakke, 1960) was used preparatively and analytically, and gradient)s were analyzed using an ISCO density gradient fractionat’or and ultraviolet1 light analyzer (Brakke, 1963). For rat,e-zonal density gradient centrifugation, gradients were prepared by storage overnight, (4”) of Spinco SW-251 tubes layered, respectively, with 4, 7-, 7-, and 7-ml amounts of 100, 200, 300, and 400 mg of

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SPOT VIRUS

241

sucrose per milliliter of various buffers as specified. These gradients were loaded wit,11 l-ml lots of the solutions to be assayed, and were centrifuged for 3.5 hours at 23,000 rpm. For quasi-equilibrium gradient centrifugation, samples mere usually t.r:msferrcd directly from rat,e-zonal density gradients t’o tubes prepared as above, but with hd amounts of 300, 400, 500, and 600 rng of sucrose/ml. Sample volumes ranged from 2 to as much as 5 ml, and were overlnyett \vith buffer to make a total of 11 ml. C’eutrifugation was for 15 hr at 23,000 rpm. For density determinat#ions, prepamt~ious of CLSV and T,\IV were centrifuged to equilibrium (16 hr at 35,000 rpm) in Aer tubes on preformed gradients made with (N, N’-diacetyl-3 , .S-di:uninoRenografin %,4-triodobenzoate, Tamir and ( ;ilvarg, 1966). These xvere made by floating I. 1-ml lots of Renografin-76 (Squibb meglumine diatrazoate inject,ion USP) , diluted \vith buffer (Tris.HCl/,lIgC& , 0.005 II) t,o O.:where. Preparafio7l 01 viral proteitl. CI,S\’ protriu was made by overnight, dialysis of purified virus obtained from quasi-equilibrium gradients, against, I M MgCl, in 0.05 df Tris. H( :I at pH 7.5. The dialyzatc was centrifuged at 37,000 rpm (Spine0 No. 40 rotor) for 1.s llr, and the supernatant fluid, containiug viral protein, was carefully removed by pipetting. Se1*olo!/y. Antisera were prepared i ti labbits by injecting samples of purifit~tl virus, both intravenously and by intramuscular injections using Freund’s incomplett* adjuvant. Gel diffusion test,ing \vas used t,o cornpare the diffusion of virus md its degrnd:~.tion products. Electrophoretic analysis. Virus purified using the standard bentonite!PE(; prowdurr was separated on a rate density gradient’ from which only the central part of the

242

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AND HADID

virus peak was removed. The final virus sample (0.8 ml) was dialyzed 20 hr at 4”, against 500 ml of 0.05 M Tris, containing 0.005 1M MgCI, (to maintain particle integrity-see below) and buffered to pH 7.5 in 3% sucrose. 0.6 ml of the dialyzate, analyzed on a further rate-zonal gradient, sedimented as a characteristic single peak: 0.2 ml of the dialyzate produced numerous lesions on inoculated leaves of C. puinoa: 0.1 ml was electrophoresed in an ISCO sucrosegradient apparatus (Brakke et al., 1968) using the dialysis buffer for making up solutions. (Electrophoresis was kindly performed by Dr. M. F. Clark.) RESULTS

Properties Evidence of purity: (a) Centrifugal homogeneity. Comparative ultraviolet absorbancy

profiles for characteristic rate-zonal and quasi-equilibrium density gradient centrifugations, of extracts from CLSV-infected and healthy plants subjected to the standard bentonite/PEG procedure, are given in Fig. 1. Though residual normal host constituents usually occurred on rate density gradients, virus bands were clearly distinguishable as

I DEPTH

FIG. 1. Upper curves are ultraviolet absorbancy profiles for rate-eons.1 density gradient centrifugations (RV, RH) of the products from purifications of extracts from CLSV-infected and healthy leaves of Chenopodium quinoa, respectively. Lower curves are UV absorbancy profiles for quasi-equilibrium density gradient centrifugation (EV, EH) of the fractions indicated (I-I) from the rate zonal density gradients.

characteristic sharp peaks. The distributions of the UV-absorbing and infective populations coincided. Characteristic filamentous virus particles also coincided with virus peaks in samples from both rate and quasiequilibrium density gradients. Samples from the latter, which were examined in detail, were free from extraneous material detectable in the electron microscope (Fig. 2A). (b) Electrophoretic homogeneity. During sucrose gradient electrophoresis virus migrated as a single electrophoretic peak with a mobility of -5.1 X low5 cm2/volt-1/sec-1 (Fig. 3). Ultraviolet light absorption. Figure 4 compares ultraviolet light absorbancy curves obtained for the 2 isolates of CLSV (C-8 and B-38E) with one obtained for the C-431 isolate of stem grooving virus prepared similarly, and collected from rate density gradient tubes in which the virus bands were well separated from detectable contaminanbs. Similar curves were obtained for the CLSV isolates using samples subjected to a subsequent, quasi-equilibrium gradient centrifugation. The Azso/~sonm ratio for the C-431 virus preparation was 1.18, in agreement with published values (de Sequeira and Lister, 1969), and typical for a rod-shaped virus. In striking contrast to this, A260~280nm values for CLSV were much higher. This ratio was determined for all preparations that appeared adequately purified from inspection of absorbancy profiles, and that showed a low level of light scattering at A3zOnm.Ratios for samples from either rate density gradients (38 samples) or quasi-equilibrium density gradients (13 samples) were, as expected, closely similar and were about 1.85. Dialysis of either kind of sample against weak buffer, to remove sucrose, inexplicably reduced the ratio to about 1.57. This reduction occurred to a similar extent whether or not infectivity and integrity were maintained during dialysis (seebelow). Thus, all AWW ratios obtained for highly purified preparations of CLSV, were, whether dialyzed or not, unusually high for an elongated simple plant virus. This could imply that CLSV contains an unusually high proportion of RNA, although this seems

PURIFICATION

OF APPLE

CHLOROTIC

LEAF

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VIRUS

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FIG. 2. Electron micrographs of CLSV. (-4) As collected from a quasi-equilibrium density gradient tube from t’he position of the virus peak. (B) Degraded by overnight dialysis against 0.01 M pH 7 phosphate buffer. (C) The same preparation subsequently adjusted to pH 5. All stained with uranyl acetate. Bar indicates approximately 0.1 p.

244

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unlikely for geometrical reasons. Alternatively, since the A280/~80nm value is a function not only of the relative amounts of RNA and protein, but also of their relative characteristics in absorbing ultraviolet light,, the high value could imply that the RNA and/or protein constituents are unusual in this regard. Particle density. The presence of an unusually high proportion of RNA should result in an unusually high particle density. Unfortunately CLSV did not ,withstand isopycnic centrifugation in cold (4”) cesium chloride gradients with an average density of 1.34 and at pH 7.6 or 8.4, whether or not 1 I I I I I I Mgz+ (0.01 M MgCh-see below) was pres6040 20 IO Chart distance in mm ent. When pretreated with 2% glutaraldehyde, the virus sedimented as a pellicle, FIG. 3. Plot of successive positions of the virus peak in sucrose gradient tonal electrophoresis of a probably containing degraded virus, which reached the same level as untreated tobacco purified preparation of CLSV (C-8 isolate) run, in 0.05 IM Tris/0.005 M MgCh buffered to pH 7.5 mosaic virus (TMV) at equilibrium (16 hr at 35,000 rpm in the Spinco SW 39 rotor at in 3% sucrose, at 12 mA in an Isco electrophoresis apparatus. The inset A shows thepform of the 4”). Untreated CLSV did not form a visible Azsdn,,,profile for t.he virus peak after 130 min. band of any sort in cesium chloride. Unlike CsCl, Renografin did not degrade the virus, and this substance u-as used to obtain an estimate of density. Preliminary experiments established that CLSV preparations made 50% with Renografin and kept at 4’ for 24 hr were as infective as control samples diluted 1: 1 with buffer before storage. Dialysis to remove the Renografin was necessary before assay: undialyzed preparations, though not damaging, caused no lesions when rubbed on leaves. In isopycnic centrifugations in Renografin gradients, CLSV and TMV were observed as light-scattering bands at 24.5 and 26 mm below the meniscus, respectively. Measurements of &onm X lo4 values (Tamir and Gilvarg, 1966) for samples either withdrawn from the visible bands or selected on the basis of infectivity (Pig. 5) indicated densities of 1.26 to 1.27 for CLSV and 1.33 for TMV. We conclude that CLSV is less dense than WAVELENGTH TMV and has a density of 1.26-1.27 in FIG. 4. Comparative UV absorbancy curves of Renografin. A previous estimate of the preparations of the C-8 and B-38-E isolates of reciprocal of the partial specific volume of CLSV and the C-431 isolate of apple stem grooving the solvated CLSV particle, as determined virus. All 3 viruses were prepared by the standard by rate-zonal density-gradient centrifugation bentonite/PEG procedure and collected from in sucrose gradients, was I.12 (Lister et al., rate-zonal density gradient tubes.

PIXIFICATION

OF ilPPLE

CHLOROTIC

LEAF

SPOT

VIRUS

o.s,-

0.6 -

-___

DEPTH

Smple

numbs,

Fro. 5. Infectivity (lesions/inoculated C. quinoa leaf) and AZM~ X lop4 plots for B-droplet samples from an equilibrium zonal density gradient centrifugation (16 hr at 35,000 rpm in Spinco SW 39 rotor) of CLSV (C-8 isolate) in Renografin.

196.5). Both this and our current densit(y estimate are consistent with the low RNA content, t,ypical of rod-shaped viruses. Thus, CLSV is likely to be unusual in regard to the constitution of its protein or RNA, ratlrer t#han in their relative proportions. Instability during dialysis. Loss of virus that occurred during manipulation of preparations of CLSV as obtained from rate or quasi-equilibrium density gradients, was traced to use of a dialysis step to remove sucrose, prior to concentrating by ultracentrifugation. Overnight st’orage in sucrose, or dialysis for 1 or 2 hr against weak buffers, did not result in serious degradation, but, overnight dialysis did. Degradation resulted in loss of infectivity, change in centrifugal charact,eristics, and obvious particle breakage. Density gradient, analysis of degraded preparations showed no virus band, but virus-associated low molecular weight material remained at the meniscus. Whereas virus sedimented with s20,w = 96 S, the degraded product sedimented wi.ibh s20,w = 17 S. Electron microscopy showed poorly defined structures interpreted as low-order polymers of viral protein subunits, and after adjustment of an aliquot to pH 5 w&h acetate buffer, such preparations showed more pronounced polymerization into ringlike structures (Fig. %B,C). It$uetze of buffer type OIL stability. Phosphate was more efficient t)han Tris in degr:diIlg

VirUs.

I’tJr

eXampk,

diquOts

Of

a

FIG. 6. U\‘ absorbancy profiles of rat,e-zonal density gradient centrifugations of aliquots of the same preparation of CLSV (C-8 isolate) dialyzed at pH 7.6 against (A) 0.065 fif Tris/HCl for 2.5 hr; (B) 0.05 .kf Tris/HCl in 0.001 M MgCl, overnight; (C) 0.05 M Tris/HCl in 0.01 ‘If MgCl, overnight; (D) 0.05 M Tris/HCl in 0.1 ill MgC12 overnight; (E) 0.05 !lf Tris/HCl overnight; and (F) 0.05 M Tris in 0.01 M M&l? overnight, followed by adding 20 rg of pancreatic ribonuclease 10 min prior to centrifugation. Inoculations of similar dilutions of the treated samples before analysis gave means of 32, 29,27,0,0, and 0 lesions per leaf of Chenopodium quinoa, respectively.

2; x <*.I

t il

pi:l_ii: A B

C

D

I

FIG. 7. UV absorbancy profiles of density gradient centrifugations of aliquots of the same preparation of CLSV (C-8 isolate) dialyzed at pH 7.6 against (A) 0.05 ICI Tris/HCl containing 0.005 M MgClz for 8 hr; (B) 0.05 M Tris/HCl for 8 hr; (C) as in B, 8 hr, followed by as in A, 12 hr; (D) as in A, 8 hr, followed by as in B, 12 hr.

preparation dialyzed overnight against Tris at 0.01 M pH 7.6 or phosphate at, 0.01 111 pH 7.0 gave 16 and 0.3 lesions per leaf, respectively. This effect initially suggested t’hat instability might result from the chelation of divalent cations of possible impor-

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tance in maintaining quaternary structure (cf. Bancroft et al., 1967, and see below). But use of Tris or other buffers at higher molarity, or for more prolonged dialysis, was found equally to result in degradation (Fig. 6-8). E$ect of added substances. Virus was efliciently preserved when dialyzed overnight against 0.05 M Tris at pH 7.6 containing 0.01 1M,0.005 M or 0.001 M MgClz (Fig. 6), or containing 0.005 1M MgS04 or 0.005 M CaClz . Little virus, and no low molecular weight viral-related products, were detected after dialysis in 0.05 M Tris in the presence of 0.1 M MgClz , suggesting that virus or viral degradation products had been precipitated (Fig. 6). In other experiments, virus was also preserved, though less efficiently, in the presence of 0.005 M spermidine. It was almost entirely degraded when dialyzed against 0.01 M Tris containing 0.05 IM NaCl or 0.0075 M ethylenediamine tetraacetate (EDTA), though not when these substances were omitted from the buffer. These results all suggest that at least near neutral pHs the integrity of the quaternary

DEPTH

FIG. 8. UV absorbancy profiles of density gradient centrifugations of the products from aliquots of a preparation of CLSV (C-S isolate) which were dialyzed overnight against-in treatments A, B, and C, respectively-MES buffer at pH 6.1 (0.05 M); TES buffer at pH 7.5 (0.05 M), and TAPS buffer at pH 8.4 (0.05 1M), and-in treatments D, E, and F, respectively-the same buffers containing 0.005 M MgCln . Products were back-dialyzed to pH 8.4 (TAPS) and analyzed on gradients made using this buffer.

structure of the viral protein coat requires the presence of a certain critical concentration of divalent cations or polyamines. Removal of these by dilution, chelation, or exchange, can cause viral degradation. Degradation resulting from dialysis was not reversed simply by supplying Mg2+, although this changed the sedimentation profile slightly. Also, provision of Mg2f ions during a preliminary dialysis reduced but did not prevent degradation during a subsequent dialysis in the absence of Mg2+ (Fig. 7). Injluence

of pH on Mg2+ eflects. Implicit in the suggestion that divalent cations play a part in maintaining protein quaternary structure for CLSV is that, as with some spherical viruses, the effect should be pHdependent. Thus, with cowpea chlorotic mottle virus it is supposed that the structural requirement for Mg2+ at pH 6.5 and above results from its ability to form bridges between mutually repulsive carboxylate ions on adjacent subunits. This requirement is reduced as pH is reduced to 5, becauseof the likelihood of hydrogen bonding between carboxylic acid side chains (Bancroft et al., 1967; Hiebert and Bancroft, 1969). That the Mg2+ requirement for CLSV particle integrity varies with pH was shown in several series of experiments in which aliquots of the same virus preparations were dialyzed against appropriate zwitterionic buffers (Good et al., 1966), used at 0.05 M and at pH levels equal to their pK values (Fig. 8). Overnight dialysis against TAPS buffer (pH 8.4) caused complete viral degradation, with the corresponding appearance of ultraviolet light-absorbing material of low molecular weight; but inclusion of 0.005 1MMgCl2 in the dialysis buffer efficiently prevented this. Dialysis against TES (pH 7.5) during the same period caused less complete degradation, and again this was completely prevented by inclusion of MgCl2 in the dialysis buffer. Interpretation of the results of dialysis against MES (pH 6.1) was complicated by the aggregation of virus that occurred at this pH. When the products of dialysis against MES alone or containing 0.005 M MgC12 were analyzed on sucrose

PURIFICATION

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gradients made with the same buffers, no amounts of ribonuclease might’ contribute to instability during extraction, storage or virus-related products were detectable. Howdialysis. This proved to be a possibilit,y, ever, this was not due to degradation, but since in several experiments pancreatic to t’he loss of aggregated virus by pelleting ribonuclease (20-40 pg/ml) rapidly degraded in the density gradient tube. Thus, virus preparations of CLSV, whether or not dialyzed to pH 6.1 sedimented normally purified using rate density gradient cenafter back-dialysis against 0.05 M TAPS Degradation was presumably (pH 8.4) containing 0.005 M MgClz . Den- trifugation. enzymatic, because addition of cytochrome sity gradient analysis on sucrose gradients made with this buffer (Fig. 8) then showed c, which is also a small basic protein, was that no degradation had occurred at pH 6.1 without effect. The presence of Mg2+ did sensitivity, as either in the presence or absence of Mg”+. not reduce ribonuclease A tendency for virus to aggregate in the assayed both by absorbancy profiles and presence of Mg*+ was also noted at pH 7.5, infectivity tests (Fig. 6). Diffusion of virus and viral products but not at pH 8.4. through czgar gels. Diffusibility through gels We interpret these results as implying provides a simple and useful basis for comthat as the pH is lowered to levels at which parison of sizes of protein molecules and CLSV tends t,o aggregate, suitable divalent macromolecules (Ackers and Steere, 1962). cations are not necessary for structural integrity. This supports the idea that a The possibility of maintaining CLSV intact], or degrading it under controlled conditions, structural mechanism similar to that already referred to for some spherical viruses applies permitted comparison of the diffusion of t,he here. Aggregation that occurred during intact virus and virus fragments, and thus a reexamination of whether CLSV is detectmaattempt’s t’o concentrate virus by pelleting was also influenced by pH. When virus ble in serological gel diffusion (Ouchtrrlony) test.s. collected from rate-zonal density gradients Tests were set up both in agar plates (0.01 Jb Tris.HCI at pH 7.6) was pelleted by ultracent’rifugation, either directly or containing Tris (0.01 LM) either alone or after dialysis against 0.01 2l4 T&s/O.01 M with MgC12 (0.005 nl), to compare the MgSOa at pH 7.6, it did not resuspend in diffusion of (a) intact virus obtained by Tris at pH 7.6 or pH 8.4. However, virus dialysis of samples from density gradient dialyzed against 0.01 ,!WTris/O.Ol M MgS04 tubes against 0.01 M Tris/0.005 M MgC12; at pH 8.4 to remove sucrose prior to pelleting (b) the same material dialyzed against, 0.05 did resuspend in Tris at pH 8.4. M Tris; and (c) viral protein made by Not all divalent cations were suitable for dialysis against 1 31 MgC12. Oxoid Ionagar preventing degradation, nor would this be No. 2 was used at concentrations of 0.5, expected. When dialyzed against 0.05 M 0.85, and l.O%, in either 0.01 &1 Tris alone Tris/0.005 &f CuCIZ at pH 7.6, the virus was or 0.01 M Tris 0.005/MgC12. NaCl at 0.85 % completely degraded, presumably as a result and sodium azide at, 0.02% were present in all tests. of cleavage of phosphate bonds in the viral The results (Fig. 9) showed clearly that RNA in the presence of Cu2+. Other divalent virus was negligible cations might be expected to have this effect diffusion of “intact” under certain conditions (Butzow and in the presence of Mg2+, though obvious in its absence, presumably because diffusion Eichhorn, 1965). Rfect of ribmuclease. The RNA of some through agar caused dilution of available plant viruses is accessible to ribonuclease divalent cations, resulting in some degradation. Viral fragments obtained in treatment while in the int’act nucleoprotein particle (Bol and Veldstra, 1969). In view of the (b) diffused much more readily, and gave possibility of an open structure for CLSV sharply defined reaction lines. Diffusion of (Lister et al., 1965), and of a requirement viral protein (treatment c) occurred tjo a for intact RNA to preserve structure, we similar extent, but resulted in a more blurred wished to know whether sensitivity to trace reaction line. For Oxoid Ionagar No. 2 at

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FIG. 9. Results of gel diffusion testing in 0.5% “Oxoid ionagar.” A, CLSV preserved intact by dialyzpH 7.6, containing 0.005 M MgClz ; B, CLSV degraded by dialyzing 8 hr against 0.05 M Tris/HCl at pH 7.6; and C, CLSV protein prepared, as described in the text, by dialysis against 1 M MgC12 Left; agar made up with 0.01 M Tris/HCl at pH 7.6, containing 0.005 M MgC12 and 0.85% NaCl: Right; same, but omitting t,he Mg2+.

ing 8 hr against 0.05 M Tris/HCl,

DEPTH

DEPTH

FIG. 10. Comparative UV absorbancy profiles of density gradient centrifugations of the viral products obtained after applying the bentonite/PEG procedure to extracts of infected Chenopodium quinoa leaves made using 0.01 M Tris/HCl buffer at pH 7.6 and containing (1) MgSOa at 0.01 M; (2) MgS04 at 0.001 M; (3) no MgS04 . The bentonite used for clarification was made either by suspension in potassium phosphate buffer (K-B) or according to the method of Dunn and Hitchborn (1965) (Mg-B). yields resulting from extraction in buffer containing MgS04 at 0, 0.01, or 0.001 M concentrations were compared. Samples were also split for clarification with potassium-bentonite or magnesium-bentonite (Dunn and Hitchborn, 1965) since Saksena and Mink (1969b) found that use of the latter improved yields. After bentonite clarification, virus was further clarified and Consequences of Some Viral Properties i?L concentrated as usual, by precipitation with PuriJication PEG followed by one cycle of differential To avoid losses through Effect of adding magnesium during virus ultracentrifugation. extraction. my+ was selected for investiga- aggregation, viral pellets were resuspended at’ each stage in Tris/HCl buffer (pH 7.6) tion of the effect of providing appropriate without added Mg2+. Absorbancy profiles divalent cations during extraction lvith for the products showed that an increase in 0.01 M Tris buffer at pH 7.6. Its potential virus yield of at least s-fold was obtainable for improving yields was striking. using 0.01 M MgS04 in extraction, as In a typical experiment (Fig. lo), virus

the concentrations

used, effective

pore radii

should exceed 120 nm (Ackers and St,eere, 1962), but the length of the intact virus particle is 600-700 nm. We conclude that the prospect’s for gel-diffusion testing with CLSV will be improved if tests are done under conditions calculated to remove divalent cations and degrade virus.

PITRIFICATION

OF APPLE’

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compared with the other t,reatments. At this level of Mg2+, .yields after clarification with either I(- or Mg-bento&e were similar; but at, lower levels, yields after clarification with Kbentonite were slightly higher. Infectivity t’cstti of unfractionated samples provided further confirmation of t,hese result,s, which contrast wit11 t’llosr of Saksena and Mink (1969b). In ot,her experiment,n, use of RIgSOl at 0.02 M in extraction afforded no improvement on the yield obtained at 0.01 M, and its use at 0.1 31 reduced virus yield. With the higher molarities of MgSO4, densit,y gradient, analysis showed that t’he virus obtained, even aft.er the usual bentonite/ PEG clarification procedure, was more heavily contaminated with undesirable nonviral host constituents, sedimenting as lightscattering mnterinl hod above and below the virus band. Use of MgS04 at 0.01 N seemed optimal both for virus yield and for minimizing this problem. Yield increases obtained varied in different experiment,s from about 5- to as much as IO-fold. Usually the improved yields obt,ained for t,he C-S isolate from newly spst,emically infected leaves approximated 1 mg!lOO g of leaf, as compared with the 0.2-0.25 mg/lOO g obtained when ?tIg’+ was not used. /Cffect

01 reducit~~~ rihonuclease

activity.

Despite the sensitivity of pure virus t,o added pancrea.tic ribonuclease, the presence of brntonit,e during blending leaves did not improve yields. Similar infectivities were obt’ained when the standard bentonite/PEG clarification procedure was applied either directly to leaf extracts, or after first blending leaves in the presence of sufficient bcntonit~c to insure a substantial degree of preliminary clarification. In t,lie first case, possible enzymatic s&on could have proceeded during the 15 min or so prior t,o adding bent’onitr : in the second, at least a rrduct ion of such a&on would be expected (l;ntrnkel-Corlrat et al., 1961). Clar$cation with butatrol lchlorofom. The effect, of added ;\I$+ in stabilizing CLSV raised t,lie cluest,ion of whether its presence would allon- use of more usual met,hods for clarification than the bemonite procedure. Previously, clarification by t,he butanol/ chloroform mr%hod of Strere (1956) had

LEAF

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VlllUS

21!)

seemed promising, though inferior il L yicltl to bentonite (Lister et al., 1965). Accordingly, it was applied to leaf extracts made with 0.01 91 Tris or 0.01 M Tris’O.O1 .I/ MgSO4, at pH 7.6. Two parts of rstract were mixed with 1 part of a 1: 1 hut:mol chloroform mixture. The aqueous l)lr:ww collected aft,er low speed centrifugat ion ww left overnight (do), clarilied by :I 1’11:(; precipitation, and then concentrated by t \vo cycles of differential ultracentrifugtt ion. Analysis of the products by density gruditnt, centrifugatJion showed that, the clnrilic:ition t Iw obtained was verv inferior to that from standard bento&ejPEG procedure. I II infectivity t,ests of t.he unfractionated prelw rations, comparative lesion numbt~r~ per inoculated leaf of C‘. quirloa bvere 0 f(lr the prep:“z~tiolrs but~anol/chloroformj.PEG (made with or without !\I$+) and 7:: for :L comparable bentonitc/PEG prep:u:rtion (made tvit,h l\lg”+). Our resuhs provide clear evidence for two possible causes of the instabiiitv of CISV ilr vitro: first, the accessibility (,f the RXA of the intact virion t)o ribonuclease, and second, :1 st’ructjurul requirement for some divalent cations. The first is lwrh:yw not surprising, because the RX:1 in intact particles of some other plant viruses is susceptible t,o ribonuclease (I301 and Vc~ltlstra, 1969). The critical requirement for divalent, cations is, however, quittl un~petted. It, is, so far as we :ue aware, unique among rod-shaped plant viruses, ad though possible structural roles for hydrogetl bonding, metal ions, and poly:~minrs lnlvc bren discussed for tobacco mosaic virw. their importance appears to bc overshadow4 by other stabilizing forces (Caspar, I!)%<). Conceivably, however, tins tlifftfrrtwc is more apparent than real, and it could tlrperid merely on the relat*ive accrssibilit~- OFstrutturally important divalrnt cation5 in tile particles of different viruses. Interestingly, this property also tlow not seem to be shared by ,\SGV, the other filament,ous virus km)\\-n to infect apples, for in earlier work this virus witjhstoocl prolonged dialysis against even 0.1 ionic strength sodium pliosphatc,~ sodium cl111britlt:

250

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AND HADIDI

electrophoresis buffer (de Sequeira and Lister, 1969), a treatment that would certainly destroy CLSV. Indeed, the sensitivity of CLSV to dialysis against even weak buffers was initially confusing, in giving us the impression that it has a much shorter in vitro longevity than ASGV. But, in the presence of appropriate amounts of Mg2+, CLSV proved much more stable than anticipated. Similarly, earlier comparisons of the properties of CLSV and ASGV in plant extracts (Lister et al., 1965) could be misleading, and may be based, for example, primarily on the relative sensitivity of the two viruses to leaf ribonucleases. It is also possible that some at least of the inhibiting effect previously noted for C. quinoa extracts with respect to CLSV (Saksena and Mink, 1969a) may be due to leaf ribonucleases. Some of the properties of the proposed inhibitor conform with this idea: it appears to be a highly stable protein and it has the capacity, unusual among inhibitors from plant extracts, to interfere with infection of the host from which it is derived. Moreover, the inhibitory effect of C. quinoa extracts is reduced by bentonite, as would be expected if ribonuclease were the inhibiting agent. The addition of Mg2+, Ca2+, and a polyamine, all improved the structural stability of CLSV, and though this does not mean that any of these are specifically involved in in vitro integrity, it is suggested that a requirement for some such substances exists. This being so, the effect of added Mg2+ in improving yields during extraction is easiest to explain by assuming that structurally necessary cations can be chelated by leaf protein or other substances normally separated from virus particles in the intact cell. The increase in yield seemsunlikely to result merely from increasing the ionic strength of the extraction buffer, for increased molarity alone did not increase yields with either Tris-HCl or phosphate buffers. On the contrary, with phosphate buffer, an increase in molarity apparently reduces virus yields (Saksena and Mink, 1969b), an effect predictably resulting from increase in the capacity for chelating divalent cations. It should be noted that with ASGV, we

have obtained no improvement in virus yield by incorporating Mg2+ in the Tris . HCl buffer used for extraction. A further striking difference between CLSV and ASGV lies in the much higher A260/zso ratio for CLSV we found when comparing highly purified preparations. While this ratio was as expected for ASGV, it was far higher for CLSV than in another recent determination (Saksena and Mink, 1969b). Possible reasons for this have already been discussed and can be subjected to direct analytical testing. We are aware that the presenceof impurities such as ribosomal derivatives in our preparations could give misleadingly high ratios, and that the reduction of the ratio upon dialysis might indicate the presence of impurities. However, the centrifugal and electrophoretic behavior of our preparations suggests a high degree of homogeneity. ACKNOWLEDGMENTS Besides those mentioned in the text, we thank Dr. J. B. Bancroft for performing analytical ultracentrifugationb and for much helpful discussion; Dr. C. E. Bracker for electron microscopy; and Dr. A. Aronson for suggesting the use of Renog&in. REFERENCES ACKERS, G. K., and STEERE, R. L. (1962). Restricted diffusion of macromolecules through agar-gel membranes. Biochim. Biophys. Acta 59, 137-149.

BANCROFT,J.B., HILLS,G. J.,and MARHHAM, R. (1967). A study of the self-assembly process in a small spherical virus. Formation of organized structures from protein subunits in vitro. Virology

31, 354-379.

BOL, J. F., and VELDSTRA, H. (1969). Degradation of alfalfa mosaic virus by pancreatic ribonuclease. Virotogy 37, 74-85. BRAKKE, M. K. (1960). Density-gradient centrifugation and its application to plant viruses. Advan.

Virus.

Res. 7, 193-224.

BRAKKE, M. K. (1963). Photometric scanning of centrifuged density gradient columns. Anal. Biochem. 5, 271-283. BRAKKE, M. K., ALLINGTON, R. W., AND LANGILLIB, F. A. (1968). Mobility measurements by photometric analysis of zone electrophoresis in a sucrose density gradient column. Anal. Biothem 25, 30-39.

BUTZOW, J.J., and EICHHORN, G. L. (1965). Interactions of metal ions with polynucleotides

PURIFICATION

OF APPLE

CHLOROTIC

and related compounds. IV. Degradation of polyribonucleotides by zinc and other divalent met,al ions. Biopolymers 3, 95-107. &SPAR, D. L. D. (1963). Assembly and stability of t.he tobacco mosaic virus particle. Advan. Protein Chem. 18, 37-121. DE SEQUEIRA, 0. A., and LISTER, R. M. (1969). Purification and relationships of Rome Hamentous viruses from apple. Phytopathology 59, 1740-1749. DUNN, D. B., and HITCHBORN, J. H. (1965). The use of bentonite in the purification of plant viruses. Virology 25, 171-192. FRAENKEL-CONRAT, H., SINGER, B., and TSUGITA A. (1961). Purification of viral RNA by means of bentonite. Virology 14, 54-58. GOOD, N. E., WINGET, C. D., WINTER, W., CONKOLLY, T. N., IZA\VA, S., and SINGH, R. M. M. (1966). Hydrogen ion buffers for biological research. Biochemistry 5, 467477. HIEBERT, E., and BANCROFT, J. B. (1969). Factors affecting the assembly of some spherical viruses. Virology 39, 296-311.

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R. M., BANCROFT, J. B., and SHAY, J. R. (1964). Chlorotic leaf spot from a mechanically transmissible virus from apple. Phytopathology 54, 1300-1301. LISTER, R. M., BANCROFT, J. B., and NADAKAVUKAREN, M. J. (1965). Some sap-transmissible viruses from apple. PhytopathoEogy 55.859-870. SAKSENA, K. N., and MINK, G. I. (1969a). Properties of an inhibitor of apple chlorotic leaf spot virus from Chenopodium quinou. Phytopathology 59, 61-63. SAKSENA, K. N., and MINK, G. I. (1969b). Purification and properties of apple chlorotic leaf .-spot virus. Phytopathology 59, 84-88. STEERE, R. L. (1956). Purification and properties of tobacco ringspot virus. Phytopathology 46,

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6M9. TAMIR,

H., and GILVARG, C. (1966). Density gradient centrifugation for the separation of sporulating forms of bacteria. J. BioZ. Chem. 241, 1085-1090.