Stability differences between the nucleoprotein components of bean pod mottle virus

VIROLOGY

27, 476-483 (1965)

Stability

Differences

between Bean

Pod Mottle

J. S. SEMANCIK Department

the Nucleoprotein

AND

Components

of

Virus

J. B. BANCROFT’

of Plant Pathology, University of California, Riverside, California, and Department and P1an.t Pathology, Purdue University, Lafayette, Zndiana Accepted August

of Botany

9, 1965

The noninfectious 91 S nucleoprotein component of bean pod mottle virus is less stable in alkaline-chloroform emulsions than the infectious 112 S component. Particle survival may be related to time of synthesis. Electrophoretic heterogeneit,y and the normal component distribution were maintained from pH 4.0 to 9.0. RNA cores of the noninfectious component displayed areas of nonuniform staining. MATERIALS

INTRODUCTION

Alkaline degradation has been utilized as an effective means of liberating protein subunits from TMV (Fraenkel-Conrat and Willaims, 1955) and wild cucumber mosaic virus (Yamazaki and Kaesberg, 1961). Characteristic products arising from dissociation of intact virus have been described (Harrington and Schachman, 1956, Bockstahler and Kaesberg, 1962). Bean pod mottle virus (BPMV) is representative of the group of ‘
AND

METHODS

BPMV was produced in Phaseolus vulgaris L. var. Cherokee Wax lo-16 days and purified by a variation of Bancroft’s (1962) method. Tissue, frozen 1 week to 1 month, was blended 1:2 (g:ml) in 0.1 M potassium phosphate buffer, pH 7.0. The aqueous phase from the chloroform-butanol clarificat,ion (Steere, 1956) was frozen overnight. After two cycles of differential centrifugation, the pellets were suspended in 0.1 Al phosphate buffer and dialyzed overnight against 0.5 II1 phosphate buffer, pH 7.0. The pellet from an additional ultracentrifugation cycle suspended in 0.1 M phosphate buffer comprised the purified preparation. Local lesion assays were made on Phaseolus vulgaris L. var. Pinto in an incomplete block design with no less than eight half-leaves per treatment. Virus preparations (O.5-1.0%) t’o be enriched with the bottom component were mixed with 5 volumes of glycine-NaOH (Gly-OH) buffer, pH 9.5 or GPS buffer (Diener, 1962) and 6 volumes of precooled reagent chloroform. An emulsion was formed by rapid mixing on a Vortex Junior agitator for 10 minutes in 2-minute intervals. Each mixing interval was followed by an equal period in an ice bath, thus maintaining the temperature at approximately 4”. The emulsion was broken by low speed centrifugation.

STABILITY

IN

NUCLEOPROTEK

The aqueous phase was centrifuged at 36,000 rpm for 1.5 hours in the No. 40 rotor of the Spinco model L centrifuge. Pellets were resuspended in 0.1 ilf phosphate buffer, pH 7.0. An alternative method involved emulsification in a small volume and deletion of the final high speed centrifugation. Infected plants were labeled with P32 (orthophosphate) as previously reported (Semancik and Bancroft, 1964). Optical density readings were taken on a Beckman DU spectrophotometer, and counts were made in a Nuclear-Chicago gas-flow counter equipped wit’h a micromil window. Analytical centrifugation was performed in a Spinco model E centrifuge. Areas of the schlieren sedimentation components were obtained by compensating planimeter without correction for the Johnston-Ogston effect (Johnston and Ogston, 1946) or radial dilution. Electrophoretic mobilities were obtained in the 2-ml Tiselius cell of the Perkin-Elmer model 38-A electrophoresis apparatus equipped with a modified Philpot-Svenson cylindrical lens system. Components to be examined by electron microscopy were fractionated as previously reported (Semancik and Bancroft, 1964) and dialyzed against 0.1 M ammonium acetate. The RNA cores were stained essentially by the method of Huxley and Zubay (1961) in a 1: 2 mixture of virus suspension to 2 % uranyl acetate. After a 3-6 hour staining period, carbon-coated Formvar grids were touched to the virus-stain drop, the excess mixture was drawn off by touching to filter paper, and rinsed by dipping in distilled water 5-10 times. The grids were viewed on a modified RCA model EMU-3B electron microscope. RESULTS

Differential Stability If purified BPMV was added to healthy tissue and repurified, the normal bottom: middle (1: 1) ratio of nucleoprotein components remained unchanged, showing that the moieties, once purified, were stable in healthy plant sap. The possibility that some population of the bottom component was degraded by nuclease act,ion to RNA-defycient particles (middle component) immedi-

OF BESK

POD

MOTTLE

T’IRCS

42

ately upon extraction to form I stable system (Semancik and Bancroft, 1964) was not obviated. Consequently, infected tissue was extracted in t,he presence of 1O-43J ZnClz (F’risch-Niggemeyer arid Reddi, 195i) and GPS buffer + chloroform (Diener, 1962), which reduced nuclease levels, measured according to Pirie (1956), by 90 ‘Y and 100 70, respectively. Virus from t,issue homogenized in ZnClz displayed the normal bottom component : middle component (B: RI) ratio; however, t’issue ext’racted in alkali + chloroform yielded preparations enriched with the bottom component’. The yield of bott’om particles was not increased over the level found when both components were present in equal amounts and the specific infect,ivity was low (Table l), considering only the bottom component has been reported to be infectious (Bancroft, 1962). This finding suggested that the formation of t,he middle moiety was not being stopped, but that already formed middle component was being destroyed. Top component, which contains little or no RNA (Bancroft, 1962), was also destroyed. This idea was verified by the preferential destruction of middle component’s in purified preparations (Table 2). Neither the chloroform emulsion treatment at neutrality nor agitation at pH 9.5 under various ionic conditions affected the nucleoprotein component ratio of 1: 1, but simultaneous emulsification in chloroform and alkali resulted in enrichment for bottom component particles (Fig. 1). This material gave single lines in Ouchterlony agar gel serological tests, indicating the degradation products, if parbialy composed of protein subunits, were lacking serological integrity. Re-extraction of preparations in alkalichloroform (Table 2, 6a, b) showed that populations of particles remaining after one extraction were not’ resistant to further treatment. Each treatment with alkali-chloroform resulted in about a 40-60 c/o recovery, indicating that, in addition to t,he majority of middle particles, a portion of the bottom component was susceptible to degradation. This progressive depletion of the noninfectious middle component was associat,ed with

478

SEMANCIK

AND TABLE

BANCROFT 1

EXTRACTION EFFECTS ON BPMV

1

260 OD280

Treatment

Expt. no.

FROM INFECTED TISSUE

(a) Healthy tissue -I- virus + 0.1 M PO+ pH 7.0 (b) Infected tissue + 0.01 M Tris, pH 7.3 + ZnCIB 10-a M (c) Infected tissue + GPS, pH 9.5 f chloroform

1.74

-

1.73 -

1.80

-

1:l

-

I:1

-

2.3:1

All bottom

ODw,a = 0.05 2

(a) Infected tissue + 0.1 M GlyOH, pH 9.5 + chloroform (b) Infected tissue + 0.1 M GlyOH, pH 9.5 (c) Infected tissue + 0.1 M 1'01, pH 7.0

1.77

10

4.3

1.69

110

9.1

1:l

1.73

90

9.2

1:l

TABLE EFFECTS OF VARIOUS TREAIMENTS Expt. no.

2

UPON PURIFIED

PREPARATIONS OF BPMV

GD 260 Infectivity (lesions/ 280 half-leaf)

Treatment

OD,,, 1

(a) Virus + 0.1 M Pod, pH 7.0 f (b) Virus + GPS, pH 9.5 (c) Control

2

(a) Virus (b) Virus

= 0.033 77 82 70

Component ratio, B:M

chloroform

1.69 1.70 1.70

pH 9.5 + chloroform pH 9.5

1.83 1.67

0D.~a = 0.06 80 97

4:l 1:1

3

(a) Virus + 0.1 M Gly-OH, pH 9.5 + chloroform (b) Virus + 0.5yo DOC + chloroform (c) Control

1.81 1.72 1.70

OD,,o = 0.02 92 58 100

3:l 1:1 -

4

(a) Virus (b) Virus

1.88 1.71

ODw,o = 0.10 5 61

4:l -

1.83

17

5:l

1.78

-

+ 0.6 M Gly-OH, + 0.6 M Gly-OH,

+ GPS, pH 9.5 + chloroform + 0.5yc DOC + chloroform

(c) Virus + GPS, pH 9.5 + 0.5yo DOC + chloroform + 0.1

M Pod, pH 7.0 + 0.5% DOC

5

(a) Virus

6

(a) Virus + 0.1 M Gly-OH, pH 9.5 + chloroform (b) Virus (6a) + 0.1 M Gly-OH, pH 9.5 + chloroform (c) Virus + 0.1 M Gly-OH, pH 9.5 (d) Virus (6~) + 0.1 M POI, pH 7.0 + chloroform (e) Control

OD,s, 1.74 1.80 1.72 1.74 1.70

= 0.05 59 27 76 60 118

1:l 1:l -

1.7:1

3:l All bottom 1:l 1.2:1 1:l

STABILITY

IN

SUCLEOPROTEIN

a decrease in specific infect,ivity. The differential stability phenomenon did not appear to be cumulative since a preparation made alkaline and emulsified with chloroform, consecutively, showed only a slight digression from the normal 1: 1 component ratio. Sodium deoxycholate (DOC), known to free viral RNA from int,act eastern equine encephalitis virus (Wecker and Richter, 1962), was only slightly effect’ive in selectively destroying the middle moiety. Yellow cowpea mosaic virus is serologitally related to BPMV and has a similar base ratio (Bancroft, unpublished) ; however, pH 9.5-chloroform treatment destroyed the virus. The differential stability response of virus components could be produced by treatment with pH 8.7 chloroform, while pH 8.5 chloroform had no effect. Effect of Aging upon Stability The portions of the preparations surviving the alkaline-chloroform t’reatment might not

FIG. 1. Ultracentrifuge schlieren pattern of BPMV after extraction in 0.6 M Gly-OH buffer, pH 9.5 (above) and 0.6 M Gly-OH buffer, pH 9.5, + chloroform (below). The photograph (bar angle = 40 degrees) was taken 10 minutes after the Spinco An E rotor reached a speed of 37,020 rpm. Sedimentation is from right to left.

OF BEAN

POD

MOTTLE

479

VIRUS

2-

0.2

400

? E 0 ul cu

01

200

2 'z 06 : L3 0 0.3 .u 'a 0

400

0.4

400

0.2

200

000

20

40

60

Sample

00

z ; 0 ; a 0

120

Number

FIG. 2. Degradation of BPMV. (A) control virus, (B) 0.1 M Gly-OH, pH 9.5, chloroform emulsification, (C) 0.6 M Gly-OH, pH 9.5, chloroform emulsification, after rate sedimentation in 100400 mg/ml sucrose density gradient for 2.5 hours at 23,000 rpm in the Spinco SW 25.1 rotor, (O-O) complete virus population, (O--O) recently made virus. Sedimentat,ion is from right to left.

necessarily be random, but particle persistence may have been related to time of synthesis. By administering a terminal 18-hour F2 label after a g-day infection period, the labeled virus in the preparation would represent the most recently synthesized portion of the population. The specific radioactivities of the bottom and middle components in control preparations, either untreated, emulsified in chloroform at pH 7.0, or shaken in GE’S at pH 9.5 were the same (Fig. 2A). The middle component of preparations enriched for bottom component by emulsification in chloroform at pH 9.5 in 0.1 M, 0.6 M Gly-OH or GPS had specific radioactivities 20-30 ‘X below those of the bottom component particles (Fig. 2B, C) indicating that the more recently formed particles were more susceptible to degradation. Stability of Electrophoretic Components In addition t,o centrifugal heterogeneity, BPMV is characterized by two electro-

480

SEMANCIK

AND

phoretic components in each sedimenting species (Bancroft, 1962). These electrophoretie componenOs conceivably could have different stabilities, related not to RNA content as with the centrifugal components, but to surface structure. Accordingly, virus dialyzed against the 0.1 ionic strength buffer series of Miller and Golder (1950), was examined by schlieren optics during electrophoresis. Both electrophoretic components were equally stable between pH 4.0 and 9.0, as were the centrifugal components, with isoelectric points of pH 4.8 and 5.3 for the “slow” and ‘(fast” components, respectively (Fig. 3). Although no physical damage was evident, an irreversible decrease in specific infectivity occurred with deviation from neutrality (Fig. 4). Dialysis at pH 10.0 for 2448 hours preferentially destroyed middle component and if such material, or preparations degraded by alkali-chloroform, were examined electrophoretically, both components appeared in the same ratio as found in controls, substantiating the earlier work of Bancroft (1962). Digression from the mobility values at pH 7.0 previously reported (Bancroft, 1962) was traced to differences in the purification schedules used. If the mobility of purified virus reversibly precipitated at pH 4-5 was taken at pH 7.0, the same lower values reported by Bancroft (1962) were found. A pH 5.0 precipitation step, not presently used,

FIG. 3. Mobility patterns of electrophoretic components of BPMYV; (0-O) small peak, (O--O) large peak.

BANCR.OFT

500 2 .: -I

400300.

cr “0 200 -I IOO-

‘4

5

6

7

8

9

PH FIG. 4. Infectivity distribution of BPMV applied at respective pH’s (0-O ) equalized at ODz6” = 0.035 and dialyzed back to pH 7.0 (O--O) equalized at OD260 = 0.10.

was employed schedule. Diferential

in

the

early

purification

Stability and Structure

The stability difference between the infective 112 S component and the noninfective 91 S component, which contains an incomplete RNA complement, might be reflected in the gross morphology of the particle. Bottom and middle particles were fractionated by density-gradient centrifugation and shadowed with palladium 4: 1 or positively stained with uranyl acetate, No morphological distinctions could be observed in the shadowed preparations (Fig. 5B, D) which were about 30 rnp in diameter. Stained middle particles showed a conspicuous area in the RNA core which did not become electron dense. The stained cores of both components were 16 f 0.5 rnp in diameter (Fig. 5A, C). The area of incomplete staining (Fig. 5C) could represent either the void formed by the missing terminal portions of RNA, susceptible sites in the RNA unstable in the presence of uranyl acetate, or differential permeability of the stain. The first alternative appears more reasonable since no comparable areas could be observed in the bottom particles even though the base composition of the RNA complements

STABILITT

Ir;

Zjt-CLE;OPIWTEIN

OF BEAN

POD

MOTTLE

\‘IIZUS

481

FIG. 5. Electron micrographs of fractionated BPMV. Uranyl acetate-stained preparat,ions; (A) hottom component, (C) middle component. Magnification: X 100,000. Shadowed preparat,ions: (B) bottom component, (D) middle component. Magnificat,ion: X 42,000.

is very similar and no excessive fragmentation of the RNA molecules was observed in either component (Semancik and Bancroft, 1964). Particles of both components were

sometimes disrupted by neutral phosphotungstic acid staining, but no difference in susceptibility between bottom and middle components could be detected.

482

SEMANCIK

AND

DISCUSSION

Alkali-chloroform preferentially destroyed the middle component during extraction from infected tissue and by treatment of purified BPMV. Since neither the alkali nor the chloroform separately produced the effect, two different sites of action were possibly involved or the pH 9.5 treat’ment could have affected the structural bonding sufficiently to allow complete disruption by the chloroform emulsion. In this regard, an increase in electrostatic repulsion between the highly charged protein subunits in alkaline conditions as well as a weakening of the hydrophobic bonding resulting from a decrease in bhe dielectric constant of the medium might be involved (Caspar, 1963). Virus stability depends on protein-protein as well as protein-RNA interactions. The different stabilities of the BI’MV components emphasized the structural dissimilarity imparted by the RNA deficiency in the middle particles. The preferential destruction of the middle component might have resulted from the rupture of the shell at a weakened area, namely, that portion lacking an underlying RNA framework. These particles were apparently more vulnerable to disruption initialIy after synthesis than after an aging period. Furthermore, if the RNAprotein binding capacities are related to nucleotide sequence as suggested by Caspar (1963), then the guanylic-uridylic acid-rich portion of the RNA chain missing from the middle and present in the bottom particles (Semancik and Bancroft, 1964) might possess strong protein-binding sites. A differential loss of components under alkaline conditions has been reported for wild cucumber mosaic virus (Yamazaki and Kaesberg, 1961); however, since stability was inversely related to RNA content either the mode of action of the alkaline and alkaline-chloroform treatment differs or the structural properties of the viruses differ. In this regard, the different stabilities of BPMV and the closely related yellow cowpea mosaic virus are germane. Paul (1963) reported that the ratio of the two centrifugal components of “true broad bean mosaic virus” was altered by different host species although the components manifested similar stability properties.

BANCROFT

The infectivities of preparations showing a preponderance of 112 S particles were low and since infectivity is associated with the bottom rather than the middle component (Bancroft, 1962), some physical derangement had probably occurred in treated bottom component particles. Since exposure to the alkali and chloroform in sequence did not seriously affect the component ratio but did produce a decrease in specific infectivity, a change in particle conformation would seem reasonable. The irreversible decrease in infectivity with deviation from neutrality might be explained by a quaternary structural change as suggested by Incardona and Kaesberg (1964). Our results imply that the alkaline-chloroform t’reatment selected for the particles containing the maximum quantity of RNA and perhaps also for subpopulations on the basis of their protein to nucleic acid binding properties. The inclusion of the complete RNA complement conferred maximum stability to alkali-chloroform, thus concurring with the X-ray diffraction evidence of Klug and Finch (1960) that RNA folding is related to structure and therefore to shell stability. These studies reemphasize that exposure to conditions such as hydrogen ion concentration, organic solvents and detergent’s used in extraction procedures are selective agents allowing purification of a virus population which does not necessarily reflect the in vivo condition. ACKNOWLEDGMENTS We wish to thank Mr. D. A. Reynolds for assistance with the electron microscopy portion of the work and Mr. L. Van Griensven. REFERENCES

and properties of bean pod mottle virus and associated

BANCROFT, J. B. (1962). Purification

centrifugal and electrophoretic components. Virology 16, 419-427. BOCKSTAHLER, L. E., and KAESBERG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2, 1-9. CASPAR, D. L. D. (1963). Assembly and stability of the tobacco mosaic virus particle. Advan. Protein Chem. 18, 37-121. DIEXER, T. 0. (1962). Isolation of an infectious, ribonuclease-sensitive fraction from tobacco

STABILITI-

IN XCCLEOPROTEIN

leaves recently inoculated with tobacco mosaic virus. Viirology 16, 140-14G. FR.ZENKEL-CONR.\T, H., and WILLI.IMS, R. C. (1955). Reconstit,ution of active tobacco mosaic virus from its inactive protein and nucleic acid component,s. Proc. Satl. Acatl. Sci. U.S. 41, 69@698. FRISCH-NIGGEMEYER, W., and REDDI, K. K. (1957). St,udies on ribonuclease in tjobacco leaves. I. Purification and properties. B&him. Biophya. Acta 26, 4046. H~RRINGTON, W. F., and SCHXHM~N, H. K. (1956). Studies on the alkaline degradation of tobacco mosaic virus. I. Ultracentrifugal analysis. Arch. Biochem. Biophys. 65, 278-295. HUXLEY, H. E., and ZUBIY, G. (1961). Preferential staining of nucleic acid-containing structures for electron microscopy. J. Biophys. Biochem. Cytol. 11, 273-296. INCARDONA, N. L., and K.IESBERG, P. (1964). A pa-induced structural change in bromegrass mosaic virus. Biophys. J. 4, 11-21. JOHNSTON, J. P., and OGSTON, A. G. (1946). A boundary anomaly found in t’he ultracentrifugal sedimentation of mixtures. I’lans. Faraday Sot. 42, 789-799. KLUG, A., and FINCH, J. T. (1960). The symmetries

OF BEAS

I’OL) MOTTLE

VIRUS

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of the protein and nucleic acid in turnip yellow mosaic virus: S-ray diffraction studies. J. No/. Bid. 2, 201-215. MILLEIZ, G. L., and GOLDEH, R. H. (1950). Butlers of pH 2 to 12 for use of electrophoresis. Arch. Biochem.

29, 420-423.

PIUL, H. L. (1963). Untersuchungen iiber das echte Ackerbohnenmosaikvirus. Phytopathol. 2. 49, 161-176. PII~IE, N. W. (1956). Some components of tobacco mosaic virus preparations made in different ways. Biochem. J. 63, 316325. SEM.~NCIK, J. S., and BANCROFT, J. B. (1964). Further characterization of t,he nucleoprotein components of bean pod mottle virus. Virology 22, 33-39. STEERE, R. L. (1956). Purification and propert.ies of tobacco ringspot virus. Phytopathology 46, 60-69. WECKER, E., and RICHTER, A. (1962). Conditions for t,he replication of infectious viral RNA. Cold Spring Harbor Symp. Quant. Biol. 2i, 137-148. Y.~M?\zAKJ, H., and KAESBERG, P. (1961). The preparation and some properties of protein subunits obtained from wild cucumber mosaic virus. Biochim. Hiophys. Acta 53, 173-180.