In vitro polymerization of winter wheat mosaic virus antigen

VIROLOGY

35, 458-472 (1968)

In Vitro

Polymerization

J. G. ATABEKOV,

Laboratory

of Bioorganic

of Winter

Wheat

Mosaic

G. A. POPOVA, N. A. KISELEV, AND G. V. PETROVSKY

Virus

A. S. KAFTANOVA,

Chemistry of Moscow State University and Institute tZcaclemy of Science, Moscow, USSR Accepted March

Antigen

of Crystallography,

20, 1.968

Paracrystals isolated from plants infected with winter wheat mosaic virus (WWMY) contain protein WWMIT antigen but do not contain nucleic acid. Some details of WWM\- protein (WWMV,) crystal structure were revealed by electron microscopy. WWM\-,, can exist in a number of molecular forms depending on solvent conditions: (a) globular form-a protein subunit, S:,,,,, = 2 S; (b) aggregate S&,, = 5 S; (c) fibrillar form-a linear aggregate of protein monomers, Si”,, = 17 S; (d) helical forman aggregate of subunits helically packed. The polymerization of WWMI‘, is reversible, and all the aggregates dissociate into 2 S-monomers at pH 9.0-10.0. INTRODUCTION

More than twenty years ago two plant virus diseases of cereals were described by Zashurilo and Sitnikova (1939) and by Suchov (1942) in the USSR. One of these diseases is caused by WWMV and the second one by oat mosaic virus (OMV) (so-called “Zakuklivanie”). Neither virus is mechanically transmissible; both may be transmitted by leafhoppers : WWMV by Psanmot&ix striatus L. (Zashurilo and Sitnikova, 1939) and OMV by Delphax striatella Fall. (Suchov, 1942). Suchov (1943) reported that some needlelike crystals believed to be composed of virus part,icIes may be separated from extracts of wheat and oat plants infected with WWMV or OMV. Schaskolskaya (1961) showed that WWMV (tested symptomatically and by the formation of microcryst,als in vitro) can be transmitted from millet to winter wheat plants and vice versa by the leafhopper P. striatus L. More recently Popova et al. (1966) demonstrated that WWMV is effectively transmitted by injection of infectious plant sap or RNA extracts t,o nonviruliferous insect vectors. Thus, the infection is caused by a virus of unknown structure.

III the present work some properties of crystalline preparations isolated from WWXV-infected plants are described. Particular aspects of this n-ork have been reported in a short communication (Atabekov et al., 1965). MATERIALS

AND

METHODS

Puri$caticm of WWMV protein, Leaves of WWAlV-infected millet and winter wheat plants were harvested in fields of t’he Kinnel Agricultural Station and stored in a deep freeze or used fresh. Preparations of WWMV, were made by the method of precipit,ation at the isoelect.ric point (IEP). The leaves were homogenized in 0.1 X phosphate buffer, pH 7.5 (3 ml/g tissue). The juice was extracted from pulp and clarified by cent,rifugation at 10,000 g for 10 minutes. The supernatant was acidified to pH 5.2 and the precipitate was immediately removed by centrifugation at 20,000 g for 10 minutes. Then the WWMV, crystallized at pH 3.S. The crystallization was followed with the microscope and then the suspension of crystals was cent,rifuged at 20,000 g for 1% 20 minutes. After resuspension of the pellet in a small volume of 0.1 34 phosphate (or 0.05 M Tris-HCl) buffer, pH 7.5-8.0, the 458

POLYMERIZATION

OF WINTER

WHEAT

solution was centrifuged at 20,000 g for 1520 minutes. The preparations of WWNV, were obtained by 5-7 cycles of IEP precipitat#ion. Yields ranging from about 20 to 80 mg of WWMV, per kilogram of fresh leaf material were obtained from the millet plants and commonly were about 50 mg. r\‘o WWMV, could be isolated from healthy plants. Careful serological and electron microscopic examination of WWMV, preparations showed that there was no contamination with TMV or any other known viruses. Anti-TMV sera did not react with WWRV, in immunodiffusion tests. Concentrations were determined by measuring UV absorption at 278 ml* and then using the absorption coefficient A1%m mfi, I~,,, = 18, which was determined from the dry weight of WWMV, water solutions of known optmica density. Ultraviolet spectra were measured with a SF-4A spectrophotometer using a cell of l-cm path length. Sedimentation analyses were carried out in a Spinco model E centrifuge. The sedimentation coefficient (X20,,) and the sedimentation constants (SIZE,2L’)were determined as usual. Viscosity of WWMV, solutions was measured at various shear st’resses (13-590 set-l) using Rubinstein’s (Rubinstein, 1945) horizontal viscomet’er. The rate of shear was calculated for water. Viscosities of WWMV, preparations were measured also in an Ostwald viscometer with a volume of 0.5 ml and a flow time for water of 146 seconds. Temperature was 20.0 f 0.05”. Electrophoretic experiments were carried out in a Zeiss (Jena) Model 35 Tiselius electrophoresis apparat’us. Double refraction of flow was studied by means of Tsvetkov’s device. The values of double refraction of flow (An) and of extinction angle (a) at different flow gradients were measured as previously described (Atabekov et al., 1965). Density-gradient centrifugations were carried out according t’o the general procedure of Brakke (1960). Each gradient column contained 15, 10, 10, and 10 ml of 0.1 131 phosphate buffer containing 100, 200, 300, and 400 g sucrose per liter, respectively. The

MOSAIC

VIRUS

ANTIGEN

459

mixture was layered (2-4 ml per tube) and, after centrfugation in a Spinico SW 25.2 rotor at 24,000 rpm for 90 minutes, the solution was fractionated. Electrofa nzinoscopy. The procedure of Brenner and Horne (1959) was used. The preparations were either stained [2-5 % phosphotungstic acid (PTA), 2 % many1 acetate, or 2% ammonium molybdate] or shadowed (with Pt/Pd). Electron micrographs were made with a JEM-6c microscope at 80 kV. Serological tests. Antisera were prepared by combined intravenous and intramuscular injections (with Freund’s complete adjuvant). Different schedules of injection were used: 30-50 mg of WWMV, in 0.1 M phosphate buffer, pH 7.0, was usually injected intravenously into each rabbit per one immunizat’ion cycle (6 injections per cycle). The tit,ers of antisera varied between 1:4 and 1: 16; thus the immunogenicity of WWRIV, is very low. Immunodiffusion and immunoelectrophoresis t’echniques (Ouchterlony, 1961) were used. Agar was prepared in the same buffer in which the analyzed antigens were dissolved. Analytical methods. Total phosphorus and nitrogen content of WWMV, were det#ermined as previously described (Atabekov et. al., 1965). Amino acid analysis. The protein was hydrolyzed with 6 N HCl at 105” for 24 hours. Excess HCl was removed by repeat,ed evaporation in vacua at 45-50°C and the samples were dissolved in citrate buffer, pH 2.2. Amino acid analyses of WWMV, were performed by Dr. V. 1’. Borzhenko wit#h a Hitachi KLA-3B automatic amino acid analyzer. The absence of cysteic acid in t,he hydrolyzates of WWNV, oxidized with performic acid was proved by the method of Hirs (1956). n’it’roprusside reaction in 8 111 guanidine chloride (Anson, 1941) was also made t,o support the absence of cysteinc in WWhIV,. Tryptophan and tyrosine were determined by the method of Goodwin and 1Iorton (1946). Nuscle protein fibrillar actin (F-actinl was isolated from rabbit muscles by the met,hod of St,raub (1942).

460

ATABEKOV

ET AL.

RESULTS

Properties of Purified WWMV,

TABLE

Preparations.

WWlLIV, preparations exhibit a protein absorption spectrum with the maximum at 276-275 rnp (Fig. 1). The spectrum in Fig. I shows in the region 300400 rnp relatively strong absorption which is probably due to the presence of pigments. The pigmentation appeared as a yellowish color in concentrated WWXIV, preparations (especially under alkaline conditions). The preparations reveal intensive biuret reaction and do not react with orcinol. The phosphorus cont,ent of WWMV-antigen preparations is not more than 0.007 %, and the total nitrogen content is about 16%. In Table 1 the results of amino acid composition analyses of 4 WWMV-protein preparations (14 hydrolyzate samples) are summarized. All preparations were isolated by the same method from WWMV-infected millet plants. At protein concentrations of about 15520 mg/ml (pH 7.2 in 0.1 M KCl) a great number of birefringent areas resembling the taktoids of TMV were seen. Under these condit,ions the protein preparations were very viscous and the oriented birefringent gel formed after flowing through the capillary. In slightly acidic solutions (pH 5.0) WWMV, formed needlelike paracrystals (Fig. 2 a) which were birefringent in crossed Nicol prisms (Fig. 2b). When heated at 50”,

FIG. 1. Ultraviolet WWMV,, preparations in 0.01 M Tris-HCI f and pH 9.0 (---).

absorption spectra of two of the same concentration 0.1 M KC1 at pH 7.0 (- - -)

AMINO

1

ACID COMPOSITION OF WWMV Amino acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Cystine Cysteine

PROTEIN

Grams of amino acid per 100 g protein 19.51 10.21 14.74 19.70 6.73 1.52 3.38 5.12 3.39 9.01 13.3 5.12 G.23 9.42 3.79 1.78

129.99

these crystals deformed and then melted. When they completely dissolved, a protein solution as described below formed. So WWMV,, microcrystals could be isolated from healthy plants. Sedimentation and Electrophoretic Properties At pH 9.0-10.0 WWMV-protein contains a basic component with a sedimentation constant of 2 S (Fig. 3 and Table 2). Below we shall refer to the 2 S protein as the “globular” (G) form of WWMV,,. The G form of WWMV, seems to be homogeneous in the synthetic boundary cell at 42,040 rpm. Nevertheless, when the G form was analyzed at 63,650 rpm in a one-sector Kel-F cell, a precipitate at the bottom of the cell was progressively formed in the course of centrifugation (Fig. 3a). It is probable that either material aggregates during centrifugation or that aggregates of widely varying size were present in solution in amounts insuflicient to form separate peaks. The sedimentation characteristics of WWXIV, change sharply after acidification. In water solution (or in 0.1 M NaCl) at pH 6.0-7.2, WWMV, sediments as a single

POLYMERIZATION

FIG. 2. Micrographs prisms

ofpolarizing

OF WINTER

of WWMV, microcrystals: microscope. X 700.

broadened peak of about 5-6 S (Kg. 3b, top component, and 3~). After addition to WWMV, of KCl, MgCl2, CaC12, or Ba&, the preparations became heterogeneous at about neutral pH’s (Table 2 and Fig. 3b, bott’om pattern), Analytical centrifugation showed that two components (with average sedimentation constants of about 5 S and 17 S) were present. The sedimentation coefficient of the 17 S component is greatly dependent’ on concen t’ration (Fig. 4) and its boundaries are hvpersharp (Fig. 3b). This is characteristic of

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(a) at pH 5.0 in water

ANTIGEN

X 300; (b) in crossed

461

Nicol

preparations cont’aining molecules with a large axial ratio and is due to a self-sharpening effect (Schachman, 1959). The aggregation of WWI\IV, is reversible: S20, w returns to the initial 2 X at pH 9.0-10.0 (even in the presence of salts). The aggregation of WWXIV, is not accompanied by an increase in opalescence. The absorption spectra of the G form (2 S) and of the heterogeneous (5 S + 17 S) preparations are almost the same (Fig. 1). During melting of WWMV, crystals and during heat’ing (40-50”) of (5 S + 17 S) mix-

462

ATABEKOV

ET AL.

TABLE SEDIMENTATION Expt.

BEHAVIOR

OF WWMV

2

PROTEIN

UNDER

AND pH CONDITIONS

SALT

SXWat pH

Cont. of protein

Type of buffer and treatment

no.

VAHIOUS

bdml)

9.GlO.O

7.0-7.2

1

Water”

8.0

2.0

-

2 3

Water 0.01 M Tris-HCl

8.0 5.0

1.8

5.0 -

+ 0.1

M NaCl

4 5 6 7 8 9 10 11 12 13

0.01 M Tris-HCl + 0.1 fif NaCl Water + 0.1 M KC1 Water + 0.1 M KC1 Water + 0.1 M BaClz Water + 0.1 111MgCl, Water + 0.01 M KC1 Water + 0.01 M BaCl? Water + 0.01 M MgC1, Water + 0.001 M CaCIz Phosphate buffer, 0.1 M

6.0 1.9 4.0 3.0 4.0 3.0 3.0 3.0 4.2 2.5

2.0 2.0

5.0, 5.0, 2.0, 4.0, 13.0 4.2, 5.2, -

14 15

Phosphate buffer, 0.1 A4 WWMV, in 0.1 M phosphate buffer was heated at 40” for 10 min Suspension of crystals was melted at 45”

2.0 4.0

-

5.7, 6.3,

14.0 36.8

4.0

-

37.8,

55.7"

16

a pH of unbufl’ered solution was adjusted by addition b Small amounts of 16 S material were also present. c The crystals were taken at pH 5.0.

tures further aggregation takes place, and 30-50 S aggregates appear (Fig. 3d and Table 2). Long fibrillar structures were found with the electron microscope in the WWMV, preparations containing 30-50 S or 17 S components (see below). Gel formation occurred at the bottom of the cell when 3050 S aggegates sedimented in the analytical centrifuge (Fig. 3d). When the 17 S component is present in WWMV, the gel plate also forms but is much smaller than with 30-50 S aggregates. The gel is compact, transparent and birefringent. Such gel pre-

of 0.01-0.1

4.P 13.0 17.4 16.4 14.0 15.0 13.4

M NaOH.

cipitates do not form when 2 S or 5 S preparations are examined. When the G form of WW,MV, was examined in free electrophoresis in 0.1 M Verona1 buffer, pH 8.6, a single peak was observed (Fig. 3e). However, in 0.1 AI phosphate buffer at pH 7.2, the aggregated WWMV, produced at least two peaks (Fig. 3f). The multiplicity of the peaks observed in free electrophoresis is in accordance with the heterogeneity of aggregated WWMV, preparations observed at neutral pH in analytical ultracentrifugations.

FIG. 3. (a-d) Sedimentation diagrams of WWMV, protein. (a) Preparation contains 6 mg/ml protein. in 0.1 M phosphate buffer, pH 10.0. The photographs were taken 12, 38, and 98 minutes after reaching a speed of 63,656 rpm. Schlieren angle, 70 degrees. (b) WWMV, preparation containing 4 mg/ml protein. Top pattern: in 0.1&l NaCl, pH 7.0; bottom pattern: in 0.1 M Tris-HCl + 0.1 M KCl, pH 7.0; 20 minutes after reaching a speed of 56,100 rpm. Schlieren angle 60 degrees. (c) WWMV, preparat,ion containing 4 mg/ml protein. Top pattern: in 0.01 M NaCl, pH 7.0; bottom pattern: in 0.05 M NaCl, pH 7.0; 33 minutes after reaching a speed of 56,100 rpm. Schlieren angle 60 degrees. (d) WWMT’, preparation containing 8 mg/ml. Top pattern: solution obtained by melting of WWMV, crystals in 0.1 M phosphate butfer pH 7.0 at 50” for 10 minrltes; bottom pattern: “5 S + 17 S” solution was heated at 50” for 10 minutes in the same buffer. Centrifugation at 29,500 rpm for 16.5 minutes. (e-f) Electrophoretic patterns of WWMT’, preparations (8 mg/ml at 8 ma). (e) In 0.1 M Verona1 buffer, pH 8.6 aft,er 0, 30, and 60 minutes; from right to left. (f) In 0.1 M phosphate buffer pH 7.2 (Fform) after 10 and 65 minutes; from right to left.

POLYJIE:RIZ~4TION

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VIRUS ANTIGEN

a

-

0 min.

4%

464

ATABEKOV

(I5 -179

ET AL.

!,

100 200 300 4cO 500' 6bo

% 7 3.0' 6 2.0,

5

mg/ml

10

FIG. 4. Concentration dependence of WWMV, sedimentation. 0, WWMV, in 0.01 M Tris-HCl + 0.1 M KC& pH 7.0; 0, WWMV, in 0.1 M KCl, pH 7.0; @, WWMV,, in 0.01 M KCl, or BaCIP, or MgCl, at pH 7.0; X, WWMV, in 0.1 M BaC12, or MgCl, at pH 7.0; ., WWMV,, at pII 8.0-9.0 in the presence of any salts.

Viscosity and Double Rejkaction oj Flow In diIute water solutions at pH 7.0 and at a concentration of 0.3 mg/ml, WWMV, showed viscosity behavior independent, of the rate of shear. Under the same solvent condition the WWMV,, solutions became non-Newtonian when the concentration was increased to 0.6 mg/ml or more (Fig. 5). Such preparations showed large double refraction of flow at concentrations of about 3 mg/mI and higher (Fig. 6, curves 1 and 2). Dilution of these preparations (lower than 2-3 mg/ml) led instantly to the disappearance of Sk-earning birefringence (Fig. 6, curve 3). At pH 5.0-9.0 WWMV, preparations were Newtonian and did not show double refraction of flow even at high protein concentrations (Fig. 5; and Fig. 6, curve 3). Double birefringence of WWMV, solutions may be restored (at least part’ly) by reacidification of preparations to pH 6..5-7.0 (Fig. 6, curve

4).

These observations suggest that anomalous viscosity and double refraction of flow of

1.0,i,_,

,

LZ3, 100 200 300 400 500 600

Set-'

set-;

FIG. 5. Dependence of relative viscosity (?/vO) on the flow gradient (see-‘) in WWMV, solutions. (a) WWMV,, in water at pH 7.2 and concentrations: (1) 0.3 mg/ml, (4) 0.64 mg/ml, (5) 1.2 mg/ml, (6) 2.7 mg/ml. WWMV, in water at; pH 9.0 and concentrations: (2) 1.2 mg/ml, (5) 2.7 mg/ml. (b) WWMV, at pH 7.2 and concentration 0.3 mg/ml in (1) water, (2) 0.01-0.1 M NaCl, (3) 0.01 M MgCl, + 0.01 k’ Versene, (6’) 0.01-0.1 ,%I KCl, (7) 0.01-0.1 M MgC12. WWMV,, at pH 9.0 and concentration 0.3 mg/ml in: (4) 0.01 X MgCl?, (5) 0.1 k’ KCl.

WWMV, preparations are due to spontaneous linear aggregation of protein subunits in solution. This aggregation is reversible and depends on protein concentration and PH. In a series of experiments the influence of some cations upon the properties of WW&IV, was investigated. It was found that addition of I(+ or hig2+ (but not of Sa+) to neutral dilute WWMV, preparations induced anomaly of viscosity and strong streaming birefringence (Figs. 5 and 6). This effect varied in individual preparations, but was always depressed at pH 8.0-9.0 (Fig. 5). The intrinsic viscosities of two separate WWMV, preparations measured in the presence of 0.1 M KC1 were 0.4 and 0.29 dl/g at pH 7.2 and 0.01 dl/g at pH 8.5, respectively. Electron Microscopy A longitudinal periodicity of 40 A and a cross-directional periodicity of 100 A were

POLYMERIZATION

I

0

OF WINTER

6

200 400 600 800 gsec-’

Uh)

1000 1200

Fro. 6. 1)oable refraction (An) in flow and extinction angle (01) dependence on flow gradient (set-1) in WWMV,, solutions. Curves 1 and %: WWMV,, in water solution at pH 7.2 and concentration 3.3 mg/ml protein. Curve 8: This line means that the double refraction effect is too low and cannot, be measiired (at, pH 8.5 and at WWMV,, coricerit,rations lower than 2.0 mg/mlj. Ciirve 4: Preparation No. 8’ after reacidification from pH 8.5 to pH 7.0. Ciirvc 5: WWMV,, in water at pH G.5 and coucent,ration 4.0 mgjml protein (another preparation). Ciirve 6 : Preparation of fibrillar miisclr protein F-actin (0.5 mg/ml) in 0.02 M Tris-HCl + 0.1 U KCl, pH 7.0. F-a&in was used here as a refereiice highly fibrillar protein. Note that iii nctiii solution the streaming birefringence is coiisiderablv higher than iii WWMV,,.

revealed in WWMV, crystals by negative &ining wit,h 2 ‘L ammonium molybdst,e (pH 5) or with 2%’ PTA (pH 4.S) (Figs. 7 and 8~). In shadowed preparations only the cross directional periodicity was visible (Fig. 7b). After heating for 1-2 minutes, large aggregates of fibrils were observed, which corresponded in size and outer shape to protein cryst,als (Fig. Sh, c). Upon longer heating the crystals disappeared completely and individual fibrils of 40 ,4 thickness could be observed (Fig. 8d). These fibrils were built of subunits. Thus, fibrillar structure of WWMV, crystals is developed with heating. Mutual packing of protein fibrils creates cross (100 A) and longitudinal (40 A) periodicities, visible on microphotographs.

WHEAT

MOSAIC

VIRUS

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465

Similar fibrillar particles (40 A thick) were also observed in ( 5 S + 17 S) protein solutions. Particles of this type are designated as “fibrillar form” of WWKIV,. A second type of particle has the shape of rigid rods about 110 A wide (Figs. 7b, 9a, b, and 10). Negative staining of these particles revealed an internal canal with a diameter of about 20-25 A (Figs. 9b and 10). Sometimes a cross periodicity of up to 35 A could be det’ectcd. This type of particle is desigform” (H-form) of nated as “helical WWMV,. H-form as well as F-form particles retained their st,ructural properties after the crystals were melted. Bot’h types of aggregates are usually-but not always-present in t’he purified WWMV, preparations in 0.1 Al phosphate buffer and in 0.1 218KC1 at pH 4%7.5; they are completely absent at pH S..‘i-9.0, when t’he transformation to Gform

occurs.

H-form particles appeared repeatedly when WWMV,, was reacidified to pH 5.0 (IEP). This observation indicates that Hform aggregates may be formed as a result of aggregation of either G-form or 5 S (or Fform) molecules. Helical particles identical to H-particles were detected also in the clarified extracts (10,000 rpm for 10 minut’es) from the infected millet and winter wheat plants. H-form and F-form particles were absent in healthy hosts. Density-Gradient

Centrifugation

Some well-known preparative procedures proved unsuccessful for virus isolation. The procedure described below allowed to obtain a light-scattering zone in density-gradient experiments. Infected millet leaves were homogenized in 0.1 2’ phosphate buffer or in 0.05 M TrisHCl + 0.1 M KC1 at pH 7.2 (1: 1, w/v) and clarified by low-speed centrifugation. The pellet of crystals obtained from these extract,s Jvas

by a single

cycle

of IEP-precipitation

resuspended in a small volume of 0.01 M Tris-HCl, pH 7.0 with 0.1 M KC1 or 0.1 M phosphat’e buffer, pH 7.0. Only one IEP precipitation mas used to minimize the probabilit#y of destruction of H particles (and, probably, of virus par-

466

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ET AL.

FIG. 7. Electron micrographs of WWMV,, microcryst.aIs (pH 5.0). (a) WWMI-, crystal negatively stained with 2(x ammonium molybdate. (b) WWMI’p crystal, shadowcast with R/I’d at an angle of 8 degrees (ratio of the shadow length artd height of the object is 1:8). (a) x 200,000; (1)) x 100,000.

POLYBIERIZATION

OF IWINTER

WHEAT

31OHAIC

\7RUS

ANTICS3

467

crystal, negatively stained wit,h 2’/;, FIG. 8. Fibrillar structrue of WWM\-, crystals. (a) WWMV, PTA. The fibril aggregate produced when WWM\‘, crystal was heated for 2 minutes at 50”. (b) Negative staining with 2% PTA. (c) Fibrils, shadowcast at an angle of 8 degrees. (d) Individual fibrils into which the crystals decay when heated. Stained with 27; lxanyl acetate. (a) and (h) X 200,000; (c) X 100.000; (d) x 300,000.

468

ATABEKOV

FIG. 9. Helical particles in WWMV, preparations. 8 degrees. (b) Particles of H-form WWMV,, stained 300,000.

titles). It is apparent that some cellular material (ribosomes and/or other cell structures) contaminated these preparations. Normal cell material isolated from healthy plants under t’he same conditions was very scanty and was analyzed as a control. Some variability was observed in the results of different experiments, but usually only one diffuse light-scattering zone was detected 26-30 mm below the meniscus (Fig. lla). A second very faint zone sometimes occurred 4046 mm below the meniscus. Electron microscopic examination of material from the 26-30 mm zone revealed rodlike particles identical to the H-form of WWMV,. No visible zone was observed in control preparations from healthy plants (Fig. llb). Material of the light-scattering zone absorbed UV light with a maximum at, 270 rnp. The same UV-absorption spectrum was

ET AL.

(a) H-form WWMV,, shadowcast at an angle of with 27” uranyl acetate. (a) X 150,000; (b) X

observed in the material obtained from the 26-30 mm position of control columns with healthy samples: It is possible that the identity of spectra was due to the presence of a small amount of UV-absorbing host material (mentioned above) contaminating both the “diseased” and “healthy” gradient columns. This contamination was not large, as no visible zone was observed in control columns. Severtheless, it was probably present in amounts suflicient to produce the UV-absorption maximum at 270 rnp in the 26-30 mm fraction from the “diseased” and “healthy” samples. Immunodiffusion tests showed that the material from the visible zone and also material of all above fractions reacted with anti-WWMV, serum. The maximum WWMV, concentration (tested by immunodiffusion tests) was located near the meniscus and was not visible by light scattering. This

POLYMERIZATION

OF WINTER

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MOSAIC

VIRUS

ANTIGEN

F ‘IG. 10. WWM\-, crystals were melted at 50” and then centrifuged at 115,000 9 for 90 minutes. The pelll et was resuspended in 0.1 1%’ phosphate buffer at pH 7.0 and examined. Negative staining with 2% PTA. X 175,000.

zone could easily be revealed serologically and was probably composed of 5 S molecules and heterogeneous I?‘-form material sedimenting slower than H part,icles of the visible zone. lSo reactions were obtained between anti-WWMV, serum and any fraction of control gradients. Serological

Properties

of WWAIV,

In some experiments (5 S + 17 S) mixtures produced tn-o precipitin lines (Fig. 12a, e). Nevertheless, such preparations usually produced a single precipitin line in immunodiffusion and immunophoresis tests. The 2 S material always produced a single precipitin line. (Fig. 12b, d). Antisera to WW?\lV-protein, purified from WWRZV-infected millet plants as a source of ant,igen, reacted with crude and clarified extracts from infected millet and n-heat plan&, but never reacted with control extracts from healthy hosts (Fig. 12~). In an earlier paper (Popova et al., 1966), we reported that WWJIV, is serologically related to the viruses of the TRIV group.

We failed to reproduce this result in the present work, although five new- antisera to WWhlV, \\-ere prepared. DISCUSSION

Preparations isolated from WWMV-infected plants contain protein-WWMV antigen but do not contain nucleic acid. It is of some interest that preparations separated by IEP precipitation from OMVinfected plants also contain a protein material of low molecular weight (Atabekov et al., 1965). The amino acid composit’ion of WWRIVprotein is significantly different from the proteins of other viruses known. WWRIV, does not contain histidine or cysteine, but contains methionine and comparatively large amounts of dicarboxylic acids, proline, and leucine (Table 1). 111 alkaline solution (pH 9.0-10.0) WWJIV, exists as a lowmolecular weight protein (C-form). In neutral water solution and in t’he presence of 0.1 M NaCl the polymerization of prot8eiu is limited and predom-

ATABEKOV

FIG. 11. Density-gradient fractionation tion. (a) Preparation from WWMI--infected

ET AL.

of a preparat,ion obtained by a single cycle of IEP precipitamillet’ plants. (b) Control preparation from healthy host.

FIG. 12. Serological reactions between antisera to WWMV, and (a) WWMT’, in 0.01 LV Tris-HCl + 0.1 32 KCl, pH 7.0; (b) the same preparation at pII 9.0; (c) 1: WWMV, preparation at pH 8.0; 2: extract in 0.1 M phosphate buffer, pH 8.0 from WWMV-infected millet plants; and 3: same from healthy millet plants. Immunophoregrams of G-form WWMV, (d) and F-from WWMV,, (e).

POLYMERIZATION

OF WINTER

inantly proceeds to 5 S-aggregate formation. The results of hydrodynamic and electron microscopic studies show that 17 S component, formed in the presence of I<+, Mgz+, Caz+, or Ba2+ (F-form), is a linear aggregate of protein subunits. It is unlikely that linear polymerization of WWMV, would yield fibrils of the same length; 17 S component consists of fibrillar molecules of different lengt’h. It can be seen that the hydrodynamic characteristics varied greatly in different (5 S + 17 S) preparations under the same conditions. This probably is due to the difference in the degree of polymerization and the length of F-form molecules formed in separate WWRIV, solutions under the same conditions. Rodlike particles with a helical structure and an internal hole (H-form) were also found in WWMV, preparations. In the course of our experiments we obtained some WWMV, preparat,ions that contained F-form particles invariably and sometimes a very few, if any, H-form particles. It is significant that sedimentation patterns and hydrodynamic features remained unaltered even in those preparations where H particles were completely absent. This result indicates that the 17 S hypersharp boundary is formed by fibrillar aggregate molecules and that H-form is a minor component of WWMV, preparations. G-form is a starting material for 5 S-aggregate molecules as well as for F- and Hform particles. This does not mean, however, that’ any particular aggregate is unfailingly formed upon polymerization direct#ly from 2 S monomers. The functional significance of different WWMV, molecular forms is not clear at the present, Gme. It is not excluded that formation of F particles is a step preceding the formation of H particles, i.e., that H particles are built from preformed F part,icles. Rapidly accumulating evidence suggests that all the information essential for secondary, tertiary, and quaternary structure formation is cont,ained in the primary structure of the proteins. In vitro reconstruction of specific quaternary structure identical to the one formjng in living cells was shown with viral (Caspar, 1963; Kiselevet al., 1966;

WHEAT

MOSAIC

VIRUS

ANTIGEN

471

Bancroft et al., 1967) and nonviral proteins (see Reithel, 1963). We cannot conclude that in vitro aggregation of WWMV, is correlated with the assembly of virus particles as we do not know the virus particles themselves. The protein could well be the virus capsid protein which has become dissociated from the RYA, or something like TMV “X”protein (Mleczkowski, 1961). Moreover, it is not clear at present whether WWMV, is a coat protein of the virus. This was the reason for naming this protein “WWMV antigen.” Nevertheless, the similarity of H particles formed in vitro to the structure of some known viruses, high concentration of WWRIV, in infected millet, and the presence of morphologically analogous particles in extracts from WWRIV-infected plants suggests that a viruslike structure is reconstructed in vitro when WWMV protein repolymerizes. ACKNOWLEDGMENTS We wish to thank Dr. M. V. Kalamkarova for the double refraction of flow analyses of WWMV,. Our thanks are also due to Dr. V. J. Chernjak for his discussions. REFERENCES A~soru, M. C. (1941). The sulfhydryl groups of egg albumin. J. Gen. Physid. 21, 399-402. ATABEKOV, J. G., POPOV.Y, G. A., and KALAMK.*Rov.\, M. V. (1965). Configurational changes of wheat, mosaic virus antigen molecules in solutions. Dokl. Akad. Sauk. USSR 163, 14991502. BANCROFT, J., HILLS, C;., and MARKHAM, R.. (1967). A study of the self-assembly process in a small spherical virus. Formation of organized structure from protein subunits in vilro. Virology 31, 354-379. BRAKKE, M. K. (1960). Density gradient centrifugation and its applicat)ion to plant vinlses. Advan. Virus Res. 7, 193-224. BRZ:NNI:I~, S., alld HORNE, R. W. (1959). A negative staining method for high resolution elect,ron microscopv of vinlses. R&him. Riophys. Acla 34, 103-110. C.ispaa, II. L. 1). (1963). Assembly and stability of the tobacco mosaic virus particle. Advan. Protein Chenz. 18, 37-121. GOODWIN, W., and MORTON, It. A. (1946). The spectrophotometric determination of tyrosine and tryptiphan in proteins. Biochem. J. 40, 628-632. HIRS, C. II. W. (1956). The oxidation of ribonuclease with performic acid. J. Biol. Chem. 219, Gll-621.

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N. A., ATABEKOV, J. G., KAFTANOVA, A. S., and NOVIKOV, V. K. (1966). Studies on virus protein repolymerization and reconstruction of some rod-shaped viruses. Bbstr. 6th Intern. Congr. Electron Microscopy, Kyoto, 147148. Maruzen, Tokyo. KLECZHOWSKI, A. (1961). Serological behaviour of tobacco mosaic virus and of its protein fragment,s. Immunology 4, 130-141. OUCHTERLONY, 0. (1961). Diffusion in gel methods for immunological analysis. II. Progr. AZZergy, 5, 78. POPOVA, G. A., PRIDANTSEVA, E. A., SCHMKOLSE. V., and AT.LBEKAYA, N. D., KUVSHINOVA, KOV, I. G. (1966). Physico-chemical and serological studies of winter wheat mosaic virus antigen and isolation of infectious viral RNA. Abstr. ZXth Intern. Congr. Microbial. p. 483, Moscow. REITHEL, F. J. (1963). The dissociation and assoKISELEV,

ET AL. ciation of protein structures. Advan. Profein. Chem. 18, 124-226. RUBINSTEIN, D. A. (1945). Measuring of the viscosity of actomyosin. Biokhimia USSR, 10, 465-470. SCHMHM~N, H. K. (1959). “Ultracentrifugation in Biochemist,ry.” Academic Press, New York. SCHASKOLSKAY~L, N. D. (1961). Identification of the millet mosaic disease. Biol. Sci. (USSR) 1, 103-109. STRBUB, F. B. (1942). Actin. Studies Inst. Illed. Chem. Univ. Szeged 2, 1-15. SUCHOV, K. S. (1942). Especial features of two viruses affecting cereals and their vectors. Mikrobiologia (USSR) 11, 4, 16&167. SUCHOV, K. S. (1943). Purification of crystal preparation of winter wheat mosaic virus. Dokl. Akad. Nauk USSR 39, 72-73. ZMHURILO, V. K., and SITNIKOV.~, G. M. (1939). Winter wheat mosaic. Dokl. Akad. Xauk LTSSR 25, 9-14.