Phytopath. Z., 85, 1—6 (1976)© 1976 Verlag Paul Parey, Berlin und HamburgISSN 0031-9481 / ASTM-Coden: PHYZA3

Institute of Microbiology and Virology, Ukrainian Academy of Sciences, Kiev

Infectivityof Red Clover Mottle Virus Nucleoprotein Components

By

L. G. LAPCHIC, L. L. KUZNETZOVA, V. S. MELNICZENKO, and E. F. KRASNOVA

With 3 figures

Received September 72, 1975

According to the classification of plant viruses of HARRISON et al. (1971)red clover mottle virus (RCMV) belongs to the Comovirus group. This groupincludes sudi representatives as cowpea mosaic virus, bean pod mottle virus,squash mosaic virus, radish mosaic virus, broad bean stain virus and true broadbean mosaic virus. The main diaracteristic feature of all these viruses is thatthe genetic information necessary for their multiplication is divided betweentwo nucleoprotein components so they are multicomponent plant viruses withdivided genome. The general information about plant viruses with dividedgenome has been summarized in review of Dr. VAN KAMMEN (10).

Up to the present there is no exact evidence that RCMV also has dividedgenome. Some beginnings have been made in this direction by Dr. VALENTAand Dr. MARCINKA (7) who have marked enhanced infectivity of combinedbottom and middle components of RCMV.

We decided to investigate the properties of the divided nucleoproteincomponents of RCMV in order to determine with what component the in-fectivity was associated. In this paper we report the experiments with RCMVwhich show that both components are necessary for the infectivity of virus.

Materials and Methods

Virus

RCMV was propagated and purified as described previously (4). The infectivity ofpurified preparations of virus and separated components was estimated by local lesion assays

Phyiopuh. Z.. Bd. 85, Heft 1 1

2 LAPCHIC, KUZNETZOVA, MELNICZENKO and KRASNOVA

on primary leaves of Phaseoius vulgaris var. Top Crop. Local lesions were counted 5 daysafter inoculation.

Separation of the virus components

Virus components were separated by centrifuging twice in sucrose density gradients (4).After the second density gradient centrifugation the divided components were concentratedby precipitation with 8% polyethylene glycol (M.W. 6000) and 0.2 M NaCl (8). The pre-cipitates were collected by centrifugation at 4000 rpm. and suspended in 0.06 M phosphatebuffer pH 7.0. Then the components were sedimented 1.5 hr at 30000 rpm. in VAC:-60]ultracentrifuge.

Determination of concentration of RCMV and its components

Concenirations were estimated using extinction coefficients (E'*,'^* ) at 260 nm 8 forpurified virus, 6 for middle component and 10 for virus bottom component. These assumptionswere based on the known percentage contents of nucleic acid and protein and by analogywith cowpea mosaic virus (5).

Serology

Antiserum against unfractionated RCMV was produced by immunization of rabbitswith highly purified virus preparations (four cycles of differential centrifugation). Rabbitspreviously bled for normal scrum were injected intravenously twice at week intervals with3.4 mg of purified virus suspended with 3 ml of saline. That followed by two intramuscularinjections at week intervals of 1.4 ml purified virus (concentration 1.4 mg/ml) emulsified withFreund's complete adjuvant. For the determination of the time when the antibody level willreadi its maximum the small test samples of the blood were taken at definite intervals afterlast immunization. Anti«:erum was prepared by removal of the clotted material and storedat +4°C until used. Antiserum titres were determined by agarose double diffusion tests in10 X 50 mm Petri dishes with 1 mm layer of I 7'> agarose gel in a humid box at a roomtemperature. The results were registered 3 days later. The lines of the precipitation werestained with 0.05 9i: amido black lOB in acetic acid during I hour and destained with 7 "^/facetic acid.

Fig. 1. Sdilieren patterns of red clover mottle virus preparations, (a) Purified virus pre-paration showing (left to right) T, M and B components. Photograph taken after 12 min.sedimentation at 30,000 rpm. Sdilieren angle 60°; (b) Middle component (M) after twocycles of sucrose density gradient centrifugation. Photograph taken after 8 min. sedimentationat 30,000 rpm. Sdilieren angle 60"'; (c) Bottom component (B) after two cycles of sucrosedensity gradient centrifugation. Photograph taken after 9 min. sedimentation at 30,000 rpm.Sdilieren angle 30°. Ultracentrifuge Spinco model E; t — +20=^ C, sedimentation is from left

to right

Infectivity of Red Clover Mottle Virus Nucleoprotein Components 3

Results

Purified preparations of RCMV separated in analytical ultracentrifugein three components: top (T), middle (M) and bottom (B) (fig. 1 a). Whenpurified RCMV was centrifugated in sucrose density gradient three light-scattering zones were revealed. These zones corresponded to the T, M andB components.

The U.V.-absorption spectra of the components of RCMV separated bytwo cycles of sucrose density gradient centrifugation give such mean ratioHWEsHo: 0.66, 1.45 and 1.67 for T, M and B respectively (fig.2). It meanstliat T component is the nucleic acid-free protein shell of the virus; M and Bare nucleoproteins. Their sedimentation coefficients S to (at infinite dilution)are 55 S, 95 S and 125 S for T, M and B components respectively. Using Reich-man's formula (6) and sedimentation coefficients given above we calculatedthat M and B components contain about 26 % and 38 % RNA respectively.

Preparations of M and B components in 0.06 M phosphate buffer pH 7.0after the first centrifugation in sucrose gradients were examined with ana-lytical ultracentrifugation and their infectivity — by local lesion assays onjirimary leaves of Phaseoius vulgaris var. Top Crop. As was expected T com-ponent was not infective, M and B components were infectious although theirmfectivity was much less high than that of unfractionated virus. The M andB mixture was highly infectious (table). It means that the low infectivity ofthe components after sucrose density gradient centrifugation was not causedby damage of virus particles in the result of centrifugation.

The results of analyticalultracentrifugation after the firstsucrose density gradient centri-fugation of M and B components ODshowed that B component con-tains particles sedimenting likeM, and M component containsparticles sedimenting like B. Itmeans that full separation wasnot adiieved and therefore thesecond cycle was performed.

1,0

0,8

0,6

0,A

0,2

Fig. 2. U.V.-ahsorption spectra of se-parated T, M and B components of red 0,0

clover mottle virus 220

\ V1 /

\

T

260 300X(nm)

LAPCHIC, KUZNETZOVA, MELNICZENKO and KRASNOVA

TableInfectivity of purified middle and bottom components and ihcir mixture

Inoculum

B IM, + B.

B.M, + B,

Concentration(/^g/ml)

5050

50 + 5050

5050 + 50

Number of lesionsa/b

50/370100/354295/200

4/36616/342

1006/350

*) Index shows the number of sucrose density gradient centrifugations after wbicb tbepreparation was examined.

a — total number of lesions in six half leaves of Phaseolus vulgaris var. Top Cropinoculated with investigated preparation; b — total number of lesions in six half leaves ofPhaseolus vulgaris var. Top Crop inoculated with unfractionated virus.

The second cycle of sucrose density gradient centrifugation did not permitto receive noninfectious M and B components: the total number of lesions insix half-leaves of Ph. vulgaris var. Top Crop produced by preparations withconcentration 50/<g/ml was for M^ 4 and 16 for Bj. Analytical centrifugationof these components gave single schlieren peak for Ma, but two for Ba (figs. 1 band c). The infectivity of Mo may be explained by small quantity which can'tbe detected by analytical centrifuging of B particles. We interpret the lowspecific infectivity of Ma and Ba components as an indication on the inabilityof our technique to produce entirely homogenous preparations. We suggestthat neither component is infective alone because if any component is infectivealone its infectivity had to increase during separation. Our experiments show-ed that as purity of eadi component increases its infectivity decreases, the high,purity of M^ being reflected in its very low infectivity.

Ma + Bj mixture was more infective than after the first cycle of densitygradient centrifugation (table). It means that the low infectivity of the com-ponents after two cycles of sucrose density gradient centrifugation was notcaused by damage of virus particles in the result of prolonged separationbecause the high infectivity was restored by mixing equal amounts (according

to Eoflo OD) of previously separated M and Bcomponents.

As it IS known great individual differ-ences exist between antiserum titres from dif-ferent rabbits. In our work ten rabbits were

Fig, 3. Immunodiffusion tests with red clover mottlevirus ccmponents and antiserum in 1 % agarose gel:well a contains antiserum to RCMV (ritre 1:256);well b — middle component (M) with concentration of0.5 mg/ml; well c — bottom component (B) with con-

centration of 0.42 mg/ml

Infectivity of Red Clover Mottle Virus Nucleoprotein Components 5

immunized according to the same schedule and the antiserum titres varied from1 : 512 to 1 : 2048 (double diffusion tests).

With the immunization scheme used in the present work maximum anti-serum titres were reached 10—20 days after the last injection. During abouttwo months the antiserum titre remained undianged then began to declaine,but it may be restored by two intravenous injections to its original level.

M and B components placed in adjacent wells in gel-diffusion plates and.intisera to unfractionated RCMV in the central well did not form spurs(fig. 3). It indicates that M and B components are serologically indistin-guishable.

Discussion

The purified infectious preparations of viruses of the cowpea mosaic virusgroup share many common properties: in the analytical ultracentrifuge theyproduced three boundaries (with the exception of true broad-bean mosaic viruswhich produced two boundaries) with sedimentation coefficients 54—60S,91 — lOOS and 112—127 S. The three components in these viruses which canbe separated by centrifugation in sucrose density gradients contain 0% (T),24—29% (M) and 33—37% (B) of RNA. Genome of these viruses exists intwo parts, one part associated with the RNA of 91—100 S middle ribonucleo-protein component and the other with the RNA of 112—127S bottom com-ponent. Neither component is infectious alone; the ribonucleic acids from Mand B components are required to initiate the infection (1, 2, 8, 9, 10).

The experiments described in this paper showed that purified prepara-tions of RCMV consist of three components with sedimentation coefficients of58S, 95 S and 125 S, whidi contain 0%, 26% and 38% RNA respectively.Local lesion assays on Phaseolus vulgaris v. Top Crop showed that middle andbottom components of RCMV after two cycles of sucrose density gradientcentrifugation had little or no infectivity individually but had a high infec-tivity, higher than that of unfractionated virus when mixed. Our data closelyresemble those found by VALENTA and MARCINKA (7); these authors alsoreported the enhanced infectivity of combined bottom and middle com-ponents of RCMV. It seemed reasonable to conclude that the infectivity of Mand B components after two cycles of sucrose density gradient centrifugationwas most likely due to the inability of this technique to produce fully separat-ed components. We supposed, therefore, that neither component was infectivealone; both they were necessary for initiation of the infection. Apparently,RCMV as other members of cowpea mosaic virus group is a plant virus withdivided genome.

Summary

Purified preparations of red clover mottle virus contain three componentswith sedimentation coefficients 55 S, 95 S and 125 S. Mean ratio Eoao/EaHo are0.66 for T, 1.45 for M and 1.67 for B. It means that T-component is the

6 LAPCHIC et al., Infectivity of Red Clover Mottle Virus Nucleoprotein Components

nucleic acid-free protein shell of the virus; M and B are nucleoproteins. M andB components contain about 26% and 3 8 % RNA respectively. The nucleo-protein components of RCMV were separated by two cycles of sucrose densitygradient centrifugation. The experiments showed that as purity of each com-ponent increases its infectivity decreases. M and B mixture was highlyinfectious.

Zusammenfassung

Die Infektiositat der Nukleoprotein-Komponentendes Schedtungs-Virus des Rotklees (Red clover mottle virus, RCMV)

Gereinigte Praparate des Scheckungs-Virus des Rotklees enthalten dreiKomponenten (T = obere, M = mittlere, B = untere) mit den Sedimentations-koeffizienten 55 S, 95 S und 125 S. Das mittlere Verhaltnis EWE.^o ist 0,66fur T, 1,45 fur M und 1,67 fiir B. Das bedeutet, die T-Komponente ist dienukleinsaurefreie Proteinhiille des Virus; M und B sind Nukleoproteine. DieM- und B-Komponenten enthalten etwa 26% bzw. 38% RNS. Die Nukleo-protcinkomponenten lassen sidi durch zwei Gange der 2uckerdichtegradienten-zentrifugierung trennen. Die Versuche zeigten, dafi die Infektiositat jeder derKomponenten mit zunehmender Reinheit abnimmt. Mischungen aus M und Bwaren hoch infektios.

Literature

1. BRUENING, G., and H. O. AGRA"«AL, 1967: Infectivity of a mixture of cowpea mosaicvirus nucleoprotein components. Virology 32, 306—320.

2. DE JAGAR, C. P., und A. VAN KAMMEN, 1970: The relationship between the componentsof cowpea mosaic "irus. III. Location of genetic information for two biological functionsin the middle component of CPMV. Virology 41, 2, 281—287.

3. HARRISON, B. D., J. T. FINCH, A. J. GIBBS, M. HOLDINGS. R. J. SHEPHERD, V. VALENTA,and C. WETTER, 1971: Sixteen groups of plant viruses. Virology 45, 2, 356—363.

4. LAPCHIC, L. G., L. L. KUZNETZOVA, V. S. MELNICHENKO, S. K. Voci LKO, and A. A.SjEDiN, 1975: Red clover mottle virus in Ukraine. Phytopath. Z. 82, 339—346.

5. NiBLETT, C. L., and J. S. SEMANCHIK, 1970: The significance of the coat protein ininfection by the electrophoretic forms of cowpea mosaic virus. Virology 41, 201-207.

6. REICHMAN, M. E., 1965: Determination of ribonucleic acid content of spherical virusesfrom sedimentation coefficients of full and empty particles. Virology 25, 1, 166—169.

7. VALENTA, V., and K. M. MARCINKA, 1969: Endianced infectivity of combined bottom andmiddle components of red clover mottle virus. Acta Virol. 12, 3, 288.

8. VAN KAMMEN, A., 1967: Purification and properties of the components of cowpea mosaicvirus. Virology 31, 4, 633—642.

9- , and L. J. L. D. VAN GRIENSVEN, 1970: The relationship between the componentsof cowpea mosaic virus. 11. Further characterization of the nucleoprotein componentsof CPMV. Virology 41, 2, 274—280.

10- • 1972: Plant viruses with a divided genome. Ann. Rev. Phytopath. 10, 125—150.

Authors' address: Institute of Microbiology and Virology, Ukrainian Academy ofSciences, Zabolotnogo street 26, Kiev — 143 (USSR).