Three nucleoprotein components of alfalfa mosaic virus necessary for infectivity

VIROLOGY

40,

419-430

(1970)

Three

Nucleoprotein

Components

of Alfalfa

for LOUS VAN VLOTEN-DOTING, AND

Biochemical

Department,

Mosaic

Virus

Necessary

Infectivity

ANNEMIEKE DINGJAN-VERSTEEGH, E. M. J. JASPARS University

Accepted

of Leiden,

September

Leiden,

The

Netherlands

23, 1969

All five nucleoprotein components of alfalfa mosaic virus 425 have been purified by several cycles of density gradient centrifugation in a zonal rotor. The purified components were homogeneous in the analytical ultracentrifuge, had characteristic particle lengths, and were noninfectious at concentrations normally used for biological assay of the virus. Infectivity could be restored to usual values by combining three of the components provided that ethylenediamine tetraacetate was added during the purification procedure. These components were: bottom component, middle component, and top component 6. None of these components could be omitted from the mixture without reducing the infectivity to very low values. On the other hand top components a and o were inactive in any combination whatsoever. Activity was not lost when one or two of the active components were exchanged with similar components from the virus isolates Caldy and I,?&. These results together with those of earlier work strongly indicate that three components contribute unique and essential pieces of information to the genome of alfalfa mosaic virus. The fact that in earlier work with purified RNA species the middle component RNA was overlooked and that top component a RNA was thought to be active instead of top component b RNA is discussed. INTRODUCTION fections on beans with combined prepara-

An

interesting phenomenon of multicomponent plant viruses is the biological interaction of their components (for revieurs, see SSinger, 196813; Bancroft, 1968). In previous publications (van VlotenDoting and Jaspars, 1967; van VlotenDoting et al., 1968), we reported the isolation

of top component a RNA and bottom component RNA from two strains of alfalfa mosaic virus (AMV). Leaves inoculated with either one of these RNAs showed no symptoms, nor did they contain viral RNA. However, a mixture of both RNAs was highly infectious, irrespective of the origin of the RNAs. It was possible to obtain a new strain of AMV by combining RNAs from different strains. Experiments indicated that the RNAs were replicated true to type. Majorana and Paul (1969) described in419 Copyright

@ 1970 by Academic

Press, Inc.

tions of the top components of one AMV strain and the bottom component of a diferent strain. Either the type of lesions was characteristic for the strain from which the top components were isolated, or a new type of lesion, not seen in the original strains, was found. The consideration of AMV as a mixture of only two functional components needed further investigation, as indications were found that the middle component was also biologically active (Wood and Bancroft, 1965; Majorana and Paul, 1969) or even essential for infectivity (van Vloten-Doting, 1968). In this paper we report experiments to elucidate the role of all the AMV components. In contrast to most of the work described earlier, the experiments were performed with the nucleoproteins instead of the RNAs. This is due to the improved

420

VAN

VLOTEN-DOTING,

DINGJAN-VERSTEEGH,

separation that can be achieved by centrifugation in zonal rotors and to the finding that AR’IV nucleoproteins retain their activity in the presence of ethylenediamine tetraacetate (EDTA). As the infectivity of nucleoproteins was 50-100 times higher than the infectivity of the RNAs, less material was needed. This enabled us to purify all components through several cycles of density gradient centrifugation. From experiments with these carefully purified nucleoproteins, it is evident that the situation in AMV is more complicated than we expected initially; in all probability three components are necessary for infection. MATERIALS

AND

METHODS

Virus culture and isolation. AMV isolate 425 of Hagedorn and Hanson (1963) was cultured and isolated as described before (van Vloten-Doting and Jaspars, 1967). Two other strains of AMV, 1344 (systemic on beans) and Caldy (Hull, 1968) were kindly supplied by Dr. R. Hull, John Innes Institute, Norwich, England. Both strains were grown in plants of Nicotiana tabacum L. var. “White Burley.” Cultivation and isolation procedure were the same as for ARIV 4’2.7,except that leaves with symptoms were harvested 6 days after inoculation. The yields per gram of leaf were about 0.7 mg for isolate 1344 and 0.3 mg for isolate Caldy. Separation of virus components. All strains were handled in the same way. Top a and bottom fraction were separated by means of differential solubility in the presence of magnesium ions (van Vloten-Doting and Jaspars, 1967). After high speed centrifugation the pellets were resuspended in 0.01 M P\TaH2P04, 0.001 M EDTA adjusted to pH 7.0 with NaOH (phosphate-EDTA buffer). Further purification of virus components was obtained by repeated density gradient centrifugation in the B XIV zonal rotor of an MSE 65 ultracentrifuge. Gradients were formed using a Varigrad (Peterson and Sober, 1959) with three vessels, one containing 250 ml of 20 mg/ml and two containing 250 ml of 120 mg/ml sucrose. Both solutions were in phosphate-EDTA buffer. The gradient was introduced with

AND

JASPARS

the rotor operating at 2500 rpm. Ten milliliters of virus solution (with a concentration not exceeding 10 mg/ml) in 10 mg of sucrose per milliliter in phosphate-EDTA buffer and an overlayer of 75 ml phosphate-EDTA buffer were introduced through the core. Separations were carried out at about 12” and 47,000 rpm. Depending on the sedimentation coefficients of the components to be purified the running time varied from 55 to 120 minutes. The rotor was then decelerated to 2500 rpm, and the contents were displaced with 200 mg of sucrose per milliliter in distilled water at about 30 ml per minute. Eighty-three fractions of 8 ml were collected, and absorbancy at 260 nm was measured. For infectivity studies these fractions were combined in groups of 2 or 3. Peak fractions were concentrated by high speed centrifugation at 4”. Virus solutions were stored at 4” in the presence of 0.001 M NaN3. Ultraviolet absorption and infectivity assay. The methods described previously were used (van Vloten-Doting and Jaspars, 1967). When large numbers of solutions had to be compared in a biological assay, samples were inoculated on S half-leaves of different plants of the group. Analytical ultracentrifugation (Dr. J. Kruseman) . Sedimentation of A3IV components was followed at 29,500 rpm at room temperature in a Spinco model E analytical ultracentrifuge, equipped with a photoelectric scanner, using light of 265 nm. Double-sector 12-mm cells with sapphire windows were used, one sector containing the virus solution and the other the buffer. The virus solution was dialyzed against 0.01 M NaHzPOh, adjusted to pH 7.0 with NaOH (phosphate buffer) containing 0.001 M NaNa. Optical densities at 265 nm were between 0.50 and 0.22. The sedimentation coefficients were corrected to 20” and water basis. At the low concentrations used (less than 0.1 mg/ml) no correction for concentration dependence was necessary (unpublished results). Electron microscopy (Dr. A. L. H. Stols). Fractions from sucrose density gradients were dialyzed against 0.001 llrl KaN3, 0.0001 &i’ EDTA (disodium salt). The preparations, containing less than 0.2 mg of virus per

THREE TABLE INFECTIVITY AFTER

STORAGE

WITH

stored

Phosphate buffer M NaN3 Phosphate-EDTA 0.001 M NaN3 tested tested

AND

0.001

buffer

AMV EDTA

TOTAL WITHOUT

Average lesions on 14 half-leaves after storing virus 2 and 4 weeks after gradient centrifugation

in:

+

COMPONENTS

1

OF GRADIENT-PURIFIED

Virus

a Virus b Virus

FUNCTIONAL

+

at 0.005 at 0.010

0 days”

14 days”

28 day@

99

0

0

54

32

92

mg/ml. mg/ml.

milliliter, were negatively stained in 2 % many1 acetate on carbon-filmed grids and were observed with a Philips EM 200 electron microscope. No special attention was paid to the magnification factor as comparative measurements were the objective. All photographs were taken within a short time at the same adjustment of the microscope. RESULTS

Stability of AMV

Nucleoproteins

The infectivity of AlUV nucleoprotein preparations was often lost upon storage after sucrose density gradient centrifugation. We found that in the presence of EDTA tllis loss did not occur. A&IV was freed from slowsedimenting material by zonal centrifugation in phosphate buffer. All components were combined and then concentrated by high speed centrifugation. The pellets were resuspended in phosphate buffer. To one half of the solution, NaN3 and EDTA were added to a final concentration of 0.001 M each; to the other half, only NaN3 was added. The final concentration of virus was 16 mg/ml. The infectivity was tested immediately and also after storage of 4” for 2 and 4 weeks by means of an incomplete block design (Kleczkowski, 1950). A typical result is shown in Table 1. To preserve the infectivity, virus was always stored in 0.001 M EDTA. The effect of EDTA 011 AMV is the subject of a sep-

arate study published).

OF

421

AMV

(Jaspars and Houwing,

Purification and Characterbatim Nucleoprotein Components

to be

of

the

Preparations of AMV 425 consist of 5 components : bottom component (B), middle component (;\I), and top components b, a, and o (Tb, Ta, To) (van Vloten-Doting and Jaspars, 1967). Purification was undertaken starting from the top a and bottom fractions of a 5-g batch of virus. To and Tb were carried through 3 cycles of zonal centrifugation, Ta and i\E through 4 cycles, and B through .5 cycles. Although the components purified in this manner had a sharp, symmetrical boundary in the analytical ultracentrifuge, it will become evident from their biological behavior that their purity is not entirely satisfactory. Since each cycle of centrifugation reduces the amount of material, further purification was impossible. Peak fractions of the final gradients, shown in Fig. 1 were dialyzed and used for the determination of sedimentation coefficients (Table 2) and particle lengths (Fig. 2). In Fig. 3 electron micrographs of all purified components are shown. Values of s&, of B, XI, Tb, and Ta were lower than those reported for these components of other AXV strains (Bancroft and Kaesberg, 1960; Kelley and Kaesberg, 1962; Hull ef al., 1969a). Especially for B and M the differences were rather large (4 S and 6 S, respectively). The preparations were homogeneous as judged from the sedimenting boundaries. Inhomogeneity was found in the length distributions, especially in that of B. This may be due partly to the fact that possibly not all the particles are lying flat (measurements were not restricted to free-lying particles) or to particles damaged during mounting or staining. Bright circles observed in photographs of all components were considered to be particles in “end on” view (Hull et al., 1969b) and were not measured. The maxima of the length distributions of B, M, Tb, Ta, and To were at 64, 51, 43, 29, and 25 nm, respectively.

~~~~

----------’

m. ,I:.,c..,.,:,’

,. I. : 1 :.I ._’ 1 ( 1.;. / ., ” ,.I i ,

,.

1,

,

.,.

(j.

‘i

+ M+Tb

f e

.___-_-I

B+M+Tb

tB+M+Tb

4 +B+Ta FIG. 1. Dependence of AMV infectivity on the qualitative and quantitative component composition of the inoculum. Optical density patterns of sucrose gradients of the 5 purified nucleoprotein components and the infectivity of the fractions of these gradients, tested uncombined and in combination. Contents of each square represent the average lesions on 8 half-leaves. One batch of bean plants was used on the same day for a and b, one for c, d, and e, and one for f. Fractions of the same gradient were diluted in the same way. The dilution factors for the gradient fractions of Bj M, Tb, Ta, and To were Wsc, ?.5s, s/44, ,i&c, and $fs, respectively, irrespective of whether the fractions were assayed singly or in two-, three-, or fourfold combinations. Graph b is a repetition of graph a with the addition of the combined peak fractions of c. Graphs d and e are repetitions of c with addition of single and combined peak fractions of a, respectively. In f, fractions are tested singly and with the addition of the combined peak fractions of d. The addition in b resulted in a final optical density of each added component of 0.004; in all other cases the final optical density of the added components was 0.010. The added component mixtures were not infectious at these optical densities, except that in f. Its infectivity was as indicated in the separate square on the left.

THREE

FUNCTIONAL

COMPONENTS

OF

423

AMV

Detection of Active Nucleoprotein Components To determine which components are active in lesion induction, infectivity was measured in the final gradients as a function of the position in the gradient. All fractions of a given gradient were diluted in the same way with phosphate-EDTA buffer. No infectivity was found in any of the gradients (Fig. la, c, and f, heavily lined squares). The peak fraction of the B gradient did not cause lesions even at a concentration of 0.015 mg/ml. A combination of all fractions of the Ta gradient with all fractions of the B gradient and of all fractions of the Tb gradient with all fractions of the M gradient did not result in infectivity either (Fig. la and c). However, many lesions appeared as a result of the infection with a combination of the noninfectious combined peak fractions of one pair of components with all combined fractions of the other pair (Fig. lb and e). Infectivity was related to the optical density in the gradients, except for the Ta gradient (Fig. lb). Moreover Ta could be omitted from the B-Ta combination added to the M-Tb combinations without a significant effect on lesion numbers (compare Fig. Id and le). No activity of To could be demonstrated either, as addition of its gradient fractions did not enhance the infectivity of a mixture of the peak fractions of B, M and Tb. In addition to Ta and To not being active in enhancing the infectivity of a mixture of B, M and Tb, these components were not able-separately or in combination-to replace one of the former 3 components either. We conclude from these results that for lesion induction each of the three nucleoprotein components B, M, and Tb have to be present in the inoculum. The nucleoproteins Ta and To have no function in this respect. It is unlikely that any other unknown nucleoprotein component, lost during purification, plays a significant role in lesion induction in beans, because the maximum infectivity of the B-M-Tb mixture in Fig. Id (about 130 lesions) could not be equaled FIG. 2. Length protein components in uranyl acetate.

distribution of AMV,

of purified negatively

nucleostained

424

V,4N

FIG. 3. Purified uranyi

acetate.

Scale

VLOTEN-DOTING,

preparations of the purified line, 100 nm.

DINGJAN-VERSTEEGH,

nucleoprotein

AND

components

JASPARS

of AMV,

negatively

stained

il

THREE

FUNCTIONAL

COMPDNENTB

OF

AMV

4%

Infectivity as a Function of the Component by a freshly harvested total virus preparaComposition of the Inoculum tion at the same optical density (0.02) on the same batch of beans (average of SO Peak fractions of the final gradients of the lesions). active components B, 11, and Tb were diluted with phosphate-EDTA buffer and combined in three pairs of two components. TABLE 2 Concentrations of both components were SEDIMENTATION COEFFICIENTS OF PURIFIED kept constant at two levels, whi!e the third NUCLEOVIZOTEIN COMPONENTS component was added to each pair in varyOF AMV ing amounts. From Fig. 4 it is evident that Mean value infectivity is on the one hand dependent on ComponentQ of s020.w the concentration of the third component, especially at lower concentrations of it, and B 0.32 95.6 95.2 on the other hand dependent on the con93.9 centration level of the other two components. 95.5 Another type of combination experiment 96.0 was performed with these purified preparaM 0.28 82.8 82.3 82.9 tions, in which the total number of particles 81.2 in each combination was kept roughly conTb 0.42 72.2 72.7 stant. We assumed that the optical density 72.8 ratio of solutions of B, 31, and Tb having 73.2 the same concentration of particles is 3 : 2 : 1. 67.3 66.1 Ta 0.43 Recent findings of Hull et al. (1969a) do 65.4 not support this assumption, but the experi65.6 ments do not suffer much from a somewhat To 0.18 57.7 57.5 variable total particle number. A typical 57.1 experiment is represented in Fig. .ia. Only 57.6 combinations of three components show u Peak fractions of the gradients shown in Fig. 1 significant infectivity, but, as was found were used. repeatedly, there is no striking maximum 6 Sedimentation at ambient temperature in of infectivity dependent on the component phosphate buffer containing NaN3 in a concentration of 0.001 M. composition, a phenomenon also observed

FIG. 4. Infect,ivity component was added plete block. All blocks

of

preparations of varying amounts. belonged to the same in

two components at fixed concentrations to which a third Lesion numbers of every line are averages from one incombatch of bean plants and were inoculated on the same day.

426

VAN

VLOTEN-DOTING,

DINGJAN-VERSTEEGH,

AND

JASPARS

b

a

FIG. 5a. Infectivity of AMV as a function of the proportions of B, M, and Tb. The contents of each hexagon represent t,he average number of lesions on 8 half-leaves caused by a specific combination. The lesion number in the central hexagon is 116. One batch of bean plants was used on the same day. At the vertices of the triangle, hexagons represent 100~~ of the preparations of the indicated purified components. From the vertex to the opposite side of the triangle the contribution of a given preparation decreases in 12 steps of 8 ?$70. The side rows of hexagons represent combinations in varying proportions of only 2 preparations. The central hexagon represents a combination of the three components in equal amounts. With the intention of keeping the particle concentration constant throughout all combinations, we used component preparations of B, M, and Tb with optical densities of 0.0225, 0.0150, and 0.0075, respectively. FIG. 5b. Infectivity as a function of the concentration of a freshly harvested total virus preparation assayed on the same batch of bean plants as in Fig. 5a. Lesion numbers are averages of 8 half-leaves.

when middle and bottom component of cowpea mosaic virus are mixed in different ratios (Bruening and Agrawal, 1967). The infectivity plateau in the center of Fig. 5a is not due to the bean leaves being saturated with lesions, as the maximum lesion number of 174 was much below the capacity of this batch of plants (Fig. 5b). Most lesion numbers in the center of Fig. 5a are higher than those caused by the freshly harvested total preparation of Fig. 5b at the same optical density. For instance the mixture of the central hexagon had an optical density of 0.015 and caused 116 lesions. Combination of Nucleoprotein Components from Diferent Strains of AMV B, M, and Tb from the strains Caldy and 1x’4 were purified by at least two cycles

of zonal centrifugation. Two- and 3-fold combinations of Caldy gave similar results as A&IV 425, whereas 1x4 is not suitable for local lesion assay on most hosts. Combinations were made, in which two components were derived from one strain and the third from a different strain, as indicated in Table 3. The infectivity of the components and of the combinations of two and three components are shown in Fig. 6. In general a clear enhancement of infectivity in heterologous combinations of components of the three isolates was found. The relatively high infectivity found in 2-fold combinations, in which one or two components ongmated from Caldy or from rs4, may be due to a lesser degree of purity. The lesions of Caldy on beans are different from those of 425 and 1gA (Hull, 1968).

THREE

FUNCTIONAL

COMPONENTS

In counting lesions of combinations with components from this isolate, no distinction was made between the types of lesions. DISCUSSION

In recent work (Desjardins and Steere, 1969), the bottom component of AMV is still considered to be infectious. Majorana and Paul (1969), although demonstrating once more that its infectivity increases considerably upon addition of middle or top components, stuck to this conception. We TABLE COMBINATION

SCHEME”

AMV

3 OF

425,

STRAINS

COMPONENTS

15/64,

OF THE

AND

CALDY

Fig.

Tb

M

425 425 Caldy 425 Caldy Caldy

425 Caldy 425 Caldy 425 Caldy

Caldy 425 425 Caldy Caldy 425

A B C D E F

425 425 15/64 425 15/61 15/64

425 15/64 425 15/64 425 15/64

15/64 425 425 15/64 15/64 425

G H I J K L

a ODzGO of M, 0.0150; B, in a 1:l or a AMV 425 were dients of Fig.

7

96

‘,

/

27

'\

1-1-o

M

/\ 73

e

oQ,6_B

s\

A ,3 144‘O, O-72-6

0 /\

/'

91

I-13-0

h

’ /“\ i’ 54‘\ o-4-0

427

AM\

can hardly accept a component as being infectious when it does not cause lesions at concentrations exceeding 0.015 mg/ml, while a total virus preparation at concentrations ten times lower causes many lesions. Nevertheless this component has a high intrinsic biological activity, as noninfectious preparations of the same upon addition of appropriate amounts of other components, also noninfectious themselves, equal or even surpass freshly harvested total virus preparations with regard to specific infectivity. From the present experiments it is evident that at t,he normally used level of concentration none of the purified components is infectious. The specific infectivity of a freshly harvested total virus preparation is obtained only by the combination of the three components B, 14, and Tb. Other combinations of two or three components show little or no activity, whereas addition of Ta and/or To do not enhance the infectivity of a mixture of B, M, and Tb (Figs. 1 and 4). Therefore we may conclude that in AMV infectivity is not associated with a single component, but with a particular combination of three components, probably because each of the active components contains some essential part of the genome not occurring in the others. In two other plant viruses, tobacco rattle virus and cowpea mosaic virus, a similar situation has been encountered (Lister, 1966; Frost et al., 1967; Bruening and Agrawal, 1967; van Kammen, 1968; Semancik

“i\

3-o

,4

6

the preparations was: Tb, 0.0075; 0.0225. Preparations were combined 1: I:1 volume ratio. Preparations of diluted peak fractions of the gra1.

"9,

0-12-o

B

OF

j

/

/"

36

'\

1-6-O

/

16

'\

O-13-0

o "/"\

d' l-3-2

2\

727,

/'

15

2\

1-11-o

FIG. 6. Infectivity of combinations of components of different strains of AMV. Infectivities of the 3fold combinations of Table 3 are indicated in the center of the triangles. Infectivities of the component preparations are at the vertices of the triangles; infectivity of Tb at the top; of B at the bottom left, and of M at the bottom right. Infectivities of a-fold combinations are at the sides of the triangles. Lesion numbers of one triangle are averages from one incomplete block. Blocks belonged to different batches of bean plants.

428

VAN

VLOTEN-DOTING,

DINGJAN-VERSTEEGH,

and Kajiyama, 1968; Sanger, 1968a; List,er and Bracker, 1969; Bruening, 1969). In AMV, part of the work has been done with RNA species, as no way was known to prevent inactivation of the nucleoproteins during purification (van Vloten-Doting and Jaspars, 1967; van Vloten-Doting et al., 1968). Bottom component RNA and top component a RNA were essential for infectivity, the latter contributing among others the information for the coat protein. The biological role of bottom and top components of AMV was confirmed by Majorana and Paul (1969) working with the nucleoproteins. The present experiments were carried out with carefully purified nucleoproteins stabilized with EDTA. Although it is desirable that preparations for biological experiments should have a higher degree of purity (a small contamination with other components has a striking effect as can be seen in Pig. 4), the experiments show unequivocally that three components are necessary for infection: the bottom component, the middle component, and the top component b. If we assume an essential biological equivalence of nucleoproteins and RNAs, the discrepancy between the present work and our earlier work with the RNAs has to be explained by contamination of the two RiL’A species with small amounts of other RNAs. The RNAs were never carried through more than two cycles of density gradient centrifugation in swing-out rotors. Consequently one may assume that they were less pure than the nucleoproteins. It is quite possible that the bottom component RXA was contaminated with some RNA from middle component. Therefore the possibility exists that the genetic information for local lesion induction in beans, being attributed to bottom component RNA, is actually located in the middle component RXA. A contamination of top component a RNA with top component b RKA, being exclusively responsible for the biological activity of the preparation, is more difficult to understand, the more so as preparations of ARIV 4’25 contain little top component b (van VlotenDoting and Jaspars, 1967) and the s value of its RNA is rather divergent from that of

AND

JASPARS

the top component a (Gillaspie and Bancroft, 1965; Hull et al., 1969a; Dr. J. F. Bol, personal communication). In future work we hope to come to a correct interpretation of the results obtained with the RNAs. Also the previous finding that sepa,rated AMV components do not cause a masked or incomplete infection on their own leads to supplementary investigations as to whether this may be the case in certain combinations of two components, e.g., bottom component and top component b. The existence of a single-stranded RNA genome consisting of more than two pieces is not unique for AMV. Recently the genomes of the animal RNA viruses influenza A and Rous sarcoma have been shown to consist of several pieces of RNA (Pons and Hirst, 1968; Duesberg, 1968a,b). However, in these cases all pieces are present in one virus particle. Though genetic experiments with influenza show that some characters recombine much more easily than others, probably depending on whether they are located on different strands or not (Burnet, 1956), no experiments have been performed to demonstrate the necessity for infectivity of all five Rn’A pieces present in the influenza particle. However, a first step in this direction was the finding of Pons and Hirst (1969) that the defective von Magnus type of influenza virus lacks one of the RNA strands. The biological significance of the multipartite nature of the genome of several R?\‘A viruses remains a subject of speculation. One may argue that it facilitates the exchange of genetic material in RNA viruses, having in general a very low recombination frequency (Cooper, 1968). On the other hand, an advantage in protein synthesis has been proposed (Jacobson and Baltimore, 1968). ACKNOWLEDGMENTS We wish to thank Professor Dr. H. Veldstra for his continuous interest and encouragement. We are indebted to Dr. J. Kruseman and Dr. A. L. H. Stols for their contributions. Mr. R. A. Heytink and Mr. B. J. M. Zonneveld skillfully assisted in part of the experimental work. Thanks

THREE

FUNCTIONAL

are also due to Miss Marianne A. Kaashoek, Miss Tineke A. Rutgers, Miss Isabel1 M. M. Vernooij, and Mr. A. G. Wesseling for isolation of virus and cultivation of plants. This work was supported in part by the Netherlands Organization for the Advancement of Pure Research (Z. W. 0.). REFERENCES J. B. (1968). Plant vir~lses: defectiveness and dependence. In “The Molecular Biology of Viruses” (L. V. Crawford and M. P. G. Stoker, eds.), pp. 229-247. Cambridge Univ. Press, London and New York. BANCROFT, J. B., and K.~XSEI~;RG, P. (1960). Macromolecular particles associated with alfalfa mosaic virus. Biochim. Biophys. Acta 39, 519528. BRUENING, G. (1969). The inheritance of top component formation in cowpea mosaic virlls.

BANZROFT,

Virology

37, 577-584. G. and AGR~~,I~, H. 0. (1967). Infectivity of a mixture of cowpea mosaic virus ribonucleoprotein components. Virology 32, 306-320. BURNET, F. M. (1956). Structure of influenza virus. Science 123, 1101-1104. COOPER, P. D. (1968). A genetic map of poliovirus temperature-sensitive mutants. Virology 35, 584-596. DESJARDINS, P. R., and STEERE, R. L. (1969). Separation of top and bottom components of alfalfa mosaic virus by combined differential and density gradient centrifugation. Arch. Ges. Virusforsch. 26, 127-137. DUESBERG, P. H. (1968a). The RNA’s of influenza virus. Proc. Xall. Acad. Sci. U.S. 59,930-937. DUESBERG, P. H. (196813). Physical properties of Rous sarcoma virus RNA. Proc. Natl. Acad. Sci. U.S. 60,1511-1518. FROST, R. R., HARRISON, B. D., and WOODS, R. D. (1967). Apparent symbiotic interact’ion between particles of tobacco rattle virus. J. Gen. Viral. 1, 57-70. GILLASPIE, A. G., and BANCROFT, J. B. (1965). Properties of ribonucleic acid from alfalfa mosaic virus and related components. Virology 27, 391-397. HAGEDORN, D. J., and HANSON, E. W. (1963). A strain of alfalfa mosaic virus severe on Trifolium pratense and Melilotus alba. Phytopathology 53, 188-192. HULL, R. (1968). Virus diseases of garden lupin in Great Britain. Ann. Appl. Biol. 61,373-380. BRUENING,

COMPONENTS

OF

AMV

429

HULL, It., HILLS, G. J., and M.~HKHAM, R. (1969a). Studies on alfalfa mosaic virrls. II. The st,ructure of the virus components. ViroZoqy37,416-428. HULL, It., HILLS, G. J., and PIASKITT, A. (1969b). Electron microscopy on in l;ivo forms of a strain of alfalfa mosaic virus. J. LTllrastruct. Kes. 25, 323-329. J.ICOI~SON, M. F., and B.\LTInqoKE, 1). (1968). Polypeptide cleavages in the formation of poliovirus proteins. Proc. ,\‘atl. Acad. Sci. CJ.S. 61, 77-84. KISLLISY, J. J., and K.\ESRISRG, P. (1962). Biophysical and biochemical properties of top component a and bottom component of alfalfa mosaic virus. Biochim. Biophys. Acta 61,865~871. KLECZKOI~SICI, A. (1950). Interpreting relationships between the concentrations of plant viruses and t,he number of local lesions. J. Gen. Microbial. 4, 53-69. LISTFX, R. M. (1966). Possible relationships of virus-specific products of tobacco rattle virus infections. Virology 28,350-353. LIST~;R, R. M., and BR.\CKI!X, C. E. (19G9). Defectiveness and dependence in three related strains of tobacco rattle virus. ViroZogy37,2(?2-275. MAJORANA, G., and PAUL, II. L. (1969). The production of new types of symptoms by mixtures of different components of two strains of alfalfa mosaic virus. ViroZogy38,145p151. PETERSON, E. A., and SOBER, H. A. (1959). Variable gradient device for chromatography. Anal. Chem. 31, 857-862. PONS, M. W., and HIRST, 6. K. (1968). Polyacrylamide gel electrophoresis of influenza virus RNA. Virology 34, 385-388. PONS, M. W., and HIRST, G. K. (1969). The singleand double-stranded RNA’s and the proteins of incomplete influenza virus. ViroZogy38,68-72. SXNGER, H. L. (1968a). Characteristics of tobacco rattle virus. I. Evidence that its two particles are functionally defective and mutually complementing. Mol. Gen. Genet. 101, 346-367. SINGER, H. L. (1968b). Defective plant viruses. In “Molecular Genetics” (H. G. Wittman and H. Schuster, Eds.), pp. 300-336. Springer, Berlin. SERZANCIK, J. S., and KAJIYAMA, M. A. (1968). Enhancement of tobacco rattle virus stable from infection by heterologous short particles. Virology 34, 170-172. VAN KAMMEN, A. (1968). The relationship between the components of cowpea mosaic virus. I. Two ribonucleoprotein particles necessary for the infectivity of CPMV. Virology 34,312-318.

430

VAN

VLOTEN-DOTING,

DINGJAN-VERSTEEGH,

VAN VLOTEN-DOTING, L. (1968). Verdeling van de genetische informatie over de natuurlijke componenten van een plantevirus. Thesis, University of Leiden. VAN VLOTEN-DOTING, L., and JASPARS, E. M. J. (1967). Enhancement of infectivity by combination of two ribonucleic acid components from alfalfa mosaic virus. Virology 33,684-693.

AND

JASPARS

VAN VLOTEN-DOTING, L., KRUSEMAN, J., and JASPARS, E. M. J. (1968). The biological function and mutual dependence of bottom component and top component a of alfalfa mosaic virus. Virology 34, 728-737. WOOD, H. A., and BANCROFT, J. B. (1965). Activation of a plant virus by related incomplete nucleoprotein particles. Virology 27,94-102.