VIROLOGY

67,1-13

Specificity

(1975)

of Protein-RNA and Protein-Protein Interaction Assembly of TMV in Vivo and in Vitro

T. I. ATABEKOVA, Laboratory

of Bioorganic

Chemistry

M. E. TALIANSKY, and Department

of Virology

AND

of Moscow

Upon

J. G. ATABEKOV State

University,

Moscow

127234,

USSR Accepted

March

24, 1975

The possibility of genomic masking and phenotypic mixing was studied in vivo (in plants mixedly infected by two TMV strains). The combinations of strains were: (1) vulgare and U2, (2) temperature-sensitive coat protein mutant Nil18 and U2. In the experiments on mixed reconstitution the conditions of TMV maturation were modeled in vitro using combinations of RNAs and proteins from the same strains. Coating of viral RNA by the coat protein of a heterologous strain (termed genomic masking in uivo and mixed particle production in uitro) could be definitely demonstrated only when a doubly infected plant (or reconstituted mixture, respectively) contained two types of viral RNA and only one type of active protein (the protein of the second, temperature-sensitive strain was inactive at 33”). On the other hand, the genomic masking and mixed-particle production was absent or very limited (within the limitation of the assay) when RNAs and functional proteins of both strains were present in a mixedly infected plant (in uiuo) or in a reconstitution mixture (in vitro). Thus, a high specificity of protein-RNA interaction exists when both RNA and protein have access to the homologous component upon TMV assembly. The absence of phenotypically mixed particles with a mosaic capsid in doubly infected plants in vivo implies that the specificity of protein-protein interactions between subunits should also be high. However, some mixed coat protein virus with mosaic capsid could be reassembled in vitro.

TMV reconstitution and mixed reconstitution of viruses from TMV protein and It has been established that the process heterologous viral RNAs. It is accepted of TMV reconstitution is initiated by the that the yield of the reconstituted nuinteraction of TMV protein in the form of a cleoprotein depends on the type of RNA 20 S-double-layer disk aggregate (Durham used (Fraenkel-Conrat and Singer, 1964; et al., 1971; Durham and Klug, 1971) with Holoubek, 1962; Matthews, 1966; Atabea specific sequence of nucleotides at the kov et al., 1970; Kado and Knight, 1970; 5’-end of TMV RNA (Butler and Klug, Breck and Gordon, 1970; Fritsh et al., 1971; Butler, 1972). The reconstitution of 1973). The reconstitution-inducing funcTMV can be inhibited by cleaving the tion can be performed effectively by RNA of a definite nucleotide sequence (in par5’-terminal nucleotides from TMV RNA (Butler and Klug, 1971; Guilley et at., ticular, by homologous TMV RNA). 1971). It could be suggested, therefore, that Although the affinity of TMV protein for the TMV RNA molecule possesses the heterologous RNAs was less than for TMV 5’-terminal reconstitution-initiating site RNA, a certain proportion of so-called displaying a high affinity for TMV protein. hybrid or mixed particles could be formed The questions concerning the specificity in vitro, consisting of TMV protein and of protein-RNA interaction have been re- heterologous viral RNA. They were similar peatedly studied in the experiments on to the intact TMV in buoyant density, INTRODUCTION

1 Copyright@ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.

2

ATABEKOVA.

TALIANSKY

sedimentation properties, serological specificity, UV-absorption spectra, and structure as revealed by electron microscopy (Atabekov et al., 1970). The existence of mixed particle formation suggested that recognition between TMV protein and RNA was not very specific. However, it must be noted that in the experiments mentioned above the incubation mixture contained one type of structural protein (usually the coat protein from TMV or its strains, or cucumber virus 4 protein) and one type of heterologous RNA. The exception was the work by Fritsh et al. (1973) who incubated TMV protein with a mixture of TMV and turnip yellow mosaic virus RNA. It was reported (Shaskolskaya et al., 1968; Sarkar, 1969; Atabekov et al., 1970a; Kassanis and Bastow, 1971a) that under conditions of mixed infection the temperature-sensitive (ts) coat protein mutant of TMV as well as mutants producing defective coat protein (Kassanis and Bastow, 1971b; Kassanis and Conti, 1971) could be complemented by different temperature-resistant (tr) helper strains. It was shown that this effect was due to hybrid particle formation, i.e., to the masking of the genome of conditionally lethal (ts) or defective virus within the coat protein of the helper TMV strain. In all the above studies the phenomenon of genomic masking was observed in conditions when, in the infected cell, there were RNAs from two strains and a functional protein from only one of them, i.e., mutant virus RNA had no access to homologous protein upon maturation of the virus particles. Therefore it was of interest to study the possibility of genomic masking and phenotypically mixed virion formation (i.e., particles with a mosaic envelope containing both parental coat proteins) under conditions of mixed infection when the infected cells contain two types of structural protein and two types of viral RNA. In other words, the specificity of interaction between viral RNA and protein and between protein subunits with each other was studied under conditions in which the proteins and RNAs presumably had the possibility of interaction with the homologous component

AND

ATABEKOV

(RNA or protein, respectively) upon the assembly of virions in uiuo in mixedly infected cells, and in vitro in reconstitution experiments. It will be shown in the present paper that the genomic masking effect in uiuo (and mixed-particle formation in uitro) could be revealed only when doubly infected plants (or reconstitution mixtures) contained one type of active coat protein and two types of viral RNA. No (or extremely small) genomic masking could be revealed in uiuo and in vitro under conditions when both RNA and protein should have access to homologous component. Some aspects of this work have been previously reported in short communications (Atabekova et al., 1972; Taliansky et al., 1974). MATERIALS

AND

METHODS

Some preparative methods (virus purification, viral RNA and protein isolation) as well as analytical procedures, and serological testing, were the same as in a previous paper (Atabekov et al., 1970a). When TMV strains were selected the following criteria were taken into consideration: (1) The strains should be antigenically distant, which allows one to use selective neutralisation of infectivity for analysing the mixed progeny; (2) The strains should be discriminable in different host plants capable of distinguishing the virus genomes; (3) One of the strains should behave, depending on the experimental conditions, either as a normal virus, or as a defective one with respect to the structural protein. Three TMV strains were selected: uulgare, Nil18 and U2. Plants of Phaseolus vulgar-is L. var Pinto produce local lesions on being inoculated by vulgare or Nil19 No visible lesions could be produced in Pinto bean inoculated by U2 under the conditions of infection used. On the other hand, Nicotiana tabacum var. Samsun EN forms local lesions upon infection with U2 and Ni118, but produces a systemic reaction in response to vulgare. Finally, Nicotiana glutinosa L. forms local lesions upon infection with any of the strains used. Thus, tobacco Samsun EN and Pinto bean plants can be used as hosts distinguishing

TMV ASSEMBLY

IN VZVO AND IN VITRO

U2 from the vulgare genome; Nil18 can be distinguished from the U2 genome in Pinto beans. Inoculations. The leaves of Samsun tobacco were mechanically inoculated with mixtures: (1) vulgare (1 pg/ml) and U2 (10 &ml) or (2) Nil18 (10 pg/ml) and U2 (1 pg/ml). The presence of both strains in the progeny from mixed infection at 24” was judged by infectivity on their respective hosts and by the results of the immunodiffusion analysis. Seven to fourteen days after infection, purified virus preparations were isolated from mixedly infected and control (infected individually by each of the viruses) plants and were used for further analysis. Virus reassembly. Reconstitution experiments were performed in 0.1 M phosphate buffer, pH 7.3, at 24” or 33”. The procedure of reconstitution was the same as in our previous work (e.g., see Atabekov et al., 1970). Neutralization. The yG-immunoglobulins were isolated from antisera by the method of Baumstark et al. (1964). Antiserum to Nil18 was absorbed with U2 and vice versa: excess virus was added to the antibody (Ab) preparation. Then the absorbed Ab preparation was layered on the top of 25% sucrose solution and centrifuged at 37,000 rpm for 2 hr in the SW 39 rotor of a Spinco L2 centrifuge to sediment and thus remove the excess virus. Antibody preparations obtained in this way, when tested at different dilutions, were noninfective unless otherwise stated. Absorbed Ab preparation was added to purified virus under test in 0.1 M phosphate buffer, pH 7.2-7.3. The mixture (lo-20 pg of virus and 100-400 pg of Ab) was kept at 4” for 20 hr and then used for inoculation tests. RESULTS

Mixed infection of vulgare and U2 in uivo. The results of the analysis of the progeny of mixed infection (uulgare + U2)

are presented in Table 1. It can be seen from these data that: 1. The preparation of antibodies to vulgare absorbed with U2 (Ab,,,) selectively neutralized the infectivity of vulgare (Ta-

3

ble 1, A) but not of U2 (Table 1, D). The preparation of Abuul was seen to be free of detectable contamination with U2 after absorption of Ab,,/ with U2 because it did not produce lesions in Samsun EN (Table 1, A). 2. The preparation of antibodies to U2 absorbed with vulgare (Ab,,) neutralized selectively U2 infectivity (Table 1, C) and had no effect on uulgare alone (Table 1, B). It must be noted that the preparations of Ab,,, used in in vivo experiments contained after absorption with vulgar-e a small residual amount of this virus, which was shown in a separate infectivity test (Ab,,, retained a small amount of infectivity for N. glutinosa and Pinto beans). This conclusion also follows from the fact that U2 after neutralization retained the ability to produce some local lesions in N. glutinosa and Pinto bean (Table 1, C). It is important to note that the contamination of vulgare in Ab,., could not affect the results of the analysis of mixed infection progeny as the mixture obtained after neutralization by Ab I.2 was used for infecting Samsun EN tobacco but not Pinto beans. On the other hand an admixture of vulgare in Ab,,, naturally increased, to a certain extent, the infectivity of the mixtures containing Ab,, for N. glutinosa. This, however, cannot affect the conclusion following from our evidence as the differentiation of vulgare from U2 was performed with two other hosts. 3. The mixed-infection progeny contains two viruses; one of the genomes (U2) can be expressed in Samsun EN tobacco and the other (vulgare) in Pinto beans (Table 1, E and F). 4. Ab[,, neutralized the infectivity of virus particles containing the U2 genome in the progeny from mixed infection, as was tested by the inoculation of Samsun EN tobacco (Table 1, E). This points to the absence (within the limitations of the assay) from the infective mixture of particles with a masked genome of the type “vulgare protein x U2 RNA.” 5. Ab,,, is not capable of neutralizing the infectivity of the progeny from the mixed infection when it was tested in Samsun EN tobacco (Table 1, F). This also indicates

4

ATABEKOVA,

TALIANSKY TABLE

COMPOSITION

OF PROGENY

Conditions of infection and neutralization

A

uulgare

uulgare + Ab,,,

OF SINGLE

AND

AND ATABEKOV 1 STRAIN INFECTION (W&Fe

MIXED

+

UT)

Mean number (a) of local lesions per half-leaf produced in:” P. uulgaris

Pinto

N. tabacum Samsun EN

I

II

III

IV

I

83 1

17 1

34 1

80 1

0 0

96 103

0 0

II

III

IV

N. glutinosa I

II

III

IV

00 00

57 5

98 1

31 1

73 1

00 00

21 24

37 46

36 38

75 89

B

vulgare uulgare + Ab, 2

39 44

c

u2 U2 + Ab,-,

0 12

273 5

317 60 62

118 1

242 54

222 42

58 12

78 19

0 0

113 115

58 60

133 135

78 79

41 41

48 46

87 83

64 0

40 92 13

100 74

192 155

117 89

30 39

41 54

153 73

73 50

77 48

D

U2

U2 + Ab,,, E

(oulgare + U21b (uulgare + U2) + Ab,.,

F

(m&are (uulgare

+ +

U21b U2) + Abuulb

218 11

140 5

90 4

183 4

90 98

“Roman numerals stand for separate neutralization experiments. (a) The figures are mean numbers for 10 half-leaves. The infectivity of every sample was compared with its respective control by inoculation of opposite half-leaves of the same leaves. Virus preparations were assayed at concentrations of 10 &ml. bProgeny virus isolated from plants mixedly infected with strains indicated.

the absence in the mixture of masked genome particles of the “uulgare protein x U2 RNA” type; in addition, this points to the absence of significant numbers of phenotypically mixed doubly neutralized particles with mosaic capsid and the genome of strain U2. 6. Ab,,, inhibits almost completely the infectivity of the mixed progeny in Pinto beans (Table 1, F) which allows one to suggest that the progeny of the mixed infection does not contain particles with a masked genome of “U2 protein x uulgare RNA” type or that at least, it contains very few of such particles, Mixed reconstitution of vulgare and U2 in oitro The results of a similar analysis of

the virus particles mixedly reassembled at 24” in a mixture containing two types of protein (uulgare + U2) and two types of RNA (vulgar-e + U2) are presented in Table 2. It can be seen from these data that: 1. Ab,,, and Ab”, selectively neutralized

most of the infectivity of uulgare and U2, respectively (Table 2, A and C). The preparations of AbVul and Ab,,, were free of contamination of heterologous virus (U2 and vulgare, respectively). 2. Abu, neutralizes most of the infectivity of the U2 in the mixedly reassembled preparations: the infectivity of the mixture tested on Samsun EN was relatively small after treatment with Abu, (Table 2, E) and approximately corresponded to that in control (U2 + AbrJ,) (Table 2, Cl. Therefore, it can be suggested that the particles with a U2 genome masked by vulgare protein (U2,,,\ x uulgare protein) are either absent from this mixture or present in comparatively small number. 3. The mode of action of AbVul, was the same, neutralizing the vulgare genome in the mixture (Table 2, F). The infectivity of the mixture for Pinto beans was markedly inhibited by Ab,,l. This observation shows that the particles with the uulgare genome masked by U2 protein (uulgare,,, x

TMV

ASSEMBLY

IN

WV0

Mixed

Inoculum”

-r t

r-

Mean

number

(Pinto)

ii-

ii -

IV -

v

Infection Vivo

of Strains

Nil 18 and U2 in

The data presented in Table 3 show that the preparations of antibodies to Nil18 absorbed with U2 (AbNil18) selectively neutralized the infectivity of Nil& and Ab,,, selectively neutralized U2. On the other hand, AbNilla was not capable of neutraliz2

REASSEMBLED

P. uulgaris

5

VITRO

(a) Analysis of progeny of the mixed infection (Nil18 + 112) propagated at 33”.

TABLE OF MIXEDLY

IN

based on the results of the infectivity experiments is difficult taking into account the contribution of interference between U2 and uulgare in the mixed inoculum. However, it may be suggested (see Table 2) that roughly 50-75s of the total amount of particles containing the U2 genome and 50-75s of the particles containing the vulgare genome are represented by normal virus (U2 and uulgare, respectively). This follows from the data on residual infectivity after neutralization of the reassembled material by Ab,,, and Abuul (Table 2, E and F).

were also absent or were present as a small proportion of the mixture. 4. Heterologous antiserum (Ab,,,) partially neutralized the infectivity of virus containing the U2 genome; the infectivity of the mixture for Samsun EN decreased considerably after Abuul treatment (Table 2, F). Taking into consideration that the mixture does not contain particles of the “uulgureprotein x U2RNA” type it seems reasonable to suppose that a considerable part of mixedly reassembled preparation is represented by doubly neutralized mixed particles containing U2 RNA wit,hin mosaic capsids. A similar conclusion can be reached on the basis of the slight neutralization of the vulgar-e genome (the infectivity of the mixture for Pinto beans decreased) by heterologous antiserum (Ab,~,) (Table 2, E). Thus, it can be suggested that mixed particles containing the vulgare genome within a mosaic capsid are present in the mixture. The quantitative estimation of normal and mixed particles with composite envelopes

U2protein)

COMPOSITION

AND

of local lesions

1 N. tabacum

--

I

-

+ U2)

(u&are

II

per half-leaf

(Samsun III

EN) IV

v-

VIRUS

produced

r

in: N. glutinosa

.-

-

I

II

III

-

-

- -

IV

u-

A

uul vu1 + Ab,,,

90 2

43 3

75 2

0 0

0 0

0 0

0 0

0 0

73 5

41 1

96 6

113 7

33 1

B

vu1 vu1 + Ab,.,

99 94

48 42

68 55

0 0

0 0

0 0

0 0

0 0

03 93

52 56

48 47

92 85

34 32

c

u2 U2 + Ab,.,

0 0

0 0

0 0

66 0

120 2

75 4

52 1

39 2

83 5

48 1

85 4

123 15

81 5

D

U2 U2 + Ab,,,

0 0

0 0

0 0

33 34

37 38

106 99

46 50

43 38

54 58

49 45

137 130

161 184

25 22

E

(U2+ uul) W2 + uul) + Ab,,,

31 22

27 19

26 18

41 25

229 35

38 7

157 4

33 4

29 7

53 29

49 20

151 67

50 24

F

W2+ vu0 KJ2 + uul) + Ab,.,

21 2 -

25 3

40 3

35 4

90 53

39 28

149 45

32 17

31 12

56 16

40 17 -

102 67

48 26

24 20

-

-

-

-

L

-

“In control the homologously reassembled uulgare and U2 were used as inoculum (vu1 and U2 in Table). Abbreviation (UQ + oun stands for the virus mixedly reassembled from the mixture of vulgare and U2 RNAs and both types of virus protein. The reassembled virus was tested at a concentration of 50 &ml. Other abbreviations are the same as in Table 1.

6

ATABEKOVA,

TALIANSKY

AND

TABLE COMPOSITION Conditions of infection and neutralization”

OF PROGENY

OF SINGLE

Temp.

P. u&ark I

A

B

c

D

E

F

G

H

Nil18 Nil18

+ Ab,,,,,

24

Nil18 Nil18

+ Ab,.,

24

u2 U2 + Ab,.,

72 1

II

3

AND MIXED-STRAIN

Infectivity:

ATABEKOV

INFECTION

mean number (Pinto)

(Nil18

of local lesions

+ U2) per half-leaf

N. tabacum (Samsun EN)

III

IV

I

31 4

36 0

76 1

69 81

61 63

II

III

AT 24” and 33O produced

in:

N. glutinosa IV

I

II

III

IV

>SOO 12

80 4

78 5

65 2

78 78

85 85

104 111

45 42

69 71

24

0 0

0 0

0 0

98 2

> 600 30

199 4

83 3

U2 +

U2 Ab,,,,,

24

0 0

0 0

0 0

89 90

133 144

51 75

72 76

(Nil18 (Nil18

+ U2)” + U2) + Abra,,,

33

25 26

7 9

34 33

100 99

172 175

116 96

86 84

(Nil18 (Nil18

+ UP)* + U2) + Ab,,,

33

28 0

7 0

40 0

84 2

109 2

62 3

72 3

(Nil18 + U2)” (Nil18 + U2) + AbN,l,a

24

45 5

24 0

33 0

164 35

99 24

88 33

24

34 37

25 20

44 42

238 168

126 73

102 59

(Nil18 (Nil18

+ U2) + U2) + Abu,

“The preparations of the virus isolated from plants inoculated by respective strains or strain mixtures lo-14 days after inoculation were used. The viruses were allowed to multiply at 24” or 33”. Virus preparations at concentrations of 20 rg/ml were used. Roman numerals stand for separate neutralization experiments. b Mixed infection.

ing Nil18 infectivity in the progeny of a mixed infection (Table 3, E). Thus the Nil18 genome in the progeny of the mixed infection was unreactive with AbNi,,,. The infectivity of Nil18 in this material could be generally neutralized by antiserum that had been prepared against strain U2 (Table 3, F). These results indicate that in the progeny of mixed infection at 33” (a restrictive temperature for Ni118) the genome of Nil18 m-strain was enclosed in the coat protein of the tr-helper strain U2. That particles with a Nil18 genome masked by U2 protein are formed, is favoured also by the immunological results,

i.e., by the absence of Nil18 antigen in the virus preparation from plants mixedly infected at 33” (Fig. 1). (b) An analysis of progeny of the mixed infection (Nil18 + U2) propagated at 24”.

The virus progeny isolated from Samsun tobacco plants mixedly infected with Nil18 and U2 at 24” contains the two viruses, judging by the data presented in Table 3. This is also supported by the results of immunodiffusion analysis with the use of specific antibodies. The infectivity of the mixed progeny for Pinto bean was almost completely inhibited by Ab NLI, 8 (Table 3, G). This indicates the absence in the

Th4V

ASSEMBLY

IN

of the particles of “Nil&,, x type. The residual infectivity of the preparation neutralized by Abm,,, does not exceed that of the control (neutralization of Nil18 or U2 by homologous antibodies in Table 3, A and C). It is clear from Table 3 that Ab”, produces no inhibiting effect on the infectivity of the mixture for Pinto bean (Table 3, H). This result lends additional support to the absence in the mixed progeny of the particles with a masked genome of the “NillSRNA x U2protein” type. The latter also indicates the absence in the mixed progeny of particles with mosaic capsids, consisting of protein subunits of the two strains (Nil18 and U2). Phenotypic mixing (“mosaic capsid”) would be revealed by the presence of doubly neutralizable virus particles, i.e., those reacting with the two types of antibodies (AbNillB and Ab,,,). The fact that the mixed infection progeny is insensitive to Ab,,, (in the Pinto bean infectivity test, which can selectively reveal the genome of Ni118) is indicative of the absence of doubly neutralizable particles containing the Nil18 genome (Table 3, H). In this case, it is difficult to prove experimentally the absence of mixed virions with a masked U2 genome in the progeny. This is first of all due to the fact that both strains (Nil18 and U2) form identical lesions in N. glutinosa and Samsun EN; thus, there is no possibility to test for U2 selectively in a mixed inoculum. The following procedure was used to identify the genome of U2 in the mixedinfection progeny. The purified preparation of virus isolated from mixedly infected (at 24”) plants was treated with Ab,,,. This should have resulted in neutralizing, in addition to U2 particles, all phenotypically mixed (doubly neutralized) particles. A preparation treated in this way could contain only Nil18 and particles of “U2KN,A x Nil18protein” type. The preparation obtained after neutralization by Ab,., was used to inoculate N. glutinosa plants; the local lesions were excised and each was homogenized in 0.1 it4 phosphate buffer, pH 7.2-7.3; the material was then used to infect Samsun tobacco plants. The in-

WV0 AND

IN VITRO

7

preparation U2protein”

FIG. 1. Immunodiffusion patterns of the material isolated from plants infected with U2 (l), Nil18 (2). and plants mixedly infected with Nil18 + U2 at 33” (3) or 24” (4). The wells 5 and 6 contained Ab,,,,, and Ab ,.2, respectively.

fected plants were kept for lo-14 days at 24” for the virus t.o increase. Then the plants were homogenized, and the homogenates were examined by immunodiffusion tests; thereby only a part of the homogenate was used, the other part being kept at -25”. It was found that out of 50 homogenates tested, 40 produced a positive reaction with Ab,,,,, but not Ab(,,, nine displayed no reaction whatever and only one reacted with the two types of antibodies. These results support the conclusion that the mixed-infection progeny contained no virions with a U2 genome masked within Nil18 protein. To make certain, the virus of the 20 homogenates which were kept at -25”, was tested wit,h respect to temperature sensitivity. As a result, it was proved unequivocally that the plants tested did contain a ts strain of TMV. Thus, the experiments with mixedly infected standard combination of TMV strains (Nil18 and U2) allowed us to conclude that the formation of masked genome particles may be observed only at nonpermissive (for Ni118) temperatures. The absence of masked genomes and phenotypic mixing in the conditions permissive for both strains suggests that the interaction of virus RNA molecules with structural protein displays an extremely high specificity. Mixed

Reconstitution

of Nil18

and U2

in Vitro (a) Analysis of virus mixedly reconstituted (Nil18 + U2) at 33”. Two types of

8

ATABEKOVA,

TALIANSKY

reconstitution experiments were performed at 33”, differing in the concentration of the virus protein (Nil18 protein + U2 protein) in the incubation mixture: (1) Both the RNAs and both proteins were mixed in equal proportions. Since the total RNA: protein ratio was 1:20, the amount of the functional protein in the mixture was only sufficient to coat one type of RNA as the protein from Nil18 (ts) was inactive at high temperature. These conditions could have been favourable for specific interaction between the homologous protein and RNA of the U2 strain; (2) When the amount of the virus proteins was doubled in the incubation mixture, the quantity of tr-strain U2 protein was sufficient to coat the RNA of both strains. The data presented in Table 4 show that considerably more infective material could be reassembled when the mixture of Nil18 and U2 RNA’s was incubated at 33” with the double amount of coat proteins. The increase of infectivity was revealed in both hosts but it was especially pronounced in Pinto bean, the plant which can selectively react to Nil18 and discriminate against the U2 genome (Table 4). It can be seen from Table 5 (rows G and H) that infectivity of the reassembled material was preferentially neutralized by AbLrz (Table 5, G) but not by AbNilla (Table 5, H), i.e., the reconstituted virus particles producing local lesions in Pinto bean contained the Nil18 genome masked by U2 protein. (b) Analysis of virus mixedly reassembled (Nil18 + U.2) at 24”. It can be seen TABLE INFECTIVITY

OF THE MATERIAL

RNA: protein ratio in incubation mixture

Expt

from Table 5 that the most of the infectivity for Pinto beans of the material mixedly reassembled at 24” can be inhibited by Ab N,118 (Table 5, F). The residual infectivity is comparable with that in the control (Nil18 neutralized by AbNills) (Table 5, D). Thus, all or almost all virus particles containing the Nil18 genome (revealed by infection on Pinto bean) can be neutralized by AbNills. This does not necessarily mean, however, that the material reassembled under these conditions contains only normal Nil18 particles. It can contain mixedly reconstituted particles with mosaic capsids and the Nil18 genome. However, this suggests that the particles with the Nil18 genome masked by U2 protein (Ni118,,,A x u2 protein) cannot be reassembled at 24” in the mixture containing two types of RNA and two types of protein. About half of infectivity in Pinto bean can be inhibited by Ab,,, (Table 5, E) which shows that the particles with mosaic capsids and Nil18 RNA are present in the reassembled material. Part of material infective for N. glutinosa is resistant to AbNilia (Table 5, F). As was mentioned above, this material cannot contain the Nil18 genome masked by U2 protein since it is not infective for Pinto bean. Therefore, this suggests that a considerable amount of homologously reassembled particles containing the U2 genome coated with U2 protein are present in the reconstituted material. These observations allow one to suggest that the following types of virus particles are present in the mixture reassembled in vitro at 24”. 4

number

I

Expt

of local lesion

5 25

41 51

Pinto

OF RNAs

Expt N.

Pinto

glutinosa

43 123

AND PROTEINS

per half-leaf

II

___ N. glutinosa

ATABEKOV

AT 33” FROM THE MIXTURE Nil18 (ts) and U2 (trl” Mean

Pinto 1:20 1:40

REASSEMBLED

AND

26 56

OF Two

STRAINS:

in: III

Expt N.

IV

Pinto

glutinosa

21 87

50 54

20 61

“The virus reassembled from the mixture of Nil18 + U2 RNAs and Nil18 + U2 proteins at 33” was used. The protein of Nil18 was inactive at 33”. The virus was reassembled at RNA:protein ratios of either 1:20 or 1:40 and used for infectivity tests at a concentration of 50 &ml. Other abbreviations are the same as in previous Tables.

TMV ASSEMBLY

TABLE COMPOSITION

--

Inoculum”

OF VIRUS MIXEDLY

Temp.

U2 + Ab,,,,, C Nil18 Nil18 + Ab,., D E

F

Nil18 Nil18 + AbNille (Nil18 + U2) (Nil18 + UP) + Abu, (Nil18 + UP) (Nil18 + U2) + Abra,,,

G (Nil18 + U2) (Nil18 + U2) + Ah 2 H

(Nil18 + U2) (Nil18 + U2) + Ab,,,,,

F

72 62

38 35

54 52

76 80

59 2

34 3

101

43

51

__-

I

IV

III

V 0 0

0 0

0 0

0 0

57 66

54 57

45 46

62 69

132 5

60 5

68 3

31 3

57 7

57

81

78

58

36

32

34

25

21

35

29

26

17

14

20

70

47

70

61

52

38

28

60

42

29

20

29

18

31

4

5

2

6

(78)127

(51)59

(55

(63) 15(67)

WW

(1)2

(5,

(128)lOO (59)41

(46

55 54

25 22

24”

52 49

64 65

24”

106 3

33”

-

0 0

81 81

33”

V

(Pinto)

P. vulgaris

49 4

55 2

IV III -65 81 2 5

24”

24”

l-

II 0 0

II

123 3

24”

+ U2) AT 24’ AND 33”

-

24”

u2

(Nil18

REASSEMBLED

N. glutinosa

I

B

5

Mean number of local lesions per half-leaf produced in:

t

A U2 U2 + Ab,,,

9

IN VZVO AND IN VITRO

Cl)2

(120)89

(2)4

032

(243) 38)35

(35)O

(4)

KW (60) 7 (4)

(39)ll

(2) 2

(3) 0

(341 (39) 10

(29) 8

(55)39

44)33 (32)0 GM) (34: (42)ll (34) 5 (53 “Abbreviations are the same as in previous Tables. Without parentheses: infectivity of the material reassembled with normal (1:20) RNA:protein ratio. In parentheses: infectivity of the material reassembled at RNA:protein ratio of 1:40.

1. Ni118,N,4 x Nil18p,,Wi,i,; 2. Nil18D,,t,i, plus U2protein x Nill&N, (mixed virions with mosaic capsid); 3. U2,,, x U2protein. The possibility is not excluded of the presence in the mixture of mixedly reconstituted particles with mosaic capsids containing the genome of U2, but such particles cannot be detected because of the absence of a host reacting selectively to U2 but not to Ni118. DISCUSSION

The specificity of protein-RNA interaction upon TMV maturation in vivo and upon reassembly of the virus particles in vitro was examined under two types of conditions: (1) mixedly infected plants (or

incubation mixtures) containing the RNAs of two TMV strains and the coat protein of only one of them; (2) analogous situations in vivo and in vitro when RNAs and the coat proteins of both strains were present, i.e., when both of structural proteins and both viral RNAs had access to both homologous components. The combination of vulgare with U2 in such types of experiments is advantageous for distinguishing, in the progeny of mixed infections, the virus particles containing the vulgare genome (local lesion production in P. vulgaris var Pinto) from virus particles containing the U2 genome (local lesion production in Samsun EN tobacco). These two TMV strains are serologically

10

ATABEKOVA,

TALIANSKY

distinct; therefore, the selective neutralization with specific antisera can be used to distinguish three types of virus particles (i.e., normal virus, phenotypically mixed virus and particles with masked genomes) in the total population of virus particles containing the U2 or vulgare genomes, respectively. It was shown here that within the limitations of the assay no (or very few) virus particles with genomes masked by heterologous coat protein could be produced upon double infection with common TMV (uulgare) and the U2 strain. Moreover, no particles with mosaic capsids, i.e., no phenotypically mixed virus containing the U2 genome, could be revealed under these conditions. These conclusions are principally in accordance with the results reported by Zaitlin (1958) who showed that the virus preparations isolated from plants mixedly infected by the Ul (vulgare) and U2 strains can be separated electrophoretitally into fractions containing virus particles similar to the original Ul and U2. These observations suggest that during TMV maturation the specificity of interactions at the level of RNA-protein is very high in vivo. The absence of phenotypically mixed particles in the progeny of mixed infection implies that the specificity of protein-protein interactions upon virus particle assembly in doubly infected cells is also very high. However, the interpretation of the data obtained with the vulgare and U2 strains, is not unambiguous for at least two reasons: (1) There is no direct evidence showing that these two viruses are capable of being introduced and of reproducing together in the same infected cell; (2) one cannot exclude that genomic masking is in principle not possible between U2 and TMV vulgare strains in vivo. This could be due, for example, to the impossibility of contacts between heterologous RNA and protein which could be localized in different sites of a mixedly infected cell. Therefore, it was essential to show that interaction between vulgare RNA and U2 protein in vivo is possible in principle. These disadvantages were overcome when the combination of strains Nil18 and

AND

ATABEKOV

U2 was used. The Nil18 RNA should be almost similar to vulgare RNA in nucleotide sequence since Nil18 is a nitrous acid-induced coat protein mutant of TMV. Nil18 is antigenically identical to TMV vulgare and has proline exchanged by leutine in residue position 20 of the coat protein (Wittmann-Liebold et al., 1965). Therefore Nil18 can be regarded as a ts-analog of m&are. Moreover, Nil18 offers some advantages over vulgare for our purposes: (1) Kassanis and Milne (1971) demonstrated that Nil18 produced unusual inclusions in infected cells at high temperatures. Thus, the presence of both viruses (Nil 18 and U2) in the same cell has been proved by the presence of two types of intracellular inclusions at 33” (amorphous, Nill8-specific and hexagonal, U2 specific) which could be seen in some cells of infected tobacco leaves (data not shown). (2) The use of Nil18 with U2 in mixed inoculations allows one to obtain two different experimental situations. One (at 33”), when two types of viral RNAs and only one type of the coat protein (U2) are functionally active, i.e., when Nil18 RNA had no access to homologous protein and should be enclosed within U2 protein. Under these conditions it was shown that the formation of mixed virus particles can occur (see Table 3). Secondly (at 24”), when both RNAs and both structural proteins were present, i.e., there was possibility of interaction between homologous components. It appeared that under these conditions phenotypic mixing or genomic masking was not found in the progeny of mixed infections (Table 3). However, it must be taken into account that this conclusion should be made with caution. It would be better to say that it is correct at the level of sensitivity of the method of infectivity neutralization used in this work. One cannot estimate the minimum number of heterologous (in respect to RNA) protein subunits within the phenotypically mixed TMV shell which are necessary to ensure the neutralization of this particle by antibodies. The degree of infectivity of the mixture should be lowered by the effect of interference between related TMV strains; the contribution of this phenomenon is

TMV

ASSEMBLY

IN WV0 AND IN VITRO

difficult to estimate. It must also be noted that the complex formed by TMV with specific antibodies retains some infectivity (Rappaport and Siegel, 1955) which is in agreement with the data presented here. Although the residual infectivity is low, it must be taken into account when evaluating the results of neutralization tests. However, it seems reasonable to assume that even if some mixed particles were formed but could not be revealed, they would be relatively few, since the same methodological approach allowed us to reveal such particles in other experimental conditions (see Tables 2, 3, and 5). The data presented above allows us to suggest that in doubly infected cells under conditions when viral RNAs and coat proteins of both strains were present, the homologous RNA and protein can specifically recognize each other. One cannot exclude, however, that genomic masking and/or phenotypic mixing could appear upon mixed infection by some other strains of TMV which are more related to each other than are vulgare and U2. It seems possible for example, that the possibility of phenotypic mixing depends on the behaviour of the virus proteins in the mixture. At least three types of situations are possible upon interaction of TMV protein with heterologous protein subunits leading to: (1) Inhibition of functionally active 2OS- disklike protein aggregate formation (Okada and Ohno, 1972); (2) Anomalous stable aggregates which are nonfunctional in virus reassembly (Novikov et al., 1974); (3) Formation of normal BOS-disks capable of reconstituting with viral RNA (TMV + U2 protein mixture). It seems that it is only feasible to analyze the third case, i.e., when active 20 S disks are formed. In this connection, the observations of Sarkar (1960) could be important, as he showed that mixed aggregates of the coat protein could be formed only when the proteins of certain TMV strains were mixed. Mixed protein aggregates could be formed using the protein preparations of any of three strains, vulgare, flavum, and dahlemense but not between the protein preparations of HR and vulgare (Sarkar, 1960). One can speculate that two different

11

situations are possible upon virus particle formation in doubly infected cell in uiuo (or upon mixed reassembly in uitro): (1) The coat proteins of two strains cannot produce mixed protein aggregates and, therefore, the mixture contains two types of aggregates each being strain specific; (2) the coat proteins of two strains produce mixed (mosaic) aggregates containing the subunits of both types. It seems logical to suggest that the formation of phenotypitally mixed particles upon virus assembly should be inevitable in this case. The phenomenon of genomic masking was reported by Rochow (1970) for different isolates of barley yellow dwarf virus. Peterson and Brakke (1973) found that a small amount of genomic masking occurred in barley plants mixedly infected by barley stripe mosaic virus (BSMV) and brome mosaic virus (BMV). The virions with BSMV genome coated by BMV protein were formed. It must be noted that in both cases the heterologous viral RNA was masked within the protein shell of an isometric virus. The data available at present suggest that the specificity of proteinRNA interaction is much less evident in mixed reassembly experiments with the coat protein of isometric viruses (Hiebert et al., 1968) than with TMV protein. Morris (1970) could not detect mixed particles containing BMV RNA coated by BSMV protein in doubly infected plants. This could be due to the high excess of BMV protein over that of BSMV protein reported by Peterson and Brakke (1973). Dodds and Hamilton (1974) demonstrated the presence of virus particles with the TMV genome masked by BSMV protein in doubly infected barley; however, no genomic masking of BSMV RNA in TMV protein was detected. In order to rationalize these results, it may be important that BSMV protein interacts nonspecifically with different RNAs on mixed reassembly (Novikov and Atabekov, unpublished). Thus, it can be assumed that the genomic masking effect took place in cells doubly infected by unrelated viruses only in the cases when protein-RNA interaction was not very specific. Kassanis and Conti (1971) and Goodman and Ross (1974)

12

ATABEKOVA,

TALIANSKY

failed to detect genomic masking in mixed infections of some unrelated viruses. The results presented in this paper show that the specificity of protein-RNA and protein-protein interaction upon virus assembly in uiuo should be very high. We made an attempt to support this conclusion in the model in vitro experiments using the reassembly mixtures containing RNAs and proteins of both TMV strains (Tables 2 and 5). Practically no (or very small amounts) of particles with masked genomes could be formed under these conditions, which is in agreement with the results of the in uiuo experiments. The absence of genomic masking in mixedly assembled virus can be interpreted as reflecting on a high efficiency for the mechanism controlling the specificity of proteinRNA interaction. Indeed, the probability of mistakes (i.e., of heterologous proteinRNA interactions in the course of reassembly in the presence of two proteins) should be very high, since TMV particles contain more than 2000 individual protein subunits packed around viral RNA. Note added in proof. When this paper was completed we found that the phenotypically mixed particles could be formed in plants mixedly infected by TMV strains aucuba and T (thermotolerant), which was due to the “mixed” protein aggregates formation. ACKNOWLEDGMENTS We express our appreciation to Dr. H. G. Wittmann for sending the U2 strain, to Miss S. Ganeva for help in some steps of this work, and to Miss T. I. Kheifets for assistance in translating the text. REFERENCES J. G., DEMENTYEVA, S. P., SCHASKOLSKAYA, N. D., and SACHAROVSKAYA, G. N. (1968). Serological study on barley stripe mosaic virus protein polymerization. II. Comparative antigenic analysis of intact virus and some stable protein intermediates. Virology 36, 601-612. ATABEKOV, J. G., NOVIKOV, V. K. VISHNICHENKO, V. K., and KAFTANOVA, A. S. (1970a). Some properties of hybrid viruses reassembled in vitro. ViroZ0g.y 41, 519-532. ATABEKOV, J. G., SCHASKOLSKAYA, N. D., ATABEKOVA, T. I., and SACHAROVSKAYA, G. A. (197Ob). Reproduction of temperature-sensitive strains of TMV under

ATABEKOV,

AND

ATABEKOV

restrictive conditions in the presence of temperature-resistant helper strain. Virology 41, 397-407. ATABEKOVA, T. I., SCHASKOLSKAY~ N. D., and ATABEKOV, J. G. (1972). Specificity of protein-protein and protein-RNA interaction upon maturation of TMV in mixedly infected plants. Biol. Nauk. 8, 116-118. BAUMSTARK, I. S., DAFFIN, R. I., and BARDAWIE, W. A. (1964). A preparative method for the separation of 7 S -r-globulin from human serum. Arch. Biochem. Biophys. 108, 514-522. BRECK, L. 0.. and GORDON, M. P. (1970). Formation, characterization and photochemical properties of a hybrid plant virus. Virology 40, 397-402. BUTLER, P. J. G., and KLUG, A. (1971). Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nature New Biol. 229, 47-50. BUTLER, P. J. G. (1972). Assembly of TMV. J. Mol. Biol. 72, 25-35. DODDS, J. A., and HAMILTON, R. I. (1974). Masking of RNA genome of tobacco mosaic virus by the protein of barley stripe mosaic virus in doubly infected barley. Virology 59, 418-427. DURHAM, A. C. H., FINCH, I. T., and KLUG, A. (1971). States of aggregation of tobacco mosaic virus protein. Nature New Biol. 229, 37-42. DURHAM, A. C. H., and KLUG, A. (1971). Polymerization of tobacco mosaic virus protein and its control. Nature New Biol. 229, 42-46. FRAENKEL-CONRAT, H., and SINGER, B. (1974). Reconstitution of tobacco mosaic virus. IV. Inhibition by enzymes and other proteins and use of polynucleotides. Virology 23, 354-362. FRITSCH, C., STUSSI, C., WITZ, J., and HIRTH, L. (1973). Specificity of TMV RNA-encapsidation: in vitro coating of heterologous RNA by TMV protein. Virology 56, 33-45. GUILLEY, H., STUSSI, C., and HIRTH, L. (1971). Influence de la phosphodiesterase de rate de port sur la reconstitution in vitro du virus de la mosaique du tabac. C. R. Acad. Sci. 272, 1181-1184. GOODMAN, R. M., and Ross, A. F. (1974). Independent assembly of virions in tobacco doubly infected by potato virus X and potato virus Y or tobacco mosaic virus. Virology 59, 314-318. HIEBERT, E., BANCROFT, J. B., and BRACKER, C. E. (1968). The assembly in vitro of some spherical viruses, hybrid viruses and other nucleoproteins. Virology 34, 492-508. HOLOUBEK, V. (1962). Mixed reconstitution between protein from common tobacco mosaic virus and ribonucleic acid from other strains. Virology 18, 401-404. KADO, C. I., and KNIGHT, C. A. (1970). Host specificity of plant viruses. II. Cucumber virus 4. Virology 40, 997-1007. KASSANIS, B., and BASTOW, C. (1971a). In uioo phenotypic mixing between two strains of tobacco mosaic virus. J. Gen. Viral. 10, 95-98. KASSANIS, B., and BASTOW, C. (1971b). Phenotypic

TMV

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IN

mixing between strains of tobacco mosaic virus. J. Gen. Virol. 11, 171-176. KASSANIS, B., and CONTI, M. (1971). Defective strains and phenotyping mixing. J. Gen. Virol. 13.361-364. KASSANIS, B., and MILNE, R. G. (1971). An unusual inclusions in plants infected with a tobacco mosaic virus mutant. J. Gen. Viral. 11, 193-195. MATTHEWS, R. E. F. (1966). Reconstitution of turnip yellow mosaic virus RNA with TMV protein subunits. Virology 30, 82-96. MORRIS, T. J. (1970). Studies on mixed infection in barley by brome mosaic virus and barley stripe mosaic virus. M. S. Thesis, MacDonald College of McGill University, Canada. Cited by Peterson and Brakke (1973). NOVIKOV, V. K., SARUKHAN-BEK, K. K., and ATABEKOV, J. G. (1974). Anomalous stable aggregates in the mixture of TMV and cucumber virus 3 proteins. Virology 62, 134-144. OKADA, J., and OHNO T. (1972). Assembly mechanism of tobacco mosaic virus particle from its ribonucleic acid and protein. Mol. Gen. Genet. 114, 205-213. PETERSON, J. F., and BRAKKE, M. K. (1973). Genomic masking in mixed infections with brome mosaic and barley stripe mosaic viruses. Virology 51, 174-182. RAPPAPORT, I., SIEGEL, A. (1955). Inactivation of tobacco mosaic virus by rabbit antiserum. J. Immunol. 74, 106-116.

WV0

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IN

VITRO

13

ROCHOW, W. F. (1970). Barley yellow dwarf virus: phenotypic mixing and vector specificity. Science 167, 875-878. SARKAR, S. (1960). Interaction and mixed aggregation of proteins from tobacco mosaic virus strains. 2. Naturforsch. 15b, 778-786. SARKAR, S. (19691. Evidence of phenotypic mixing between two strains of tobacco mosaic virus. Mol. Gen. Genet. 105, 87-90. SCHASKOLSKAYA, N. D., ATABEKOV, J. G., SACHAROVSKAYA, G. N., and DZHAVAKHIA, V. G. (1968). Reproduction of temperature-sensitive strain of TMV under nonpermissive conditions in the presence of helper strain. Biol. Nauk. (USSR) 8, 101-105. TALIANSKY, M. E., ATABEKOVA, T. I., and ATABEKOV, J. G. (1974). Absence of genomic masking upon mixed infection of cells by two TMV strains. Proc. Agricultural Acad. USSR 6, h--7. WITTMANN-LIEBOLD, B., JALJREGUI-ADELL, T.. and WITTMANN, H. G. (1965). Die primaire Proteinstruktur Temperatursensitiver Mutanten des ‘Tabakmosaikvirus. II. Chemisch indusierte Mutanten. 2. Naturforsch. 20b, 1235-1249. ZAITLIN, M. (1958). Continuous filter paper electrophoresis of tobacco mosaic virus. J. Chromatogr. 1, 186-199.