VIROLOGY
‘76, 701-708 (1977)
The Formation of Phenotypically Mixed Particles upon Mixed Assembly of Some Tobacco Mosaic Virus (TMV) Strains M. E. TALIANSKY, Laboratory
of Bioorganic
Chemistry
T. I. ATABEKOVA, and Department
Accepted
AND
of Virology, U.S.S.R. September
Moscow
J. G. ATABEKOV’ State
University,
Moscow
117234.
5,1976
A correlation is established between the potential formation of phenotypically mixed virus particles upon joint infection of plants with certain tobacco mosaic virus (TMV) strains and the capability of the coat proteins of these strains to form hybrid aggregates in an in vitro mixture.It has been demonstrated that proteins ofaucaba and T (thermotolerant) TMV strains form hybrid 20 S aggregates in an in vitro mixture. Upon joint infection with these strains, the formation of phenotypically mixed particles takes place. Proteins of the U2 and uulgare strains, when mixed, do not form hybrid 20 S aggregates and no phenotypic mixing is found upon joint infection with these strains. It was previously reported [Atabekova, T. I., Taliansky, M. E., and Atabekov (1975). Virology 67, l-131 that, on mixed in vitro reconstitution of U2 and vulgure, a certain proportion of mixed particles (mosaic capsidl was formed. Here we show that the effectiveness of generating mosaic capsids at the initial stages of mixed reconstitution is markedly lower than at the later stages.It is postulated that the probability of nonspecific protein-RNA and protein-protein interactions is higher at the step of elongation than at initiation of TMV reconstitution. INTRODUCTION
but active protein of only one (tr). Quite different results were obtained upon mixed infection of plants with two normal (trl TMV strains (U2 and vulgure) and when ts mutant TMV (Nil181 was used together with U2 at a temperature permissive for Nil18 (i.e., under conditions such that Nil18 coat protein was functionally active). Under these conditions, the coat proteins and viral RNAs of both strains had access to their homologous components. Neither genomic masking nor the phenomenon of phenotypic mixing (i.e., formation of viral particles containing a capsid composed of the protein subunits of both strains) was revealed (Atabekova et al., 1975). This is basically consistent with the data of Zaitlin (1958) who demonstrated the electrophoretic separation of virus progeny fmm mixed infections of Ul (uulgare) and U2 into two components corresponding to the original strains.
It is known that, upon mixed infection of plants with two strains of tobacco mosaic virus (TMV), one of which produces a temperature-sensitive (ts) and the other a temperature-resistant (tr> coat protein, their complementation may take place under conditions nonpermissive for the ts mutant alone (Schaskolskaya et al., 1968; Sarkar, 1969; Atabekov et al., 1970b; Kassanis and Bastow, 1971; Atabekova et al., 1975). This type of interaction results in the formation of particles with a so-called masked genome, i.e., particles consisting of ts mutant RNA and tr protein, It must be noted that in this case, RNA of the ts mutant has no access to homologous (ts) structural protein, since the doubly infected cell contains the RNA of each strain ’ Author to whom requests for reprints should be addressed at: Laboratory of Bioorganic Chemistry, Moscow State University, Moscow 117234, USSR. 701 Copyright All rights
0 1977 by Academic Press, of reproduct,ion in any form
Inc. reserved.
ISSN
00426322
702
TALIANSKY,
ATABEKOVA
These observations suggest that the lack of genomic masking and phenotypic mixing is due to a high specificity of the RNAprotein and protein-protein interaction during the assembly of TMV in Co, However, it could not be concluded that these suggestions would hold for all combinations of viruses, and it is possible that for other TMV strains the specificity of protein-protein or protein-RNA interaction would not be as high. Preliminary information obtained by 0. S. Kapitsa (personal communication) suggested the possibility of formation of phenotypically mixed particles upon joint infection of tobacco plants with aucuba and thermotolerant (T) TMV strains. It has been shown by Sarkar (1960) that hybrid protein aggregates consisting of two heterologous types of subunits could be formed by mixing the coat protein preparations of only certain TMV strains. We supposed that the potential for the formation of phenotypically mixed particles in viuo was determined by strain-specific differences in viral coat proteins which conditioned their capacity for hybrid aggregate formation. Although no evidence of phenotypic mixing was detected in double infections with U2 and uulgare, a small number of phenotypically mixed particles was still formed upon their mixed in vitro reconstitution (Atabekova et al., 1975). However, it can be suggested that the specificity of RNA-protein interaction was highest when the reconstitution initiation site (RIS) of the RNA molecule was encapsidated, since it is known that RIS of TMV RNA is responsible for the recognition of the protein (Butler and Klug, 1971). In the present work it is shown that the proteins of the aucubu and T strains of TMV form hybrid aggregates in vitro and that joint infections with these strains lead to the formation of phenotypically mixed particles. The proteins of U2 and uulgure strains, in contrast, produce no hybrid aggregates in vitro, and no phenotypic mixing was found in uiuo with these strains. During mixed in vitro reconstitution of U2 and uulgure a certain proportion of particles with a mosaic capsid was formed.
AND
ATABEKOV
The efficiency of the formation was lowest at the first stages of TMV RNA encapsidation. MATERIALS
AND
METHODS
Some preparative methods (virus purification, viral RNA and protein isolation), as well as analytical procedures, serologiradioactivity procedures, cal testing, methods of virus reconstitution, and electron microscopy, were the same as those described previously (Atabekov et al., 1970a,b; Atabekova et al., 1975). TMV strains. The uucubu and T strains used for mixed infection are serologically related but not identical. The uucubu strain could be differentiated in tobacco plants (Nicotiunu tubucum L. var. Samsun EN) which produced local lesions with uucubu but not with T strain infection. Nicotiunul glutinosu L. plants produced lesions upon infection with either strain. Serologically distinct TMV strains U2 and uulgure were also used. Partial reconstitution. The incubation proceeded under standard conditions but only for 90 sec. The reaction was terminated by adding to the incubation mixture an equal volume of water cooled to 0” and containing pancreatic ribonuclease (1 pg/ 20 pg of RNA). Ultrasonic treatment of the reconstituted material. The treatment was per-
formed in an MSE disintegrator (20 kHz, 500 W, lo-15 min). The material was then examined in an electron microscope. Radioactivity measurement. Radioactivity was measured in a liquid scintillation counter. Each sample (0.2 ml) containing 1% sodium dodecyl sulfate (SDS) was mixed with 10 ml of a scintillation cocktail consisting of 1 liter of dioxane, 200 ml of ethyl alcohol, 60 g of naphthalene, 5 g of PPO (2,5diphenyloxazole), and 2.2 g of POPOP (1,4-bis(2(5-phenyloxazolyl)benzene) . Precipitation of antigens. Precipitation of the coat proteins (4 mg/ml) and their mixtures with specific antisera (diluted 1:4 to 18, initial end point titer 1:128 to 1:256) was carried out in 0.1 M phosphate buffer (pH 7.3) for 4-6 hr at 30”. The precipitate was removed by centrifugation, and as-
TMV
MIXED
says for the presence of residual antigen in the supernatant were conducted by immunodiffision techniques (Ouchterlony, 1962). Precipitation of the fragments of the 14Clabeled reconstituted virus (10 pg) mixed with unlabeled fragments of U2 (60-80 pg) by antibodies to T strain (400-600 wg) was performed in 0.01 M Na-phosphate buffer for 24-48 hr at 4”. The precipitate was isolated by centrifugation and dissolved in 1% SDS. SDS was also added to the supernatant to a final concentration of 1%. The radioactivity of those samples was measured. RESULTS
Analysis of the Progeny of Mixed Infection with aucuba and T Strains
It can be seen from Table 1 that the preparation of immunoglobulin G (IgG) from antiserum to T strain absorbed with aucuba (AbT) selectively neutralized the infectivity of T strain (Table 1, A), but not of aucuba (Table 1, D). Similarly, the preparation of IgG isolated from antiseTABLE ANALYSIS Inoculum”
OF THE PROGENY Infectivity
FROM
703
ASSEMBLY
rum to aucuba
and absorbed with the puritied T strain preparation (Abaucuba) specifically neutralized the infectivity of the aucuba (Table 1, C) but not the T strain (Table 1, B). The infectivity of the virus preparation from the mixed infection (aucuba + T) inN. tabacum var. Samsun EN (the plant selectively identifying the aucuba genome) was almost completely inhibited by Abaucuba. This suggests that the progeny of the joint infection contains very few or no particles with a masked genome of the “aucuba RNA x T protein” type (Table 1, F). On the other hand, the lower infectivity of inoculum for Samsun EN tobacco plants in the presence of AbT (Table 1, E) indicates that in the progeny of the mixed infection (aucuba + T) there is a considerable proportion of phenotypitally mixed particles containing the aucuba genome and a protein capsid composed of both aucuba and T proteins. It can be seen from Table 1 (G) that the decreasing infectivity of the inoculum for Samsun EN tobacco plants is not the consequence of nonspecific trapping of aucuba 1 MIXED
(the mean
INFECTIONS number
(aucuba
of local in
N. glutinosa
lesions
N. tabacum
I
II
III
I
II
T + Ab-aucuba aucuba aucuba + Ab-aucuba D aucuba
44 2 51 56 61 3 59
72 2 65 45 54 3 51
44 2 32 38 49 3 32
0 0 0 0 63 5 50
0 0 0 0 86 8 128
aucuba + AbT CT + aucuba) CT + aucuba) + AbT F (T + aucuba) CT + aucuba) + Abaucuba G T + aucuba” T + aucuba + AbT
68 49 19 50 10 70 33
48 18 10 45 14 -
43 39 14 65 32 -
55 46 8 20 1 112 107
95 26 12 31 4 -
AT T + AbT BT C
E
+ T) per half-leaf)
produced
var.
Samsun
EN
III
IV 0 0 0 0
v
-
0 0 0 0 33 3 36
0 0 0 0 -
27 12 30 5 -
34 51 24 50 12 -
55 19 37 2 -
” Virus preparations used for inoculation were isolated from N. tabacum var. Samsun infected with aucuba, T, or their mixture. The concentration of virus preparations used for antibody neutralization was 20 pg/ml. AbT and Ab-aucuba concentrations were 200 Kg/ml. Roman numerals stand for separate experiments performed. The values are mean numbers for 8-10 half-leaves. The infectivity of every sample was compared with its respective control by inoculation of the opposite half-leaf of the same leaf. b Artificial mixture of aucuba and T preparations.
704
TALIANSKY,
ATABEKOVA
particles in the T-AbT precipitate, since the infectivity of the artificial mixture (aucubu + T) does not decrease in the presence of AbT. Thus, the formation of significant numbers of phenotypically mixed particles with the aucuba genome in a joint infection with aucuba and T strains distinguishes this combination from the mixture (U2 + uulgure) described previously (Atabekova et al., 1975), where no such phenomenon was observed. Exuminution of Hybrid Aggregate Formution in the Mixtures of (uucubu + T) and (vulgure + U2) Coat Proteins
We have investigated the possibility of hybrid aggregate formation by mixing the proteins of uucubu with T and of U2 with uulgure in 0.1 M phosphate buffer, pH 7.3, 24”. Under these conditions, TMV protein is known to form 20 S aggregates (the socalled double disk containing 34 protein subunits) (Durham et al., 1971; Rodionova et al., 1973). The 20 S aggregates are essential for the in vitro and, presumably,
AND
the in uiuo assembly of virus particles (Butler and Klug, 1971). It has been previously shown by us that U2 and uulgure proteins produce 20 S disks both in individual preparations and in the mixture (Taliansky et al., 1975). Similar results were obtained when the proteins of uucubu, T, and a mixutre of the two were examined (data not shown). The serological approach was used to examine the possibility of hybrid 20 S aggregate formation. The 20 S protein preparations of uucubu and T strains and their mixtures were treated with specific antisera and the immunoprecipitates were removed by centrifugation; then the amounts of residual uucubu and T antigens were estimated in the supernatants. The results of the immunoprecipitation of antigens in controls and in the mixture of the virus proteins are shown in Table 2. It can be seen that antiserum to vulgure absorbed by U2 (As-vulgure) reacted specifically with vulgure protein (Table 2). The titer of U2 antigen in the U2 protein preparation did not change significantly after treatment with As-uulgure.
TABLE IMMUNOPRECIPITATION Type
OF THE COAT PROTEINS
of antigen
2
OF TMV Residual
STRAINS titer
U2” u2 vulgar-e U2 f vulgare U2 + As-vulgare’ vulgare + As-vulgare (I-72 f vulgare) + As-vulgare
ATABEKOV
1024 0 1024
512 0 512
1024 -
512
512 -
512
256
TAKEN
of antigen
AWNE
OR IN A MIXTURE
in the supernatant
vulgare
T
0 512 256
-b -
aucuba
-
-
2 4
256
T aucuba CT + aucuba)
-
T + Asaucuba aucuba + Asaucuba CT + aucuba) +*Asaucuba
-
64 0 64 32 4
0 32 32 64 8
4 4
a Determination of the titer of antigen was performed with the specific antisera (endpoint titer 128 to 256) diluted 1:4 to 1:8. b Dash (-), not tested. c The preparations of U2, vulgare, T, and aucuba coat proteins or their mixtures (4 mg/ml) were incubated in 0.1 M phosphate buffer, pH 7.2, 24”, and then were treated with the specific antisera in the same buffer for 4-6 hr at 30”. Titer of antigens was determined in the supernatant after removal of the precipitate by low speed centrifugation.
TMV
MIXED
Neither did the titer of U2 antigen in the supernatant change much upon the reaction of As-vulgare with a U2 and vulgare protein mixture (Table 2). It is suggestive, therefore, that no hybrid 20 S aggregates (i.e., aggregates containing the protein subunits of both strains) were present in the mixture of the coat protein preparations of U2 and vulgare (or the amount of such hybrid 20 S disks was very small). This is consistent with the finding that phenotypically mixed particles are lacking in the progeny obtained from the mixed U2 and vulgare infection (Atabekova et al ., 1975). Quite different results were obtained with the proteins of the aucuba and T strains. It follows from Table 2 that antiserum to aucuba absorbed with T antigen (Asaucuba) interacts specifically with the aucuba protein, sharply reducing its content in the supernatant, but having no marked effect on T protein titer. On the other hand, the titer of both T and aucuba antigens decreases significantly after the treatment of the mixture of T and aucuba proteins with As-aucuba. It can be suggested that the nonspecific trapping of homologous T disks by aucuba-As-aucuba immunoprecipitate does not take place since in an analogous situation U2 antigen does not interact with the vulgare-As-vulgare immunoprecipitate (see Table 2). These results seem to suggest that, when proteins of the aucuba and T strains are mixed, a considerable number of hybrid 20 S aggregates is formed, and these contain the protein subunits of both strains. This is in agreement with evidence for phenotypically mixed particle formation during a double infection with aucuba and T strains (see above).
705
ASSEMBLY
1975). It is possible that the frequency with which heterologous (with respect to RNA) protein was incorporated into the mosaic capsid was not uniform throughout the RNA molecule, with the lowest affinity being located at the RIS responsible for the protein recognition. If true, it might be expected that small partially reconstituted virus fragments (PRVF) arising from limited reconstitution should be distinguishable from comparable but wholly reconstituted virus by the amount of heterologous protein included per unit of rod length. We examined the efficiency of mosaic capsid formation at different stages of mixed reassembly of U2 and vulgare. The frequency of mixed capsid formation in PRVF upon limited (90 set) reassembly was compared with that of particles reassembled in the normal time. The results of electron microscopy of the reassembled material obtained are presented as histograms in Fig. 1. The length of PRVF particles was not more than 75-90
42
t 36 -
A
30-
24 18 -
II
Efficiency of Mixed Particle (Mosaic Capsid) Production at Different Stages of Mixed Reconstitution (U2 + vulgare)
Although upon double infection with U2 and vulgare strains there was no evidence of phenotypic mixing, a certain proportion of mixed particles was nevertheless observed during the joint in vitro reconstitution of these strains (Atabekova et al.,
mm FIG. 1. Distribution
Pm)
of the lengths of the jointly reconstituted preparations (U2 + uulgure).(A) Nucleoprotein formed under conditions of limited reassembly (PRVF). (B) Nucleoprotein formed under usual conditions (14-16hr). (0 Sonicated preparation B (20 kHz, 500 W, 10 min).
706
TALIANSKY,
ATABEKOVA
nm, generally being 30-45 nm (Fig 1A). The length of particles reconstituted under usual conditions (14-16 hr) reaches 300 nm (Fig. 1B). A considerable fraction of the reconstituted particles was 60-70 nm in length, which is in accordance with the data of Stussi et al. (1969). To evaluate the degree of mixing of the heterologous proteins (U2 and uulgure) in PRVF and in the mixed reconstituted virus we used 14C-labeled uulgure and unlabeled U2 proteins. For the comparative analysis of PRVF and mixedly reconstituted virus the latter was fragmented by sonication; the regime of the treatment was such that the length distribution of the fragments did not substantially differ from that of PRVF (Fig. 10. The fragments obtained were treated with IgG preparations from antiserum to U2 absorbed by vulgar-e (AbU2). The incorporation of 14C-labeled uulgare protein into the immunoprecipitate with AbU2 indicated the presence of mixed particles containing the protein subunits of both uulgare and U2. The amount of the 14C-labeled vulgar-e protein precipitated by AbU2 corresponded to the degree of mixing of heterologous proteins in the reassembled material (Table 3). The degree of mixing of heterologous proteins both in the whole virus and in PRVF varied in separate experiments, probably depending on the properties of the protein preparation used. However, it turned out that in a given experiment the degree of heterologous protein incorporation into the whole virus was markedly higher than in PRVF. In three experiments the phenomenon of protein mixing in PRVF was not detected at all (Table 3). This testifies to the fact that the interaction of the heterologous protein with RNA during the first stages of mixed reassembly (U2 + uulgure) occurs at a considerably lower frequency than during the late stages. It was found that, after storage of reconstituted preparations of mixed virus (U2 + vulgare) at 4" for 2-3 weeks, the degree of the mixing of heterologous proteins decreased markedly (from 38 to 0% in preparation I and from 22 to 0% in preparation
AND
ATABEKOV TABLE
3
A COMPARATIVE ANALYSIS OF THE PRODUCTS OF PARTIAL AND COMPLETE, MIXED (U2 + uulgare) RECONSTITUTION Products of mixed reassembly
Amount of W-labeled uulgure protein bound by AbUQ (o/o of total of W-labeled protein)”
Completely reconstituted virus (ultrasonically fragmented) PRVF
I
II
III
IV
v
38
-
21
22
15
18
13
0
0
0
a Determined according to the equation [(n,/N,) - (n&V,)] x lOO%, where N is total radioactivity (counts per minute) of the mixedly reassembled preparation (U2 + uulgare), n, is the radioactivity of the immunoprecipitate obtained after treatment of the preparation examined (U2 + uulgare) with AbU2; N, is total radioactivity of the control Wlabeled vulgare preparation; nz is radioactivity of the precipitate obtained after treatment of vulgare material with AbU2. The precipitation reaction was carried out for 24-48 hr at 4” with ‘*C-labeled reconstituted virus (10 pg of mixed reconstituted virus or reconstituted vulgure), fragments of unlabeled reconstituted U2 (60-80 pg), and 300-650 pg of antibodies. Thus, nonspecific trapping was estimated. The reported values correspond to the average of four different samples. Roman numerals stand for separate experiments.
IV). It is likely that during prolonged incubation phenotypically mixed particles are destroyed. DISCUSSION
Upon double infection of plants with the aucuba and T strains of TMV, a significant proportion of phenotypically mixed particles is formed (Table 1). However, no evidence of in uiuo phenotypic mixing was observed for another (U2 and uulgure) combination of TMV strains (Zaitlin, 1958; Atabekova et al., 1975). It seems that the formation of phenotypically mixed particles depends on the nature of viral proteins in the mixture. We visualize that at least three types of interaction between heterologous protein subunits are possible, leading to: (1) inhibition of functionally active 20 S disk-like protein aggregate formation (Okada and Ohno, 1972); (2) formation of stable 20 S aggregates which are nonfunc-
TMV
MIXED
tional in virus reassembly (Novikov et al., 1974); and (3) formation of normal 20 S disks capable of reconstituting with viral RNA (U2 + vulgare, U, + Nil& aucuba + T) (Taliansky et al., 1975). In the former two cases, inhibition of virion assembly takes place (Ohashi et al., 1969; Novikov et al., 1974). The occurrence of phenotypic mixing upon the double infection with such viruses does not seem plausible. In the third type, the 20 S disks formed by recombining the coat proteins may theoretically be homologous or hybrid (i.e., made by the protein subunits of one or both types). It was shown by Sarkar (1960) that hybrid aggregates of A protein may arise by mixing the proteins of vulgar-e, fluvum, and dahlemense, but not of vulgure and HR. We obtained similar results upon mixing the proteins under the conditions for 20 S disk formation. Hybrid 20 S disks were formed upon mixing uucubu and T proteins, but there were few if any of them when U2 and vulgure proteins were mixed (Table 2). Thus, the presence of hybrid protein 20 S aggregates (uucubu + T) in a doubly infected cell should inevitably lead to the formation of phenotypically mixed particles, and this we did observe among the progeny of a mixed (uucuba + T) infection. On the other hand, no phenotypic mixing was revealed in double infections with U2 + vulgure (Atabekova et al., 1975) which is in accordance with the above data showing that the coat proteins of these strains do not form hybrid protein aggregates. However, the presence of two types (vulgure and U2) of homologous 20 S aggregates in the mixture does not rule out the possibility of phenotypically mixed particle formation during in vitro reassembly. The mixed capsids produced under these conditions could be built up of two different types of 20 S disks, each of which contains the protein subunits of only one strain (U2 or vulgure). The formation of hybrid (mosaic) aggregates was also observed during the mixed in vitro repolymerization of the protein preparations of these strains at pH 5.5 (Rentschler, 1967). However, the frequency of interaction be-
ASSEMBLY
707
tween heterologous protein subunits during the mixed reconstitution (U2 and vulgure) may still be expected to be lower than that between homologous subunits. Otherwise, it would hardly be possible to explain the formation of the significant amount of homologous virus particles under these conditions (Atabekova et al., 1975). Thus, it may be inferred that, although the interaction between heterologous subunits (U2 and vulgure) is not, in principle, ruled out during the mixed in vitro reconstitution, it is predominantly the interaction between homologous subunits that takes place. We showed that the interaction of heterologous protein with RNA at the early stages of the reconstitution (during the first 90 set) occurs at a lower frequency than at the later stages (Table 3). These results suggest that the frequency of errors of heterologous pro(i.e., incorporation tein) upon reassembly is considerably lower inside RIS and in the adjacent sites of RNA than outside RIS. The actual length of the RIS of TMV RNA which displays a higher affinity for the virus coat protein is not known. It is reasonable to assume, however, that the RIS should not be very long, as it is considerably shorter than an RNA fragment encapsidated into the PRVF used in this work. In this respect, the low efficiency of mosaic capsid formation within PRVF (see Table 3) suggests that the length of the fragment of the virus RNA coated with only homologous protein subunits in the course of mixed reconstitution exceeds that of RIS. It must be noted, however, that the proportion of RNA molecules of the reconstitution sample involved in the reassembly during first 90 set was not estimated. Therefore one cannot rule out that the frequency of errors (inclusion of heterologous protein) is higher at the later stages of initiation of virus reassembly. The results obtained do not exclude the possibility that the TMV RNA molecule contains additional sites (at a certain distance from RIS) capable of controlling the specificity of protein-RNA and proteinprotein interaction. Our preliminary observations suggest
708
TALIANSKY,
ATABEKOVA
that in vitro reassembled particles with a mosaic capsid (U2 + uulgure) are unsta- , ble. One cannot rule out, therefore, that the lack of phenotypic mixing in plants doubly infected with U2 and uulgure was due to the inherent instability of such particles which, though having been formed, were broken upon subsequent treatments. REFERENCES J. G., NOVIKOV, V. K., VISCHNICHENKO, V. K., and KAFTANOVA, A. S. (1970a). Some properties of hybrid viruses reassembled in vitro. Virology 41, 519-532. ATABEKOV, J. G., SCHASKOL~KAYA N. D., ATABEKOVA, T. I., and SACHAROVSKAYA, G. N. (1970b). Reproduction of temperature-sensitive strains of TMV under restrictive conditions in the presence of temperature-resistant helper strain. Virology ATABEKOV,
41, 397-407. ATABEKOVA, KOV, J. G.
T. I., TALIANSKY, M. E., and ATABE(1975). Specificity of protein-RNA and protein-protein interactions upon assembly of TMV in vivo and in vitro. Virology 67, l-13. BUTLER, P. J. G., and KLUG, A. (19711. Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nature New Biol. 229, 47-50. DURHAM, A., FINCH, J. T., and KLUG, A. (1971). States of aggregation of tobacco mosaic virus protein. Nature New Biol. 229, 37-42. KASSANIS, B., and BASTOW, C. (1971). Phenotypic mixing between strains of tobacco mosaic virus. J. Gen. Viral. 11, 171-176. NOVIKOV, V. K., SARUKHAN-BEK, O., and ATABEKOV, J. G. (1974). Anomalous stable aggregates in mixture of TMV and cucumber virus 3 protein. Virology 62. 134-144. OHASHI, Y., OHNO, T., Nosu, Y., and OKADA, Y. (1969). Reconstitution of tobacco mosaic virus in
AND ATABEKOV by coat proteins of other strains. 919-924. Y., (1972). Assembly mechanisms of tobacco mosaic virus particle from its ribonucleic acid and protein. Mol. Gen. Genet. 114, 205-213. OUCHTERLONY, 0. (1962). Diffusion in gel methods for immunological analysis. II. Progr. Allergy 6, 30-154. RENTSCHLER, L. (1967). Aminosaurequenzen und physikochemisches Verhalten des Hullproteins eines Wildstammes des Tabakmosaikvirus. 2. Aggregationsverhalten und Ladungsverteilung im Vergleich zu den Stammen vulgare und dahlemense. Mol. Gen. Genet. 100, 96-108. RODIONOVA, N. P., VESENINA, N. E., ATABEKOVA, T. I., DZHAVAKHIA, V. G., and ATABEKOV, J. G. (1973). Further studies on the reconstitution of TMV and incomplete nucleoprotein complex. Virology 51, 24-33. SARKAR, S. (1960). Interaction and mixed aggregation of proteins from tobacco mosaic virus strains. vitro. Proc. OKADA,
Inhibition
Japan.
Acad. 45, and OHNO, T.
Z. Naturforsch. 15b, 778-786. SARKAR, S. (1969). Evidence
of phenotypic mixing between two strains of tobacco mosaic virus. Mol.
Gen. Genet. 105, 87-90. SCHASKOLSKAYA, N. D., ATABEKOV, OVSKAYA, G. N., and DZHAVAKHIA,
J. G., SACHARV. G. (1968). Replication of temperature-sensitive strain of tobacco mosaic virus under nonpermissive conditions in the presence of helper strain. Biol. Sci. USSR 8, 101-105. STUSSI, C., LEBEURIER, J., and HIRTH, L. (1969). Partial reconstitution of tobacco mosaic virus. Virology 38, 16-25. TALIANSKY, M. E., ATABEKOVA, T. I., and ATABEKOV, J. G. (1975). Mixed reconstitution of two strains of TMV. Proc. Agr. Acad. USSR 5, 23-25. ZAITLIN, M. (1958). Continuous filter paper electrophoresis of tobacco mosaic virus. J. Chromatogr. 1, 186-199.
‘76, 701-708 (1977)
The Formation of Phenotypically Mixed Particles upon Mixed Assembly of Some Tobacco Mosaic Virus (TMV) Strains M. E. TALIANSKY, Laboratory
of Bioorganic
Chemistry
T. I. ATABEKOVA, and Department
Accepted
AND
of Virology, U.S.S.R. September
Moscow
J. G. ATABEKOV’ State
University,
Moscow
117234.
5,1976
A correlation is established between the potential formation of phenotypically mixed virus particles upon joint infection of plants with certain tobacco mosaic virus (TMV) strains and the capability of the coat proteins of these strains to form hybrid aggregates in an in vitro mixture.It has been demonstrated that proteins ofaucaba and T (thermotolerant) TMV strains form hybrid 20 S aggregates in an in vitro mixture. Upon joint infection with these strains, the formation of phenotypically mixed particles takes place. Proteins of the U2 and uulgare strains, when mixed, do not form hybrid 20 S aggregates and no phenotypic mixing is found upon joint infection with these strains. It was previously reported [Atabekova, T. I., Taliansky, M. E., and Atabekov (1975). Virology 67, l-131 that, on mixed in vitro reconstitution of U2 and vulgure, a certain proportion of mixed particles (mosaic capsidl was formed. Here we show that the effectiveness of generating mosaic capsids at the initial stages of mixed reconstitution is markedly lower than at the later stages.It is postulated that the probability of nonspecific protein-RNA and protein-protein interactions is higher at the step of elongation than at initiation of TMV reconstitution. INTRODUCTION
but active protein of only one (tr). Quite different results were obtained upon mixed infection of plants with two normal (trl TMV strains (U2 and vulgure) and when ts mutant TMV (Nil181 was used together with U2 at a temperature permissive for Nil18 (i.e., under conditions such that Nil18 coat protein was functionally active). Under these conditions, the coat proteins and viral RNAs of both strains had access to their homologous components. Neither genomic masking nor the phenomenon of phenotypic mixing (i.e., formation of viral particles containing a capsid composed of the protein subunits of both strains) was revealed (Atabekova et al., 1975). This is basically consistent with the data of Zaitlin (1958) who demonstrated the electrophoretic separation of virus progeny fmm mixed infections of Ul (uulgare) and U2 into two components corresponding to the original strains.
It is known that, upon mixed infection of plants with two strains of tobacco mosaic virus (TMV), one of which produces a temperature-sensitive (ts) and the other a temperature-resistant (tr> coat protein, their complementation may take place under conditions nonpermissive for the ts mutant alone (Schaskolskaya et al., 1968; Sarkar, 1969; Atabekov et al., 1970b; Kassanis and Bastow, 1971; Atabekova et al., 1975). This type of interaction results in the formation of particles with a so-called masked genome, i.e., particles consisting of ts mutant RNA and tr protein, It must be noted that in this case, RNA of the ts mutant has no access to homologous (ts) structural protein, since the doubly infected cell contains the RNA of each strain ’ Author to whom requests for reprints should be addressed at: Laboratory of Bioorganic Chemistry, Moscow State University, Moscow 117234, USSR. 701 Copyright All rights
0 1977 by Academic Press, of reproduct,ion in any form
Inc. reserved.
ISSN
00426322
702
TALIANSKY,
ATABEKOVA
These observations suggest that the lack of genomic masking and phenotypic mixing is due to a high specificity of the RNAprotein and protein-protein interaction during the assembly of TMV in Co, However, it could not be concluded that these suggestions would hold for all combinations of viruses, and it is possible that for other TMV strains the specificity of protein-protein or protein-RNA interaction would not be as high. Preliminary information obtained by 0. S. Kapitsa (personal communication) suggested the possibility of formation of phenotypically mixed particles upon joint infection of tobacco plants with aucuba and thermotolerant (T) TMV strains. It has been shown by Sarkar (1960) that hybrid protein aggregates consisting of two heterologous types of subunits could be formed by mixing the coat protein preparations of only certain TMV strains. We supposed that the potential for the formation of phenotypically mixed particles in viuo was determined by strain-specific differences in viral coat proteins which conditioned their capacity for hybrid aggregate formation. Although no evidence of phenotypic mixing was detected in double infections with U2 and uulgare, a small number of phenotypically mixed particles was still formed upon their mixed in vitro reconstitution (Atabekova et al., 1975). However, it can be suggested that the specificity of RNA-protein interaction was highest when the reconstitution initiation site (RIS) of the RNA molecule was encapsidated, since it is known that RIS of TMV RNA is responsible for the recognition of the protein (Butler and Klug, 1971). In the present work it is shown that the proteins of the aucubu and T strains of TMV form hybrid aggregates in vitro and that joint infections with these strains lead to the formation of phenotypically mixed particles. The proteins of U2 and uulgure strains, in contrast, produce no hybrid aggregates in vitro, and no phenotypic mixing was found in uiuo with these strains. During mixed in vitro reconstitution of U2 and uulgure a certain proportion of particles with a mosaic capsid was formed.
AND
ATABEKOV
The efficiency of the formation was lowest at the first stages of TMV RNA encapsidation. MATERIALS
AND
METHODS
Some preparative methods (virus purification, viral RNA and protein isolation), as well as analytical procedures, serologiradioactivity procedures, cal testing, methods of virus reconstitution, and electron microscopy, were the same as those described previously (Atabekov et al., 1970a,b; Atabekova et al., 1975). TMV strains. The uucubu and T strains used for mixed infection are serologically related but not identical. The uucubu strain could be differentiated in tobacco plants (Nicotiunu tubucum L. var. Samsun EN) which produced local lesions with uucubu but not with T strain infection. Nicotiunul glutinosu L. plants produced lesions upon infection with either strain. Serologically distinct TMV strains U2 and uulgure were also used. Partial reconstitution. The incubation proceeded under standard conditions but only for 90 sec. The reaction was terminated by adding to the incubation mixture an equal volume of water cooled to 0” and containing pancreatic ribonuclease (1 pg/ 20 pg of RNA). Ultrasonic treatment of the reconstituted material. The treatment was per-
formed in an MSE disintegrator (20 kHz, 500 W, lo-15 min). The material was then examined in an electron microscope. Radioactivity measurement. Radioactivity was measured in a liquid scintillation counter. Each sample (0.2 ml) containing 1% sodium dodecyl sulfate (SDS) was mixed with 10 ml of a scintillation cocktail consisting of 1 liter of dioxane, 200 ml of ethyl alcohol, 60 g of naphthalene, 5 g of PPO (2,5diphenyloxazole), and 2.2 g of POPOP (1,4-bis(2(5-phenyloxazolyl)benzene) . Precipitation of antigens. Precipitation of the coat proteins (4 mg/ml) and their mixtures with specific antisera (diluted 1:4 to 18, initial end point titer 1:128 to 1:256) was carried out in 0.1 M phosphate buffer (pH 7.3) for 4-6 hr at 30”. The precipitate was removed by centrifugation, and as-
TMV
MIXED
says for the presence of residual antigen in the supernatant were conducted by immunodiffision techniques (Ouchterlony, 1962). Precipitation of the fragments of the 14Clabeled reconstituted virus (10 pg) mixed with unlabeled fragments of U2 (60-80 pg) by antibodies to T strain (400-600 wg) was performed in 0.01 M Na-phosphate buffer for 24-48 hr at 4”. The precipitate was isolated by centrifugation and dissolved in 1% SDS. SDS was also added to the supernatant to a final concentration of 1%. The radioactivity of those samples was measured. RESULTS
Analysis of the Progeny of Mixed Infection with aucuba and T Strains
It can be seen from Table 1 that the preparation of immunoglobulin G (IgG) from antiserum to T strain absorbed with aucuba (AbT) selectively neutralized the infectivity of T strain (Table 1, A), but not of aucuba (Table 1, D). Similarly, the preparation of IgG isolated from antiseTABLE ANALYSIS Inoculum”
OF THE PROGENY Infectivity
FROM
703
ASSEMBLY
rum to aucuba
and absorbed with the puritied T strain preparation (Abaucuba) specifically neutralized the infectivity of the aucuba (Table 1, C) but not the T strain (Table 1, B). The infectivity of the virus preparation from the mixed infection (aucuba + T) inN. tabacum var. Samsun EN (the plant selectively identifying the aucuba genome) was almost completely inhibited by Abaucuba. This suggests that the progeny of the joint infection contains very few or no particles with a masked genome of the “aucuba RNA x T protein” type (Table 1, F). On the other hand, the lower infectivity of inoculum for Samsun EN tobacco plants in the presence of AbT (Table 1, E) indicates that in the progeny of the mixed infection (aucuba + T) there is a considerable proportion of phenotypitally mixed particles containing the aucuba genome and a protein capsid composed of both aucuba and T proteins. It can be seen from Table 1 (G) that the decreasing infectivity of the inoculum for Samsun EN tobacco plants is not the consequence of nonspecific trapping of aucuba 1 MIXED
(the mean
INFECTIONS number
(aucuba
of local in
N. glutinosa
lesions
N. tabacum
I
II
III
I
II
T + Ab-aucuba aucuba aucuba + Ab-aucuba D aucuba
44 2 51 56 61 3 59
72 2 65 45 54 3 51
44 2 32 38 49 3 32
0 0 0 0 63 5 50
0 0 0 0 86 8 128
aucuba + AbT CT + aucuba) CT + aucuba) + AbT F (T + aucuba) CT + aucuba) + Abaucuba G T + aucuba” T + aucuba + AbT
68 49 19 50 10 70 33
48 18 10 45 14 -
43 39 14 65 32 -
55 46 8 20 1 112 107
95 26 12 31 4 -
AT T + AbT BT C
E
+ T) per half-leaf)
produced
var.
Samsun
EN
III
IV 0 0 0 0
v
-
0 0 0 0 33 3 36
0 0 0 0 -
27 12 30 5 -
34 51 24 50 12 -
55 19 37 2 -
” Virus preparations used for inoculation were isolated from N. tabacum var. Samsun infected with aucuba, T, or their mixture. The concentration of virus preparations used for antibody neutralization was 20 pg/ml. AbT and Ab-aucuba concentrations were 200 Kg/ml. Roman numerals stand for separate experiments performed. The values are mean numbers for 8-10 half-leaves. The infectivity of every sample was compared with its respective control by inoculation of the opposite half-leaf of the same leaf. b Artificial mixture of aucuba and T preparations.
704
TALIANSKY,
ATABEKOVA
particles in the T-AbT precipitate, since the infectivity of the artificial mixture (aucubu + T) does not decrease in the presence of AbT. Thus, the formation of significant numbers of phenotypically mixed particles with the aucuba genome in a joint infection with aucuba and T strains distinguishes this combination from the mixture (U2 + uulgure) described previously (Atabekova et al., 1975), where no such phenomenon was observed. Exuminution of Hybrid Aggregate Formution in the Mixtures of (uucubu + T) and (vulgure + U2) Coat Proteins
We have investigated the possibility of hybrid aggregate formation by mixing the proteins of uucubu with T and of U2 with uulgure in 0.1 M phosphate buffer, pH 7.3, 24”. Under these conditions, TMV protein is known to form 20 S aggregates (the socalled double disk containing 34 protein subunits) (Durham et al., 1971; Rodionova et al., 1973). The 20 S aggregates are essential for the in vitro and, presumably,
AND
the in uiuo assembly of virus particles (Butler and Klug, 1971). It has been previously shown by us that U2 and uulgure proteins produce 20 S disks both in individual preparations and in the mixture (Taliansky et al., 1975). Similar results were obtained when the proteins of uucubu, T, and a mixutre of the two were examined (data not shown). The serological approach was used to examine the possibility of hybrid 20 S aggregate formation. The 20 S protein preparations of uucubu and T strains and their mixtures were treated with specific antisera and the immunoprecipitates were removed by centrifugation; then the amounts of residual uucubu and T antigens were estimated in the supernatants. The results of the immunoprecipitation of antigens in controls and in the mixture of the virus proteins are shown in Table 2. It can be seen that antiserum to vulgure absorbed by U2 (As-vulgure) reacted specifically with vulgure protein (Table 2). The titer of U2 antigen in the U2 protein preparation did not change significantly after treatment with As-uulgure.
TABLE IMMUNOPRECIPITATION Type
OF THE COAT PROTEINS
of antigen
2
OF TMV Residual
STRAINS titer
U2” u2 vulgar-e U2 f vulgare U2 + As-vulgare’ vulgare + As-vulgare (I-72 f vulgare) + As-vulgare
ATABEKOV
1024 0 1024
512 0 512
1024 -
512
512 -
512
256
TAKEN
of antigen
AWNE
OR IN A MIXTURE
in the supernatant
vulgare
T
0 512 256
-b -
aucuba
-
-
2 4
256
T aucuba CT + aucuba)
-
T + Asaucuba aucuba + Asaucuba CT + aucuba) +*Asaucuba
-
64 0 64 32 4
0 32 32 64 8
4 4
a Determination of the titer of antigen was performed with the specific antisera (endpoint titer 128 to 256) diluted 1:4 to 1:8. b Dash (-), not tested. c The preparations of U2, vulgare, T, and aucuba coat proteins or their mixtures (4 mg/ml) were incubated in 0.1 M phosphate buffer, pH 7.2, 24”, and then were treated with the specific antisera in the same buffer for 4-6 hr at 30”. Titer of antigens was determined in the supernatant after removal of the precipitate by low speed centrifugation.
TMV
MIXED
Neither did the titer of U2 antigen in the supernatant change much upon the reaction of As-vulgare with a U2 and vulgare protein mixture (Table 2). It is suggestive, therefore, that no hybrid 20 S aggregates (i.e., aggregates containing the protein subunits of both strains) were present in the mixture of the coat protein preparations of U2 and vulgare (or the amount of such hybrid 20 S disks was very small). This is consistent with the finding that phenotypically mixed particles are lacking in the progeny obtained from the mixed U2 and vulgare infection (Atabekova et al ., 1975). Quite different results were obtained with the proteins of the aucuba and T strains. It follows from Table 2 that antiserum to aucuba absorbed with T antigen (Asaucuba) interacts specifically with the aucuba protein, sharply reducing its content in the supernatant, but having no marked effect on T protein titer. On the other hand, the titer of both T and aucuba antigens decreases significantly after the treatment of the mixture of T and aucuba proteins with As-aucuba. It can be suggested that the nonspecific trapping of homologous T disks by aucuba-As-aucuba immunoprecipitate does not take place since in an analogous situation U2 antigen does not interact with the vulgare-As-vulgare immunoprecipitate (see Table 2). These results seem to suggest that, when proteins of the aucuba and T strains are mixed, a considerable number of hybrid 20 S aggregates is formed, and these contain the protein subunits of both strains. This is in agreement with evidence for phenotypically mixed particle formation during a double infection with aucuba and T strains (see above).
705
ASSEMBLY
1975). It is possible that the frequency with which heterologous (with respect to RNA) protein was incorporated into the mosaic capsid was not uniform throughout the RNA molecule, with the lowest affinity being located at the RIS responsible for the protein recognition. If true, it might be expected that small partially reconstituted virus fragments (PRVF) arising from limited reconstitution should be distinguishable from comparable but wholly reconstituted virus by the amount of heterologous protein included per unit of rod length. We examined the efficiency of mosaic capsid formation at different stages of mixed reassembly of U2 and vulgare. The frequency of mixed capsid formation in PRVF upon limited (90 set) reassembly was compared with that of particles reassembled in the normal time. The results of electron microscopy of the reassembled material obtained are presented as histograms in Fig. 1. The length of PRVF particles was not more than 75-90
42
t 36 -
A
30-
24 18 -
II
Efficiency of Mixed Particle (Mosaic Capsid) Production at Different Stages of Mixed Reconstitution (U2 + vulgare)
Although upon double infection with U2 and vulgare strains there was no evidence of phenotypic mixing, a certain proportion of mixed particles was nevertheless observed during the joint in vitro reconstitution of these strains (Atabekova et al.,
mm FIG. 1. Distribution
Pm)
of the lengths of the jointly reconstituted preparations (U2 + uulgure).(A) Nucleoprotein formed under conditions of limited reassembly (PRVF). (B) Nucleoprotein formed under usual conditions (14-16hr). (0 Sonicated preparation B (20 kHz, 500 W, 10 min).
706
TALIANSKY,
ATABEKOVA
nm, generally being 30-45 nm (Fig 1A). The length of particles reconstituted under usual conditions (14-16 hr) reaches 300 nm (Fig. 1B). A considerable fraction of the reconstituted particles was 60-70 nm in length, which is in accordance with the data of Stussi et al. (1969). To evaluate the degree of mixing of the heterologous proteins (U2 and uulgure) in PRVF and in the mixed reconstituted virus we used 14C-labeled uulgure and unlabeled U2 proteins. For the comparative analysis of PRVF and mixedly reconstituted virus the latter was fragmented by sonication; the regime of the treatment was such that the length distribution of the fragments did not substantially differ from that of PRVF (Fig. 10. The fragments obtained were treated with IgG preparations from antiserum to U2 absorbed by vulgar-e (AbU2). The incorporation of 14C-labeled uulgare protein into the immunoprecipitate with AbU2 indicated the presence of mixed particles containing the protein subunits of both uulgare and U2. The amount of the 14C-labeled vulgar-e protein precipitated by AbU2 corresponded to the degree of mixing of heterologous proteins in the reassembled material (Table 3). The degree of mixing of heterologous proteins both in the whole virus and in PRVF varied in separate experiments, probably depending on the properties of the protein preparation used. However, it turned out that in a given experiment the degree of heterologous protein incorporation into the whole virus was markedly higher than in PRVF. In three experiments the phenomenon of protein mixing in PRVF was not detected at all (Table 3). This testifies to the fact that the interaction of the heterologous protein with RNA during the first stages of mixed reassembly (U2 + uulgure) occurs at a considerably lower frequency than during the late stages. It was found that, after storage of reconstituted preparations of mixed virus (U2 + vulgare) at 4" for 2-3 weeks, the degree of the mixing of heterologous proteins decreased markedly (from 38 to 0% in preparation I and from 22 to 0% in preparation
AND
ATABEKOV TABLE
3
A COMPARATIVE ANALYSIS OF THE PRODUCTS OF PARTIAL AND COMPLETE, MIXED (U2 + uulgare) RECONSTITUTION Products of mixed reassembly
Amount of W-labeled uulgure protein bound by AbUQ (o/o of total of W-labeled protein)”
Completely reconstituted virus (ultrasonically fragmented) PRVF
I
II
III
IV
v
38
-
21
22
15
18
13
0
0
0
a Determined according to the equation [(n,/N,) - (n&V,)] x lOO%, where N is total radioactivity (counts per minute) of the mixedly reassembled preparation (U2 + uulgare), n, is the radioactivity of the immunoprecipitate obtained after treatment of the preparation examined (U2 + uulgare) with AbU2; N, is total radioactivity of the control Wlabeled vulgare preparation; nz is radioactivity of the precipitate obtained after treatment of vulgare material with AbU2. The precipitation reaction was carried out for 24-48 hr at 4” with ‘*C-labeled reconstituted virus (10 pg of mixed reconstituted virus or reconstituted vulgure), fragments of unlabeled reconstituted U2 (60-80 pg), and 300-650 pg of antibodies. Thus, nonspecific trapping was estimated. The reported values correspond to the average of four different samples. Roman numerals stand for separate experiments.
IV). It is likely that during prolonged incubation phenotypically mixed particles are destroyed. DISCUSSION
Upon double infection of plants with the aucuba and T strains of TMV, a significant proportion of phenotypically mixed particles is formed (Table 1). However, no evidence of in uiuo phenotypic mixing was observed for another (U2 and uulgure) combination of TMV strains (Zaitlin, 1958; Atabekova et al., 1975). It seems that the formation of phenotypically mixed particles depends on the nature of viral proteins in the mixture. We visualize that at least three types of interaction between heterologous protein subunits are possible, leading to: (1) inhibition of functionally active 20 S disk-like protein aggregate formation (Okada and Ohno, 1972); (2) formation of stable 20 S aggregates which are nonfunc-
TMV
MIXED
tional in virus reassembly (Novikov et al., 1974); and (3) formation of normal 20 S disks capable of reconstituting with viral RNA (U2 + vulgare, U, + Nil& aucuba + T) (Taliansky et al., 1975). In the former two cases, inhibition of virion assembly takes place (Ohashi et al., 1969; Novikov et al., 1974). The occurrence of phenotypic mixing upon the double infection with such viruses does not seem plausible. In the third type, the 20 S disks formed by recombining the coat proteins may theoretically be homologous or hybrid (i.e., made by the protein subunits of one or both types). It was shown by Sarkar (1960) that hybrid aggregates of A protein may arise by mixing the proteins of vulgar-e, fluvum, and dahlemense, but not of vulgure and HR. We obtained similar results upon mixing the proteins under the conditions for 20 S disk formation. Hybrid 20 S disks were formed upon mixing uucubu and T proteins, but there were few if any of them when U2 and vulgure proteins were mixed (Table 2). Thus, the presence of hybrid protein 20 S aggregates (uucubu + T) in a doubly infected cell should inevitably lead to the formation of phenotypically mixed particles, and this we did observe among the progeny of a mixed (uucuba + T) infection. On the other hand, no phenotypic mixing was revealed in double infections with U2 + vulgure (Atabekova et al., 1975) which is in accordance with the above data showing that the coat proteins of these strains do not form hybrid protein aggregates. However, the presence of two types (vulgure and U2) of homologous 20 S aggregates in the mixture does not rule out the possibility of phenotypically mixed particle formation during in vitro reassembly. The mixed capsids produced under these conditions could be built up of two different types of 20 S disks, each of which contains the protein subunits of only one strain (U2 or vulgure). The formation of hybrid (mosaic) aggregates was also observed during the mixed in vitro repolymerization of the protein preparations of these strains at pH 5.5 (Rentschler, 1967). However, the frequency of interaction be-
ASSEMBLY
707
tween heterologous protein subunits during the mixed reconstitution (U2 and vulgure) may still be expected to be lower than that between homologous subunits. Otherwise, it would hardly be possible to explain the formation of the significant amount of homologous virus particles under these conditions (Atabekova et al., 1975). Thus, it may be inferred that, although the interaction between heterologous subunits (U2 and vulgure) is not, in principle, ruled out during the mixed in vitro reconstitution, it is predominantly the interaction between homologous subunits that takes place. We showed that the interaction of heterologous protein with RNA at the early stages of the reconstitution (during the first 90 set) occurs at a lower frequency than at the later stages (Table 3). These results suggest that the frequency of errors of heterologous pro(i.e., incorporation tein) upon reassembly is considerably lower inside RIS and in the adjacent sites of RNA than outside RIS. The actual length of the RIS of TMV RNA which displays a higher affinity for the virus coat protein is not known. It is reasonable to assume, however, that the RIS should not be very long, as it is considerably shorter than an RNA fragment encapsidated into the PRVF used in this work. In this respect, the low efficiency of mosaic capsid formation within PRVF (see Table 3) suggests that the length of the fragment of the virus RNA coated with only homologous protein subunits in the course of mixed reconstitution exceeds that of RIS. It must be noted, however, that the proportion of RNA molecules of the reconstitution sample involved in the reassembly during first 90 set was not estimated. Therefore one cannot rule out that the frequency of errors (inclusion of heterologous protein) is higher at the later stages of initiation of virus reassembly. The results obtained do not exclude the possibility that the TMV RNA molecule contains additional sites (at a certain distance from RIS) capable of controlling the specificity of protein-RNA and proteinprotein interaction. Our preliminary observations suggest
708
TALIANSKY,
ATABEKOVA
that in vitro reassembled particles with a mosaic capsid (U2 + uulgure) are unsta- , ble. One cannot rule out, therefore, that the lack of phenotypic mixing in plants doubly infected with U2 and uulgure was due to the inherent instability of such particles which, though having been formed, were broken upon subsequent treatments. REFERENCES J. G., NOVIKOV, V. K., VISCHNICHENKO, V. K., and KAFTANOVA, A. S. (1970a). Some properties of hybrid viruses reassembled in vitro. Virology 41, 519-532. ATABEKOV, J. G., SCHASKOL~KAYA N. D., ATABEKOVA, T. I., and SACHAROVSKAYA, G. N. (1970b). Reproduction of temperature-sensitive strains of TMV under restrictive conditions in the presence of temperature-resistant helper strain. Virology ATABEKOV,
41, 397-407. ATABEKOVA, KOV, J. G.
T. I., TALIANSKY, M. E., and ATABE(1975). Specificity of protein-RNA and protein-protein interactions upon assembly of TMV in vivo and in vitro. Virology 67, l-13. BUTLER, P. J. G., and KLUG, A. (19711. Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nature New Biol. 229, 47-50. DURHAM, A., FINCH, J. T., and KLUG, A. (1971). States of aggregation of tobacco mosaic virus protein. Nature New Biol. 229, 37-42. KASSANIS, B., and BASTOW, C. (1971). Phenotypic mixing between strains of tobacco mosaic virus. J. Gen. Viral. 11, 171-176. NOVIKOV, V. K., SARUKHAN-BEK, O., and ATABEKOV, J. G. (1974). Anomalous stable aggregates in mixture of TMV and cucumber virus 3 protein. Virology 62. 134-144. OHASHI, Y., OHNO, T., Nosu, Y., and OKADA, Y. (1969). Reconstitution of tobacco mosaic virus in
AND ATABEKOV by coat proteins of other strains. 919-924. Y., (1972). Assembly mechanisms of tobacco mosaic virus particle from its ribonucleic acid and protein. Mol. Gen. Genet. 114, 205-213. OUCHTERLONY, 0. (1962). Diffusion in gel methods for immunological analysis. II. Progr. Allergy 6, 30-154. RENTSCHLER, L. (1967). Aminosaurequenzen und physikochemisches Verhalten des Hullproteins eines Wildstammes des Tabakmosaikvirus. 2. Aggregationsverhalten und Ladungsverteilung im Vergleich zu den Stammen vulgare und dahlemense. Mol. Gen. Genet. 100, 96-108. RODIONOVA, N. P., VESENINA, N. E., ATABEKOVA, T. I., DZHAVAKHIA, V. G., and ATABEKOV, J. G. (1973). Further studies on the reconstitution of TMV and incomplete nucleoprotein complex. Virology 51, 24-33. SARKAR, S. (1960). Interaction and mixed aggregation of proteins from tobacco mosaic virus strains. vitro. Proc. OKADA,
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Acad. 45, and OHNO, T.
Z. Naturforsch. 15b, 778-786. SARKAR, S. (1969). Evidence
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Gen. Genet. 105, 87-90. SCHASKOLSKAYA, N. D., ATABEKOV, OVSKAYA, G. N., and DZHAVAKHIA,
J. G., SACHARV. G. (1968). Replication of temperature-sensitive strain of tobacco mosaic virus under nonpermissive conditions in the presence of helper strain. Biol. Sci. USSR 8, 101-105. STUSSI, C., LEBEURIER, J., and HIRTH, L. (1969). Partial reconstitution of tobacco mosaic virus. Virology 38, 16-25. TALIANSKY, M. E., ATABEKOVA, T. I., and ATABEKOV, J. G. (1975). Mixed reconstitution of two strains of TMV. Proc. Agr. Acad. USSR 5, 23-25. ZAITLIN, M. (1958). Continuous filter paper electrophoresis of tobacco mosaic virus. J. Chromatogr. 1, 186-199.
