VIROLOGY 65.215225
(1975)
Coat Protein Virus:
Is Required
Biological
for Infectivity
Equivalence
of the Coat Proteins
Streak and Alfalfa LGUS Department
of Biochemistq,
VAN
of Tobacco
Mosaic
Streak
of Tobacco
Viruses
VLOTEN-DOTING
State University,
Wassenaarseweg
Accepted January
64, Leiden,
The Netherlands
28, 1975
The four nucleoprotein components of tobacco streak virus (TSV) were purified by zonal centrifupation. Each component contains mainly one RNA species. Infectivity studies indicate that the three fastest-sedimenting nucleoproteins are required for viral replication. A mixture of the three largest RNA’s however, is not infectious hut can he activated by the viral coat protein or by the smallest TSV-RNA. The same situation exists in alfalfa mosaic virus (AMV). The coat proteins of TSV and AMV, which are rather different as judged by serology and tryptic digestion products, cannot only activate their own genome but also that of each other. The nucleoprotein components of the two viruses, however, could not be substituted for each other. TSV-RNA is as efficient as AMV-RNA in withdrawing protein subunits from AMV nucleoprotein. However, TSV nucleoprotein could not be uncoated by its own RNA or by AMV-RNA. Nevertheless, the affinity of TSV-RNA for AMV coat protein and the functional equivalence of the two coat proteins point to a possible common origin of these two viruses.
that the genome is tripartite (for a review see Jaspars, 1974). Fulton (1970) showed that for TSV the highest specific infectivity was associated with the combination of the three fastestsedimenting nucleoprotein components and that hybrid strains could be obtained by inoculating components of different strains (Fulton, 1972). In 1971 our group found that the AMV genome requires its coat protein or the mRNA for the coat protein for infectivity (Bol et al., 1971). Since that time we have been interested in the comparison of viruses with a tripartite genome, especially in relation to the question whether or not the coat protein of these viruses plays a biological role. The bromo- and cucumoviruses were found not to require their coat protein for infectivity (Lane and Kaesberg, 1971; Bancroft, 1972; Lot et al., 1974). Another difference between AMV, on the
INTRODUCTION
Tobacco streak virus (TSV) preparations contain either three or four nucleoprotein components, according to various investigators (Fulton, 1970, 1972; Lister and Bancroft, 1970; Clark and Lister, 1971). When TSV-RNA is analyzed by electrophoresis on polyacrylamide gels four or five RNA’s are found (Clark and Lister, 1971). The RNA pattern of TSV, consisting of three RNA’s with molecular weights of about 10” and a small RNA of about 3 x lo5 daltons, is rather similar to that of cowpea chlorotic mottle virus (CCMV) (Fowlks and Young, 1970; Bancroft, 1971), bromegrass mosaic virus (BMV) (Fowlks and Young, 1970; Lane and Kaesberg, 1971), alfalfa mosaic virus (AMV) (Bol et al., 1971) and cucumber mosaic virus (CMV) (Kaper and West, 1972; Peden and Symons, 1973; Lot et ai., 1974). For all these viruses, evidence has been obtained 215 Copyright 0 19’75 by Academic Press, Inc. All rights of reproduction in any form reserved.
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one hand, and the bromo- and cucumoviruses, on the other, is that the AMV nucleoprotein components show heterogeneity in size (Gibbs et al., 1963) being bacilliform particles of different lengths, while the components of the other viruses each have identical icosahedral protein shells (Kuhn, 1964; Bockstahler and Kaesberg, 1962; Murant, 1965). Lister et al. (1972) showed that the TSV components are spheres of different sizes. Therefore, I wondered whether the coat protein of TSV might be adapted to a biological function rather than to a very stringent role in particle structure. The particle-size heterogeneity together with the characteristic RNA pattern prompted me to investigate the infectivity requirements of this virus. In this paper I present results of experiments which show that the TSV genome in all probability is tripartite and that this genome is only active in the presence of either TSV coat protein or AMV coat protein. Furthermore it is demonstrated that both TSV-RNA and AMV-RNA are capable of withdrawing protein subunits from AMV particles. TSV particles are more stable in that they do not lose subunits to either TSV- or AMV-RNA. MATERIALS
AND
METHODS
Virus and virus culture. The TSV used in this study was TSV strain WC, an inoculum of which was kindly supplied by Dr. R. W. Fulton, Madison, WI. The virus was grown for 5 days in plants of Nicotiana tabacum L., cultivars Samsun NN, White Burley or Bright Yellow, or for 10 days in N. glutinosa x N. clevelandii (Christie, 1969). Seed of Christie’s hybrid was a gift from Dr. A. 0. Jackson, Purdue University, Lafayette, IN. Preparation of viral nucleoproteins. Two methods of TSV isolation were used. Procedure 1 of Clark and Lister (1971) was used but with 2-mercaptoethanol instead of thioglycolate. The nucleoprotein material obtained after the second high speed centrifugation was resuspended in 0.1 M sodium acetate, pH 5.0, containing 0.001 M ethylenediaminetetraacetic acid (EDTA) and 0.001 M NaN, (NEN buffer). The yield varied between 0.003 and 0.009 mg of
virus per g of fresh leaf. Different batches of virus behaved rather irreproducibly with regard to sedimentation in sucrose density gradients, and quite often the material did not penetrate into a polyacrylamide gel on electrophoresis. In the electron microscope, cell material was found to be present. I presume that the virus was bound to cell material, probably to membrane fragments. Therefore I decided to isolate the virus in the presence of Triton X-100. Lister and Bancroft (1970) stated that addition of 0.5% Triton X-100 did not affect the yield of TSV. We found that addition of 1% Triton X-100 did improve the yield and purity of our virus preparations. The procedure adopted finally is a modification of the method described for AMV by Van Vloten-Doting and Jaspars (1972). Each 120 g of leaves were blended with 120 ml of 0.1 M KH,PO, containing 0.01 M EDTA, 0.02 M 2-mercaptoethanol and 2% Triton X-100, adjusted to pH 7.2 with KOH. The slurry was rehomogenized with 120 ml of 1: 1 chloroform-butanol for 1 min. The emulsion was broken by centrifugation. To the water layer, a solution of 30% polyethylene glycol (Serva; MW, 20,000) in distilled water was added to a final concentration of 5%. The pellets obtained after low speed centrifugation were resuspended in one-tenth of the original volume of 0.01 M NaH,PO,, 0.001 M EDTA and 0.001 M NaN,, adjusted to pH 7.0 with NaOH (PEN buffer). After two cycles of low and high speed centrifugation the pellets were dissolved and stored at 4” in PEN buffer. The yield varied between 0.07 and 0.30 mg of virus per g of fresh leaf. The highest yields were obtained from Christie’s hybrid. Nucleoprotein material of AMV strain 425, CMV strain S and BMV was isolated as described by Van Vloten-Doting and Jaspars (1972), by Lot et al. (1974), and by Bockstahler and Kaesberg (1962, 1965), respectively. Nucleoprotein material of turnip yellow mosaic virus (TYMV) was a gift from Dr. C. W. A. Pley. of TSV-RNA. Nucleic acid Extraction was obtained from TSV nucleoprotein by phenol extraction at 60”. To a virus solu-
EQUIVALENCE
OF TSV AND AMV PROTEINS
tion (10 mg/ml), sodium dodecylsulphate (SDS) to 0.6% and bentonite to 0.1 mg/ml were added. This mixture was added dropwise to an equal volume of preheated phenol placed on a Vortex mixer. The mixture was kept at 60” for 5 min. After low speed centrifugation the aqueous phase was collected and the procedure repeated. The phenol was removed either by shaking with ether or by precipitating the nucleic acid with ethanol followed by ether treatment of the dissolved pellet. The ether was removed by a stream of nitrogen gas. The yield was about 80’7r and the RNA seemed to be intact (Fig. 1). When the same procedure was used at 4”, the yield was very low, 2-lo%, and most of the RNA seemed to have been degraded. Extraction of other RNA’s. CMV-RNA and TYMV-RNA were prepared as described by Lot et al. (1974) and Stols and Veldstra (1965), respectively. AMV-RNA, BMV-RNA and tobacco leaf RNA were prepared as described by Van Vloten-Doting and Jaspars (1972). Preparation of TSV protein. Different methods to obtain TSV coat protein were
0.0260
moblllty
-
FIG. 1. Densitogram of a polyacrylamide with TSV-RNA isolated at 60”.
gel run
217
tried: Dialysis against 1.0 M CaCl,, 0.02 M Tris, pH 7.5, and 0.001 M 2-mercaptoethano1 (Shepherd et al., 1968); dissociation of the virus by 0.5 M MgCl,, 0.001 M 2-mercaptoethanol (Kruseman et al., 1971) and dissociation of the virus by 66% acetic acid (Fraenkel-Conrat, 1957), all without much success. Finally we used a modification of the method of Shepherd et al. (1968), dialyzing the virus against 1.0 M CaCl,, 0.1 M Tris, pH 9.0, and 0.001 M 2-mercaptoethanol. Only in this case was complete dissociation of the virus obtained. Preparation of other proteins. AMV and CMV coat proteins were prepared according to Kruseman et al. (1971) but with the omission of 2-mercaptoethanol. BMV coat protein was prepared according to Yamazaki and Kaesberg (1963). Sucrose density gradient centrifugation. TSV nucleoproteins and AMV bottom component were purified by one or two cycles of centrifugation in a Spinco Ti 15 zonal rotor. Isokinetic sucrose density gradients (lo-30% in PEN buffer) were used. The time of centrifugation was adapted to the component to be purified. RNA-nucleoprotein mixtures were run in an SW 27 rotor as described by Van Vloten-Doting and Jaspars (1972). TSV-RNA was run in an SW 27 rotor for 24 hr at 25,000 rpm and 4”. The composition of the gradients was 50-200 mg/ml of sucrose in 0.01 M Tris, pH 7.8, 0.06 M KC1 and 0.01 mg/ml of bentonite. The RNA was heated at 60” for 5 min before loading it onto the gradient. Infectiuity assay. Local lesion tests for both viruses were performed on primary leaves of Phaseolus vulgaris L., cultivars “Berna” or “Noord Hollandse Bruine,” as described for AMV in 1967 by Van VlotenDoting and Jaspars. Polyacrylamide-gel electrophoresis. Electrophoresis of TSV nucleoprotein and TSV-RNA was performed as described by Bol et al. (1971) except that urea was omitted in the case of RNA. Sometimes, TSV-RNA was run immediately after dissociating the nucleoprotein with 1% SDS. Ultraviolet absorption. Concentrations were determined assuming A$ (260 nm) values of 51 (Fulton and Potter, 1971) and 239 (Ghabrial and Lister. 1974) in the case
218
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VAN VLOTEN-DOTING
of nucleoproteins and RNA, respectively. The RNA content was taken as 14% (Ghabrial and Lister, 1974). For TSV coat protein, A:& (280 nm) was taken as 70. Peptide mapping and limited tryptic digestion. These were performed by Ing. A. Neeleman, G. M. A. van Beynum and F. Th. Brederode, respectively. The methods described by Bol et al. (1974) were used. RESULTS
Nomenclature of TSV Nucleoproteins TSV-RNA’s
and
Originally Fulton (1970) found four components of TSV and designated them, in order of decreasing sedimentation velocity, c,b,a and T. Later they were renamed by Fulton (1972) as follows: b, bottom component; a, middle component; T, top component; while c was not described any further. Lister and Bancroft (1970) and Clark and Lister (1971) described three components: BV, bottom component; MV, middle component; and TV, top component. Lister et al. (1972) mentioned that sometimes, especially when the virus was cultivated in Christie’s hybrid, an additional component called super top component, STV, was present. In my hands, material of TSV-WC usually contained four components, which I assume to be identical to the BV, MV, TV and STV described by Lister and co-workers, while the three fastest-sedimenting components could correspond to b, a, and T of Fulton. However, I am not completely sure of this and should prefer to number the components, in order of decreasing sedimentation velocity, NP 1, NP 2, NP 3 and NP 4. Clark and Lister (1971) described four RNAs, BN, MN, TN’ and TTN, while sometimes an additional RNA, TN”, was found between TN’ and TTN. We found mostly four RNA’s (RNA 1, RNA 2, RNA 3 and RNA 4, in order of increasing electrophoretic mobility on polyacrylamide gels); sometimes an additional RNA was found ahead of RNA 3. This nomenclature has the advantage that it corresponds with that used for the bromo- and cucumoviruses.
Biological Activity of TSV Nucleoproteins Figure 2A shows the sedimentation pattern of TSV nucleoprotein isolated from Christie’s hybrid. Fig. 2B represents the infectivity of the peak fractions and of the combinations of these fractions. The infectivity of NP 1 and NP 2 was very low, while NP 3 and NP 4 showed no infectivity at all. Combination of NP 1 with either NP 2 or NP3 had five to seven times higher infectivities than NP 1 alone. The combination of NP 1 with NP 2 and NP 3 was two to seven times as infectious as the twofold combinations. Addition of NP 4 to the threefold combination did increase the infectivity slightly; however, this was not reproducible. When all components had been purified by a second cycle of sucrose density gradient purification in a zonal rotor the infectivity of the twofold combinations was much lower; addition of a third component to a mixture of two components enhanced the infectivity considerably (Fig. 2C). NP 4 is apparently inert (Fig. 2D). The RNA content of the purified components was analyzed by electrophoresis on polyacrylamide gels in the presence of SDS. Each component was found to contain mainly one species of RNA (results not shown). The simplest explanation for the results presented above is that the TSV genome consists of three RNA’s (RNA 1, RNA 2 and RNA 3), each encapsidated in one specific component (NP 1, NP 2 and NP 3). Molecular Species Required for Infectivity Assuming that the TSV genome is tripartite, the situation in TSV could be like that in BMV (Lane and Kaesberg, 1971), CCMV (Bancroft, 1971) or CMV (Peden and Symons, 1973; Lot et al., 1974) where the combination of the three RNA’s is infectious itself, or it could be like that in AMV (Bol et al., 1971) where the three RNA’s need to be activated by the coat protein. It has been shown that AMV top component a RNA (comparable in size with RNA 4 of the other viruses) is also capable of activating the AMV genome (Bol et al., 1971). Like RNA 4 of BMV
EQUIVALENCE
219
OF TSV AND AMV PROTEINS
0. D
D
T , .
FK. 2. Purification of a TSV preparation in order to determine the components required for infectivity. (A), Optical density profile of a sucrose density gradient in which 20 mg of a TSV preparation was centrifuged. Centrifugation in a Spinco B IV zonal rotor for 5.25 hr at 31,500 rpm and 4’. (B), Infectivity of the peak fractions and the combinations of the peak fractions from (A). (Cl, Infectivity of components and combinations of components purified by two cycles of sucrose density gradient centrifugation in a zonal rotor. (D), The same as (C) except that to all solutions NP4 had been added. Infectivities of components at the vertices, infectivities of the twofold combinations at the sides and infectivities of the threefold combinations at the center of the triangles are shown. Lesion numbers of one triangle are averages (B) or totals (C) and (D) from one incomplete block. Blocks belonged to different batches of plants. Concentration of all components in all combinations of (B) is 1.0 &ml, (Cl, 0.5 &ml and (D), 0.5 &ml.
(Shih and Kaesberg, 1973) and probably RNA 4 of other bromo- and cumumoviruses (Davies and Kaesberg, 19741 top component a RNA of AMV contains the genetic information for the coat protein (Van Ravenswaay Claasen et al., 1967; and other work from our laboratory, to be published) and is apparently translated in the cell. Preparations of TSV-RNA do contain only low amounts of RNA 4. It is unknown whether RNA 4 of TSV functions as messenger for the coat protein. Even if this RNA in the cell were translated into coat protein, the amount of RNA 4 in a total RNA preparation might be limiting. If the coat protein were required for infectivity its addition to an unfractionated RNA prepa-
ration might still enhance infectivity. Figure 3 shows that indeed the infectivity of unfractionated TSV-RNA increases upon the addition of increasing amount of TSV coat protein. In order to investigate whether a combination of the three largest TSV-RNA’s can also be activated by TSV-RNA 4, a total RNA preparation was run on a sucrose density gradient. Figure 4 shows that the fractions of this gradient are not or are only slightly infectious. However, when to each fraction an equal volume of fraction 20 was added, infectivity was found in that region of the gradient where RNA’s 1, 2, and 3 overlap. Together, the results from Figs. 3 and 4 suggest that TSV-RNA 4 contains the genetic information for the coat pro-
LOUS
VAN VLOTEN-DOTING
the homologous as well as in the heterologous combinations (results not shown). The activity of TSV coat protein was lost upon heating at 60” for 5 min. It was shown earlier (Bol et al., 1971) that the activity of AMV coat protein was sensitive to the same treatment. Differences Between TSV Coat Protein and AMV Coat Protein
10 number
20 of protein RNA
30
40
molecules
molecule
FIG. 3. Comparison of the capability of different viral coat proteins to activate the TSV genome. TSV-RNA (100 &ml) was combined with solutions of TSV coat protein (O-O), AMV coat protein (OA), BMV or CMV coat protein (W-m). For the molecular weights of these coat proteins values of 30,000, 24,800, 20,300 and 24,200 were used (Ghabrial and Lister, 1974; Kraal et al., 1972; Stubbs and Kaesberg, 1967; Hill and Shepherd, 1972). Lesion numbers are averages from seven half-leaves.
tein, as do the comparable AMV and BMV.
The fact that TSV coat protein and AMV coat protein can both serve the same function could mean that (part) of these proteins are identical. The following experiments were performed to investigate this possibility. Serology. Neither our TSV nucleoprotein preparations nor our TSV protein preparations gave precipitation lines with antiserum raised against AMV nucleoprotein. Limited trypsin digestion. Bol et al. (1974) showed that 27 amino acids can be removed from the N-terminus of the AMV coat protein by incubating AMV particles at pH 7.0 with 1 &ml of trypsin for 20 hr
r -
260
RNA species of
Equivalence of TSV Coat Protein and AMV Coat Protein in the Activation of the Genomes of TSV and AMV Figure 3 demonstrates that TSV-RNA can be activated by TSV coat protein and by AMV coat protein, while the addition of BMV coat protein or CMV coat protein has no effect at all. When AMV-RNA instead of TSV-RNA was used the same results were found (not shown). In all cases the kind of lesion was typical for the virus from which the RNA had been used. Instead of the isolated coat proteins, their presumed mRNA’s (TSV-RNA 4 and AMV top component a RNA) or the purified nucleoprotein components could also be used in
5
Fraction
number
FIG. 4. Activation of RNA 1 + RNA 2 + RNA 3 by RNA 4. Optical density profile (-) of a sucrose density gradient in which 0.4 mg of TSV-RNA was run. Infectivity of fractions after addition of an equal volume of buffer (O-O) and after addition of an equal volume of fraction 20 (RNA 4) (x-x ). Lesion numbers are averages from seven half-leaves.
EQUIVALENCE
at 25”. Incubation of TSV particles under the same conditions had a more pronounced effect on the size of its coat protein but the conversion was not complete. At a trypsin concentration of 100 &ml, all TSV subunits were converted to a much smaller product (Fig. 5). It is curious that the AMV and TSV coat proteins do migrate with the same mobility since the molecular weights are different, viz., 24,800 and 30,000, respectively (Kraal et al., 1972; Ghabrial and Lister, 1974). Tryptic fingerprints. TSV and AMV coat protein were digested by trypsin. Figure 6 shows that the peptide maps of the two proteins are very different. However, a few spots might be identical and will be investigated further. Attempts to Construct TSV and AMV
Hybrids
221
OF TSV AND AMV PROTEINS
Between
For both TSV (Fulton, 1972) and AMV (Van Vloten-Doting et al., 1968, 1970; Dingjan-Versteegh et al., 1972, 1974; Bol and Lak-Kaashoek, unpublished results) it has been shown that combinations of components from different virus strains are infectious and yield hybrid strains. Bancroft (1972) demonstrated that a hybrid virus could be made from two components of BMV and one component of CCMV. The reverse combination was not
FIG. 5. Comparison of the effect of trypsin on AMV coat protein and TSV coat protein From both viruses 2 mg/ml of nucleoprotein was incubated for 20 hr at 25” with the indicated concentration of trypsin in 0.05 M Tris, 0.001 M EDTA and 0.001 M NaN, at pH 7.0. The incubation was stopped by the addition of SDS. The upper band of all gels represents a reference protein added to the samples.
infectious. The hybrid infected only those plants that could function as host for both parent viruses. I determined whether the infectivity of a combination of two components of TSV did increase upon the addition of a third component of AMV. The same was done with the reverse combinations. The mixtures were inoculated on Berna beans, since these plants produce large local lesions with TSV and small ones upon infection with AMV. In these experiments NP 1, NP 2 and NP 3 of TSV were taken to be analogous to bottom component, middle component and top component b of AMV, respectively. Since the infectivity of the threefold combinations was never higher than that of the twofold combinations, I had to conclude that either the components of TSV and AMV are not interchangeable at all or that the infectivity of hybrids between these viruses is much lower than that of either virus or its interstrain hybrids. Interaction between RNA ‘s
Nucleoproteins
and
In a previous publication (Van VlotenDoting and Jaspars, 1972) it was shown that, upon addition of AMV-RNA to AMV nucleoprotein, some coat protein molecules from the nucleoprotein become associated with the viral RNA. The reaction is highly specific, and it was postulated that AMVRNA has a few sites with strong affinity for AMV coat protein. Since these high-affinity sites might be connected with the biological role of the AMV coat protein, I thought it worthwhile to investigate whether TSV-RNA would react with TSV and AMV nucleoproteins and whether AMV-RNA would react with TSV nucleoprotein. AMV or TSV nucleoproteins were run on sucrose density gradients after incubation with different RNA’s or with buffer. From Figs. 7A-C it is evident that the AMV and TSV-RNA’s react in the same way with AMV nucleoprotein. In both cases the infectivity in the RNA regions of gradients run with the mixtures was much higher than the infectivity of a comparable region of a gradient run with RNA alone, suggest-
222
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VAN VLOTEN-DOTING
Fm. 6. Tryptic “fingerprints” of TSV coat protein (A) and AMV coat protein (B). Experimental details as described by Bol et al. (1974). Chromatography was in the horizontal direction, from right to left, high voltage electrophoresis was in the vertical direction, cathode at the top.
O.D.
D
_,r,
Fraction number Fro. 7. Effect of AMV- and TSV-RNA on AMV and TSV nucleoprotein (-1. (A), 100 pg component; (B,C), 100 pg of AMV bottom component incubated with 100 rg AMV-RNA respectively: (D), 100 pg of TSV NP 3; (E,F), 100 ag of TSV NP 3 incubated with 100 bg of TSV-RNA, respectively. In all cases incubation was for 32 hr at 4” in 0.5 ml of PEN buffer. - run in sister tubes; (B,E), 100 pg of AMV-RNA and (C,F), 100 plugof TSV-RNA, respectively.
of AMV bottom and TSV-RNA, AMV-RNA and -, RNA controls
EQUIVALENCE
OF TSV AND AMV PROTEINS
ing the presence of coat protein in the former. Furthermore the residual nucleoprotein was sensitive to bean leaf sap, suggesting the disintegration of the viral structure. Figures 7D-F show that, in the gradients run with mixtures of TSV nucleoprotein with either TSV-RNA or AMV-RNA incubated under the same conditions, the nucleoprotein peaks did not disappear but got a shoulder on the meniscus side. In this case the infectivity of the RNA regions of the gradients did not increase, and the infectivity of the nucleoproteins did not become sensitive to leaf sap. Other RNA’s (BMV-RNA, CMV-RNA or tobacco leaf RNA) did not change the pattern of either nucleoprotein under these conditions (results not shown). DISCUSSION
The results obtained with the TSV nucleoproteins can best be explained by the assumption that the TSV genome consists of three RNA’s, each separately encapsidated. Fulton (1970) demonstrated that his component b (probably our NP 1) had a low but significant intrinsic infectivity. The dilution curve of purified component b was found to be rather steep, suggesting that two or more particles cooperate in the formation of each infection. Furthermore, Fulton (1972) showed that there exists redundancy in the genetic information among the particle types of TSV. In our opinion these results can be explained by the assumption that each nucleoprotein component contains, in addition to its main RNA species, small amounts of other RNA species. Evidence for this has been reported by Clark and Lister (1971). There could be a variation in the amounts of minor RNA’s of a given nucleoprotein component depending on the cultivation circumstances. For the infectivity experiments in this report, the virus has been cultivated in Christie’s hybrid at 23”, while Fulton cultivated it in Nicotiana tabacum L., in N. rustica L. or in Datura stramonium L. at 24-25”. Recently Heytink and Jaspars (1974) showed that the bottom component of
223
AMV (comparable to NP 1 of TSV) can contain, besides bottom RNA, traces of the smaller AMV-RNA’s. The intrinsic infectivity that has been claimed for the bottom component of AMV by Majorana and Paul (1969) and by Desjardins and Steere (1969) may be due to the presence of M-RNA and Tb-RNA (comparable to TSV-RNA 2 and 3) in pseudo-bottom particles. As with AMV, only the three fastestsedimenting nucleoproteins of TSV appear to be necessary for infectivity. By separating the TSV-RNA’s by sucrose density gradient centrifugation it was possible to demonstrate that the combination of the three largest RNA’s was not infectious by itself. However, infectivity was found when either TSV-RNA 4 or AMV-top component a RNA or TSV coat protein or AMV coat protein was added. This finding explains why Clark and Lister (1971) found that nucleic acid preparations of TSV, even when they had been carefully prepared and appeared intact, showed only very low infectivities. The equivalence of RNA species 4 and the coat protein of TSV may be explained by the assumption that the RNA contains the genetic information for the coat protein. The equivalence between AMV coat protein and TSV coat protein is more difficult to understand. According to different criteria, immunology, limited trypsin digestion and peptide mapping, the two proteins cannot have many amino acid sequences in common. The possibility that there are a few critical identical amino acid sequences is under investigation. The results do not exclude the possibility that the peptide chains are folded in such a way that similar groups are found on the surface of the coat protein. It has been proposed that in AMV the coat protein fulfills its function by associating with specific sites on the RNA (Van Vloten-Doting and Jaspars, 1972). It is remarkable also that TSV-RNA has a very high affinity for AMV coat protein, because it is able to withdraw subunits from AMV particles. Apparently there is some kind of cross-recognition between TSV-RNA and AMV coat protein, on the one hand, and AMV-RNA and TSV coat protein, on the other. Bol et al. (1975) have
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VAN VLOTEN-DOTING
shown that there exists no detectable homology in the primary sequence of the RNA’s of TSV and AMV, although it has to be remembered that the method used (competitive hybridization) would not reveal the presence of very short identical regions. Also the interaction might depend on secondary and tertiary structures rather than on primary structure. The fact that under identical circumstances AMV nucleoprotein loses protein subunits to RNA, in contrast to TSV nucleoprotein, probably reflects a difference in stability (compare Fig. 7). Both nucleoproteins seem to be primarily stabilized by RNA-protein interactions according to criteria defined by Kaper and Geelen (1971). However, in AMV nucleoprotein the subunits seem to be less tightly bound to their RNA than in TSV nucleoprotein. This difference in stability is also reflected by the difficulties encountered during the preparation of TSV coat protein and TSVRNA. The difficulty of extracting intact TSV-RNA has already prompted Clark and Lister (1971) to suggest a particular kind of structural relationship between TSV protein and RNA in the particle. The high affinity for AMV coat protein displayed by TSV-RNA could result in the occurrence of genomic masking and phenotypic mixing in doubly infected plants. Preliminary experiments have already indicated that at least TSV-RNA can be encapsidated by AMV coat protein (unpublished results). It is remarkable that, although the coat proteins of TSV and AMV can be substituted for each other in the onset of infectivity, up to now it was impossible to construct hybrids between the viruses. Even the combinations in which only the top component b of AMV had been exchanged for NP 3 of TSV, or vice versa, were not infectious. This could mean that top component b RNA of AMV and RNA 3 of TSV do contain besides the information for the coat protein (Dingjan-Versteegh et al., 1972; Fulton, 1972) the information for some other protein which is not interchangeable. This protein might be a replicase subunit. Also it is quite possible that the different virus functions are not dis-
tributed in the same way over the three viral RNA’s. ACKNOWLEDGMENTS I express my gratitude to all the members of our laboratory who contributed to this work. The Netherlands Organization of Pure Research (ZWO) supported part of the work. REFERENCES BANCROFT, J. B. (1971). The significance of the multicomponent nature of cowpea chlorotic mottle virus RNA. Virology 45, 830-834. BANCROFT,J. B. (1972). A virus made from parts of the genomes of brome mosaic and cowpea chlorotic mottle virus. J. Gen. Vi&. 14, 223-228. BOCKSTAHLEH, L. E., and KAESBERG, P. (1962). The molecular weight and other biophysical properties of bromegrass mosaic virus. Biophys. J. 2, l-9. BOCKSTAHLEH, L. E., and KAESBERG, P. (1965). Infectivity studies of bromegrass mosaic virus RNA. Virology 27, 418-425. BOL, J. F., VAN VLOTEN-DOTING, L., and JASPAHS, E. M. J. (1971). A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus. Virology 46, 73-85. BOL, J. F., KRAAL, B., and BREDERODE,F. TH. (1974). Limited proteolysis of alfalfa mosaic virus: Influence on the structural and biological function of the coat protein. Virology 58, 101-110. BOL, J. F., BREDERODE, F. TH., JANZE, G. C., and RAUH, D. C. (1975). Studies on sequence homology between the RNAs of alfalfa mosaic virus. Virology, in press. CHRISTIE, S. R. (1969). Nicotiana hybrid developed as a host for plant viruses. Plant. Dis. Rep. 53, 939-941. CLARK, M. F., and LISTER, R. M. (1971). Preparation and some properties of the nucleic acid of tobacco streak virus. Virology 45, 61-74. DAVIES, J. W., and KAESBERG, P. (1974). Translation of virus mRNA: Protein synthesis directed by several virus RNAs in a cell-free extract from wheat germ. J. Gen. Viral. 25, 11-20. DESJARDINS, P. R., and STEERE, R. L. (1969). Separation of top and bottom components of alfalfa mosaic virus by combined differential and density gradient centrifugation. Arch. Gesamte Virusforsch. 26, 127-137. DINGJAN-VERSTEEGH, A. M., VAN VLOTEN-DOTING, L., and JASPARS, E. M. J. (1972). Alfalfa mosaic virus hybrids constructed by exchanging nucleoprotein components. Virology 49, 716-722. DINGJAN-VERSTEEGH, A. M., VAN VLOTEN-DOTING, L., and JASPARS,E. M. J. (1974). Confirmation of the constitution of alfalfa mosaic virus hybrid genomes by backcross experiments. Virology 59, 328-330.
EQUIVALENCE
OF TSV AND AMV PROTEINS
FOWLKS, E., and YOUX, R. J. (1970). Detection of heterogeneity in plant viral RNA by polyacrylamide gel electrophoresis. Virology 42, 548-550. FULTON, R. W. (1970). The role of particle heterogeneity in infection by tobacco streak virus. Virolog!: 41, 288-294. FULTON, R. W. (1972). Inheritance and recombination of strain-specific characters in tobacco streak virus. Virology 50, 810-820. FULTON, R. W., and POTTER, K. T. (1971). Factors affecting the proportion of nucleoprotein components of tobacco streak virus. Virology 45,734-W. FRAENKEL-CONHAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Virology 4, l-4. GHABHIAL, S. A.. and LISTER, R. M. (1974). Chemical and physicochemical properties of two strains of tobacco streak virus. Virology 57, l-10. GIBBS, A. J., NIXON, H. L., and WOODS, R. D. (1963). Properties of purified preparations of lucerne mosaic virus. Virology 19, 441-449. HE’I.TINK, R. A., and JASPARS, E. M. J. (1974). RNA contents of abnormally long particles of certain strains of alfalfa mosaic virus. Virolog.y 59,371~382. HII.I., J. H., and SHEPHF.RD,R. J. (1972). Molecular weights of plant virus coat proteins by polyacrylamide gel electrophoresis. Virology 47, 817-822. JASPAHS,E. M. J. (19741. Plant viruses with a multipartite Rename. Aduan. Virus Res. 19, 37-149. KAPER, J. M., and GEELEN, J. L. M. C. (1971). Studies on the stabilizing forces of simple RNA viruses. II. Stability, Dissociation and Reassembly of cucumber mosaic virus. J. Mol. Biol. 56, 277-294. KAPER, J. M.. and WES.I., C. K. (1972). Polyacrylamide gel separation and molecular weight determination of the components of cucumber mosaic virus RNA. Prep. Biochem. 2, 251-263. KRAAL.. B., DE GRAAF, J. M., BAKKER, T. A., VAN BEYNC’M, G. M. A., GOEDHART, M., and BOSCH, L. (1972). Structural studies on the coat protein of alfalfa mosaic virus. Eur. J. Biochem. 28, 20-29. KRUSEMAN, J., KRAAL, B., JASPARS,E. M. J.. BOL, J. F., BREDEKODE, F. T., and VEI.DS.~RA, H. (1971). Molecular weight of the coat protein of alfalfa mosaic virus. Biochemistry 10, 447-454. KUHN, C. W. (1964). Purification, serology and properties of a new cowpea virus. Phytopathology 54, 853-857. LANE, C. L., and KAESBERC, P. (1971). Multiple genetic components in bromegrass mosaic virus. Nature New Biol. 232, 40-43. LISTEH, R. M., and BANCKOFT, J. B. (1970). Alteration of tobacco streak virus component ratios as influenced by host and extraction procedure. Phytopatholog.~ 60, 689-694. LISTER, R. M., GHABRIAI., S. A., and SAKSENA, K. N.
225
(1972). Evidence that particle size heterogeneity is the cause of centrifugal heterogeneity in tobacco streak virus. Virology 49, 290-299. LOT. H., MARCHOUX, G., MARROL-, J., KAPER, J. M., WEST, C. K., VAN VLOTEN-DOTING, L., and HULL, R. (1974). Evidence for three functional RNA species in several strains of cucumber mosaic virus. J. Gen. ViFOl. 22, 81-93. MAJORANA, G.. and PAUL, H. L. (1969). The production of new types of symptoms by mixtures of different components of two strains of alfalfa mosaic virus. Virology 38, 145-151. MURANT, A. F. (1965). The morphology of cucumber mosaic. Virology 26, 538-544. PEDEN, K. W. C., and SYMONS, R. H. (1973). Cucumber mosaic virus contains a functionally divided genome. Virolog?: 53, 487-492. SHEPHERD,R. J., WAKEMAN, R. J., and GHABRIAI., S. A. (1968). Preparation and properties of the protein and nucleic acid components of pea enation mosaic virus. Virology 35, 255-267. SHIH, D. S., and KAESBEHG, P. (1973). Translation of brome mosaic viral ribonucleic acid in a cell-free system derived from wheat embryo. PFOC. Nat. Acad. Sci. USA 70, 1799-1803. STOLS, A. L. H., and VELDSTRA, H. (1965). Interactions of turnip yellow mosaic virus with quaternary ammonium salts. Virology 25, 508-515. STUBBS, J. D., and KAESBERC,, P. (1964). A protein subunit of bromegrass mosaic virus. J. Mol. Biol. 8, 314-323. VAN VLOTEN-DOTING:, L., and JASPARS,E. M. J. (1967). Enhancement of infectivity by combination of two ribonucleic acid components from alfalfa mosaic virus. Virology 33, 684-693. VAN VLOTEN-DOTING, L., KRLXEMAN, J., and JASPARS, E. M. J. (1968). The biological function and mutual dependence of bottom component and top component a of alfalfa mosaic virus. Virology 34, 728-737. VAN VLOTEN-DOTING:, L., DINGJAN-VERSTEEGH, A. M., and JASPAHS.E. M. J. (1970). Three nucleoprotein components of alfalfa mosaic virus necessary for infectivity. Virology 40, 419-430. VAN VLOTEN-DOTING, L.. and JASPAKS,E. M. J. (1972). The uncoating of alfalfa mosaic virus by its own RNA. Virology 48, 699-708. VAN RAVENSB’AAYCLAASEX, J. C., VAN LEEI.WEN, A. B. J., DUYTS, G. A. H., and BOSCH, L. (1967). In vitro translation of alfalfa mosaic virus RNA. J. Mol. Biol. 23, 535544. YAMAZAKI, H., and KAESBERG, P. (1963). Degradation of bromegrass mosaic virus with calcium chloride and the isolation of its protein and nucleic acid. J. Mol. Viol. 7. 760-762.
(1975)
Coat Protein Virus:
Is Required
Biological
for Infectivity
Equivalence
of the Coat Proteins
Streak and Alfalfa LGUS Department
of Biochemistq,
VAN
of Tobacco
Mosaic
Streak
of Tobacco
Viruses
VLOTEN-DOTING
State University,
Wassenaarseweg
Accepted January
64, Leiden,
The Netherlands
28, 1975
The four nucleoprotein components of tobacco streak virus (TSV) were purified by zonal centrifupation. Each component contains mainly one RNA species. Infectivity studies indicate that the three fastest-sedimenting nucleoproteins are required for viral replication. A mixture of the three largest RNA’s however, is not infectious hut can he activated by the viral coat protein or by the smallest TSV-RNA. The same situation exists in alfalfa mosaic virus (AMV). The coat proteins of TSV and AMV, which are rather different as judged by serology and tryptic digestion products, cannot only activate their own genome but also that of each other. The nucleoprotein components of the two viruses, however, could not be substituted for each other. TSV-RNA is as efficient as AMV-RNA in withdrawing protein subunits from AMV nucleoprotein. However, TSV nucleoprotein could not be uncoated by its own RNA or by AMV-RNA. Nevertheless, the affinity of TSV-RNA for AMV coat protein and the functional equivalence of the two coat proteins point to a possible common origin of these two viruses.
that the genome is tripartite (for a review see Jaspars, 1974). Fulton (1970) showed that for TSV the highest specific infectivity was associated with the combination of the three fastestsedimenting nucleoprotein components and that hybrid strains could be obtained by inoculating components of different strains (Fulton, 1972). In 1971 our group found that the AMV genome requires its coat protein or the mRNA for the coat protein for infectivity (Bol et al., 1971). Since that time we have been interested in the comparison of viruses with a tripartite genome, especially in relation to the question whether or not the coat protein of these viruses plays a biological role. The bromo- and cucumoviruses were found not to require their coat protein for infectivity (Lane and Kaesberg, 1971; Bancroft, 1972; Lot et al., 1974). Another difference between AMV, on the
INTRODUCTION
Tobacco streak virus (TSV) preparations contain either three or four nucleoprotein components, according to various investigators (Fulton, 1970, 1972; Lister and Bancroft, 1970; Clark and Lister, 1971). When TSV-RNA is analyzed by electrophoresis on polyacrylamide gels four or five RNA’s are found (Clark and Lister, 1971). The RNA pattern of TSV, consisting of three RNA’s with molecular weights of about 10” and a small RNA of about 3 x lo5 daltons, is rather similar to that of cowpea chlorotic mottle virus (CCMV) (Fowlks and Young, 1970; Bancroft, 1971), bromegrass mosaic virus (BMV) (Fowlks and Young, 1970; Lane and Kaesberg, 1971), alfalfa mosaic virus (AMV) (Bol et al., 1971) and cucumber mosaic virus (CMV) (Kaper and West, 1972; Peden and Symons, 1973; Lot et ai., 1974). For all these viruses, evidence has been obtained 215 Copyright 0 19’75 by Academic Press, Inc. All rights of reproduction in any form reserved.
216
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VLOTEN-DOTING
one hand, and the bromo- and cucumoviruses, on the other, is that the AMV nucleoprotein components show heterogeneity in size (Gibbs et al., 1963) being bacilliform particles of different lengths, while the components of the other viruses each have identical icosahedral protein shells (Kuhn, 1964; Bockstahler and Kaesberg, 1962; Murant, 1965). Lister et al. (1972) showed that the TSV components are spheres of different sizes. Therefore, I wondered whether the coat protein of TSV might be adapted to a biological function rather than to a very stringent role in particle structure. The particle-size heterogeneity together with the characteristic RNA pattern prompted me to investigate the infectivity requirements of this virus. In this paper I present results of experiments which show that the TSV genome in all probability is tripartite and that this genome is only active in the presence of either TSV coat protein or AMV coat protein. Furthermore it is demonstrated that both TSV-RNA and AMV-RNA are capable of withdrawing protein subunits from AMV particles. TSV particles are more stable in that they do not lose subunits to either TSV- or AMV-RNA. MATERIALS
AND
METHODS
Virus and virus culture. The TSV used in this study was TSV strain WC, an inoculum of which was kindly supplied by Dr. R. W. Fulton, Madison, WI. The virus was grown for 5 days in plants of Nicotiana tabacum L., cultivars Samsun NN, White Burley or Bright Yellow, or for 10 days in N. glutinosa x N. clevelandii (Christie, 1969). Seed of Christie’s hybrid was a gift from Dr. A. 0. Jackson, Purdue University, Lafayette, IN. Preparation of viral nucleoproteins. Two methods of TSV isolation were used. Procedure 1 of Clark and Lister (1971) was used but with 2-mercaptoethanol instead of thioglycolate. The nucleoprotein material obtained after the second high speed centrifugation was resuspended in 0.1 M sodium acetate, pH 5.0, containing 0.001 M ethylenediaminetetraacetic acid (EDTA) and 0.001 M NaN, (NEN buffer). The yield varied between 0.003 and 0.009 mg of
virus per g of fresh leaf. Different batches of virus behaved rather irreproducibly with regard to sedimentation in sucrose density gradients, and quite often the material did not penetrate into a polyacrylamide gel on electrophoresis. In the electron microscope, cell material was found to be present. I presume that the virus was bound to cell material, probably to membrane fragments. Therefore I decided to isolate the virus in the presence of Triton X-100. Lister and Bancroft (1970) stated that addition of 0.5% Triton X-100 did not affect the yield of TSV. We found that addition of 1% Triton X-100 did improve the yield and purity of our virus preparations. The procedure adopted finally is a modification of the method described for AMV by Van Vloten-Doting and Jaspars (1972). Each 120 g of leaves were blended with 120 ml of 0.1 M KH,PO, containing 0.01 M EDTA, 0.02 M 2-mercaptoethanol and 2% Triton X-100, adjusted to pH 7.2 with KOH. The slurry was rehomogenized with 120 ml of 1: 1 chloroform-butanol for 1 min. The emulsion was broken by centrifugation. To the water layer, a solution of 30% polyethylene glycol (Serva; MW, 20,000) in distilled water was added to a final concentration of 5%. The pellets obtained after low speed centrifugation were resuspended in one-tenth of the original volume of 0.01 M NaH,PO,, 0.001 M EDTA and 0.001 M NaN,, adjusted to pH 7.0 with NaOH (PEN buffer). After two cycles of low and high speed centrifugation the pellets were dissolved and stored at 4” in PEN buffer. The yield varied between 0.07 and 0.30 mg of virus per g of fresh leaf. The highest yields were obtained from Christie’s hybrid. Nucleoprotein material of AMV strain 425, CMV strain S and BMV was isolated as described by Van Vloten-Doting and Jaspars (1972), by Lot et al. (1974), and by Bockstahler and Kaesberg (1962, 1965), respectively. Nucleoprotein material of turnip yellow mosaic virus (TYMV) was a gift from Dr. C. W. A. Pley. of TSV-RNA. Nucleic acid Extraction was obtained from TSV nucleoprotein by phenol extraction at 60”. To a virus solu-
EQUIVALENCE
OF TSV AND AMV PROTEINS
tion (10 mg/ml), sodium dodecylsulphate (SDS) to 0.6% and bentonite to 0.1 mg/ml were added. This mixture was added dropwise to an equal volume of preheated phenol placed on a Vortex mixer. The mixture was kept at 60” for 5 min. After low speed centrifugation the aqueous phase was collected and the procedure repeated. The phenol was removed either by shaking with ether or by precipitating the nucleic acid with ethanol followed by ether treatment of the dissolved pellet. The ether was removed by a stream of nitrogen gas. The yield was about 80’7r and the RNA seemed to be intact (Fig. 1). When the same procedure was used at 4”, the yield was very low, 2-lo%, and most of the RNA seemed to have been degraded. Extraction of other RNA’s. CMV-RNA and TYMV-RNA were prepared as described by Lot et al. (1974) and Stols and Veldstra (1965), respectively. AMV-RNA, BMV-RNA and tobacco leaf RNA were prepared as described by Van Vloten-Doting and Jaspars (1972). Preparation of TSV protein. Different methods to obtain TSV coat protein were
0.0260
moblllty
-
FIG. 1. Densitogram of a polyacrylamide with TSV-RNA isolated at 60”.
gel run
217
tried: Dialysis against 1.0 M CaCl,, 0.02 M Tris, pH 7.5, and 0.001 M 2-mercaptoethano1 (Shepherd et al., 1968); dissociation of the virus by 0.5 M MgCl,, 0.001 M 2-mercaptoethanol (Kruseman et al., 1971) and dissociation of the virus by 66% acetic acid (Fraenkel-Conrat, 1957), all without much success. Finally we used a modification of the method of Shepherd et al. (1968), dialyzing the virus against 1.0 M CaCl,, 0.1 M Tris, pH 9.0, and 0.001 M 2-mercaptoethanol. Only in this case was complete dissociation of the virus obtained. Preparation of other proteins. AMV and CMV coat proteins were prepared according to Kruseman et al. (1971) but with the omission of 2-mercaptoethanol. BMV coat protein was prepared according to Yamazaki and Kaesberg (1963). Sucrose density gradient centrifugation. TSV nucleoproteins and AMV bottom component were purified by one or two cycles of centrifugation in a Spinco Ti 15 zonal rotor. Isokinetic sucrose density gradients (lo-30% in PEN buffer) were used. The time of centrifugation was adapted to the component to be purified. RNA-nucleoprotein mixtures were run in an SW 27 rotor as described by Van Vloten-Doting and Jaspars (1972). TSV-RNA was run in an SW 27 rotor for 24 hr at 25,000 rpm and 4”. The composition of the gradients was 50-200 mg/ml of sucrose in 0.01 M Tris, pH 7.8, 0.06 M KC1 and 0.01 mg/ml of bentonite. The RNA was heated at 60” for 5 min before loading it onto the gradient. Infectiuity assay. Local lesion tests for both viruses were performed on primary leaves of Phaseolus vulgaris L., cultivars “Berna” or “Noord Hollandse Bruine,” as described for AMV in 1967 by Van VlotenDoting and Jaspars. Polyacrylamide-gel electrophoresis. Electrophoresis of TSV nucleoprotein and TSV-RNA was performed as described by Bol et al. (1971) except that urea was omitted in the case of RNA. Sometimes, TSV-RNA was run immediately after dissociating the nucleoprotein with 1% SDS. Ultraviolet absorption. Concentrations were determined assuming A$ (260 nm) values of 51 (Fulton and Potter, 1971) and 239 (Ghabrial and Lister. 1974) in the case
218
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VAN VLOTEN-DOTING
of nucleoproteins and RNA, respectively. The RNA content was taken as 14% (Ghabrial and Lister, 1974). For TSV coat protein, A:& (280 nm) was taken as 70. Peptide mapping and limited tryptic digestion. These were performed by Ing. A. Neeleman, G. M. A. van Beynum and F. Th. Brederode, respectively. The methods described by Bol et al. (1974) were used. RESULTS
Nomenclature of TSV Nucleoproteins TSV-RNA’s
and
Originally Fulton (1970) found four components of TSV and designated them, in order of decreasing sedimentation velocity, c,b,a and T. Later they were renamed by Fulton (1972) as follows: b, bottom component; a, middle component; T, top component; while c was not described any further. Lister and Bancroft (1970) and Clark and Lister (1971) described three components: BV, bottom component; MV, middle component; and TV, top component. Lister et al. (1972) mentioned that sometimes, especially when the virus was cultivated in Christie’s hybrid, an additional component called super top component, STV, was present. In my hands, material of TSV-WC usually contained four components, which I assume to be identical to the BV, MV, TV and STV described by Lister and co-workers, while the three fastest-sedimenting components could correspond to b, a, and T of Fulton. However, I am not completely sure of this and should prefer to number the components, in order of decreasing sedimentation velocity, NP 1, NP 2, NP 3 and NP 4. Clark and Lister (1971) described four RNAs, BN, MN, TN’ and TTN, while sometimes an additional RNA, TN”, was found between TN’ and TTN. We found mostly four RNA’s (RNA 1, RNA 2, RNA 3 and RNA 4, in order of increasing electrophoretic mobility on polyacrylamide gels); sometimes an additional RNA was found ahead of RNA 3. This nomenclature has the advantage that it corresponds with that used for the bromo- and cucumoviruses.
Biological Activity of TSV Nucleoproteins Figure 2A shows the sedimentation pattern of TSV nucleoprotein isolated from Christie’s hybrid. Fig. 2B represents the infectivity of the peak fractions and of the combinations of these fractions. The infectivity of NP 1 and NP 2 was very low, while NP 3 and NP 4 showed no infectivity at all. Combination of NP 1 with either NP 2 or NP3 had five to seven times higher infectivities than NP 1 alone. The combination of NP 1 with NP 2 and NP 3 was two to seven times as infectious as the twofold combinations. Addition of NP 4 to the threefold combination did increase the infectivity slightly; however, this was not reproducible. When all components had been purified by a second cycle of sucrose density gradient purification in a zonal rotor the infectivity of the twofold combinations was much lower; addition of a third component to a mixture of two components enhanced the infectivity considerably (Fig. 2C). NP 4 is apparently inert (Fig. 2D). The RNA content of the purified components was analyzed by electrophoresis on polyacrylamide gels in the presence of SDS. Each component was found to contain mainly one species of RNA (results not shown). The simplest explanation for the results presented above is that the TSV genome consists of three RNA’s (RNA 1, RNA 2 and RNA 3), each encapsidated in one specific component (NP 1, NP 2 and NP 3). Molecular Species Required for Infectivity Assuming that the TSV genome is tripartite, the situation in TSV could be like that in BMV (Lane and Kaesberg, 1971), CCMV (Bancroft, 1971) or CMV (Peden and Symons, 1973; Lot et al., 1974) where the combination of the three RNA’s is infectious itself, or it could be like that in AMV (Bol et al., 1971) where the three RNA’s need to be activated by the coat protein. It has been shown that AMV top component a RNA (comparable in size with RNA 4 of the other viruses) is also capable of activating the AMV genome (Bol et al., 1971). Like RNA 4 of BMV
EQUIVALENCE
219
OF TSV AND AMV PROTEINS
0. D
D
T , .
FK. 2. Purification of a TSV preparation in order to determine the components required for infectivity. (A), Optical density profile of a sucrose density gradient in which 20 mg of a TSV preparation was centrifuged. Centrifugation in a Spinco B IV zonal rotor for 5.25 hr at 31,500 rpm and 4’. (B), Infectivity of the peak fractions and the combinations of the peak fractions from (A). (Cl, Infectivity of components and combinations of components purified by two cycles of sucrose density gradient centrifugation in a zonal rotor. (D), The same as (C) except that to all solutions NP4 had been added. Infectivities of components at the vertices, infectivities of the twofold combinations at the sides and infectivities of the threefold combinations at the center of the triangles are shown. Lesion numbers of one triangle are averages (B) or totals (C) and (D) from one incomplete block. Blocks belonged to different batches of plants. Concentration of all components in all combinations of (B) is 1.0 &ml, (Cl, 0.5 &ml and (D), 0.5 &ml.
(Shih and Kaesberg, 1973) and probably RNA 4 of other bromo- and cumumoviruses (Davies and Kaesberg, 19741 top component a RNA of AMV contains the genetic information for the coat protein (Van Ravenswaay Claasen et al., 1967; and other work from our laboratory, to be published) and is apparently translated in the cell. Preparations of TSV-RNA do contain only low amounts of RNA 4. It is unknown whether RNA 4 of TSV functions as messenger for the coat protein. Even if this RNA in the cell were translated into coat protein, the amount of RNA 4 in a total RNA preparation might be limiting. If the coat protein were required for infectivity its addition to an unfractionated RNA prepa-
ration might still enhance infectivity. Figure 3 shows that indeed the infectivity of unfractionated TSV-RNA increases upon the addition of increasing amount of TSV coat protein. In order to investigate whether a combination of the three largest TSV-RNA’s can also be activated by TSV-RNA 4, a total RNA preparation was run on a sucrose density gradient. Figure 4 shows that the fractions of this gradient are not or are only slightly infectious. However, when to each fraction an equal volume of fraction 20 was added, infectivity was found in that region of the gradient where RNA’s 1, 2, and 3 overlap. Together, the results from Figs. 3 and 4 suggest that TSV-RNA 4 contains the genetic information for the coat pro-
LOUS
VAN VLOTEN-DOTING
the homologous as well as in the heterologous combinations (results not shown). The activity of TSV coat protein was lost upon heating at 60” for 5 min. It was shown earlier (Bol et al., 1971) that the activity of AMV coat protein was sensitive to the same treatment. Differences Between TSV Coat Protein and AMV Coat Protein
10 number
20 of protein RNA
30
40
molecules
molecule
FIG. 3. Comparison of the capability of different viral coat proteins to activate the TSV genome. TSV-RNA (100 &ml) was combined with solutions of TSV coat protein (O-O), AMV coat protein (OA), BMV or CMV coat protein (W-m). For the molecular weights of these coat proteins values of 30,000, 24,800, 20,300 and 24,200 were used (Ghabrial and Lister, 1974; Kraal et al., 1972; Stubbs and Kaesberg, 1967; Hill and Shepherd, 1972). Lesion numbers are averages from seven half-leaves.
tein, as do the comparable AMV and BMV.
The fact that TSV coat protein and AMV coat protein can both serve the same function could mean that (part) of these proteins are identical. The following experiments were performed to investigate this possibility. Serology. Neither our TSV nucleoprotein preparations nor our TSV protein preparations gave precipitation lines with antiserum raised against AMV nucleoprotein. Limited trypsin digestion. Bol et al. (1974) showed that 27 amino acids can be removed from the N-terminus of the AMV coat protein by incubating AMV particles at pH 7.0 with 1 &ml of trypsin for 20 hr
r -
260
RNA species of
Equivalence of TSV Coat Protein and AMV Coat Protein in the Activation of the Genomes of TSV and AMV Figure 3 demonstrates that TSV-RNA can be activated by TSV coat protein and by AMV coat protein, while the addition of BMV coat protein or CMV coat protein has no effect at all. When AMV-RNA instead of TSV-RNA was used the same results were found (not shown). In all cases the kind of lesion was typical for the virus from which the RNA had been used. Instead of the isolated coat proteins, their presumed mRNA’s (TSV-RNA 4 and AMV top component a RNA) or the purified nucleoprotein components could also be used in
5
Fraction
number
FIG. 4. Activation of RNA 1 + RNA 2 + RNA 3 by RNA 4. Optical density profile (-) of a sucrose density gradient in which 0.4 mg of TSV-RNA was run. Infectivity of fractions after addition of an equal volume of buffer (O-O) and after addition of an equal volume of fraction 20 (RNA 4) (x-x ). Lesion numbers are averages from seven half-leaves.
EQUIVALENCE
at 25”. Incubation of TSV particles under the same conditions had a more pronounced effect on the size of its coat protein but the conversion was not complete. At a trypsin concentration of 100 &ml, all TSV subunits were converted to a much smaller product (Fig. 5). It is curious that the AMV and TSV coat proteins do migrate with the same mobility since the molecular weights are different, viz., 24,800 and 30,000, respectively (Kraal et al., 1972; Ghabrial and Lister, 1974). Tryptic fingerprints. TSV and AMV coat protein were digested by trypsin. Figure 6 shows that the peptide maps of the two proteins are very different. However, a few spots might be identical and will be investigated further. Attempts to Construct TSV and AMV
Hybrids
221
OF TSV AND AMV PROTEINS
Between
For both TSV (Fulton, 1972) and AMV (Van Vloten-Doting et al., 1968, 1970; Dingjan-Versteegh et al., 1972, 1974; Bol and Lak-Kaashoek, unpublished results) it has been shown that combinations of components from different virus strains are infectious and yield hybrid strains. Bancroft (1972) demonstrated that a hybrid virus could be made from two components of BMV and one component of CCMV. The reverse combination was not
FIG. 5. Comparison of the effect of trypsin on AMV coat protein and TSV coat protein From both viruses 2 mg/ml of nucleoprotein was incubated for 20 hr at 25” with the indicated concentration of trypsin in 0.05 M Tris, 0.001 M EDTA and 0.001 M NaN, at pH 7.0. The incubation was stopped by the addition of SDS. The upper band of all gels represents a reference protein added to the samples.
infectious. The hybrid infected only those plants that could function as host for both parent viruses. I determined whether the infectivity of a combination of two components of TSV did increase upon the addition of a third component of AMV. The same was done with the reverse combinations. The mixtures were inoculated on Berna beans, since these plants produce large local lesions with TSV and small ones upon infection with AMV. In these experiments NP 1, NP 2 and NP 3 of TSV were taken to be analogous to bottom component, middle component and top component b of AMV, respectively. Since the infectivity of the threefold combinations was never higher than that of the twofold combinations, I had to conclude that either the components of TSV and AMV are not interchangeable at all or that the infectivity of hybrids between these viruses is much lower than that of either virus or its interstrain hybrids. Interaction between RNA ‘s
Nucleoproteins
and
In a previous publication (Van VlotenDoting and Jaspars, 1972) it was shown that, upon addition of AMV-RNA to AMV nucleoprotein, some coat protein molecules from the nucleoprotein become associated with the viral RNA. The reaction is highly specific, and it was postulated that AMVRNA has a few sites with strong affinity for AMV coat protein. Since these high-affinity sites might be connected with the biological role of the AMV coat protein, I thought it worthwhile to investigate whether TSV-RNA would react with TSV and AMV nucleoproteins and whether AMV-RNA would react with TSV nucleoprotein. AMV or TSV nucleoproteins were run on sucrose density gradients after incubation with different RNA’s or with buffer. From Figs. 7A-C it is evident that the AMV and TSV-RNA’s react in the same way with AMV nucleoprotein. In both cases the infectivity in the RNA regions of gradients run with the mixtures was much higher than the infectivity of a comparable region of a gradient run with RNA alone, suggest-
222
LOUS
VAN VLOTEN-DOTING
Fm. 6. Tryptic “fingerprints” of TSV coat protein (A) and AMV coat protein (B). Experimental details as described by Bol et al. (1974). Chromatography was in the horizontal direction, from right to left, high voltage electrophoresis was in the vertical direction, cathode at the top.
O.D.
D
_,r,
Fraction number Fro. 7. Effect of AMV- and TSV-RNA on AMV and TSV nucleoprotein (-1. (A), 100 pg component; (B,C), 100 pg of AMV bottom component incubated with 100 rg AMV-RNA respectively: (D), 100 pg of TSV NP 3; (E,F), 100 ag of TSV NP 3 incubated with 100 bg of TSV-RNA, respectively. In all cases incubation was for 32 hr at 4” in 0.5 ml of PEN buffer. - run in sister tubes; (B,E), 100 pg of AMV-RNA and (C,F), 100 plugof TSV-RNA, respectively.
of AMV bottom and TSV-RNA, AMV-RNA and -, RNA controls
EQUIVALENCE
OF TSV AND AMV PROTEINS
ing the presence of coat protein in the former. Furthermore the residual nucleoprotein was sensitive to bean leaf sap, suggesting the disintegration of the viral structure. Figures 7D-F show that, in the gradients run with mixtures of TSV nucleoprotein with either TSV-RNA or AMV-RNA incubated under the same conditions, the nucleoprotein peaks did not disappear but got a shoulder on the meniscus side. In this case the infectivity of the RNA regions of the gradients did not increase, and the infectivity of the nucleoproteins did not become sensitive to leaf sap. Other RNA’s (BMV-RNA, CMV-RNA or tobacco leaf RNA) did not change the pattern of either nucleoprotein under these conditions (results not shown). DISCUSSION
The results obtained with the TSV nucleoproteins can best be explained by the assumption that the TSV genome consists of three RNA’s, each separately encapsidated. Fulton (1970) demonstrated that his component b (probably our NP 1) had a low but significant intrinsic infectivity. The dilution curve of purified component b was found to be rather steep, suggesting that two or more particles cooperate in the formation of each infection. Furthermore, Fulton (1972) showed that there exists redundancy in the genetic information among the particle types of TSV. In our opinion these results can be explained by the assumption that each nucleoprotein component contains, in addition to its main RNA species, small amounts of other RNA species. Evidence for this has been reported by Clark and Lister (1971). There could be a variation in the amounts of minor RNA’s of a given nucleoprotein component depending on the cultivation circumstances. For the infectivity experiments in this report, the virus has been cultivated in Christie’s hybrid at 23”, while Fulton cultivated it in Nicotiana tabacum L., in N. rustica L. or in Datura stramonium L. at 24-25”. Recently Heytink and Jaspars (1974) showed that the bottom component of
223
AMV (comparable to NP 1 of TSV) can contain, besides bottom RNA, traces of the smaller AMV-RNA’s. The intrinsic infectivity that has been claimed for the bottom component of AMV by Majorana and Paul (1969) and by Desjardins and Steere (1969) may be due to the presence of M-RNA and Tb-RNA (comparable to TSV-RNA 2 and 3) in pseudo-bottom particles. As with AMV, only the three fastestsedimenting nucleoproteins of TSV appear to be necessary for infectivity. By separating the TSV-RNA’s by sucrose density gradient centrifugation it was possible to demonstrate that the combination of the three largest RNA’s was not infectious by itself. However, infectivity was found when either TSV-RNA 4 or AMV-top component a RNA or TSV coat protein or AMV coat protein was added. This finding explains why Clark and Lister (1971) found that nucleic acid preparations of TSV, even when they had been carefully prepared and appeared intact, showed only very low infectivities. The equivalence of RNA species 4 and the coat protein of TSV may be explained by the assumption that the RNA contains the genetic information for the coat protein. The equivalence between AMV coat protein and TSV coat protein is more difficult to understand. According to different criteria, immunology, limited trypsin digestion and peptide mapping, the two proteins cannot have many amino acid sequences in common. The possibility that there are a few critical identical amino acid sequences is under investigation. The results do not exclude the possibility that the peptide chains are folded in such a way that similar groups are found on the surface of the coat protein. It has been proposed that in AMV the coat protein fulfills its function by associating with specific sites on the RNA (Van Vloten-Doting and Jaspars, 1972). It is remarkable also that TSV-RNA has a very high affinity for AMV coat protein, because it is able to withdraw subunits from AMV particles. Apparently there is some kind of cross-recognition between TSV-RNA and AMV coat protein, on the one hand, and AMV-RNA and TSV coat protein, on the other. Bol et al. (1975) have
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VAN VLOTEN-DOTING
shown that there exists no detectable homology in the primary sequence of the RNA’s of TSV and AMV, although it has to be remembered that the method used (competitive hybridization) would not reveal the presence of very short identical regions. Also the interaction might depend on secondary and tertiary structures rather than on primary structure. The fact that under identical circumstances AMV nucleoprotein loses protein subunits to RNA, in contrast to TSV nucleoprotein, probably reflects a difference in stability (compare Fig. 7). Both nucleoproteins seem to be primarily stabilized by RNA-protein interactions according to criteria defined by Kaper and Geelen (1971). However, in AMV nucleoprotein the subunits seem to be less tightly bound to their RNA than in TSV nucleoprotein. This difference in stability is also reflected by the difficulties encountered during the preparation of TSV coat protein and TSVRNA. The difficulty of extracting intact TSV-RNA has already prompted Clark and Lister (1971) to suggest a particular kind of structural relationship between TSV protein and RNA in the particle. The high affinity for AMV coat protein displayed by TSV-RNA could result in the occurrence of genomic masking and phenotypic mixing in doubly infected plants. Preliminary experiments have already indicated that at least TSV-RNA can be encapsidated by AMV coat protein (unpublished results). It is remarkable that, although the coat proteins of TSV and AMV can be substituted for each other in the onset of infectivity, up to now it was impossible to construct hybrids between the viruses. Even the combinations in which only the top component b of AMV had been exchanged for NP 3 of TSV, or vice versa, were not infectious. This could mean that top component b RNA of AMV and RNA 3 of TSV do contain besides the information for the coat protein (Dingjan-Versteegh et al., 1972; Fulton, 1972) the information for some other protein which is not interchangeable. This protein might be a replicase subunit. Also it is quite possible that the different virus functions are not dis-
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