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Replication of tobacco mosaic virus
VIROLOGY
63, 75-83 (1973)
Replication IV. Further AT,RERT fh?VO,Tfm.ent
of Tobacco
Characterization
SIEGEL3,
of Agricultural
of Viral
MILTON
AND
The University
January
Virus
Related
ZAITLIN,
&ochemistry, Accepted
Mosaic
RNAsls2
C. T. DUDAa
of Arizona,
Tucson 86791
30, 1.973
Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5’ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form. INTRODUCTION
RNA preparations from tissue infected with tobacco mosaic virus (TMV) contain several species of RNA that are absent from extracts of healthy tissue. In addition to viral RNA, there are the replicative intermediate (RI), replicative form (RF), and a low molecular weight single-stranded species of about 350,000 daltons (LMC) (cf. Jackson et al., 1972). The present work was designed to determine whether LMC might be a fragment of viral RNA. We have found that it is, and we have determined some of its 1 This investigation was supported in part by grants from the National Science Foundation and Contract AT(ll-l)-873 from the Atomic Energy Commission. 2 University of Arizona Agricultural Experiment Station Technical Paper No. 2030. 3 Present address: Department of Biology, Wayne State University, Detroit, Michigan 48202. 75 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
properties. We have also characterized further the other virus-related RNA species. MATERIALS
AND METHODS
Labeling and incubation of plant materials. Either separated tobacco (Nicotiana t&cum L. Turkish Samsun) cells prepared according to Jensen et al. (1971) or tobacco leaves cut into strips ca. 2 mm wide were used in these experiments. Inoculation and plant maintenance was as described before (Jackson et al., 1972). When cells were used, they were labeled with 3H-uridine as described by Jackson et al. (1972) and incubated at 25” in the light (450 ft-c) for periods indicated in the experimental section. The leaf strips, 1 g, were labeled by immersion in 5 ml of 0.01 M KHTPO, containing 0.5 mCi [5-3Hluridine and, in some experiments, 200 pg actinomycin D (Calbiochem). The tissue was then infiltrated with the suspending medium and incubated under fluo-
76
SIEGEL,
ZAITLIN,
rescent light (ca. 700 ft-c) for indicated periods. Extraction of RSA. Following the incubation period, the cells were sediment’ed at 80 g, washed once with incubation buffer (Francki et al., 1971), and frozen with liquid nitrogen; leaf strips were placed in a stainless steel kitchen strainer, washed thoroughly with tap water, placed in a mortar and frozen with liquid nitrogen. The leaf st’rips were pulverized while frozen, and the following was added: 3 ml of a 85 : 15 phenolcresol solution, 0.1% in 8-hydroxyquinoline and 3 ml TNE buffer (0.1 M Tris, 0.1 M NaCl, 0.01 M Naz EDTA, pH 7) containing 2% sodium dodecyl sulfate. Cells received the same reagents but were not pulverized. The mixtures were stirred as thawing took place and then were shaken vigorously before being centrifuged to separate the phases. The aqueous phase was reextracted with the phenol mixture and nucleic acids were precipitated by adding 1.5 volumes of isopropanol and a few drops of 3 M Na acetate, pH 4.0. The precipitates were redissolved in 1 ml of half-strength STJ#lg buffer (STMg buffer is 0.2 M NaCl, 0.01 M Tris, 0.01 M MgCl, , pH 7.6), 25 pg pancreatic DNase were added (Worthington, RNase free), and the extract was incubated at 37” for 20 min. A lo-p1 portion of the extract was taken for determination of TCA-precipitable radioactivity and depending upon the amount of radioactivity present, a sample was either taken directly for electrophoresis or the RNA was first concentrated by ethanol precipitation into a smaller volume of one-third strength STMg. Electrophoresis. Fifty to 100 ~1 of a radioactive RNA preparation was loaded onto a 11 cm 1.8% polyacrylamideeO.5% agarose gel and electrophoresis was conducted as described (Jackson et al., 1971) at 5 mA/gel for 0.5 hr beyond the time a bromophenol blue dye marker migrated off the gel (ca. 4.5 hr). The gels were then frozen. Extraction of RNA from polyacrylamide gels. The frozen gels were sliced with a manifold of razor blades spaced 1.07 mm apart. RNA was extracted from the gel slices by placing 2 (sometimes 3) consecutive slices in tubes containing 0.5 ml PE buffer (0.01 M Na2HP04, 0.005 M Na2EDTA,
AND
DUDA
pH 7). The tubes were shaken in an ice water bath for several hours and allowed to sit at 4” overnight. The 0.5 ml of liquid was removed and the gel slices were washed with another 0.5 ml portion of PE buffer for 2-4 hr, and this was removed and combined with the first extract. About half the counts applied to the gel were extracted by this method. At least some of the nonrecovered radioactivity is accounted for by low molecular weight RNA species which were extracted but which were allowed to run off the gel during electrophoresis. That all of the larger species of RNA were extract,ed proportionally by this method was indicated by the pattern of radioactivity extracted from the gel slices compared to the pattern obtained by total radioactivity determination of slices of sister gels after total digestion with HaOz . Analysis of gel extracts and hybridization studies. Aliquots of 0.05 to 0.2 ml were removed from the gel slice extracts for different analytic procedures as follows : a. Determination of total radioactivity extracted from the gel: Generally 0.05 ml was taken and pipetted directly into a vial, adding 0.45 ml NCS reagent (Amersham/ Searle) and scintillation fluid as described below. b. Determinat’ion of RNasc resistance before heat denaturation: Usually O.l-ml samples were removed from the gel extracts and sufficient concentrated buffer was added to make the salt concentration at least 2 X STE (STE buffer is 0.1 M NaCl, 0.05 IL! Tris, 0.001 M EDTA, pH 8) and the volume close to 0.2 ml. Ten microliters of an RNase solution (1 mg/ml pancreatic ribonuclease A, Worthington and 0.05 mg/ml Tl ribonuclease, Worthington) were added and the samples were incubated at 37” for 20 min. The samples then were transferred quantit’at’ively to 3 RIM filter disks (Whatman 1 in) and counted as described below. The counting efficiency of this filter disk plating procedure was determined to be the same as that of (a) above, so results from the two methods may be compared directly. Before the remainder of the analyses were performed the tubes containing the gel extracts were immersed in boiling water for 2 min and then cooled in ice water to ensure
TMV-RELATED
heat denaturation (melting) of any doublestranded RNA that might be present. c. Determination of RNase resistance after melting: The same procedure was followTed as in (b) above. d. Self annealing: The procedure as in (b) was followed except that after the salt concentration was increased and before ribonuclease treatment, 10 ~1 of water was added and the preparation was incubated at 70” for 4 hr. e. Annealing in the presence of TMV RNA: Except as indicated in the text, the procedure in (d) was followed except that 10 ~1 of water containing 0.05 pg TMV RNA (phenol extracted from virions) was added before the 70” incubation. f. Annealing in the presence of DS RNA: The procedure as in (e) was followed except that instead of TMV RNA, the 10 ~1 of water contained 0.1 pg of melted DS TMV RNA (prepared according to Jackson et al., 1971). g. Reconstitution with TMV protein: Solutions (0.2 ml) containing 50 ~1 of gel slice extract adjusted to 0.1 M NaqP207, pH 7.2, and 170 pg TMV protein (prepared according to Fraenkel-Conrat, 1957) were incubated for 18 hr at room temperature, treated with RNase as in the other assays, transferred to 3 MM disks, and counted. h. Assay for the presence of polyadenylic acid sequences. Three assay methods were used: (1) Aliquots of the gel slice extracts (0.2 ml) were mixed with 10 ml of high salt buffer (0.5 M KCI, 10 mM Tris, 1 mM MgClz ) pH 7.6) and filtered through 24 mm nitrocellulose filter membranes (B-6, Schleicher and Schuell) according to Lee et al. (1971). After two washes with the high salt buffer the filters were dried and counted. (2) Aliquots (0.1 ml) of the gel slice extracts were added to 5 ml of binding buffer (0.01 M Tris, 0.12 M NaCl, pH 7.5) and filtered through glass fiber filters (Whatman, GF/C, 24 mm) which had been embedded with 0.1 mg polyuridylic acid (Schwartz/Mann) and irradiated with UV according to Sheldon et al. (1972). (3) Aliquots (0.2 ml) of the gel slice extracts were made to 2 X STE and 0.1 #Zi [3H]polyuridylic acid [7 mCi/ mmole P, Schwartz/Mann] was added. The
77
RNA8
samples were then treated as in assay d (Gillespie et al., 1972). Determination of radioactivity. Radioactivity was determined in assay a by adding 10 ml of a PPO-POPOP scintillation fluid and counting in a Packard Tri-Carb, Model 3320 scintillation spectrometer. Cold trichloroacetic acid-precipitable radioactivity remaining after treatment in assays b through g, and h2 and h3 was determined by applying total samples to two 25 mm filter paper disks (Whatman 3MM) and washing them according to Byfield and Scherbaum (1966). The material remaining on the disks was digested for 0.5 hr at 50” in 0.5 ml of a mixture of 9 parts NCS reagent (Amersham-Searle) to 1 part water and count’ed in 10 ml PPO-POPOP scintillation fluid. Radioactivity in assay hl was determined by immersing the filter membrane in 10 ml of scintillation fluid, without NCS treatment, and counting. The results of the analyses performed on aliquots of the gel extracts were adjusted to make them equivalent to the original volume of the extracts. In order to determine the total radioactivity contained in gel slices (as in Fig. l), 3 consecutive slices were placed in glass scintillation vials containing 0.75 ml H,Oz and were incubated overnight at 50” for digestion. Ten milliliters of a PPO-POPOP scintillation fluid containing 33 % Triton X-100 and 67% toluene were added before counting. RESULTS
Several experiments were performed both with separated cells and leaf strips. All experiments yielded the same type of result. The following data were obtained from an experiment in which leaf strips from uninfected and from 6 day-infected leaves were infiltrated with 3H-uridine with or without actinomycin D and incubated for 2 hr before RNA was extracted and subjected to electrophoresis. Distribution of Radioactive RNA acrylamide Gels
in Po(y-
Figure 1 shows the distribution of radioactivity in RNA contained in gels of TMV-
78
SIEGEL,
ZAITLIN,
AND DUDA
FRACTION NUMBER
FIQ. 1. Distribution of radioactivity in polyacrylamide gels containing RNA extracted from TMYinfected leaf tissue treated with actinomycin D, and comparable untreated tissue. Two l-g samples of leaf tissue infected for 6 days were vacuum infiltrated (3 times) with 5 ml of a solution containing 0.01 M KHePOa , 500 &i 8H-uridine, and in one case 40 rg/ml actinomycin D. The tissue was incubated under a fluorescent light (ca. 700 ft-c) for 2 hr at room temperature. RNA extracts were prepared, gels were run and sliced, and 3 consecutive 1.07-mm slices were digested with Hz02 as described in Materials and Methods. The top of the gel is on the left in this and all other gels presented. Actinomycin D treated (o-0); not treated with actinomycin D (e-----O).
infected leaf tissue which had been incubated with and without actinomycin D treatment. The patterns from diseased and uninfected leaf tissue (not shown here) are similar to those reported by Jackson et al. (1972) for separated cells. Treatment with actinomycin D appears to be more effective when applied to leaf strips than to separated cells, possibly because the inhibitor may be protected from light inactivation upon infiltration into intracellular spaces. The data confirm our previous observation (Jackson et al., 1972) that incorporation of a radioactive precursor into RNA species that are unique to infected tissue was not inhibited by actinomycin D. The unique species are replicative form (RF) at fractions 5 and 6, TMV RNA at fraction 14, and the low-molecular-weight component (LMC) at fractions 29 and 30. Replicative intermediate was not detected in these extracts
because
of the
long
incorporation
period (Jackson et al., 1972) Analysis of Extracts from Electrophoretic Gels Containing RNA from TMV Infected and Uninfected Actinom,ycin D-Treated Leaf Strips The PE buffer-extracts of gel slices were analyzed in a number of ways in order to
characterize further the RNA species unique to TMV infection. The description of the results that follow and the figures depicting them (Figs. 2 and 3) represent aliquots of the same extracts and thus are compared directly. It, appears that very little RNA becomes labeled in uninfected, actinomycin D-treated tissue (Fig. 2A) but, in fact, much of what does get labeled is probably of low molecular weight and runs off the gel during electrophoresis, for in this experiment there is onethird as much radioactivity in the healthy, actinomycin D-treated RNA preparation as in a comparable amount of untreated healthy tissue. It is clear, however, that the TMV gel is qualitatively different, exhibiting the same 3 virus-related RNAs seen in Fig. 1, i.e., RF peaking at fraction 8, TMV RNA peaking at fraction 22, and the LMC at fraction 45. The results shown in Fig. 2B were obtained when aliquots of the eluates of the gel slices were treated with RNase before and after melting. It is evident that the material present in fractions 7-9, called RF, is the only RNase-resistant RNA in the preparation; it is RNase resistant before melting and virtually completely digested by RNase after
TMV-RELATED
RNAse
SENSITIVITY
P
uI
- BEFORE YELTH(G I, AFTER MELTWG
% o
-o-WWFECTED
I-
TMV RN.4 3
TOTAL
FRACTION
0
79
RNAs
anneal with the TMV RNA at fraction 22. It is to be noted that the amount of ribonuclcase resistance in fraction 8 following annealing was less than would be predicted if an appreciable amount of the material in this fraction were originally a doublestranded structure. This proved to result from inefficient anncaling conditions due to the low concentration of t.he RNA; by in-
COUNTS
NUMBER
2. Analysis of radioactivity extracted FIG. from actinomycin D-treated, TMV-infected, and uninfected leaf tissue. A portion of the RNA extract from rtctinomycin D-treated TMV-infected tissue used in the experiment depicted in Fig. 1 and a comparable RNA extract from uninfected tissue were electrophorcsed on gels and the gel slices (2 per fract.ion) wcrc extracted in PE buffer as described in Materials and hfethods. Figure 2A shows the t.otal radioactivity extracted from each 2-slice fraction from TMV-infected and uninfected tissues and 213the effect of RNase treatment on every second fraction of those extracts, and on selected tubes of the 1’1fV extract after heating them for 90 set and quick cooling ~-9described in Materials and Methods. Sotc that the ordinates differ by a factor of 10 in 2A and 2B.
melting, suggesting that it is indeed doublcstranded RF. The conclusion that the RXase-rwistant mat,crial in the RF region is double-stranded is supported by the rcannenling experiment (Fig. 3A) because the RNA in this region recovers KX\‘ase resistance when allowed to reanncnl. This experiment also demonstrates that the RF region is the only one which contains complementary RSA strands because this is the only region which displays RNase resistance after the annealing trcatis ruled out, for mctnt . The possibility instance, that significant amounts of singlestranded KXA complementary to and of the same size as TMV RSA had been encapsidated with viral protcin and might thcreforc
:P 6i 5-
ANNEAL DOUBLE
FRACTION
WITH MELTED STRAMED RNA
NUMBER
FIG. 3. Annealing and reconstitution experiments with aliquots of the gel extracts depicted in Fig. 2A. Aliquots of extracts eluted from the gel were either self-annealed (A) ; annealed in the presence of 0.05 fig TMV RNA (B); annealed in the presence of 0.1 M melted DS TMY RX-4 (C); or reconstituted with an excess of TMV protein (D). After RNnse treatment,, RIKase-resistant RNA was dctermincd aa described in Mstcrials and Methods. The recovered radioactivities were recalculated to be equivalent to the total volume of each fraction, so that Figs. 2.4 and 13and Figs. 3.4 through 3D are comparable, even though all the ordinates do not. have the same scale.
80
SIEGEL,
ZAITLIN,
creasing the concentration of fraction 8 four-fold during the annealing, the amount of RNA converted to RNase resistance increased to about half the amount present before melting (data not shown). The result of annealing the gel fraction extracts in the presence of TMV RNA (Fig. 3B) reveals that RF at fraction 8 contains the only material in the gel complementary to viral RNA, for this is the only region which cont.ains material which anneals to TMV RNA. The small peak shown in the TMV RNA region (fraction 22) was not consistently observed and we believe has no significance. Figure 3C presents data of the annealing of the gel fraction extracts in the presence of melted DS TMV RNA. This reagent contains both viral RNA and RNA which is complementary to viral RNA and, thus, is capable of detecting both types of RNA in the gel extracts. A comparison of Figs. 3B and 3C reveals that DS TMV RNA anneals with LMC in addition to its reaction with TMV RNA and RF, whereas TMV RNA anneals only with RF. The conclusion is clear that LMC is complementary to a component of DS TMV RNA and is most likely a fragment of TMV RNA. The results depicted in Fig. 3C are informative in another sense; LMC is not the only fragment which is most likely viral RNA in the gel extracts, although it appears to be the only one present in significant amounts of a definitive size. Appreciable annealing to the viral RNA complementary strand takes place with material of a broad size range, but smaller than TMV RNA, in fractions 28 to 38. The amount of this material varies in different experiments and may have its origin in a breakage of TMV RNA during extraction, or possibly it may represent incompletely synthesized viral RNA. Experiments with separated cells have so far yielded greater amounts of the polydisperse TMV RNA than have leaf strips, but we do not know whether this reflects a difference in virus replication or extraction conditions. Symmetry
of Labeling of RF Strands
Comparison of the amount of radioactivity rendered RNase resistant when the RF fraction is annealed in the presence of TMV RNA and melted DS TMV RNA yields in-
AND
DUDA
formation concerning the symmetry of labeling of the two strands of the RF fraction because both strands should anneal in the presence of DS TMV RNA whereas only the complementary strand anneals with TMV RNA. In order to obtain a valid estimate of symmetry, however, it is necessary to ensure that annealing is performed efficiently in the presence of an excess of additives. To this end twice the usual concentration of RF (fraction 9) was annealed in the presence of lo-fold the usual concentration of TMV and DS TMV RNA (0.5 and 1.0 pg, respectively). Under these conditions, 1225 cpm became resistant to RNase in the presence of TMV RNA whereas 3045 cpm did so in the presence of DS TMV RNA. Since twice as much RNA became resistant when RF was annealed with DS TMV RNA, these data indicate that during the 2hr incubation period of leaf strips both strands of RF were labeled almost equally. Whether the same would be true of a shorter labeling period remains to be determined. Reconstitution of LMC with TMV
protein
Having demonstrated that LMC is a definite sized fragment of TMV RNA, the gel extracts were incubated with an excess of TMV protein in order to determine whether LMC might combine with TMV protein to reconstitute an RNase resistant nucleoprotein rod. As seen in Fig. 3D, material in the RF, TMV RNA, and polydisperse TMV RNA regions becomes resistant to RNase degradation under these conditions but LMC (fraction 45) does not.4 Several investigators (e.g., Butler and Klug, 1971; Okada and Ohno, 1972) have demonstrated that the 5’ end of TMV RNA is essential for initiation of 4 About one-half of the counts assayed in the RF region were found not to result from reconstituted nucleoprotein, but rather they reflected self-annealed RF, because the reconstitution was performed under high salt conditions which protect RF from RNase. In a separate experiment, the solutions from fractions 8, 22, and 30 were diluted with water before RNase treatment. The counts obtained in the RF region fell to about one-half the value obtained in high salt while those in the TMV RNA region and at fraction 30 were unaffected, showing that there was also self-annealing in the RF region under reconstitution conditions.
TMV-RELATED
nucleoprotein reconstitution; thus, we feel that LMC is probably not a homogeneous fragment of RNA which contains the 5’ end of viral RNA. It may be a homogeneous fragment from some other region of the viral RNA or it may represent a heterogeneous population of fragments all of a similar size, it might have the 5’ end but be unable to associate for some other reason, or the level of reconstitution might be too low to detect’. to see Experiment)s were conducted whether L32C was encapsidated with viral protein in vivo, possibly as a short virus rod. TMV labeled with 3H-uridine was isolat,ed from tissue incubated as described in Materials and 14ethods and purified by differential centrifugation. RNA was isolated from the virus and was subjected to gel clect,rophoresis; the gels were scanned optically using the linear transport accessory of the Gilford Model 240 spectrophometer and sliced for determination of radioactivity. In these experiment’s there was no suggestion of either radioactivity or UV absorbing material in the LYIC region of the gel. However, we reasoned that if short virus rods containing LMC were present in the leaf, t’he technique of isolation of virus by differential centrifugation would tend to discriminate aga.inst them because they would not sediment as efficiently as t’he longer rods. Accordingly, we examined encapsidated RNA isolated without high speed ccntrifugat’ion. Tissue infected for 4 days was incubated in the presence of 3H-uridine for 7 hr. The tissue was then ground in a mortar with M/15 phosphate buffer and clarified at 20,000 g for 10 min. The clarified supernatant was treated with RNase A (10 pg/ml) and RNase Tl (0.5 pg/ml) at 37” for 1.5 hr, followed by Yronase digestion (0.2 mg/ml for a further 1.5 hr to help eliminate the RNase. RNA was then extracted with phenol and analyzed by gel electrophoresis. Once again there was no suggestion of either counts or UV absorbing material in the LMC region, supporting the conclusion that L?tlC is not encapsidatcd in, viuo. LMC may be the same as the “rapidly labeled RNA” identified by Babos (1969, 1971) as being associated with ribosomes in T,1’IV-infected leaves. If this is so it may function as a messenger RNA. Recently,
81
RNAs
evidence has accumulated to indicate that RNA possesses eukaryote messenger poly (A) sequences (i.e., Lee et al., 1971). In order to determine whether LMC, or any of the other viral RNA components contain such poly (A) sequences, the gel extracts were assayed by the three methods described in Materials and Methods, but neither LMC, TMV RNA, nor RF were found to possess sufficient poly(A) sequences t,o be detectable. If LMC functions as messenger, it does so without a discernible polyadenylate sequence. In addition, TMV RNA, if lacking a polyadenylate sequence, differs from some other viral RNAs that have been tested, such as those of poliovirus, eastern equine encephalitis virus, Sindbis, Columbia SK, and some of the RNA tumor viruses (Armstrong et al., 1972; Green and Cartas, 1972; Johnston and Bose, 1972; Gillespie et al., 1972). DISCUSSION
The experiments described here permit the conclusion that LMC is a fragment of TMV RNA. We could detect no aggregation with TlllV protein to form a nucleoprotein; thus it probably lacks the 5’ end of TMV RNA, unless the reconstitution is not detectable or is prevented by some other mechanism. The function, if any, of this component remains unknown although it could be a messenger, possibly for the viral coat protein. If so, it functions without a poly(A) sequence, characteristic of other eukaryotic messenger RNAs. Babos (1969, 1971) has identified a rapidly labeled RNA of about the same size as LMC associated with ribosomes of TMVinfected leaves and if LMC and this component are the same, its association with ribosomes lends weight to the speculation that it may be a specialized messenger RNA. Other examples of viral RNA fragments have been observed; one is the 20 S RNA found in cells which produce murine sarcoma virus but which is absent from transformed non-virus-producing cells (Tsuchida et al., 1972). Another is the 2.8 X 105d RNA component found in brome mosaic virus (Shih et al., 1972), which apparently is not necessary for infection but appears to contain part of the sequence specifying coat protein (Stubbs and Kaesberg, 1967). In contrast to
82
SIEGEL,
ZAITLIN,
LMC, the brome mosaic virus RNA fragment is encapsidated along with another viral RNA component into one of the virion components of this split-genome virus. Another small encapsidated RNA, from top component a of alfalfa mosaic virus, is necessary for infection along with three other separately encapsidated virion RNAs, but the addition of small amounts of coat protein to the inoculum will substitute for it. Bol and van Vloten-Doting (1973) have suggested that the function of this small RNA is to specify a few protein molecules which are essential to initiate infection, An RNA which, like LMC is probably not a constituent of the virion, has been discovered by Romero (1972) in leaves infected with broadbean mottle virus. Its function, like that of LMC, remains obscure. Other conclusions about the high molecular weight virus-related RNA components extracted from infected leaves are that the RF component contains the only negative strand found in the cell and both positive and negative strands of RF become about equally labeled during a 2-hr incubation period in the presence of radioactive precursor. This is somewhat surprising, for one would expect a more rapid Iabeling of viral RNA on the assumption that the negative strand in RF is the template for viral plus strand synthesis. Perhaps we would observe asynchronous synthesis with shorter labeling periods, or it may be that RF is not an intermediate in a direct path to viral RNA synthesis, as we have discussed previously (Jackson et al., 1972). LMC is consistently observed in infected tissue, but we have occasionally had suggestions that other TMV RNA fragments of definite size may also be present. It has been difficult so far to obtain clear-cut evidence for such components because of the presence of a variable amount of TMV RNA fragments of polydisperse size. We are puzzled about the origin of these fragments. They may represent breakage of TMV RNA during RNA extraction or possibly incomplete particles whose synthesis was interrupted by terminat.ion of the experiment. A comment is necessary concerning hybridization of the RNA in gel extracts. We have indicated that at times our annealing
AND
DUDA
conditions were less than optimal. We do not consider this to be a hazard as long as interpretation of data is based on a knowledge of experimental conditions. One type of pitfall that must be guarded against is met when one hybridizes 3H-TMV RNA extracted from gels, such as those described herein, with melted DS TMV RNA. The results are reliable only when the melted DS TMV RNA is in excess during the annealing reaction. It is essential to ensure that this, indeed, is the case. ACKNOWLEDGMENTS We are pleased to acknowledge technical assistance of Mary Petti Smith.
the capable and Ruth C.
REFERENCES ARMSTRONG, J., EDMONDS, M., NAKAZATO, H., PHILLIPS, B., and VAUGHAN, M. (1972). Polyadenylic acid sequences in the virion RNA of poliovirus and eastern equine encephalitis virus. Science 176,526-528. BABOS, P. (1969). Rapidly labeled RNA associated with ribosomes of tobacco leaves infected with tobacco mosaic virus. Pz?oZogy 39,893-900. BABOS, P. (1971). TMV-RNA associated with ribosomes of tobacco leaves infected with TMV. Virology 43, 597-606. BOL. J. F., and VAN VLOTEN-DOTING, L. (1973). The function of top component a RNA in the initiation of infection by alfalfa mosaic virus. Virology 61, 102-108. BUTLER, P., and KLUG, A. (1971). Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nature (London) New Biol. 229,47-50. BYFIELD, J. E., and SCHERBAUM, 0. H. (1966). A rapid radioassay for cellular suspensions. Anal. Biochem. 17,434443. FR.LENKEL-CONRAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Vi’irology 4, l-4. FRANCKI, R. I. B., ZAITLIN, M., and JENSEN, R. G. (1971). Metabolism of separated leaf cells. II. Uptake and incorporation of protein and ribonucleic acid precurors by tobacco cells. Plant Physiol. 48,14-18. GILLESPIE, D., MARSHALL, S., and GALLO, R. (1972). RNA of RNA tumour viruses contains poly A. Nature (London) New Biol. 236,227-231. GREEN, M., and CARTAS, M. (1972). The genome of RNA tumor viruses contains polyadenylic acid sequences. Proc. Nat. Acad. Sci. U.S. 69, 791-794.
TMV-RELATED A., MITCHELL, D., and SIEGEL, A. (1971). Replication of tobacco mosaic virus. I. Isolation and characterization of doublestranded forms of ribonucleic acid. Virology 45,182-191. JACKSON, A., ZAITLIN, M., SIEGEL, A., and FRANCKI, R. I. B. (1972). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48,655-&X JENSEN, R. G., FRANCKI, R. I. B., and ZAITLIN, M. (1971). Metabolism of separated leaf cells. I. Preparation of photosynthetically active cells from tobacco. Plant Physiol. 48,9-13. JOHNSTON, R. E., and BOSE, H. R. (1972). Correlation of messenger RNA function with adenylaterich segments in the genomes of single-stranded RNA viruses. Proc. Nat. Acad. Sci. U.S. 69, 1514-1516. LEE, S., MENDECKI, J., and BRAWERMAN, G (1971). A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma
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RNAs
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180 ascites cells. Proc. Nat. Acad. Sci. U.S. 68, 1331-1335. OKADA, Y., and OHNO, T. (1972). Assembly mechanism of tobacco mosaic virus particle from its ribonucleic acid and protein. Mol. Gen. Genet. 114,X&213. ROMERO, J. (1972). RNA synthesis in broadbean leaves infected with broadbean mottle virus. Virology 48, 591-594. SHELDON, R., JURALE, C., and KATES, J. (1972). Detection of polyadenylic acid sequences in viral and eukaryotic RNA. Proc. Nat. Acad. Sci. U.S. 69,417421. SHIH, D. S., LANE, L., and KAESBERG, P. (1972). Origin of the small component of brome mosaic virus RNA. J. Mol. Biol. 64, 353-362. STUBBS, J., and KAESBERG, P. (1967). Amino acid incorporation in an Escherichia coli cell-free system directed by bromegrass mosaic virus ribonucleic acid. Virology 33, 38.5397. TSUCHIDA, N., ROBIN, M., and GREEN, M. (1972). Viral RNA subunits in cells transformed by RNA tumor viruses. Science 176, 1418-1420.
63, 75-83 (1973)
Replication IV. Further AT,RERT fh?VO,Tfm.ent
of Tobacco
Characterization
SIEGEL3,
of Agricultural
of Viral
MILTON
AND
The University
January
Virus
Related
ZAITLIN,
&ochemistry, Accepted
Mosaic
RNAsls2
C. T. DUDAa
of Arizona,
Tucson 86791
30, 1.973
Experiments were performed to characterize the viral related RNA species which appear in extracts of tobacco mosaic virus (TMV)-infected tissue and, in particular, the low molecular weight (ca. 350,000 daltons) component, LMC. It was determined that LMC is probably not a component of the virus rod but is a fragment of unincapsidated TMV RNA. Synthesis of LMC in diseased tissue is not inhibited by the presence of actinomycin D. Because LMC would not reconstitute into a rod with TMV protein, it was considered not to contain a detectable amount of the 5’ terminus of TMV RNA. Polyadenylic acid sequences could not be detected by three analytical methods in any RNA component. In addition to LMC, which is homogeneous in size, TMV RNA fragments polydisperse in size are also present in leaf tissue extracts. In contrast, RNA complementary to TMV RNA was present in extracts only as a component of the double-stranded TMV replicative form and was not found free in the infected cell. Neither fragments of replicative form nor single-stranded TMV complementary RNA could be detected. In addition, the only RNA fraction of uniform size found to contain both an RNA species and its complement was the TMV replicative form. INTRODUCTION
RNA preparations from tissue infected with tobacco mosaic virus (TMV) contain several species of RNA that are absent from extracts of healthy tissue. In addition to viral RNA, there are the replicative intermediate (RI), replicative form (RF), and a low molecular weight single-stranded species of about 350,000 daltons (LMC) (cf. Jackson et al., 1972). The present work was designed to determine whether LMC might be a fragment of viral RNA. We have found that it is, and we have determined some of its 1 This investigation was supported in part by grants from the National Science Foundation and Contract AT(ll-l)-873 from the Atomic Energy Commission. 2 University of Arizona Agricultural Experiment Station Technical Paper No. 2030. 3 Present address: Department of Biology, Wayne State University, Detroit, Michigan 48202. 75 Copyright All rights
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
properties. We have also characterized further the other virus-related RNA species. MATERIALS
AND METHODS
Labeling and incubation of plant materials. Either separated tobacco (Nicotiana t&cum L. Turkish Samsun) cells prepared according to Jensen et al. (1971) or tobacco leaves cut into strips ca. 2 mm wide were used in these experiments. Inoculation and plant maintenance was as described before (Jackson et al., 1972). When cells were used, they were labeled with 3H-uridine as described by Jackson et al. (1972) and incubated at 25” in the light (450 ft-c) for periods indicated in the experimental section. The leaf strips, 1 g, were labeled by immersion in 5 ml of 0.01 M KHTPO, containing 0.5 mCi [5-3Hluridine and, in some experiments, 200 pg actinomycin D (Calbiochem). The tissue was then infiltrated with the suspending medium and incubated under fluo-
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SIEGEL,
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rescent light (ca. 700 ft-c) for indicated periods. Extraction of RSA. Following the incubation period, the cells were sediment’ed at 80 g, washed once with incubation buffer (Francki et al., 1971), and frozen with liquid nitrogen; leaf strips were placed in a stainless steel kitchen strainer, washed thoroughly with tap water, placed in a mortar and frozen with liquid nitrogen. The leaf st’rips were pulverized while frozen, and the following was added: 3 ml of a 85 : 15 phenolcresol solution, 0.1% in 8-hydroxyquinoline and 3 ml TNE buffer (0.1 M Tris, 0.1 M NaCl, 0.01 M Naz EDTA, pH 7) containing 2% sodium dodecyl sulfate. Cells received the same reagents but were not pulverized. The mixtures were stirred as thawing took place and then were shaken vigorously before being centrifuged to separate the phases. The aqueous phase was reextracted with the phenol mixture and nucleic acids were precipitated by adding 1.5 volumes of isopropanol and a few drops of 3 M Na acetate, pH 4.0. The precipitates were redissolved in 1 ml of half-strength STJ#lg buffer (STMg buffer is 0.2 M NaCl, 0.01 M Tris, 0.01 M MgCl, , pH 7.6), 25 pg pancreatic DNase were added (Worthington, RNase free), and the extract was incubated at 37” for 20 min. A lo-p1 portion of the extract was taken for determination of TCA-precipitable radioactivity and depending upon the amount of radioactivity present, a sample was either taken directly for electrophoresis or the RNA was first concentrated by ethanol precipitation into a smaller volume of one-third strength STMg. Electrophoresis. Fifty to 100 ~1 of a radioactive RNA preparation was loaded onto a 11 cm 1.8% polyacrylamideeO.5% agarose gel and electrophoresis was conducted as described (Jackson et al., 1971) at 5 mA/gel for 0.5 hr beyond the time a bromophenol blue dye marker migrated off the gel (ca. 4.5 hr). The gels were then frozen. Extraction of RNA from polyacrylamide gels. The frozen gels were sliced with a manifold of razor blades spaced 1.07 mm apart. RNA was extracted from the gel slices by placing 2 (sometimes 3) consecutive slices in tubes containing 0.5 ml PE buffer (0.01 M Na2HP04, 0.005 M Na2EDTA,
AND
DUDA
pH 7). The tubes were shaken in an ice water bath for several hours and allowed to sit at 4” overnight. The 0.5 ml of liquid was removed and the gel slices were washed with another 0.5 ml portion of PE buffer for 2-4 hr, and this was removed and combined with the first extract. About half the counts applied to the gel were extracted by this method. At least some of the nonrecovered radioactivity is accounted for by low molecular weight RNA species which were extracted but which were allowed to run off the gel during electrophoresis. That all of the larger species of RNA were extract,ed proportionally by this method was indicated by the pattern of radioactivity extracted from the gel slices compared to the pattern obtained by total radioactivity determination of slices of sister gels after total digestion with HaOz . Analysis of gel extracts and hybridization studies. Aliquots of 0.05 to 0.2 ml were removed from the gel slice extracts for different analytic procedures as follows : a. Determination of total radioactivity extracted from the gel: Generally 0.05 ml was taken and pipetted directly into a vial, adding 0.45 ml NCS reagent (Amersham/ Searle) and scintillation fluid as described below. b. Determinat’ion of RNasc resistance before heat denaturation: Usually O.l-ml samples were removed from the gel extracts and sufficient concentrated buffer was added to make the salt concentration at least 2 X STE (STE buffer is 0.1 M NaCl, 0.05 IL! Tris, 0.001 M EDTA, pH 8) and the volume close to 0.2 ml. Ten microliters of an RNase solution (1 mg/ml pancreatic ribonuclease A, Worthington and 0.05 mg/ml Tl ribonuclease, Worthington) were added and the samples were incubated at 37” for 20 min. The samples then were transferred quantit’at’ively to 3 RIM filter disks (Whatman 1 in) and counted as described below. The counting efficiency of this filter disk plating procedure was determined to be the same as that of (a) above, so results from the two methods may be compared directly. Before the remainder of the analyses were performed the tubes containing the gel extracts were immersed in boiling water for 2 min and then cooled in ice water to ensure
TMV-RELATED
heat denaturation (melting) of any doublestranded RNA that might be present. c. Determination of RNase resistance after melting: The same procedure was followTed as in (b) above. d. Self annealing: The procedure as in (b) was followed except that after the salt concentration was increased and before ribonuclease treatment, 10 ~1 of water was added and the preparation was incubated at 70” for 4 hr. e. Annealing in the presence of TMV RNA: Except as indicated in the text, the procedure in (d) was followed except that 10 ~1 of water containing 0.05 pg TMV RNA (phenol extracted from virions) was added before the 70” incubation. f. Annealing in the presence of DS RNA: The procedure as in (e) was followed except that instead of TMV RNA, the 10 ~1 of water contained 0.1 pg of melted DS TMV RNA (prepared according to Jackson et al., 1971). g. Reconstitution with TMV protein: Solutions (0.2 ml) containing 50 ~1 of gel slice extract adjusted to 0.1 M NaqP207, pH 7.2, and 170 pg TMV protein (prepared according to Fraenkel-Conrat, 1957) were incubated for 18 hr at room temperature, treated with RNase as in the other assays, transferred to 3 MM disks, and counted. h. Assay for the presence of polyadenylic acid sequences. Three assay methods were used: (1) Aliquots of the gel slice extracts (0.2 ml) were mixed with 10 ml of high salt buffer (0.5 M KCI, 10 mM Tris, 1 mM MgClz ) pH 7.6) and filtered through 24 mm nitrocellulose filter membranes (B-6, Schleicher and Schuell) according to Lee et al. (1971). After two washes with the high salt buffer the filters were dried and counted. (2) Aliquots (0.1 ml) of the gel slice extracts were added to 5 ml of binding buffer (0.01 M Tris, 0.12 M NaCl, pH 7.5) and filtered through glass fiber filters (Whatman, GF/C, 24 mm) which had been embedded with 0.1 mg polyuridylic acid (Schwartz/Mann) and irradiated with UV according to Sheldon et al. (1972). (3) Aliquots (0.2 ml) of the gel slice extracts were made to 2 X STE and 0.1 #Zi [3H]polyuridylic acid [7 mCi/ mmole P, Schwartz/Mann] was added. The
77
RNA8
samples were then treated as in assay d (Gillespie et al., 1972). Determination of radioactivity. Radioactivity was determined in assay a by adding 10 ml of a PPO-POPOP scintillation fluid and counting in a Packard Tri-Carb, Model 3320 scintillation spectrometer. Cold trichloroacetic acid-precipitable radioactivity remaining after treatment in assays b through g, and h2 and h3 was determined by applying total samples to two 25 mm filter paper disks (Whatman 3MM) and washing them according to Byfield and Scherbaum (1966). The material remaining on the disks was digested for 0.5 hr at 50” in 0.5 ml of a mixture of 9 parts NCS reagent (Amersham-Searle) to 1 part water and count’ed in 10 ml PPO-POPOP scintillation fluid. Radioactivity in assay hl was determined by immersing the filter membrane in 10 ml of scintillation fluid, without NCS treatment, and counting. The results of the analyses performed on aliquots of the gel extracts were adjusted to make them equivalent to the original volume of the extracts. In order to determine the total radioactivity contained in gel slices (as in Fig. l), 3 consecutive slices were placed in glass scintillation vials containing 0.75 ml H,Oz and were incubated overnight at 50” for digestion. Ten milliliters of a PPO-POPOP scintillation fluid containing 33 % Triton X-100 and 67% toluene were added before counting. RESULTS
Several experiments were performed both with separated cells and leaf strips. All experiments yielded the same type of result. The following data were obtained from an experiment in which leaf strips from uninfected and from 6 day-infected leaves were infiltrated with 3H-uridine with or without actinomycin D and incubated for 2 hr before RNA was extracted and subjected to electrophoresis. Distribution of Radioactive RNA acrylamide Gels
in Po(y-
Figure 1 shows the distribution of radioactivity in RNA contained in gels of TMV-
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SIEGEL,
ZAITLIN,
AND DUDA
FRACTION NUMBER
FIQ. 1. Distribution of radioactivity in polyacrylamide gels containing RNA extracted from TMYinfected leaf tissue treated with actinomycin D, and comparable untreated tissue. Two l-g samples of leaf tissue infected for 6 days were vacuum infiltrated (3 times) with 5 ml of a solution containing 0.01 M KHePOa , 500 &i 8H-uridine, and in one case 40 rg/ml actinomycin D. The tissue was incubated under a fluorescent light (ca. 700 ft-c) for 2 hr at room temperature. RNA extracts were prepared, gels were run and sliced, and 3 consecutive 1.07-mm slices were digested with Hz02 as described in Materials and Methods. The top of the gel is on the left in this and all other gels presented. Actinomycin D treated (o-0); not treated with actinomycin D (e-----O).
infected leaf tissue which had been incubated with and without actinomycin D treatment. The patterns from diseased and uninfected leaf tissue (not shown here) are similar to those reported by Jackson et al. (1972) for separated cells. Treatment with actinomycin D appears to be more effective when applied to leaf strips than to separated cells, possibly because the inhibitor may be protected from light inactivation upon infiltration into intracellular spaces. The data confirm our previous observation (Jackson et al., 1972) that incorporation of a radioactive precursor into RNA species that are unique to infected tissue was not inhibited by actinomycin D. The unique species are replicative form (RF) at fractions 5 and 6, TMV RNA at fraction 14, and the low-molecular-weight component (LMC) at fractions 29 and 30. Replicative intermediate was not detected in these extracts
because
of the
long
incorporation
period (Jackson et al., 1972) Analysis of Extracts from Electrophoretic Gels Containing RNA from TMV Infected and Uninfected Actinom,ycin D-Treated Leaf Strips The PE buffer-extracts of gel slices were analyzed in a number of ways in order to
characterize further the RNA species unique to TMV infection. The description of the results that follow and the figures depicting them (Figs. 2 and 3) represent aliquots of the same extracts and thus are compared directly. It, appears that very little RNA becomes labeled in uninfected, actinomycin D-treated tissue (Fig. 2A) but, in fact, much of what does get labeled is probably of low molecular weight and runs off the gel during electrophoresis, for in this experiment there is onethird as much radioactivity in the healthy, actinomycin D-treated RNA preparation as in a comparable amount of untreated healthy tissue. It is clear, however, that the TMV gel is qualitatively different, exhibiting the same 3 virus-related RNAs seen in Fig. 1, i.e., RF peaking at fraction 8, TMV RNA peaking at fraction 22, and the LMC at fraction 45. The results shown in Fig. 2B were obtained when aliquots of the eluates of the gel slices were treated with RNase before and after melting. It is evident that the material present in fractions 7-9, called RF, is the only RNase-resistant RNA in the preparation; it is RNase resistant before melting and virtually completely digested by RNase after
TMV-RELATED
RNAse
SENSITIVITY
P
uI
- BEFORE YELTH(G I, AFTER MELTWG
% o
-o-WWFECTED
I-
TMV RN.4 3
TOTAL
FRACTION
0
79
RNAs
anneal with the TMV RNA at fraction 22. It is to be noted that the amount of ribonuclcase resistance in fraction 8 following annealing was less than would be predicted if an appreciable amount of the material in this fraction were originally a doublestranded structure. This proved to result from inefficient anncaling conditions due to the low concentration of t.he RNA; by in-
COUNTS
NUMBER
2. Analysis of radioactivity extracted FIG. from actinomycin D-treated, TMV-infected, and uninfected leaf tissue. A portion of the RNA extract from rtctinomycin D-treated TMV-infected tissue used in the experiment depicted in Fig. 1 and a comparable RNA extract from uninfected tissue were electrophorcsed on gels and the gel slices (2 per fract.ion) wcrc extracted in PE buffer as described in Materials and hfethods. Figure 2A shows the t.otal radioactivity extracted from each 2-slice fraction from TMV-infected and uninfected tissues and 213the effect of RNase treatment on every second fraction of those extracts, and on selected tubes of the 1’1fV extract after heating them for 90 set and quick cooling ~-9described in Materials and Methods. Sotc that the ordinates differ by a factor of 10 in 2A and 2B.
melting, suggesting that it is indeed doublcstranded RF. The conclusion that the RXase-rwistant mat,crial in the RF region is double-stranded is supported by the rcannenling experiment (Fig. 3A) because the RNA in this region recovers KX\‘ase resistance when allowed to reanncnl. This experiment also demonstrates that the RF region is the only one which contains complementary RSA strands because this is the only region which displays RNase resistance after the annealing trcatis ruled out, for mctnt . The possibility instance, that significant amounts of singlestranded KXA complementary to and of the same size as TMV RSA had been encapsidated with viral protcin and might thcreforc
:P 6i 5-
ANNEAL DOUBLE
FRACTION
WITH MELTED STRAMED RNA
NUMBER
FIG. 3. Annealing and reconstitution experiments with aliquots of the gel extracts depicted in Fig. 2A. Aliquots of extracts eluted from the gel were either self-annealed (A) ; annealed in the presence of 0.05 fig TMV RNA (B); annealed in the presence of 0.1 M melted DS TMY RX-4 (C); or reconstituted with an excess of TMV protein (D). After RNnse treatment,, RIKase-resistant RNA was dctermincd aa described in Mstcrials and Methods. The recovered radioactivities were recalculated to be equivalent to the total volume of each fraction, so that Figs. 2.4 and 13and Figs. 3.4 through 3D are comparable, even though all the ordinates do not. have the same scale.
80
SIEGEL,
ZAITLIN,
creasing the concentration of fraction 8 four-fold during the annealing, the amount of RNA converted to RNase resistance increased to about half the amount present before melting (data not shown). The result of annealing the gel fraction extracts in the presence of TMV RNA (Fig. 3B) reveals that RF at fraction 8 contains the only material in the gel complementary to viral RNA, for this is the only region which cont.ains material which anneals to TMV RNA. The small peak shown in the TMV RNA region (fraction 22) was not consistently observed and we believe has no significance. Figure 3C presents data of the annealing of the gel fraction extracts in the presence of melted DS TMV RNA. This reagent contains both viral RNA and RNA which is complementary to viral RNA and, thus, is capable of detecting both types of RNA in the gel extracts. A comparison of Figs. 3B and 3C reveals that DS TMV RNA anneals with LMC in addition to its reaction with TMV RNA and RF, whereas TMV RNA anneals only with RF. The conclusion is clear that LMC is complementary to a component of DS TMV RNA and is most likely a fragment of TMV RNA. The results depicted in Fig. 3C are informative in another sense; LMC is not the only fragment which is most likely viral RNA in the gel extracts, although it appears to be the only one present in significant amounts of a definitive size. Appreciable annealing to the viral RNA complementary strand takes place with material of a broad size range, but smaller than TMV RNA, in fractions 28 to 38. The amount of this material varies in different experiments and may have its origin in a breakage of TMV RNA during extraction, or possibly it may represent incompletely synthesized viral RNA. Experiments with separated cells have so far yielded greater amounts of the polydisperse TMV RNA than have leaf strips, but we do not know whether this reflects a difference in virus replication or extraction conditions. Symmetry
of Labeling of RF Strands
Comparison of the amount of radioactivity rendered RNase resistant when the RF fraction is annealed in the presence of TMV RNA and melted DS TMV RNA yields in-
AND
DUDA
formation concerning the symmetry of labeling of the two strands of the RF fraction because both strands should anneal in the presence of DS TMV RNA whereas only the complementary strand anneals with TMV RNA. In order to obtain a valid estimate of symmetry, however, it is necessary to ensure that annealing is performed efficiently in the presence of an excess of additives. To this end twice the usual concentration of RF (fraction 9) was annealed in the presence of lo-fold the usual concentration of TMV and DS TMV RNA (0.5 and 1.0 pg, respectively). Under these conditions, 1225 cpm became resistant to RNase in the presence of TMV RNA whereas 3045 cpm did so in the presence of DS TMV RNA. Since twice as much RNA became resistant when RF was annealed with DS TMV RNA, these data indicate that during the 2hr incubation period of leaf strips both strands of RF were labeled almost equally. Whether the same would be true of a shorter labeling period remains to be determined. Reconstitution of LMC with TMV
protein
Having demonstrated that LMC is a definite sized fragment of TMV RNA, the gel extracts were incubated with an excess of TMV protein in order to determine whether LMC might combine with TMV protein to reconstitute an RNase resistant nucleoprotein rod. As seen in Fig. 3D, material in the RF, TMV RNA, and polydisperse TMV RNA regions becomes resistant to RNase degradation under these conditions but LMC (fraction 45) does not.4 Several investigators (e.g., Butler and Klug, 1971; Okada and Ohno, 1972) have demonstrated that the 5’ end of TMV RNA is essential for initiation of 4 About one-half of the counts assayed in the RF region were found not to result from reconstituted nucleoprotein, but rather they reflected self-annealed RF, because the reconstitution was performed under high salt conditions which protect RF from RNase. In a separate experiment, the solutions from fractions 8, 22, and 30 were diluted with water before RNase treatment. The counts obtained in the RF region fell to about one-half the value obtained in high salt while those in the TMV RNA region and at fraction 30 were unaffected, showing that there was also self-annealing in the RF region under reconstitution conditions.
TMV-RELATED
nucleoprotein reconstitution; thus, we feel that LMC is probably not a homogeneous fragment of RNA which contains the 5’ end of viral RNA. It may be a homogeneous fragment from some other region of the viral RNA or it may represent a heterogeneous population of fragments all of a similar size, it might have the 5’ end but be unable to associate for some other reason, or the level of reconstitution might be too low to detect’. to see Experiment)s were conducted whether L32C was encapsidated with viral protein in vivo, possibly as a short virus rod. TMV labeled with 3H-uridine was isolat,ed from tissue incubated as described in Materials and 14ethods and purified by differential centrifugation. RNA was isolated from the virus and was subjected to gel clect,rophoresis; the gels were scanned optically using the linear transport accessory of the Gilford Model 240 spectrophometer and sliced for determination of radioactivity. In these experiment’s there was no suggestion of either radioactivity or UV absorbing material in the LYIC region of the gel. However, we reasoned that if short virus rods containing LMC were present in the leaf, t’he technique of isolation of virus by differential centrifugation would tend to discriminate aga.inst them because they would not sediment as efficiently as t’he longer rods. Accordingly, we examined encapsidated RNA isolated without high speed ccntrifugat’ion. Tissue infected for 4 days was incubated in the presence of 3H-uridine for 7 hr. The tissue was then ground in a mortar with M/15 phosphate buffer and clarified at 20,000 g for 10 min. The clarified supernatant was treated with RNase A (10 pg/ml) and RNase Tl (0.5 pg/ml) at 37” for 1.5 hr, followed by Yronase digestion (0.2 mg/ml for a further 1.5 hr to help eliminate the RNase. RNA was then extracted with phenol and analyzed by gel electrophoresis. Once again there was no suggestion of either counts or UV absorbing material in the LMC region, supporting the conclusion that L?tlC is not encapsidatcd in, viuo. LMC may be the same as the “rapidly labeled RNA” identified by Babos (1969, 1971) as being associated with ribosomes in T,1’IV-infected leaves. If this is so it may function as a messenger RNA. Recently,
81
RNAs
evidence has accumulated to indicate that RNA possesses eukaryote messenger poly (A) sequences (i.e., Lee et al., 1971). In order to determine whether LMC, or any of the other viral RNA components contain such poly (A) sequences, the gel extracts were assayed by the three methods described in Materials and Methods, but neither LMC, TMV RNA, nor RF were found to possess sufficient poly(A) sequences t,o be detectable. If LMC functions as messenger, it does so without a discernible polyadenylate sequence. In addition, TMV RNA, if lacking a polyadenylate sequence, differs from some other viral RNAs that have been tested, such as those of poliovirus, eastern equine encephalitis virus, Sindbis, Columbia SK, and some of the RNA tumor viruses (Armstrong et al., 1972; Green and Cartas, 1972; Johnston and Bose, 1972; Gillespie et al., 1972). DISCUSSION
The experiments described here permit the conclusion that LMC is a fragment of TMV RNA. We could detect no aggregation with TlllV protein to form a nucleoprotein; thus it probably lacks the 5’ end of TMV RNA, unless the reconstitution is not detectable or is prevented by some other mechanism. The function, if any, of this component remains unknown although it could be a messenger, possibly for the viral coat protein. If so, it functions without a poly(A) sequence, characteristic of other eukaryotic messenger RNAs. Babos (1969, 1971) has identified a rapidly labeled RNA of about the same size as LMC associated with ribosomes of TMVinfected leaves and if LMC and this component are the same, its association with ribosomes lends weight to the speculation that it may be a specialized messenger RNA. Other examples of viral RNA fragments have been observed; one is the 20 S RNA found in cells which produce murine sarcoma virus but which is absent from transformed non-virus-producing cells (Tsuchida et al., 1972). Another is the 2.8 X 105d RNA component found in brome mosaic virus (Shih et al., 1972), which apparently is not necessary for infection but appears to contain part of the sequence specifying coat protein (Stubbs and Kaesberg, 1967). In contrast to
82
SIEGEL,
ZAITLIN,
LMC, the brome mosaic virus RNA fragment is encapsidated along with another viral RNA component into one of the virion components of this split-genome virus. Another small encapsidated RNA, from top component a of alfalfa mosaic virus, is necessary for infection along with three other separately encapsidated virion RNAs, but the addition of small amounts of coat protein to the inoculum will substitute for it. Bol and van Vloten-Doting (1973) have suggested that the function of this small RNA is to specify a few protein molecules which are essential to initiate infection, An RNA which, like LMC is probably not a constituent of the virion, has been discovered by Romero (1972) in leaves infected with broadbean mottle virus. Its function, like that of LMC, remains obscure. Other conclusions about the high molecular weight virus-related RNA components extracted from infected leaves are that the RF component contains the only negative strand found in the cell and both positive and negative strands of RF become about equally labeled during a 2-hr incubation period in the presence of radioactive precursor. This is somewhat surprising, for one would expect a more rapid Iabeling of viral RNA on the assumption that the negative strand in RF is the template for viral plus strand synthesis. Perhaps we would observe asynchronous synthesis with shorter labeling periods, or it may be that RF is not an intermediate in a direct path to viral RNA synthesis, as we have discussed previously (Jackson et al., 1972). LMC is consistently observed in infected tissue, but we have occasionally had suggestions that other TMV RNA fragments of definite size may also be present. It has been difficult so far to obtain clear-cut evidence for such components because of the presence of a variable amount of TMV RNA fragments of polydisperse size. We are puzzled about the origin of these fragments. They may represent breakage of TMV RNA during RNA extraction or possibly incomplete particles whose synthesis was interrupted by terminat.ion of the experiment. A comment is necessary concerning hybridization of the RNA in gel extracts. We have indicated that at times our annealing
AND
DUDA
conditions were less than optimal. We do not consider this to be a hazard as long as interpretation of data is based on a knowledge of experimental conditions. One type of pitfall that must be guarded against is met when one hybridizes 3H-TMV RNA extracted from gels, such as those described herein, with melted DS TMV RNA. The results are reliable only when the melted DS TMV RNA is in excess during the annealing reaction. It is essential to ensure that this, indeed, is the case. ACKNOWLEDGMENTS We are pleased to acknowledge technical assistance of Mary Petti Smith.
the capable and Ruth C.
REFERENCES ARMSTRONG, J., EDMONDS, M., NAKAZATO, H., PHILLIPS, B., and VAUGHAN, M. (1972). Polyadenylic acid sequences in the virion RNA of poliovirus and eastern equine encephalitis virus. Science 176,526-528. BABOS, P. (1969). Rapidly labeled RNA associated with ribosomes of tobacco leaves infected with tobacco mosaic virus. Pz?oZogy 39,893-900. BABOS, P. (1971). TMV-RNA associated with ribosomes of tobacco leaves infected with TMV. Virology 43, 597-606. BOL. J. F., and VAN VLOTEN-DOTING, L. (1973). The function of top component a RNA in the initiation of infection by alfalfa mosaic virus. Virology 61, 102-108. BUTLER, P., and KLUG, A. (1971). Assembly of the particle of tobacco mosaic virus from RNA and disks of protein. Nature (London) New Biol. 229,47-50. BYFIELD, J. E., and SCHERBAUM, 0. H. (1966). A rapid radioassay for cellular suspensions. Anal. Biochem. 17,434443. FR.LENKEL-CONRAT, H. (1957). Degradation of tobacco mosaic virus with acetic acid. Vi’irology 4, l-4. FRANCKI, R. I. B., ZAITLIN, M., and JENSEN, R. G. (1971). Metabolism of separated leaf cells. II. Uptake and incorporation of protein and ribonucleic acid precurors by tobacco cells. Plant Physiol. 48,14-18. GILLESPIE, D., MARSHALL, S., and GALLO, R. (1972). RNA of RNA tumour viruses contains poly A. Nature (London) New Biol. 236,227-231. GREEN, M., and CARTAS, M. (1972). The genome of RNA tumor viruses contains polyadenylic acid sequences. Proc. Nat. Acad. Sci. U.S. 69, 791-794.
TMV-RELATED A., MITCHELL, D., and SIEGEL, A. (1971). Replication of tobacco mosaic virus. I. Isolation and characterization of doublestranded forms of ribonucleic acid. Virology 45,182-191. JACKSON, A., ZAITLIN, M., SIEGEL, A., and FRANCKI, R. I. B. (1972). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48,655-&X JENSEN, R. G., FRANCKI, R. I. B., and ZAITLIN, M. (1971). Metabolism of separated leaf cells. I. Preparation of photosynthetically active cells from tobacco. Plant Physiol. 48,9-13. JOHNSTON, R. E., and BOSE, H. R. (1972). Correlation of messenger RNA function with adenylaterich segments in the genomes of single-stranded RNA viruses. Proc. Nat. Acad. Sci. U.S. 69, 1514-1516. LEE, S., MENDECKI, J., and BRAWERMAN, G (1971). A polynucleotide segment rich in adenylic acid in the rapidly-labeled polyribosomal RNA component of mouse sarcoma
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180 ascites cells. Proc. Nat. Acad. Sci. U.S. 68, 1331-1335. OKADA, Y., and OHNO, T. (1972). Assembly mechanism of tobacco mosaic virus particle from its ribonucleic acid and protein. Mol. Gen. Genet. 114,X&213. ROMERO, J. (1972). RNA synthesis in broadbean leaves infected with broadbean mottle virus. Virology 48, 591-594. SHELDON, R., JURALE, C., and KATES, J. (1972). Detection of polyadenylic acid sequences in viral and eukaryotic RNA. Proc. Nat. Acad. Sci. U.S. 69,417421. SHIH, D. S., LANE, L., and KAESBERG, P. (1972). Origin of the small component of brome mosaic virus RNA. J. Mol. Biol. 64, 353-362. STUBBS, J., and KAESBERG, P. (1967). Amino acid incorporation in an Escherichia coli cell-free system directed by bromegrass mosaic virus ribonucleic acid. Virology 33, 38.5397. TSUCHIDA, N., ROBIN, M., and GREEN, M. (1972). Viral RNA subunits in cells transformed by RNA tumor viruses. Science 176, 1418-1420.