73, 363-371
VlROLOGY
A Messenger
ALBERT
(1976)
RNA for Capsid Protein Isolated Virus-Infected Tissue’
SIEGEL,2
V. HARI,
ILENE
MONTGOMERY,3
Biology
Department,
Wayne State University, Accepted April
Detroit,
from Tobacco
AND
KATHRYN
Michigan
Mosaic
KOLACZ
48202
22,1976
A low molecular weight virus-related RNA component (LMC) was isolated from extracts of TMV-infected leaf tissue by employing a combination of the techniques of sucrose density gradient sedimentation and polyacrylamide gel electrophoresis. It was found that LMC has an estimated molecular weight of 2.5 x lo5 and acts as a template for the synthesis of TMV capsid protein in a wheat germ-derived cell-free protein synthesizing system. Evidence is presented for the possible existence of other unique virus-related RNA species in extracts of infected tissue. INTRODUCTION
The tobacco mosaic virus (TMV) genome is a single, infectious, 2 x lo6 dalton single-stranded RNA molecule, similar to that of the animal picornaviruses. The latter, although polycistronic, are translated into a polyprotein which is processed by specific cleavage into a number of smaller functional proteins (Jacobson et al., 1970; Kiehn and Holland, 1970). Presumably the TMV genome is also polycistronic, containing a gene for the capsid protein, which accounts for less than a tenth of the RNA, and probably at least one other gene, a good candidate being that for a rather large protein responsible, at least in part, for replication of the genome (Zaitlin et al., 1973). The question arises as to whether TMV RNA is translated without pause, as in the case of the animal picornaviruses, into a total genome sized product which is subsequently cleaved, or whether, perhaps, monocistronic messengers are generated during infection as is ‘This investigation was supported in part by a grant from the National Science Foundation, and by ERDA Contract No. E(ll-H-2334. 2 Author to whom requests for reprints should be addressed. 3 Present address: Department of Immunology and Microbiology, Wayne State University, Detroit, Michigan 48202. 363 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
the case for at least one of the genomic components of the bromo- and alfalfa mosaic virus groups (Shih and Kaesberg, 1973). A suggestion that at least one monocistronic messenger RNA might be generated during TMV infection comes from the observation that a unique low molecular weight (ca. 350,000) RNA species (LMC) with the same nucleotide sequence as that of at least a portion of viral RNA is present in extracts of TMV-infected tissue (Jackson et al., 1972; Siegel et al., 1973). This paper describes the isolation of sufficient LMC to use as template in a cell-free protein synthesizing system. It was found that LMC stimulates the incorporation of labeled precursor into an acid-insoluble product and further, that it is a special messenger for TMV capsid protein. Results similar to this have been obtained independently by Knowland et al. (1975). In the course of isolating LMC, we detected the presence of additional unique RNAs which might possibly represent other species of viral messenger RNA. MATERIALS
Conditions
for
AND
infection
METHODS
and
labeling.
Young, rapidly growing Nicotiana tabacum L. cultivar Samsun plants, about 6in. tall, were inoculated by abrading ex-
364
SIEGEL
panded leaves with a M/15 Sorenson’s phosphate buffer solution containing 10 pg/ml of TMV (the Ul strain) and 50 mg/ ml of Celite (Johns Manville Hyflo Superccl). Either the first leaves to become systemically infected or the inoculated leaves were detached at indicated times after inoculation and were sliced into ,1-mm wide strips. These were infiltrated with a volume of liquid equal to the wet weight of the leaf tissue. The infiltration liquid was 0.01 M KH,PO, and contained, per gram of tissue, 40-60 pg of actinomycin D (Sigma), 200 &i of [3H]uridine (Nuclear Dynamics, La Jolla, Calif.), 300 pg of Cephaloridine (Eli Lilly), and 10 pg of Rimocidin (Pfizer). The infiltrated tissue was incubated in the dark at temperatures and for times detailed in the Results section, washed with water, and frozen in liquid nitrogen. The tissue was either pulverized immediately or stored at -78” until a more convenient time. Extraction of RNA. Frozen leaf tissue in a mortar was pulverized to a fine powder with a pestle in the presence of liquid nitrogen. The following two mixtures were then added per gram fresh weight of tissue: (A) 0.9 ml of TNE-saturated phenol, 0.1 ml of m-cresol, 0.01 g of &hydroxyquinoline; and (B) 1 ml of TNE (0.1 M Tris, 0.1 M NaCl, 0.01 M Na,EDTA, pH 7, at 20”) containing 1% sodium dodecyl sulfate, 1 mg of bentonite and vanadyl sulfate-guanosine monophosphate complex, prepared and used as described by Gray (1974). The mixture was stirred as it thawed and formed a slurry. The aqueous phase was recovered after breaking the emulsion by centrifugation at 10,000 i-pm for 10 min in the Sorvall RC-2B centrifuge. Both the aqueous and phenol phases were reextracted with phenol and buffer, respectively, and the resultant aqueous phases combined and allowed to sit overnight at 0 after addition of LiCl to 2 M in order to effect precipitation of the larger singlestranded RNA species. The precipitate was sedimented at 10,000 r-pm for 20 min and was resuspended in H,O. The RNA was usually reprecipitated by addition of 2.5 volumes of ethanol and again resuspended in H,O after collection of a pellet by centrifugation.
ET AL.
Fractionation of leaf RNA. RNA extracted from infected or control healthy leaves was made to a concentration of 1 mglml and 1 ml was layered on a 5 to 20% linear sucrose gradient in E buffer (0.04 M Tris, 0.02 M Na acetate, 1 mit4 Na,EDTA, pH 7.2) containing vanadyl sulfate-guanosine complex at l/lo the concentration in the extraction buffer. The gradients in 1 x 3.5-in. tubes were centrifuged at 4” for 18 hr at 25,000 rpm in the Spinco SW-27 rotor. One-milliliter fractions were collected while monitoring for absorbance at 260 nm with the aid of an Isco (Lincoln, Nebr.) density gradient fractionator and uv monitor, and the radioactivity of the fractions was determined by transferring small samples to scintillation vials containing a toluene-based scintillation fluid and 7% NCS (Amersham-Searle). Those fractions from the infected preparation which showed high specific radioactivity relative to comparable fractions of the healthy preparation were combined, the RNA precipitated with ethanol, and subjected to further purification by electrophoresis on cylindrical gels. Polyacrylamide
gel
electrophoresis.
Electrophoresis on cylindrical, 6-mm diameter, polyacrylamide gels was carried out essentially as described by Bishop et al. (1967)) after which the gels were soaked in H,O for at least an hour and scanned for absorbance at 260 nm with the aid of a Gilford Model 2400-S spectrophotometer equipped with the linear transport accessory. The gels were then frozen, sliced into l-mm-thick
disks with the aid of a bank of
razor blades, and each two consecutive gel slices were soaked in 0.5 ml of 0.1 M NaCl overnight at 0” in order to elute the RNA. Samples were taken from the eluates for radioactivity determination or, alternatively, the elution procedure was omitted and total radioactivity contained in gel slices was determined following either digestion with 30% H,O, or soaking in a 9:l
NCS:water mixture. Electrophoresis in slab gels was performed in an apparatus similar to that
described by Studier (1973) supplied by Aquebogue (Aquebogue, New York) or in the Bio-Rad (Richmond, Calif.) slab-gel apparatus, Model 220. The RNA samples
MESSENGER
RNA
FOR
were run on linear 2.2-4 or 5% gradient gels prepared from acrylamide stock solutions at an acrylamide-bis ratio of 30:1.5 for the 2.2% gel solution and 3O:O.a for the heavier 4 or 5% solution. Both the 2.2 and 45% gel stock solutions contained 1% ammonium persulfate and TEMED at a concentration of 106 ~1 per 100 ml of 2.2% gel solutions and 53 ~1 per 100 ml of the 4 or 5% solutions. The more concentrated gel solution also contained 15% sucrose. All stock solutions were kept on ice and the gradient was generated using a simple sucrose gradient device (Buchler). All operations were carried out in the cold. When l.Bmm-thick gradient gels were made in the Bio-Rad apparatus, 14.5 ml of the 2.2% gel solution and 13.6 ml of the 4 or 5% gel solution were used. Further, 1 ml of 2.2% gel solution was layered on the top of the gradient. Electrophoresis was carried out using E buffer (Bishop et al., 1967) at 35 mA constant current until the voltage reached 125 V, after which this voltage was maintained constant. Samples were usually run for 1 hr after the marker dye (bromophenol blue) had run off the gel. Protein samples were run in slab gels, prepared according to Laemmli (1970) using 12.5% separating gels and 5% stacking gels. The acrylamide-bis cross-linking ratio was reduced to 30:0.15 in order to obtain sharper bands and to eliminate cracking of gels during drying (Knowland, 1974). Electrophoresis was carried out at 35 mA constant current until voltage reached 125 V, after which this voltage was maintained constant until the marker dye reached the front. Marker proteins and 14C-labeled TMV capsid protein were run as standards. After electrophoresis, tritiated RNA and protein bands were detected by fluorography as described by Bonner and Laskey (1974) using Kodak Royal X O-Mat film which had been preflashed to a density of 0.15 (Laskey and Mills, 1975). Cell-free protein synthesis. A wheat germ-derived cell-free protein synthesizing system was prepared and used as described by Davies and Kaesberg (19731, except that phosphoenolpyruvate was omitted because in our hands it was found to inhibit the reaction. Except as other-
TMV
CAPSID
PROTEIN
365
wise noted, 2 &!i of [3Hlleucine (45 Ci/ mmol) was added for each 100 ~1 of reaction mixture which contained each of the other 19 amino acids at a concentration of 25 pm. Reactions were run at 30” and the extent of incorporation of leucine into acidinsoluble product was determined by spotting either 5 or 10 ~1 of reaction mix onto Whatman 3 MM 2.3-cm filter disks that had been prewetted with 5% trichloroacetic acid containing 2 g/liter of nonradioactive leucine and washing the disks with the leucine containing 5% trichloroacetic acid four times at 90”, twice with ethanol, once with 1:l ethanol:ethyl ether mixture, and finally with ethyl ether. The disks were then dried and counted in a toluenebased scintillation fluid containing 7% of a 9:l NCS:H,O mixture. For analysis of the reaction product, the reaction mix was made to 200 to 400 ~1 and at the termination of the incubation (usually 1 hr) was treated for an additional 20 min with 20 ~1 of a solution containing 1 mg/ml of pancreatic ribonuclease (Worthington) and 15,000 units/ml Tl ribonuclease (P-L Biochemicals). Five milliliters of 5% trichloroacetic acid containing 1% casamino acids were added and the precipitated protein was collected after 1 hr at 0” by centrifugation. The pellet was washed twice with the precipitating solution at 0” and twice more with ice-cold acetone after which it was dried in uacuo. The protein was dissolved in Laemmli sample buffer (Laemmli, 1970) for electrophoresis and in phosphate-buffered saline containing 0.1% SDS for the gel diffusion serological test. RESULTS
Partial purification of LMC by band sedimentation on sucrose density gradients. The object of this study was to determine whether an RNA component, LMC, present in extracts of TMV-infected tissue is a unique monocistronic messenger RNA, and if so, what the nature of its gene product might be. In previous work this component had been detected as a radioactive band on polyacrylamide gel electropherograms because it incorporated radioactive precursor ( 13Hluridine) even in the presence of actinomycin D. After several unsuccessful attempts to accumulate
366
SIEGEL
enough material for characterization by elution of the radioactive LMC band from 6-mm diameter gels, it was decided first to obtain an LMC rich fraction by band sedimentation of a relatively large amount of tissue RNA through a sucrose density gradient. This was accomplished as described in the Materials and Methods section, and the results of one such experiment are shown in Fig. 1. Note the peak labeled A, of high radioactivity and relatively low absorbance. It appears consistently in infected, but not in healthy, tissue extracts and has the appropriate estimated sedimentation for the LMC component. Thus, the fractions constituting this peak were combined, precipitated with 2.5 volumes of ethanol, and resuspended in a small vol-
J FIG. 1. Sucrose density gradient centrifugation of an RNA extract from TMV-infected tissue. The first systemic leaves to become infected were harvested 4 days after inoculation and were infiltrated with an equal weight of 0.01 M KH,PO, containing 40 wg/ml of actinomycin D and 200 &i/ml of 13Hluridine. RNA was extracted from an 18-hr, dark incubation period and 1 mg was layered on a 5-20% linear sucrose gradient. Centrifugation was in the Spinco SW-27 rotor at 25,000 rpm for 18 hr. The peaks labeled A and B are unique to infected tissue and were collected and saved for further analysis. Sedimentation is to the right.
ET AL
ume of H,O for further fractionation. In some, but not all, experiments a second peak of high radioactivity and relatively low absorbance, labeled B in Fig. 1, was noted between the two large ribosomal absorbance peaks. Fractions constituting this material were also collected for further analysis. Further purification of LMC by polyacrylamide gel electrophoresis. The material collected as peak A after sucrose density gradient centrifugation was subjected to electrophoresis on cylindrical polyacrylamide gels. These- were scanned for absorbance, sliced, and eluates of the gel slices were tested for radioactivity. As seen in Fig. 2, the radioactivity appears in one sharply defined peak which is taken to be LMC. Note also that enough LMC is present to yield an absorbance as well as a radioactive peak. The major absorbance peak in the electropherogram is judged to be the smallest (16 S) ribosomal RNA. The rate of movement of RNA species of known molecular weights (brome mosaic virus RNA) was determined on parallel gels, and using this information, an estimate of 250,000 daltons was made for the size of LMC. This is somewhat smaller than a previous estimate (Jackson et al., 1972) and may in part provide an explanation for the failure by others (Aoki and Takebe, 1975) to observe the LMC component. The gel-slice eluates which contained radioactivity were combined from several gels and the resultant solution was freed from contaminating substances by passing it through a hydroxyapatite column (Diener and Smith, 1973) under which conditions RNA is absorbed to the column and much of the contaminating material passes through. The RNA was recovered from the column with 0.12 M KPB (KPB is an equimolar mixture of KH,PO, and K,HPO,) and it was further purified and greatly concentrated by loading on a hydroxyapatite minicolumn (3-mm diameter, 4-mm high) at 0.03 M KPB and eluting it with a minimal volume of 0.12 M KPB. The two-step purification and concentration procedure was found to be necessary, because during the first step a sufficient amount of hydroxyapatite must be used so
MESSENGER
9-
3%
RNA
FOR
1
GEL 16 !
B-
7-
BMV 1t2 1
BMV 3
IMV
i
1
6-
IO
20
30
40
FRACTION
FIG. 2. Gel electrophoresis of the material contained in peak A of Fig. 1. The fractions labeled A in Fig. 1 were combined, concentrated, and subjected to electrophoresis on a 6-mm diameter 3% gel. The major radioactive peak is assumed to represent the LMC RNA described by Jackson et al. (1972). Migration is to the right. Arrows indicate the positions of brome mosaic virus and the 16 S chloroplast RNA species on parallel gels.
that it is all not poisoned by a component in the gel-eluted LMC solution. LMC as a messenger RNA in the wheat germ-derived cell-free protein synthesizing system. The radioactive material recovered from the minihydroxyapatite column was diluted with an equal amount of H,O to reduce the KPB concentration and was tested as a template in the wheat germ reaction mixture. The data in Table 1 show that both the LMC and TMV RNA stimulate incorporation of [3Hlleucine into acid-insoluble material, presumably polypeptides. The nature of the LMC and TMV RNAstimulated products was examined by subjecting the incubated reaction mixtures to
TMV
CAPSID
367
PROTEIN
polyacrylamide slab gel electrophoresis. The fluorograph of one such gel is shown in Fig. 3 where it can be seen that the LMC-stimulated material contains a radioactive protein of the same size as TMV capsid protein in addition to some smaller material. In contrast, the TMV RNA stimulated mixture, in agreement with previous work (Efron and Marcus, 1973; Roberts et al., 19741, contains many polypeptides, many of which are larger than capsid protein. The material in the LMCstimulated wheat germ system was tested for reaction to an antiserum prepared against TMV capsid protein. Figure 4 shows the result of a gel-diffusion assay in which both control 14C-labeled capsid protein and the LMC-stimulated material react with the antiserum. We conclude that LMC has the coding capacity for TMV capsid protein because it stimulates the wheat germ system to produce a protein of the same size and with the same antigenic specificity as the capsid protein. Conditions for biosynthesis of LMC. Actinomycin D was employed initially to suppress incorporation of labeled precursor into host RNA during incubation of TMVin the presence of infected tissue [3H]uridine. The inhibitor was found not only to be efficient for this purpose, particularly when incubation was carried out in the dark, but in addition, more labeled precursor became incorporated into LMC in its presence than in its absence, as can be seen in Fig. 5. The double effect of actinomycin D makes its employment parTABLE Time (min)
None 0 60
1
TRANSLATION OF LMC In Vitro” Template
9206 24,000
TMV RNA (1 pg) 1,400
220,000
LMC 3,600 168,000
a Either nothing, 1 pg of TMV RNA, or LMC extracted from 1 g of infected leaf tissue was added to 100 ~1 of wheat germ reaction mixture containing 2 Z.&i of PH]leucine as described in Materials and Methods. b TCA-insoluble counts per minute.
368
SIEGEL
ET AL.
tected in electropherograms of RNA extracts as soon as 12 hr after inoculation. In a parallel experiment, young expanding leaves were harvested 42 hr after lower leaves had been inoculated and these were also incubated under the same conditions. The leaves were harvested at a time when they had been only recently invaded by virus, several hours before vein-clearing symptoms appeared. Here again (Fig. 6) the LMC could be detected at a relatively short time (2 hr) after the leaves were harvested. Other unique RNA components present in TMV-infected tissue. Unique RNA species, other than LMC, were also noted in extracts of infected tissue. The radioactive
FIG. 3. SDS-gel electrophoresis of radioactive peptides appearing in a wheat germ cell-free system when stimulated with LMC and TMV RNA. The channels contain: (a) W-labeled TMV capsid protein (360 cpm); (b) 3H-labeled in vitro products stimulated by 10 pg of TMV RNA (45,000 cpm); (c) same as (b), but twice the amount (90,000 cpm); (d) SHlabeled endogenous products (8,000 cpm); (el 3Hlabeled in vitro products stimulated by 1 pg of TMV RNA (45,000 cpm); (0 empty; (gl 3H-labeled in vitro products stimulated by LMC (20,000 cpm).
titularly valuable for the experiments reported in this paper, because, together with the newly adopted technique of fluorography, the detection of LMC and other viral-specific tritiated RNA components is greatly facilitated. Experiments were conducted to determine when during infection the LMC could be first detected. To this end, leaves were detached 3 hr after inoculation and were incubated for different periods of time in the presence of actinomycin D and C3Hluridine. Under these conditions, where only a low proportion of the cells are infected, LMC can, nevertheless, be de-
FIG. 4. Fluorograph of a gel diffusion reaction between the 3H-labeled peptides generated in an LMC-stimulated wheat germ system and rabbit anti-TMV capsid serum. The wells were loaded with 14C-labeled capsid protein (CP), LMC-stimulated product (LMC), unlabeled capsid protein (U), and antiserum (B). The bands were allowed to develop for 3 days after which the gel (1.5% agarose) was soaked for 0.5 hr with five successive changes of phosphate buffered saline (pH 7.0) to remove nonprecipitated radioactivity. The gel was then prepared for fluorography as described in Materials and Methods, except that methanol was used to dehydrate the gel instead of DMSO (Laskey and Mills, 1975).
MESSENGER
RNA
FOR
peak labeled B in Fig. 1 is an example. It has an apparent size intermediate between the two major ribosomal RNA species and when the material in the fractions constituting this peak was collected and analyzed on acrylamide disk gels, a peak of radioactivity was found to corn&-ate with the large chloroplastic 23 S RNA (Fig. 7) indicating a molecular weight of 1.1 x 106. The detection of this component
TMV
CAPSID
PROTEIN
369
appears to require particular conditions because it has not always been observed as clearly as shown in Figs. 1 and 7, although it probably is the same as the band to which the arrow points in Figs. 5 and 6. DISCUSSION
The experiments presented here demonstrate the presence of a species of RNA, LMC, in extracts of TMV-infected tissue which acts in vitro as a special messenger for the capsid protein. It has a molecular
LMC
FIG. 5. Fluorograph of RNA extracted from healthy and TMV-infected tissue. The first leaves to become systemically infected or comparable healthy leaves were harvested 5 days after inoculation. These were either infiltrated with [3H]uridine and actinomycin D or with ‘[3H]uridine alone and were incubated in the dark for 18 hr. The RNA was extracted and 10 pg were subjected to electrophoresis on a 2.2-5% polyacrylamide slab gel. (1) Healthy, no actinomycin D (170,000 cpm); (2) healthy, with actinomycin D (15,000 cpm); (3) infected, no actinomycin D (175,000 cpm); (4) infected, with actinomycin D (150,000 cpm). The arrow indicates a putative TMVrelated RNA of ca. 1.1 x 10” daltons.
FIG. 6. Fluorograph of RNA samples extracted from infected leaves after different time periods. Systemically invaded leaves were detached 42 hr after inoculation and were infiltrated with [3H]uridine and actinomycin D. Leaf samples were taken for RNA extraction after different periods of incubation in the dark and 10 pg of each RNA was subjected to electrophoresis on a 2.2-5% polyacrylamide gel. The numbers under the channels indicate the hours of incubation. The channel labeled H contains 2 pg of RNA labeled in the absence of actinomycin D and extracted from healthy tissue; the upper and lower heavily labeled bands are, respectively, 25 and 18 S ribosomal RNA. The arrow indicates a component suspected of being a virus-specific RNA of 1.1 x- 10” daltons.
370
SIEGEL ,
41.8%
GEL
3-
‘;
‘I’
‘1” 1p
\
FRACTION
FIG. 7. Gel electrophoresis of the material contained in peak B of Fig. 1. The fractions labeled B in Fig. 1 were combined, concentrated, and subjected to electrophoresis on a 6-mm diameter 1.8% polyacrylamide-0.5% agarose gel. Most of the radioactivity migrated with the 23 S chloroplast ribosomal RNA.
weight of ca. 250,000 and the same nucleotide sequence as a portion of the viral RNA. Our findings confirm those of Knowland et al. (1975) who also demonstrated that a low-molecular weight component present in extracts of infected tissue directs the synthesis of capsid protein both in vitro and in oocytes ofxenopus laevis. We have found no evidence that LMC is encapsidated in our studies with the Ul strain. However, it probably is similar to the component which does become encapsidated during infection with the cowpea strain (Morris, 1974) and which also has been shown to direct the in vitro synthesis of capsid protein (Bruening et al., 1976; Higgins et al., 1976). One can think of at least two mechanisms for the origin of LMC. It may be either split from a strand of viral RNA or
ET AL.
it may be an incomplete transcript of a complementary RNA strand. At the present time the choice between these two or other possible alternatives is speculative. It is likely that LMC does represent the in vivo messenger for capsid protein rather than being a nonfunctional adjunct to TMV replication because its presence can be detected rather early in the infection process and because evidence is available that a low molecular weight RNA which bears resemblance to LMC is present in polysomes (Babos, 1971; Beachy and Zaitlin, 1975). The demonstration that at least one gene product is translated from a monocistronic messenger RNA raises the question as to whether monocistronic messenger RNA may be generated for other genes on the virion genome. Evidence is presented that new RNA species other than LMC may be generated during infection. Whether these represent specific virus related mRNA and if so, for what gene product they may code, remains to be determined. It is known that, among other products, the synthesis of two large proteins of molecular weights ca. 135,000 and 165,000 is stimulated in TMV-infected tissue (Zaitlin and Hariharasubramanian, 1972; Sakai and Takebe, 1974; Paterson and Knight, 1975) and similar large proteins are made under the direction of TMV RNA in oocytes (Knowland, 1974) and in a reticulocyte cell-free system (Knowland et al., 1975). It is possible that the ca. 1.1 x lo6 dalton RNA species, to which the arrow points in Figs. 5 and 6, might be just large enough to code for one of the two large proteins, one of which may be a viral-coded RNA replicase (Zaitlin et al., 1973). However, little is still known about what genes, other than those for capsid protein, might be present in the TMV genome, and whether monocistronic messages might exist for these. Thus, further work is necessary to clarify the translation strategy of the TMV genome and its control. REFERENCES S., and TAKEBE, I. (1975). Replication of tobacco mosaic virus in tobacco mesophyll protoplasts inoculated in uitro. Virology 65, 343-354. BABOS, P. (1971). TMV-RNA associated with riboAOKI,
MESSENGER
RNA
FOR
somes of tobacco leaves infected with TMV. Virology 43, 597-606. BEACHY, R. N., and ZAITLIN, M. (1975). Replication of tobacco mosaic virus. VI. Replicative intermediate and TMV-RNA related RNAs associated with polyribosomes. Virology 63, 84-97. BISHOP, D., CLAYBROOK, J., and SPIEGELMAN, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26, 373-387. BONNER, W., and LASKEY, R. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 44, 279-288. BRUENING, G., BEACHY, R., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in uiuo translation of the ribonucleic acids of the cowpea strain of tobacco mosaic virus. Virology 71, 498-517. DAVIES, J. W., and KAESBERG, P. (1973). Translation of virus mRNA: Synthesis of bacteriophage QB proteins in a cell-free extract from wheat embryo. J. Virol. 12, 1434-1441. DIENER, T. O., and SMITH, D. R. (1973). Potato spindle tuber viroid. IX. Molecular-weight determination by gel electrophoresis of formylated RNA. Virology 53, 359-365. EFRON, D., and MARCUS, A. (1973). Translation of TMV-RNA in a cell free wheat embryo system. Virology 53, 343-348. GRAY, J. C. (1974). The inhibition of ribonuclease activity and the isolation of polysomes from leaves of the french bean, Phaseolus vulgaris. Arch. Biothem. Biophys. 163, 343-348. HIGGINS, T., GOODWIN, P., and WHITFELD, P. (1976). Occurrence of short particles in beans infected with the cowpea strain of TMV. II. Evidence that the short particles contain the cistron for coat protein. Virology 71, 486-497. 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, 655-665. JACOBSON, M., Asso, J., and BALTIMORE, D. (1970). Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49, 657-668. KIEHN, E., and HOLLAND, J. (1970). Synthesis and cleavage of enterovirus polypeptides in mammalian cells. J. Virol. 5, 358-371.
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CAPSID
PROTEIN
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KNOWLAND, J. (1974). Protein synthesis directed by the RNA from a plant virus in normal animal cell. Genetics 78, 383-394. KNOWLAND, J., HUNTER, T., HUNT, T., and ZIMMERN, D. (1975). Translation of tobacco mosaic virus RNA and isolation of the messenger for TMV coat protein. Les Colloques de l’lnstitut National de la Sante et de la Recherche Medicale. 47, 211-216. LAEMMLI, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. LASKEY, R., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14Cin polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. MORRIS, T. J. (1974). Two nucleoprotein components associated with the cowpea strain of TMV. Proc. Amer. Phytopathol. Sot. 1, 83. PATERSON, R., and KNIGHT, C. A. (1975). Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus. Virology 64, 10-22. ROBERTS, B. E., PATERSON, B., and SPERLING, R. (1974). The cell-free synthesis and assembly of viral specific polypeptides into TMV particles. Virology 59, 307-313. SAKAI, F., and TAKEBE, I. (1974). Protein synthesis in tobacco mesophyll protoplasts induced by tobacco mosaic virus infection. Virology 62,426-433. SHIH, C., and KAESBERG, P. (1973). Translation of brome mosaic viral ribonucleic acid in a cell free system derived from wheat embryo. Proc. Nut. Acad. Sci. USA 70, 1799-1805. SIEGEL, A., ZAITLIN, M., and DUDA, C. T. (1973). Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs. Virology 53, 75-83. STUDIER, F. W. (1973). Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. ZAITLIN, M., DUDA, C. T., and PETTI, M. (1973). Replication of tobacco mosaic virus. V. Properties of the bound and soluble replicase. Virology 53, 300-311. ZAITLIN, M., and HARIHARASUBRAMANIAN, V. (1972). A gel electrophoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 47,.296-305.
VlROLOGY
A Messenger
ALBERT
(1976)
RNA for Capsid Protein Isolated Virus-Infected Tissue’
SIEGEL,2
V. HARI,
ILENE
MONTGOMERY,3
Biology
Department,
Wayne State University, Accepted April
Detroit,
from Tobacco
AND
KATHRYN
Michigan
Mosaic
KOLACZ
48202
22,1976
A low molecular weight virus-related RNA component (LMC) was isolated from extracts of TMV-infected leaf tissue by employing a combination of the techniques of sucrose density gradient sedimentation and polyacrylamide gel electrophoresis. It was found that LMC has an estimated molecular weight of 2.5 x lo5 and acts as a template for the synthesis of TMV capsid protein in a wheat germ-derived cell-free protein synthesizing system. Evidence is presented for the possible existence of other unique virus-related RNA species in extracts of infected tissue. INTRODUCTION
The tobacco mosaic virus (TMV) genome is a single, infectious, 2 x lo6 dalton single-stranded RNA molecule, similar to that of the animal picornaviruses. The latter, although polycistronic, are translated into a polyprotein which is processed by specific cleavage into a number of smaller functional proteins (Jacobson et al., 1970; Kiehn and Holland, 1970). Presumably the TMV genome is also polycistronic, containing a gene for the capsid protein, which accounts for less than a tenth of the RNA, and probably at least one other gene, a good candidate being that for a rather large protein responsible, at least in part, for replication of the genome (Zaitlin et al., 1973). The question arises as to whether TMV RNA is translated without pause, as in the case of the animal picornaviruses, into a total genome sized product which is subsequently cleaved, or whether, perhaps, monocistronic messengers are generated during infection as is ‘This investigation was supported in part by a grant from the National Science Foundation, and by ERDA Contract No. E(ll-H-2334. 2 Author to whom requests for reprints should be addressed. 3 Present address: Department of Immunology and Microbiology, Wayne State University, Detroit, Michigan 48202. 363 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
the case for at least one of the genomic components of the bromo- and alfalfa mosaic virus groups (Shih and Kaesberg, 1973). A suggestion that at least one monocistronic messenger RNA might be generated during TMV infection comes from the observation that a unique low molecular weight (ca. 350,000) RNA species (LMC) with the same nucleotide sequence as that of at least a portion of viral RNA is present in extracts of TMV-infected tissue (Jackson et al., 1972; Siegel et al., 1973). This paper describes the isolation of sufficient LMC to use as template in a cell-free protein synthesizing system. It was found that LMC stimulates the incorporation of labeled precursor into an acid-insoluble product and further, that it is a special messenger for TMV capsid protein. Results similar to this have been obtained independently by Knowland et al. (1975). In the course of isolating LMC, we detected the presence of additional unique RNAs which might possibly represent other species of viral messenger RNA. MATERIALS
Conditions
for
AND
infection
METHODS
and
labeling.
Young, rapidly growing Nicotiana tabacum L. cultivar Samsun plants, about 6in. tall, were inoculated by abrading ex-
364
SIEGEL
panded leaves with a M/15 Sorenson’s phosphate buffer solution containing 10 pg/ml of TMV (the Ul strain) and 50 mg/ ml of Celite (Johns Manville Hyflo Superccl). Either the first leaves to become systemically infected or the inoculated leaves were detached at indicated times after inoculation and were sliced into ,1-mm wide strips. These were infiltrated with a volume of liquid equal to the wet weight of the leaf tissue. The infiltration liquid was 0.01 M KH,PO, and contained, per gram of tissue, 40-60 pg of actinomycin D (Sigma), 200 &i of [3H]uridine (Nuclear Dynamics, La Jolla, Calif.), 300 pg of Cephaloridine (Eli Lilly), and 10 pg of Rimocidin (Pfizer). The infiltrated tissue was incubated in the dark at temperatures and for times detailed in the Results section, washed with water, and frozen in liquid nitrogen. The tissue was either pulverized immediately or stored at -78” until a more convenient time. Extraction of RNA. Frozen leaf tissue in a mortar was pulverized to a fine powder with a pestle in the presence of liquid nitrogen. The following two mixtures were then added per gram fresh weight of tissue: (A) 0.9 ml of TNE-saturated phenol, 0.1 ml of m-cresol, 0.01 g of &hydroxyquinoline; and (B) 1 ml of TNE (0.1 M Tris, 0.1 M NaCl, 0.01 M Na,EDTA, pH 7, at 20”) containing 1% sodium dodecyl sulfate, 1 mg of bentonite and vanadyl sulfate-guanosine monophosphate complex, prepared and used as described by Gray (1974). The mixture was stirred as it thawed and formed a slurry. The aqueous phase was recovered after breaking the emulsion by centrifugation at 10,000 i-pm for 10 min in the Sorvall RC-2B centrifuge. Both the aqueous and phenol phases were reextracted with phenol and buffer, respectively, and the resultant aqueous phases combined and allowed to sit overnight at 0 after addition of LiCl to 2 M in order to effect precipitation of the larger singlestranded RNA species. The precipitate was sedimented at 10,000 r-pm for 20 min and was resuspended in H,O. The RNA was usually reprecipitated by addition of 2.5 volumes of ethanol and again resuspended in H,O after collection of a pellet by centrifugation.
ET AL.
Fractionation of leaf RNA. RNA extracted from infected or control healthy leaves was made to a concentration of 1 mglml and 1 ml was layered on a 5 to 20% linear sucrose gradient in E buffer (0.04 M Tris, 0.02 M Na acetate, 1 mit4 Na,EDTA, pH 7.2) containing vanadyl sulfate-guanosine complex at l/lo the concentration in the extraction buffer. The gradients in 1 x 3.5-in. tubes were centrifuged at 4” for 18 hr at 25,000 rpm in the Spinco SW-27 rotor. One-milliliter fractions were collected while monitoring for absorbance at 260 nm with the aid of an Isco (Lincoln, Nebr.) density gradient fractionator and uv monitor, and the radioactivity of the fractions was determined by transferring small samples to scintillation vials containing a toluene-based scintillation fluid and 7% NCS (Amersham-Searle). Those fractions from the infected preparation which showed high specific radioactivity relative to comparable fractions of the healthy preparation were combined, the RNA precipitated with ethanol, and subjected to further purification by electrophoresis on cylindrical gels. Polyacrylamide
gel
electrophoresis.
Electrophoresis on cylindrical, 6-mm diameter, polyacrylamide gels was carried out essentially as described by Bishop et al. (1967)) after which the gels were soaked in H,O for at least an hour and scanned for absorbance at 260 nm with the aid of a Gilford Model 2400-S spectrophotometer equipped with the linear transport accessory. The gels were then frozen, sliced into l-mm-thick
disks with the aid of a bank of
razor blades, and each two consecutive gel slices were soaked in 0.5 ml of 0.1 M NaCl overnight at 0” in order to elute the RNA. Samples were taken from the eluates for radioactivity determination or, alternatively, the elution procedure was omitted and total radioactivity contained in gel slices was determined following either digestion with 30% H,O, or soaking in a 9:l
NCS:water mixture. Electrophoresis in slab gels was performed in an apparatus similar to that
described by Studier (1973) supplied by Aquebogue (Aquebogue, New York) or in the Bio-Rad (Richmond, Calif.) slab-gel apparatus, Model 220. The RNA samples
MESSENGER
RNA
FOR
were run on linear 2.2-4 or 5% gradient gels prepared from acrylamide stock solutions at an acrylamide-bis ratio of 30:1.5 for the 2.2% gel solution and 3O:O.a for the heavier 4 or 5% solution. Both the 2.2 and 45% gel stock solutions contained 1% ammonium persulfate and TEMED at a concentration of 106 ~1 per 100 ml of 2.2% gel solutions and 53 ~1 per 100 ml of the 4 or 5% solutions. The more concentrated gel solution also contained 15% sucrose. All stock solutions were kept on ice and the gradient was generated using a simple sucrose gradient device (Buchler). All operations were carried out in the cold. When l.Bmm-thick gradient gels were made in the Bio-Rad apparatus, 14.5 ml of the 2.2% gel solution and 13.6 ml of the 4 or 5% gel solution were used. Further, 1 ml of 2.2% gel solution was layered on the top of the gradient. Electrophoresis was carried out using E buffer (Bishop et al., 1967) at 35 mA constant current until the voltage reached 125 V, after which this voltage was maintained constant. Samples were usually run for 1 hr after the marker dye (bromophenol blue) had run off the gel. Protein samples were run in slab gels, prepared according to Laemmli (1970) using 12.5% separating gels and 5% stacking gels. The acrylamide-bis cross-linking ratio was reduced to 30:0.15 in order to obtain sharper bands and to eliminate cracking of gels during drying (Knowland, 1974). Electrophoresis was carried out at 35 mA constant current until voltage reached 125 V, after which this voltage was maintained constant until the marker dye reached the front. Marker proteins and 14C-labeled TMV capsid protein were run as standards. After electrophoresis, tritiated RNA and protein bands were detected by fluorography as described by Bonner and Laskey (1974) using Kodak Royal X O-Mat film which had been preflashed to a density of 0.15 (Laskey and Mills, 1975). Cell-free protein synthesis. A wheat germ-derived cell-free protein synthesizing system was prepared and used as described by Davies and Kaesberg (19731, except that phosphoenolpyruvate was omitted because in our hands it was found to inhibit the reaction. Except as other-
TMV
CAPSID
PROTEIN
365
wise noted, 2 &!i of [3Hlleucine (45 Ci/ mmol) was added for each 100 ~1 of reaction mixture which contained each of the other 19 amino acids at a concentration of 25 pm. Reactions were run at 30” and the extent of incorporation of leucine into acidinsoluble product was determined by spotting either 5 or 10 ~1 of reaction mix onto Whatman 3 MM 2.3-cm filter disks that had been prewetted with 5% trichloroacetic acid containing 2 g/liter of nonradioactive leucine and washing the disks with the leucine containing 5% trichloroacetic acid four times at 90”, twice with ethanol, once with 1:l ethanol:ethyl ether mixture, and finally with ethyl ether. The disks were then dried and counted in a toluenebased scintillation fluid containing 7% of a 9:l NCS:H,O mixture. For analysis of the reaction product, the reaction mix was made to 200 to 400 ~1 and at the termination of the incubation (usually 1 hr) was treated for an additional 20 min with 20 ~1 of a solution containing 1 mg/ml of pancreatic ribonuclease (Worthington) and 15,000 units/ml Tl ribonuclease (P-L Biochemicals). Five milliliters of 5% trichloroacetic acid containing 1% casamino acids were added and the precipitated protein was collected after 1 hr at 0” by centrifugation. The pellet was washed twice with the precipitating solution at 0” and twice more with ice-cold acetone after which it was dried in uacuo. The protein was dissolved in Laemmli sample buffer (Laemmli, 1970) for electrophoresis and in phosphate-buffered saline containing 0.1% SDS for the gel diffusion serological test. RESULTS
Partial purification of LMC by band sedimentation on sucrose density gradients. The object of this study was to determine whether an RNA component, LMC, present in extracts of TMV-infected tissue is a unique monocistronic messenger RNA, and if so, what the nature of its gene product might be. In previous work this component had been detected as a radioactive band on polyacrylamide gel electropherograms because it incorporated radioactive precursor ( 13Hluridine) even in the presence of actinomycin D. After several unsuccessful attempts to accumulate
366
SIEGEL
enough material for characterization by elution of the radioactive LMC band from 6-mm diameter gels, it was decided first to obtain an LMC rich fraction by band sedimentation of a relatively large amount of tissue RNA through a sucrose density gradient. This was accomplished as described in the Materials and Methods section, and the results of one such experiment are shown in Fig. 1. Note the peak labeled A, of high radioactivity and relatively low absorbance. It appears consistently in infected, but not in healthy, tissue extracts and has the appropriate estimated sedimentation for the LMC component. Thus, the fractions constituting this peak were combined, precipitated with 2.5 volumes of ethanol, and resuspended in a small vol-
J FIG. 1. Sucrose density gradient centrifugation of an RNA extract from TMV-infected tissue. The first systemic leaves to become infected were harvested 4 days after inoculation and were infiltrated with an equal weight of 0.01 M KH,PO, containing 40 wg/ml of actinomycin D and 200 &i/ml of 13Hluridine. RNA was extracted from an 18-hr, dark incubation period and 1 mg was layered on a 5-20% linear sucrose gradient. Centrifugation was in the Spinco SW-27 rotor at 25,000 rpm for 18 hr. The peaks labeled A and B are unique to infected tissue and were collected and saved for further analysis. Sedimentation is to the right.
ET AL
ume of H,O for further fractionation. In some, but not all, experiments a second peak of high radioactivity and relatively low absorbance, labeled B in Fig. 1, was noted between the two large ribosomal absorbance peaks. Fractions constituting this material were also collected for further analysis. Further purification of LMC by polyacrylamide gel electrophoresis. The material collected as peak A after sucrose density gradient centrifugation was subjected to electrophoresis on cylindrical polyacrylamide gels. These- were scanned for absorbance, sliced, and eluates of the gel slices were tested for radioactivity. As seen in Fig. 2, the radioactivity appears in one sharply defined peak which is taken to be LMC. Note also that enough LMC is present to yield an absorbance as well as a radioactive peak. The major absorbance peak in the electropherogram is judged to be the smallest (16 S) ribosomal RNA. The rate of movement of RNA species of known molecular weights (brome mosaic virus RNA) was determined on parallel gels, and using this information, an estimate of 250,000 daltons was made for the size of LMC. This is somewhat smaller than a previous estimate (Jackson et al., 1972) and may in part provide an explanation for the failure by others (Aoki and Takebe, 1975) to observe the LMC component. The gel-slice eluates which contained radioactivity were combined from several gels and the resultant solution was freed from contaminating substances by passing it through a hydroxyapatite column (Diener and Smith, 1973) under which conditions RNA is absorbed to the column and much of the contaminating material passes through. The RNA was recovered from the column with 0.12 M KPB (KPB is an equimolar mixture of KH,PO, and K,HPO,) and it was further purified and greatly concentrated by loading on a hydroxyapatite minicolumn (3-mm diameter, 4-mm high) at 0.03 M KPB and eluting it with a minimal volume of 0.12 M KPB. The two-step purification and concentration procedure was found to be necessary, because during the first step a sufficient amount of hydroxyapatite must be used so
MESSENGER
9-
3%
RNA
FOR
1
GEL 16 !
B-
7-
BMV 1t2 1
BMV 3
IMV
i
1
6-
IO
20
30
40
FRACTION
FIG. 2. Gel electrophoresis of the material contained in peak A of Fig. 1. The fractions labeled A in Fig. 1 were combined, concentrated, and subjected to electrophoresis on a 6-mm diameter 3% gel. The major radioactive peak is assumed to represent the LMC RNA described by Jackson et al. (1972). Migration is to the right. Arrows indicate the positions of brome mosaic virus and the 16 S chloroplast RNA species on parallel gels.
that it is all not poisoned by a component in the gel-eluted LMC solution. LMC as a messenger RNA in the wheat germ-derived cell-free protein synthesizing system. The radioactive material recovered from the minihydroxyapatite column was diluted with an equal amount of H,O to reduce the KPB concentration and was tested as a template in the wheat germ reaction mixture. The data in Table 1 show that both the LMC and TMV RNA stimulate incorporation of [3Hlleucine into acid-insoluble material, presumably polypeptides. The nature of the LMC and TMV RNAstimulated products was examined by subjecting the incubated reaction mixtures to
TMV
CAPSID
367
PROTEIN
polyacrylamide slab gel electrophoresis. The fluorograph of one such gel is shown in Fig. 3 where it can be seen that the LMC-stimulated material contains a radioactive protein of the same size as TMV capsid protein in addition to some smaller material. In contrast, the TMV RNA stimulated mixture, in agreement with previous work (Efron and Marcus, 1973; Roberts et al., 19741, contains many polypeptides, many of which are larger than capsid protein. The material in the LMCstimulated wheat germ system was tested for reaction to an antiserum prepared against TMV capsid protein. Figure 4 shows the result of a gel-diffusion assay in which both control 14C-labeled capsid protein and the LMC-stimulated material react with the antiserum. We conclude that LMC has the coding capacity for TMV capsid protein because it stimulates the wheat germ system to produce a protein of the same size and with the same antigenic specificity as the capsid protein. Conditions for biosynthesis of LMC. Actinomycin D was employed initially to suppress incorporation of labeled precursor into host RNA during incubation of TMVin the presence of infected tissue [3H]uridine. The inhibitor was found not only to be efficient for this purpose, particularly when incubation was carried out in the dark, but in addition, more labeled precursor became incorporated into LMC in its presence than in its absence, as can be seen in Fig. 5. The double effect of actinomycin D makes its employment parTABLE Time (min)
None 0 60
1
TRANSLATION OF LMC In Vitro” Template
9206 24,000
TMV RNA (1 pg) 1,400
220,000
LMC 3,600 168,000
a Either nothing, 1 pg of TMV RNA, or LMC extracted from 1 g of infected leaf tissue was added to 100 ~1 of wheat germ reaction mixture containing 2 Z.&i of PH]leucine as described in Materials and Methods. b TCA-insoluble counts per minute.
368
SIEGEL
ET AL.
tected in electropherograms of RNA extracts as soon as 12 hr after inoculation. In a parallel experiment, young expanding leaves were harvested 42 hr after lower leaves had been inoculated and these were also incubated under the same conditions. The leaves were harvested at a time when they had been only recently invaded by virus, several hours before vein-clearing symptoms appeared. Here again (Fig. 6) the LMC could be detected at a relatively short time (2 hr) after the leaves were harvested. Other unique RNA components present in TMV-infected tissue. Unique RNA species, other than LMC, were also noted in extracts of infected tissue. The radioactive
FIG. 3. SDS-gel electrophoresis of radioactive peptides appearing in a wheat germ cell-free system when stimulated with LMC and TMV RNA. The channels contain: (a) W-labeled TMV capsid protein (360 cpm); (b) 3H-labeled in vitro products stimulated by 10 pg of TMV RNA (45,000 cpm); (c) same as (b), but twice the amount (90,000 cpm); (d) SHlabeled endogenous products (8,000 cpm); (el 3Hlabeled in vitro products stimulated by 1 pg of TMV RNA (45,000 cpm); (0 empty; (gl 3H-labeled in vitro products stimulated by LMC (20,000 cpm).
titularly valuable for the experiments reported in this paper, because, together with the newly adopted technique of fluorography, the detection of LMC and other viral-specific tritiated RNA components is greatly facilitated. Experiments were conducted to determine when during infection the LMC could be first detected. To this end, leaves were detached 3 hr after inoculation and were incubated for different periods of time in the presence of actinomycin D and C3Hluridine. Under these conditions, where only a low proportion of the cells are infected, LMC can, nevertheless, be de-
FIG. 4. Fluorograph of a gel diffusion reaction between the 3H-labeled peptides generated in an LMC-stimulated wheat germ system and rabbit anti-TMV capsid serum. The wells were loaded with 14C-labeled capsid protein (CP), LMC-stimulated product (LMC), unlabeled capsid protein (U), and antiserum (B). The bands were allowed to develop for 3 days after which the gel (1.5% agarose) was soaked for 0.5 hr with five successive changes of phosphate buffered saline (pH 7.0) to remove nonprecipitated radioactivity. The gel was then prepared for fluorography as described in Materials and Methods, except that methanol was used to dehydrate the gel instead of DMSO (Laskey and Mills, 1975).
MESSENGER
RNA
FOR
peak labeled B in Fig. 1 is an example. It has an apparent size intermediate between the two major ribosomal RNA species and when the material in the fractions constituting this peak was collected and analyzed on acrylamide disk gels, a peak of radioactivity was found to corn&-ate with the large chloroplastic 23 S RNA (Fig. 7) indicating a molecular weight of 1.1 x 106. The detection of this component
TMV
CAPSID
PROTEIN
369
appears to require particular conditions because it has not always been observed as clearly as shown in Figs. 1 and 7, although it probably is the same as the band to which the arrow points in Figs. 5 and 6. DISCUSSION
The experiments presented here demonstrate the presence of a species of RNA, LMC, in extracts of TMV-infected tissue which acts in vitro as a special messenger for the capsid protein. It has a molecular
LMC
FIG. 5. Fluorograph of RNA extracted from healthy and TMV-infected tissue. The first leaves to become systemically infected or comparable healthy leaves were harvested 5 days after inoculation. These were either infiltrated with [3H]uridine and actinomycin D or with ‘[3H]uridine alone and were incubated in the dark for 18 hr. The RNA was extracted and 10 pg were subjected to electrophoresis on a 2.2-5% polyacrylamide slab gel. (1) Healthy, no actinomycin D (170,000 cpm); (2) healthy, with actinomycin D (15,000 cpm); (3) infected, no actinomycin D (175,000 cpm); (4) infected, with actinomycin D (150,000 cpm). The arrow indicates a putative TMVrelated RNA of ca. 1.1 x 10” daltons.
FIG. 6. Fluorograph of RNA samples extracted from infected leaves after different time periods. Systemically invaded leaves were detached 42 hr after inoculation and were infiltrated with [3H]uridine and actinomycin D. Leaf samples were taken for RNA extraction after different periods of incubation in the dark and 10 pg of each RNA was subjected to electrophoresis on a 2.2-5% polyacrylamide gel. The numbers under the channels indicate the hours of incubation. The channel labeled H contains 2 pg of RNA labeled in the absence of actinomycin D and extracted from healthy tissue; the upper and lower heavily labeled bands are, respectively, 25 and 18 S ribosomal RNA. The arrow indicates a component suspected of being a virus-specific RNA of 1.1 x- 10” daltons.
370
SIEGEL ,
41.8%
GEL
3-
‘;
‘I’
‘1” 1p
\
FRACTION
FIG. 7. Gel electrophoresis of the material contained in peak B of Fig. 1. The fractions labeled B in Fig. 1 were combined, concentrated, and subjected to electrophoresis on a 6-mm diameter 1.8% polyacrylamide-0.5% agarose gel. Most of the radioactivity migrated with the 23 S chloroplast ribosomal RNA.
weight of ca. 250,000 and the same nucleotide sequence as a portion of the viral RNA. Our findings confirm those of Knowland et al. (1975) who also demonstrated that a low-molecular weight component present in extracts of infected tissue directs the synthesis of capsid protein both in vitro and in oocytes ofxenopus laevis. We have found no evidence that LMC is encapsidated in our studies with the Ul strain. However, it probably is similar to the component which does become encapsidated during infection with the cowpea strain (Morris, 1974) and which also has been shown to direct the in vitro synthesis of capsid protein (Bruening et al., 1976; Higgins et al., 1976). One can think of at least two mechanisms for the origin of LMC. It may be either split from a strand of viral RNA or
ET AL.
it may be an incomplete transcript of a complementary RNA strand. At the present time the choice between these two or other possible alternatives is speculative. It is likely that LMC does represent the in vivo messenger for capsid protein rather than being a nonfunctional adjunct to TMV replication because its presence can be detected rather early in the infection process and because evidence is available that a low molecular weight RNA which bears resemblance to LMC is present in polysomes (Babos, 1971; Beachy and Zaitlin, 1975). The demonstration that at least one gene product is translated from a monocistronic messenger RNA raises the question as to whether monocistronic messenger RNA may be generated for other genes on the virion genome. Evidence is presented that new RNA species other than LMC may be generated during infection. Whether these represent specific virus related mRNA and if so, for what gene product they may code, remains to be determined. It is known that, among other products, the synthesis of two large proteins of molecular weights ca. 135,000 and 165,000 is stimulated in TMV-infected tissue (Zaitlin and Hariharasubramanian, 1972; Sakai and Takebe, 1974; Paterson and Knight, 1975) and similar large proteins are made under the direction of TMV RNA in oocytes (Knowland, 1974) and in a reticulocyte cell-free system (Knowland et al., 1975). It is possible that the ca. 1.1 x lo6 dalton RNA species, to which the arrow points in Figs. 5 and 6, might be just large enough to code for one of the two large proteins, one of which may be a viral-coded RNA replicase (Zaitlin et al., 1973). However, little is still known about what genes, other than those for capsid protein, might be present in the TMV genome, and whether monocistronic messages might exist for these. Thus, further work is necessary to clarify the translation strategy of the TMV genome and its control. REFERENCES S., and TAKEBE, I. (1975). Replication of tobacco mosaic virus in tobacco mesophyll protoplasts inoculated in uitro. Virology 65, 343-354. BABOS, P. (1971). TMV-RNA associated with riboAOKI,
MESSENGER
RNA
FOR
somes of tobacco leaves infected with TMV. Virology 43, 597-606. BEACHY, R. N., and ZAITLIN, M. (1975). Replication of tobacco mosaic virus. VI. Replicative intermediate and TMV-RNA related RNAs associated with polyribosomes. Virology 63, 84-97. BISHOP, D., CLAYBROOK, J., and SPIEGELMAN, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26, 373-387. BONNER, W., and LASKEY, R. (1974). A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 44, 279-288. BRUENING, G., BEACHY, R., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in uiuo translation of the ribonucleic acids of the cowpea strain of tobacco mosaic virus. Virology 71, 498-517. DAVIES, J. W., and KAESBERG, P. (1973). Translation of virus mRNA: Synthesis of bacteriophage QB proteins in a cell-free extract from wheat embryo. J. Virol. 12, 1434-1441. DIENER, T. O., and SMITH, D. R. (1973). Potato spindle tuber viroid. IX. Molecular-weight determination by gel electrophoresis of formylated RNA. Virology 53, 359-365. EFRON, D., and MARCUS, A. (1973). Translation of TMV-RNA in a cell free wheat embryo system. Virology 53, 343-348. GRAY, J. C. (1974). The inhibition of ribonuclease activity and the isolation of polysomes from leaves of the french bean, Phaseolus vulgaris. Arch. Biothem. Biophys. 163, 343-348. HIGGINS, T., GOODWIN, P., and WHITFELD, P. (1976). Occurrence of short particles in beans infected with the cowpea strain of TMV. II. Evidence that the short particles contain the cistron for coat protein. Virology 71, 486-497. 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, 655-665. JACOBSON, M., Asso, J., and BALTIMORE, D. (1970). Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49, 657-668. KIEHN, E., and HOLLAND, J. (1970). Synthesis and cleavage of enterovirus polypeptides in mammalian cells. J. Virol. 5, 358-371.
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CAPSID
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KNOWLAND, J. (1974). Protein synthesis directed by the RNA from a plant virus in normal animal cell. Genetics 78, 383-394. KNOWLAND, J., HUNTER, T., HUNT, T., and ZIMMERN, D. (1975). Translation of tobacco mosaic virus RNA and isolation of the messenger for TMV coat protein. Les Colloques de l’lnstitut National de la Sante et de la Recherche Medicale. 47, 211-216. LAEMMLI, U. K. (1970). Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. LASKEY, R., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14Cin polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341. MORRIS, T. J. (1974). Two nucleoprotein components associated with the cowpea strain of TMV. Proc. Amer. Phytopathol. Sot. 1, 83. PATERSON, R., and KNIGHT, C. A. (1975). Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus. Virology 64, 10-22. ROBERTS, B. E., PATERSON, B., and SPERLING, R. (1974). The cell-free synthesis and assembly of viral specific polypeptides into TMV particles. Virology 59, 307-313. SAKAI, F., and TAKEBE, I. (1974). Protein synthesis in tobacco mesophyll protoplasts induced by tobacco mosaic virus infection. Virology 62,426-433. SHIH, C., and KAESBERG, P. (1973). Translation of brome mosaic viral ribonucleic acid in a cell free system derived from wheat embryo. Proc. Nut. Acad. Sci. USA 70, 1799-1805. SIEGEL, A., ZAITLIN, M., and DUDA, C. T. (1973). Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs. Virology 53, 75-83. STUDIER, F. W. (1973). Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. ZAITLIN, M., DUDA, C. T., and PETTI, M. (1973). Replication of tobacco mosaic virus. V. Properties of the bound and soluble replicase. Virology 53, 300-311. ZAITLIN, M., and HARIHARASUBRAMANIAN, V. (1972). A gel electrophoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 47,.296-305.
