VIROLOGY

65,343-354( 1975)

Replication

of Tobacco

Mesophyll SHIGEJI Institute

for Plant

Mosaic

Protoplasts AOKI’

Virus RNA in Tobacco

Inoculated

AND ITARU

TAKEBE

Virus Research, 959 Aobacho, Accepted

December

in Vitro

Chiba 280, Japan

30, 1974

Synthesis of tobacco mosaic virus (TMV)-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated in uitro. Three species of TMV-specific RNA were separated by polyacrylamide gel electrophoresis, and were identified as single-stranded viral RNA, its replicative form (RF), and replicative intermediate (RI) by examining their chromatographic behavior on cellulose, solubility in 1 M NaCl, and susceptibility to denaturation by dimethylsulfoxide. The molecular weight of RF was shown to be about 3.8 x 10’ by coelectrophoresis with rice dwarf virus RNA. Time course of the synthesis of RF and RI as well as the results of pulse-chase experiments were consistent with the possible precursor role of these structures in the synthesis of TMV-RNA. No other forms of TMV-specific RNA were detected in infected protoplasts. Kinetics of the replication of TMV-RNA in synchronously infected protoplasts showed the presence of three successive phases in virus replication. During the initial phase, viral RNA replicated exponentially and was encapsidated 4-5 hr later, so that most of the viral RNA synthesized remained free or only partially coated. The rate of viral RNA replication became linear at the end of the initial phase and remained so throughout the subsequent phases. Active formation of virus particles continued during the intermediate phase to encapsidate the bulk of viral RNA synthesized by this time. In the final phase, the synthesis of viral RNA was closely followed by encapsidation. INTRODUCTION

Synthesis of tobacco mosaic virus (TMV) RNA in infected leaf tissues has been studied extensively (Engler and Schramm, 1959; Diener, 1962; Sarker, 1965; Kubo, 1966; Wollum et al., 1967; Fraser, 1973). Relatively little information has been obtained, however, on the replication of viral RNA in individual infected cells, because the tissue systems used in these studies consists of cells which are heterogenous with respect to the stage of infection. Nilsson-Tillgren et al. (1969) developed a method to partially synchronize systemic infection of young tobacco leaves, and the synthesis of TMV-RNA in such leaves has been described in detail (Nilsson-Tillgren, 1969; Kielland-Brandt and Nilsson-Tillgren, 197313). However, the I Present address: Department of Microbiology, Nippon Dental College, 1-8 Hamauracho, Niigata 951, Japan.

system developed by these workers has a serious drawback in that the exact time of infection of individual cells is not known. On the other hand, protoplasts isolated from the mesophyll tissue of tobacco can be inoculated in vitro with TMV to give rise to a synchronous infection in nearly all the cells (Takebe and Otsuki, 1969; Ostuki et al., 1972a). Since the spread of infection from one cell to another is excluded (Takebe et al., 1971), it is possible with this system to follow a single cycle of virus replication in individual cells. We have studied some basic aspects of TMV-RNA replication in this system, and the results are presented in this paper. MATERIALS

AND

METHODS

Preparation and inoculation of protoplasts. The palisade tissue of healthy Nicotiana tabacum cv. Xanthi plants was the source of protoplasts. The methods for 343

344

AOKI

AND TAKEBE

the isolation (Aoki and Takebe, 1969) and for the inoculation (Otsuki et al., 1972a) of protoplasts were described previously. A common strain of TMV (strain OM) was used throughout this study. Assays using the fluorescent antibody technique (Otsuki and Takebe, 1969; Otsuki et al., 1972b) showed that 80-90% of the protoplasts were infected in this study.

addition of 2.5 vol of cold ethanol, After storage at -20°C overnight, the precipitate was collected by centrifugation and was dissolved in 0.2-0.5 ml of 0.15 M sodium acetate (pH 6.0) containing 0.5% SDS, and RNA was precipitated by adding 2.5 vol of cold ethanol. RNA purified by two to three cycles of differential precipitation was collected by centrifugation, and ethanol was Culture of inoculated protoplasts and removed by evacuation. For gel electropholabeling with 32P-phosphate. Inoculated resis, RNA was dissolved in 50-100 ~1 protoplasts were washed aseptically with of electrophoresis buffer containing 10% 0.7 M mannitol solution containing 0.1 sucrose (RNase-free, Nakarai Chemicals mM CaCl, and were suspended in a culture Ltd.), and lo-50 ~1 aliquots were electromedium at a density of 2-4 x lo5 cells/ml. phorezed. The composition of the medium was as If DNase digestion was necessary, RNA before (Aoki and Takebe, 1969) except that dissolved in 0.05 M Tris buffer, pH 7.6, 6-benzyladenine was omitted (Otsuki et containing 2 mM magnesium acetate (Loal., 1972b). Light and temperature condi- ening et al., 1969) was treated with DNase tions of culture were described previously (RNase-free, Worthington Biochemical (Otsuki et al., 1972b). In the experiments Corp.) at a concentration of 5-10 &ml for in which the protoplasts were to be labeled 10 min at 25°C. RNA was then reextracted with 32P, KH,PO, was omitted from the with phenol and precipitated with ethanol. Polyacrylamide gel electrophoresis of medium and carrier-free 32P-orthophosphate (Daiichi Pure Chemicals, Ltd.) was RNA. Acrylamide and bis-acrylamide were added to 20-50 pCi/ml. When necessary, purified essentially according to the actinomycin D (Merck Sharp and Dohme method of Loening (1967). Gels (0.7 x 9 International or Calbiochem) and/or chlor- cm) containing 2.4% acrylamide and 0.12% amphenicol were added to the medium to bis-acrylamide were polymerized in acrythe concentrations of 10 and 100 &ml, late tubes. n-Propylamine, reported to inrespectively. After an appropriate time of crease the mobility of RNA (Oberg and labeling, protoplasts were collected by cen- Philipson, 1969), was included at 1 mM in trifugation, washed with 0.7 M mannitol the electrophoresis buffer of Loening solution and were stored at -20°C until (1968). Gels were prerun for l-3 hr at a use for the extraction of RNA. Usually, 2-4 constant current of 5 mA/gel. x lo6 protoplasts were enough for the RNA (lo-50 pg) carefully layered over analysis of RNA. For the isolation and the gel was electrophoresed for 4-6 hr at a characterization of double-stranded RNAs, constant current of 5 mA/gel at 5°C. Gels 2-4 x 10’ protoplasts were used as starting were then washed in distilled water and material. were scanned by a Joyce Loebl ChromoExtraction of RNA. Frozen protoplasts scan either at 265 nm, or at 595 nm after (2-4 x lo6 cells) were solubilized with 1.25 staining with 0.2% methylene blue in 0.4 M ml of 1% sodium dodecylsulfate (SDS) in acetate buffer, pH 5.0 (Peacock, and Dingglycine buffer (0.1 M glycine, 0.1 M NaCl, man, 1967). For radioactivity measure5 mM EDTA, pH 9.5; Stinger and Knight, ments, gels were cut with a razor blade into 1963) containing 1% bentonite, and were slices of 1 or 1.5 mm thickness using the shaken with an equal volume of water- apparatus devised by Iandolo (1970). 32Psaturated redistilled phenol for 10 min at radioactivity of dried slices was measured room temperature. The emulsion was bro- by a low background gas flow counter ken by centrifugation at 4°C and the aque- (Aloka model PDC801). ous phase was extracted twice more with a Fractionation of virus-specific RNAs. half-volume of phenol. RNA was precipi- S2P-labeled RNA extracted from infected tated from the final aqueous phase by the protoplasts was fractionated by cellulose

TMV-RNA

REPLICATION

chromatography and by precipitation in 1 M NaCl (Franklin, 1966). RNA extracted from 3.5 x 10’ protoplasts was dissolved in 1.3 ml of STE buffer (0.1 M NaCl, 0.05 M Tris, 1 mM EDTA, pH 6.9) and ethanol was added to a final concentration of 35%. The solution was applied to a column (1 x 20 cm) of cellulose (Whatman CF-11) washed previously with STE buffer, and was eluted successively with STE buffer containing 35% ethanol (60 ml), with the same buffer containing 15% ethanol (90 ml) and finally with STE buffer only (60 ml). After the radioactivity in each 3.0 ml fraction was measured, single-stranded RNAs eluted with 15% ethanol (which should contain TMV-RNA) were pooled and were precipitated by adding 2.5 vol of cold ethanol. Double-stranded RNAs eluted with buffer only were also pooled and to this was added carrier RNA extracted from uninfected protoplasts. Onequarter volume of 5 M NaCl was then added to the solution of double-stranded RNAs and the mixture was stored overnight at 5°C. Precipitate formed by this treatment (which should contain replicative intermediate) was collected by centrifugation. RNA remained soluble in 1 M NaCl (which should contain replicative form) was precipitated with 2.5 vol of ethanol after the addition of nonradioactive RNA from healthy protoplasts as carrier. A portion of the 1 M NaCl-insoluble RNA was dissolved in 2 x SSC (SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH, 7.0) and was treated with RNase (5 x crystallized, Sigma Chemical Co.) at 5 pg/ml for 10 min at 25°C. The solution was deproteinized by phenol extraction and RNA was precipitated with cold ethanol. of double-stranded RNAs Denaturation with dimethylsulfoxide. 32P-labeled RNA extracted from infected protoplasts was treated with DNase and was fractionated by cellulose chromatography and by precipitation in 1 M NaCl as described above. Radioactive RF or RI thus obtained was dissolved in 0.01 M Tris buffer, pH 7.2, containing 1 mM EDTA (Iglewski and Franklin, 1967). Nine volumes of dimethylsulfoxide were added to the RNA solution and the mixture was incubated for 15 min

345

IN PROTOPLASTS

at 37°C. The solution was then cooled rapidly in ice water to prevent renaturation, and RNA was precipitated with cold ethanol. Determination of nucleotide composition of RF. 32P-labeled RNA was prepared from infected protoplasts by various procedures (see the legend of Table 1) and was electrophoresed. Gel slices corresponding to the region of RF were incubated in 0.5 ml of 0.3 M KOH for 18 hr at 37°C to hydrolyze RNA. The slices were then washed twice with 0.5 ml of distilled water, and to the combined hydrolysate was added the hydrolysate of 5-7 mg yeast RNA. The mixture was neutralized with perchloric acid, and the precipitate formed was removed by centrifugation. The supernatant solution was applied to a column (0.9 x 20 cm) of Dowex 1 x 2 (200-400 mesh, formate form). Four ribonucleotides were eluted with increasing concentrations of formic acid according to the method of Osawa et al. (1958). The elution of nucleotides was monitored by measuring the absorbancy of eluates at 260 nm. Fractions containing each nucleotide were combined quantitatively and 32P-radioactivity was measured by a low background gas flow counter. Determination of virus content. Extracts of infected protoplasts were prepared as described in a previous paper (Aoki and Takebe, 1969), and infectivity in the extracts was assayed after appropriate dilution on 8-10 half-leaves of N. tabacum cv. Xanthi nc. A purified TMV solution (0.1 pg/ml) was inoculated on the opposite half-leaves to standardize the assay and to express the results in terms of the number of virus particles in the extracts. RESULTS

Virus-specific RNA species synthesized in infected protoplasts. In order to identify virus-specific RNA species, protoplasts inoculated with TMV were cultured in the presence of actinomycin D and newly synthesized RNA was labeled by adding 32Porthophosphate to the medium. RNA was extracted from the protoplasts 20 hr postinfection (p.i.) and was analyzed by electrophoresis in polyacrylamide gels. Protoplasts inoculated with ultraviolet-inac-

AOKI

346

AND TAKEBE TABLE

NUCLE~TIDE COMPOSITION OF T-LABELED

RF ISOLATED FROM TMV-INFECTED

PROTOPLASTS’

Mole percent

Nucleotide TMV-RNAb

AMP GMP CMP UMP

1

29.8 25.3

18.5 26.3

32P-RF from protoplasts

RF

28.0 22.0 22.0 28.0

Expt l*

Expt 2d

Expt 3’

Expt 4’

28.4 21.5 21.7 28.4

28.3 21.2 21.2 29.3

28.1 21.6 22.3 28.0

28.5 21.4 20.9 29.2

a 32P-labeled RNA prepared from infected protoplasts by various methods (d-f) was electrophorezed in polyacrylamide gels. Gel slices from the RF region were digested with KOH, and the radioactivity distribution in four nucleotides was determined as described in Materials and Methods. b Data by Knight (1952). c Theoretical values calculated from the nucleotide composition of TMV-RNA. d Infected protoplasts were labeled with 32P-phosphate (50 pCi/ml) between 1 and 17 hr p.i. RNA extracted from 3 x lo7 protoplasts was chromatographed on a cellulose column. Eluate with buffer containing no ethanol was pooled, and RNA was precipitated with ethanol. e Infected protoplasts were labeled with a2P-phosphate (6.7 pCi/ml) between 0 and 17 hr pi. in the presence of actinomycin D and chloramphenicol. RNA extracted from 2.4 x 10’ protoplasts was treated with DNase, deproteinized with phenol, and was precipitated with cold ethanol. ‘Infected protoplasts were labeled with 32P-phosphate (50 rCi/ml) between 4 and 11 hr p.i. in the presence of chloramphenicol. RNA from 2.6 x 10’ protoplasts was treated with DNase and was dissolved in 1 M NaCl solution. RNA remained soluble was precipitated with ethanol and was treated with RNase (50 &ml) in 5 x SSC for 20 min at 25”. RNA was precipitated with cold ethanol after deproteinization with phenol.

tivated virus were used as the control and are referred to as uninfected protoplasts in this paper. Figure 1 compares the electrophoresis profiles of RNA extracted from uninfected (A) and infected (B) protoplasts. Small amounts of 32P-radioactivity were incorporated by uninfected protoplasts into 25s and 18s cytoplasmic ribosomal RNAs (rRNA) (Fig. 1A) as well as into transfer RNA (data not shown). Little, if any, radioactivity was found in 23s and 16s chloroplast rRNAs or in DNA. The low turnover rate of chloroplast rRNAs in tobacco mesophyll protoplasts has been shown also by labeling with [‘4C]uracil (Sakai and Takebe, 1970). It should be noticed that no radioactive RNA larger than 25s rRNA is detectable in uninfected protoplasts (Fig. 1A). Infection by TMV did not markedly affect 32P-incorporation into cytoplasmic rRNAs, whereas it resulted in the synthesis of three new RNA species (Fig. 1B). RNA with the highest radioactivity (slices 1623) was detectable also by staining with methylene blue (Fig. 1B) and was identified as TMV-RNA, since its mobility was

10 : 05 ; Et

a g

15

10

5

0

20 Slice

40 Number

60

FIG. 1. Gel electrophoresis profiles of RNA extracted from uninfected and infected protoplasts. Protoplasts were cultured at a density of 1.9 x lo5 cells/ml and were labeled with 3zP-phosphate (20 bCi/ml) between 8 and 20 hr p.i. Actinomycin D had been added to the medium at 6 hr p.i. RNA (14 rg) extracted from uninfected (A) and infected (B) protoplasts was subjected to gel electrophoresis. (-) Absorbance at 595 nm; (-•-) 3zP-radioactiuity.

TMV-RNA

REPLICATION

identical to that of RNA extracted from the virus. RNAs in the smaller radioactivity peaks (slices l-7 and slices 10-12) were identified as double-stranded RNA species containing viral RNA, as will be described below. Jackson et al. (1972) reported that, in addition to the three TMV-specific RNA species demonstrated in infected protoplasts, two more TMV-related RNAs occur in infected leaves, one larger than TMVRNA and the other with a molecular weight of 3.5 x 105. The latter RNA was designated as low molecular weight component and was later shown to be a fragment of TMV-RNA (Siegel et al., 1973). This component was not found in infected protoplasts (Fig. 2A). RNA from infected protoplasts occasionally showed small radioactivity peaks in low molecular weight regions of gel (Fig. 2B). These peaks are most probably due to partial breakdown of host or viral RNAs during extraction, since their appearance was always accompanied by more or less marked degradation of 23s chloroplast rRNA (Fig. 2B). Fractionation and analysis of virusspecific RNAs. 32P-labeled RNA from infected protoplasts was fractionated into single- and double-stranded species by cellulose chromatography (Franklin, 1966). Electrophoresis profiles of total (unfractionated) RNA, of RNA eluted with buffer containing 15% ethanol, and of RNA eluted with buffer only are shown in Fig. 3A, B, and C, respectively. TMV-RNA and cytoplasmic rRNAs were recovered in the 15% ethanol fraction (Fig. 3B) which should contain single-stranded RNAs, whereas the two other virus-specific RNAs were present exclusively in the fraction eluted with buffer containing no ethanol (Fig. 3C) indicating their double-stranded nature. The two virus-specific double-stranded RNAs in the buffer fraction could be separated from each other on the basis of their difference in the solubility in NaCl solution. The faster moving component of virus-specific double-stranded RNAs predominated in the 1 M NaCl soluble fraction, forming upon electrophoresis a sharp symmetrical band (Fig. 3D). On the other hand, the slower moving component was recovered as NaCl-precipitable RNA and

347

IN PROTOPLASTS

6-

TMV RNA F

(A) 25s 23s

25s .-: 2 2

40 60 Slice Number Frc. 2. Gel electrophoresis profiles of RNA preparations from infected protoplasts. (A) Preparation with little breakdown of 23s chloroplast rRNA. Infected protoplasts at 4.0 x lo5 cells/ml were labeled with 32P-phosphate (25 &i/ml) between 0 and 12 hr p.i. in the presence of actinomycin D. (B) Preparation with marked breakdown of 23s chloroplast rRNA. Infected protoplasts at 0.7 x 10’ cells/ml were labeled with “ZP-phosphate (25 &i/ml) between 0 and 14 hr p.i. in the presence of chloramphenicol. (. .) Absorbance at 265 nm; (-•-) “P-radioactivity. 0

showed a tailing toward the regions of lower molecular weights (Fig. 3E). Treatment of the latter component with RNase yielded a structure with the mobility of the faster moving component together with some smaller forms (Fig. 3F). The results compiled in Fig. 3 indicated that the faster moving component had a purely double-stranded structure of uniform size, whereas the slower moving component had the same basic structure to which single-stranded tails are attached. The TMV-specific double-stranded RNAs synthesized in infected protoplasts were thus shown to have the structures assigned to replicative form (RF) and replicative intermediate (RI) of bacteriophage RNA (Ralph, 1969). Identification of virus-specific doublestranded RNAs as RF and RI. The faster moving component of TMV-specific double-stranded RNAs was isolated from a2Plabeled protoplasts and was denatured by incubation with dimethylsulfoxide. Fig-

348

AOKI AND TAKEBE

obtained with separate samples were 3.8 f 0.2 x lo’, in good agreement to the theoretical molecular weight of TMV-RNA RF (4 x 106). It is clear in Fig. 5 that the molecular weight-mobility relationship is markedly different between single- and double-stranded RNAs. This observation raises some doubt about the accuracy of (El r; (B) the molecular weight determination of RF 2 -20 E a ,” using single-stranded RNAs as standards u4-h h A (Jackson et al., 1971). Table 1 shows the nucleotide composition of 32P-labeled faster moving component prepared by several different procedures. The results agreed well with the -2 theoretical nucleotide composition of 1 TMV-RNA RF irrespective of the proce20 40 60 0 20 40 60 dure of preparation. These results indicate Slice Number that both plus and minus strands are FIG. 3. Gel electrophoresis of virus-specific RNAs equally labeled with 32P and that the fractionated by cellulose chromatography and by amount of nonlabeled parental TMV-RNA precipitation in salt solution. Infected protoplasts at in this structure is very small, if any. 3.5 x IO5 cells/ml were labeled with 32P-phosphate (50 The experiments described in this and &i/ml) between 1.5 and 12.5 hr p.i. in the presence of the foregoing section leave little doubt actinomycin D and chloramphenicol. Labeled RNA that the faster moving double-stranded extracted from the protoplasts was fractionated as RNA produced in infected protoplasts repdescribed in Materials and Methods. (A) Total (unresents the RF of TMV-RNA. It follows fractionated) RNA; (B) RNA eluted from cellulose column with 15% ethanol; (C) RNA eluted from then that the slower moving doublecellulose column with buffer; (D) 1 M NaCl soluble stranded RNA is RI, since it has singleRNA from C; (E) 1 M NaCl insoluble RNA from C; stranded tails of varying length and is (F) E after RNase treatment. converted into RF by RNase (Fig. 3F). ure 4A and B compare the electrophoretic Furthermore, denaturation of this compomobility of the component before and after nent with dimethylsulfoxide yielded predenaturation. The main product of dena- dominantly an RNA with the size of TMVturation had the size of TMV-RNA (Fig. RNA together with smaller RNAs which 4B) indicating that the faster moving com- probably derived from the single-stranded tails (Fig. 4C and D). An accurate determiponent contains full length TMV-RNA and RNA complementary to it. However, nation of the molecular weight of RI was only 29% of the original radioactivity was difficult because of its heterogeneity in size and because of the unavailability of proper recovered as the full length single-stranded molecular weight standards. A modal value molecules. The molecular weight of the faster mov- estimated by coelectrophoresis with RDVing component was measured by coelectro- RNA was 6.7 f 0.6 x lo6 (Fig. 5). Time course of synthesis of virus-specific phoresis with RNA of rice dwarf virus (RDV). RDV-RNA consists of 12 segments RNAs. Separation of virus-specific RNAs by means of gel electrophoresis made it of double-stranded RNA and the molecular weight of each segment has been deter- possible to quantitate the 32Pin each RNA mined (Reddy et al., 1974). The size of species. In order to study the time course of 32P-labeled faster moving component was TMV-RNA replication during synchronous estimated by extrapolating the linear rela- infection, the production of virus-specific tionship between the mobility of RDV- RNAs was followed over the periods of RNA segments and the logarithm of their virus replication in protoplasts. Protoplasts inoculated with TMV were molecular weights (Fig. 5). The values

TMV-RNA

REPLICATION (‘3

IN PROTOPLASTS RI

(D) DMSO-hated

RI

20 Number

60

Slice

349

40

80

FIG. 4. Gel electrophoresis profiles of RF and RI before and after denaturation with dimethylsulfoxide. Infected protoplasts were labeled with 32P-phosphate between 1 and 17 hr p.i. RF and RI were isolated, denatured with dimethylsulfoxide and electrophoresed as described in Materials and Methods. Authentic unlabeled TMV-RNA as marker was added to the samples before electrophoresis. Profiles A and B, RF before and after denaturation; C and D, RI before and after denaturation. (-----) Absorbance at 595 nm; (-•-) 82P-radioactivity.

cultured in the presence of actinomycin D and 32P-phosphate. Samples of protoplasts were withdrawn at intervals after inoculation, and radioactive RNA extracted from them was electrophoresed to determine the radioactivity in virus-specific RNAs. With protoplasts sampled at 8 hr p.i. or later, it was also possible to determine the amount of TMV-RNA by measuring the absorbance at 265 nm of gel regions containing the RNA, with known amounts of TMV-RNA on sister gels as the standard. The radioactivity in TMV-RNA was proportional to the absorbance, indicating that the specific radioactivity of this RNA reached a constant level before 8 hr p.i. The quantity of virus particles was determined in a parallel experiment by comparing the infectivity in extracts of protoplasts with that of a purified TMV preparation. Figure 6 summarizes the time course of the incorporation of 32P into TMV-RNA, RF and RI, as well as the synthesis of complete virus particles (infectivity). Immediately after inoculation, about 1000 inoculum particles adsorbed per protoplast, and their infectivity decreased during the subsequent hours, probably as the result of uncoating. A rapid increase in the amount of virus started at 6 hr p.i. and

proceeded exponentially until 10 hr p.i. The rate of increase then slowed down gradually and became linear. Viral RNA synthesis was detectable as early as 4 hr p.i. and was exponential in its rate until about 8 hr p.i. Viral RNA synthesis should have started earlier than 4 hr p.i., since the estimated amount of viral RNA synthesized by this time exceeds lo3 molecules per protoplast. The rate of RNA replication dropped rather sharply at 10 hr p.i. and remained linear in later periods. The results shown in Fig. 6 clearly indicate that the course of TMV replication in synchronously infected protoplasts consists of three successive phases which differ in the balance between viral RNA synthesis and particle formation. In the initial phase (O-10 hr p.i.), both RNA synthesis and particle formation start and progress exponentially. However, there is a time lag of 4-5 hr between these processes, so that viral RNA production far outstrips virus particle formation. A large fraction of viral RNA synthesized in this phase should therefore exist in free or partially coated forms. In the intermediate phase (lo-20 hr p.i.), virus particle formation continues at a near maximal rate, whereas the rate of viral RNA synthesis is greatly reduced.

350

AOKI AND TAKEBE

Most of viral RNA is encapsidated into virus by the end of this phase. The final phase (20-35 hr p.i.) is characterized by parallel production of RNA and particles. The amount of viral RNA present in this phase corresponds roughly to the number of virus particles. Synthesis of RF and RI followed a course 05similar to that of viral RNA (Fig. 6). At the earliest detectable time, RF and RI comprised about 40% of total radioactivity in virus-specific RNAs, and this ratio graduO’Oally decreased to 10% by 35 hr p.i. There Dtslance Moved (cm) was no increase in the level of RF in the FIG. 5. Estimation of molecular weight of RF and final phase, while RI increased roughly in RI by coelectrophoresis with rice dwarf virus (RDV) parallel to viral RNA. RNA. 32P-labeled RF and RI were isolated from inThe accumulation of free or partially fected protoplasts by cellulose chromatography and coated forms of viral RNA in the initial 1 M NaCl treatment. Nonradioactive RDV-RNA and RNA from healthy protoplasts were added to the phase of TMV replication was also demonradioactive RF and RI, and the mixture was electrostrated by comparing the amount of fully phoresed in a 2.4% gel for 6.25 hr at a constant curencapsidated with that of total viral RNA rent of 5 mA/gel. RF and RI were located by radioby infectivity assay. As shown in Table 2, activity measurement of gel slices, RDV-RNA segonly 4% of the total viral RNA at 9 hr p.i. ments and tobacco leaf rRNAs were located by abwas present in a form which survived the sorbance at 595 nm after staining the gel with methylincubation with cell homogenate, indicatene blue. (0) RF and RI; (0) 12 segments of RDVing that most of the viral RNA is not RNA; (X 1 rRNAs of tobacco leaf cells. encapsidated at this stage. In contrast, the bulk of viral RNA was present in complete virus particles at 24 hr p.i. (Table 2). Pulse-chase experiment. It is generally assumed that the replication of bacteriophage and animal virus RNA involves double-stranded intermediate RNAs (for review see Ralph, 1969). There are also some evidences suggesting that RF and RI are precursors of TMV-RNA (Jackson et al., 1972; Kielland-Brandt and NilssonTillgren, 1973b). An attempt was, therefore, made to examine the possible precursor-product relationship between RF, RI, b’ I 1 and TMV-RNA by pulse-chase experiments. [3H]uridine was used to label RNA in these experiments, since preliminary studies showed that such experiments with FIG. 6. Time course of synthesis of virus-specific RNAs and of virus particle formation in protoplasts. 32P are complicated by the presence in Inoculated protoplasts were suspended in 260 ml of tobacco leaf cells of non-RNA, phosphorculture medium containing actinomycin D at a density ous-containing large molecules (possibly of 4.2 x lo5 cells/ml. The suspension was divided into polyphosphate). two equal halves, and 32P-phosphate was added to Actinomycin D was added to the culture one-half at 30 rCi/ml. The two halves were culof infected protoplasts 19 hr p.i. and this tured under the same conditions, and 10 ml samples were withdrawn at intervals. Samples from the 32P- was followed 1 hr later by the administration of [3H]uridine (16 Ci/mmole) at a labeled culture were used for analysis of virus-speconcentration of 10 pCi/ml. After 30 min of cific RNAs and those from the nonlabeled culture pulse labeling, the culture was divided into for infectivity assay. .a

-

‘0

i

‘i

I

I

TMV-RNA TABLE

REPLICATION

351

IN PROTOPLASTS

2

PROPORTION OF,TMV-RNA PRESENTIN COMPLETE VIRUSPARTICLESAT Two DIFFERENTTIMES AFTER INOCULATION OF PROTOPLASTS~ Hours p.i.

9 24

Infe;$$y

by

In$f~$;ec&y

TMV-RNA

(A)

in virus particles (B)

581 * 185 577 * 274

22 * 12 505 * 178

Ratio B/A

0.04 0.88

’ TMV-inoculated protoplasts were cultured in the presence of actinomycin D. At 9 and 24 hr p.i., 4 x lo6 protoplasts were harvested and divided into two equal halves. One-half was solubilized with 2.5 ml of SDS-bentonite-glycine buffer (see Materials and Methods), extracted twice with phenol to give total TMV-RNA. The other half was homogenized with 1.25 ml of 0.1 M phosphate buffer, pH 7.0, in a VirTis Micro Homogenizer, and the homogenate was incubated for 1 hr at 25°C to destroy any free or partially coated TMV-RNA. The incubated homogenate was mixed with 1.25 ml of the SDS-bentonite-glycine buffer and was extracted with phenol to give TMVRNA which was present in virus particles. RNAs from the two halves were precipitated with ethanol, dissolved in 1 ml of the phosphate buffer, and their infectivity was compared on eight opposite halfleaves of N. tabacum cv. Xanthi nc. RNAs from protoplasts sampled at 24 hr p.i. were diluted fivefold for infectivity assay. The figures represent the mean of number of lesions per half-leaf with standard error.

two equal halves; one-half was allowed further to incorporate [3H]uridine (continuous labeling), while the other half was exposed to cold uridine 6400 times in excess of [3H]uridine (chase). RNA was extracted from the protoplasts at intervals after the pulse labeling, separated by gel electrophoresis and the radioactivity in TMV-specific RNAs was measured (Fig. 7). Over 50% of the total radioactivity in virus-specific RNAs was found in RF and RI immediately after pulse labeling. Whereas the radioactivity in RF and RI increased steadily during continuous labeling, it slightly decreased during chase (Fig. 7). However, the radioactivity was not chased out completely from either RF or RI. The radioactivity in TMV-RNA increased during the chase, and this increase was much greater than could be accounted for by the radioactivity liberated from RF and RI.

17 0

I I( Id 2 3 1 Tlme(Hours)

II

Fro. 7. Incorporation of [3H]uridine into virus specific RNAs during chase and continuous labeling. Infected protoplasts were cultured in 100 ml medium at a density of 1.8 x lo5 cells/ml. The culture was pulse-labeled for 30 min at 20 hr p.i. by adding [5-3H] uridine (16 Ci/mmole, Daiichi Pure Chemicals) to 10 rCi/ml. Actinomycin D (10 pg/ml) had been added to the culture 1 hr before pulse-labeling. The pulse-labeled culture was divided into two equal halves, and one-half was cultured further (continuous labeling), while the other half received cold uridine to a concentration of 4 mM (chase). Samples of 10 ml were withdrawn at 0.5, 1.5. and 3.5 hr after the addition of [3H]uridine, and RNA extracted from them was analyzed by gel electrophoresis. Gel slices were digested with 0.2 ml of 35% H,O1 at 50°C overnight, and 3H-radioactivity was measured in 10 ml Kinard’s scintillation fluid using a liquid scintillation spectrometer (Aloka LSC-502). (-) Continuous labeling; (-----) chase; (x) TMV-RNA; (0) RF; (0) RI. DISCUSSION

Single-stranded viral RNA, its RF and RI were identified in tobacco mesophyll protoplasts actively synthesizing TMV. No other forms of virus-specific RNA could be demonstrated in these cells. In particular, the “low molecular weight component” of TMV-RNA (Jackson et al., 1972; Siegel et al., 1973) could not be found in infected protoplasts. RNA species smaller than rRNAs were occasionally detectable in preparations from infected protoplasts, but these are probably products of partial degradation of host or viral RNA during extraction, since they occurred only in such preparations in which the breakdown of 23s chloroplast rRNA was apparent (Fig. 2B). Bourque et al. (1973) showed that phenol extraction of tobacco leaves does not usually eliminate RNA-degrading enzyme(s) completely. The results of our study with

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the protoplast system do not support the hypothesis that the low molecular weight component of TMV-RNA actively participates in the in viuo replication of TMV (Beachy and Zaitlin, 1975). A possibility cannot be excluded, however, that this component turns over rapidly and has escaped detection in our experiments in which RNA was labeled for rather long times. Double-stranded forms of TMV-RNA have been isolated from infected tissues by many workers (Ralph, 1969; NilssonTillgren, 1970; Jackson et al., 1971, 1972; Kielland-Brandt and Nilsson-Tillgren, 1973a). Shipp and Haselkorn (1964) estimated that a cell of infected tobacco leaves contain 102-lo3 viral equivalents of doublestranded TMV-RNA or one molecule of such RNA per lo3 virus particles. The absolute amounts of RF and RI in protoplasts could not be determined, because they are detectable only by radioactivity. However, the fact that their radioactivity comprise lo-40% of total radioactivity in viral RNA species (Fig. 6) suggests that the double-stranded forms may occur in protoplasts in higher concentrations than in leaf tissues (Shipp and Haselkorn, 1964; Burdon et al, 1964). This discrepancy may be due to a real difference in the two systems or to the different methods of isolation. The time course of synthesis of doublestranded TMV-RNAs in protoplasts (Fig. 6) and the results of pulse-chase experiment (Fig. 7) are not inconsistent with the generally accepted idea that RI, and possibly also RF, is involved in the in uivo replication of TMV-RNA. However, the data obtained in pulse-chase experiments were far from satisfactory to demonstrate conclusively the precursor role of these structures for TMV-RNA synthesis. Only a part of the radioactivity incorporated into RF and RI by pulse labeling was released during chase, and this could not quantitatively account for the radioactivity incorporated into TMV-RNA (Fig. 7). Using separated cells from infected leaves, Jackson et al. (1972) were able to chase out the label of RI completely but that of RF only partially. The radioactivity chased from RF and RI in the experiments of these

workers was also not sufficient to account for the radioactivity incorporated into TMV-RNA during the same time. These results suggest that mature tobacco leaf cells contain a relatively large pool of RNA precursors which interferes with the quantitative transfer of radioactivity in pulsechase experiments. Oberg and Philipson (1969) showed that the large precursor pool in mammalian cells prevents the chasing out of label from double-stranded intermediate RNAs of poliovirus. Kinetics of the synthesis of TMVspecific RNAs in individual cells could be studied using synchronously infected protoplasts and was correlated with the production of progeny virus particles. A most noteworthy facet of TMV replication emerging from this study is that virus replication goes through three succesive phases which are distinct in the balance between viral RNA synthesis and virus particle formation. The initial phase is characterized by an exponential synthesis of viral RNA followed after an interval of 4-5 hr by a similarly exponential production of virus particles. A large amount of free or partially coated viral RNA accumulates in this phase. Sarkar (1965) observed a temporary accumulation of RNase-sensitive infectious material in TMV-infected tobacco leaves. The continued production of virus particles at a near maximal rate in the subsequent intermediate phase catches up viral RNA synthesis which is now greatly reduced in its rate. The “excess” viral RNA is thus fully encapsidated by the end of this phase. In the final phase of virus replication, the synthesis of viral RNA is closely followed by the formation of particles, indicating that viral RNA synthesized in this phase is rapidly assembled into particles. The initially exponential replication of viral RNA becomes linear at 8-10 hr p.i. with concomitant slowdown of the synthesis of double-stranded RNA forms (Fig. 6). What mechanism is responsible for this switchover is a matter of speculation at the moment. It is possible that viral coat protein plays a role in regulating the replication of RNA. The formation of virus particles obviously depends on the synthe-

TMV-RNA

REPLICATION

sis of coat protein, and, in fact, Sakai and Takebe (1974) showed that the course of TMV production in protoplasts closely follows that of coat protein synthesis. When the amount of coat protein reaches a certain level, it may act to regulate the replication of viral RNA in various ways. For example, coat protein of RNA bacteriophages is known to have a repressor function for the translation of the replicase cistron (Kozak and Nathans, 1972). Possible mechanisms regulating the replication and the translation of TMV-RNA will be duscussed in some detail in a separate paper (Takebe et al., 1975). ACKNOWLEDGMENT We are indebted to the late Dr. Tadashi Kodama for kindly supplying purified rice dwarf virus. REFERENCES AOKI, S., and TAKEBE, I. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus ribonucleic acid. Virology 39, 439-448. BEACHY, R. N., and ZAITLIN, M. (1975). Replication of tobacco mosaic virus. VI. Replicative intermediate and TMV-RNA-related RNAs associated with polysomes. Virology 63,84-97. BOURQUE, D. P., HAGILADI, A., and NAYLOR, A. W. (1973). A general method for extracting intact chloroplast and cytoplasmic ribosomal RNA from higher plant leaves. Biochem. Biophys. Res. Commm. 51,993-999. BURDON, R. H., BILLETER, M. A., WEISSMANN, C., WARNER, R. C., OCHOA, S., and KNIGHT, C. A. (1964). Replication of viral RNA. V. Presence of a virus-specific double-stranded RNA in leaves infected with tobacco mosaic virus. Proc. Nat. Acad. Sci. US 52, 768-775. DIENER, T. 0. (1962). Isolation of an infectious, ribonuclease-sensitive fraction from tobacco leaves recently inoculated with tobacco mosaic virus. Virology 16, 140-146. ENGLER, R., and SCHRAMM, G. (1959). Die Bildung der infektiiisen Ribonucleinslure wiihrend der Vermehrung des Tabakmosaikvirus. Z. Naturforschg. 15b, 38-45. FRANKLIN, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage Rl7. Proc. Nat. Acad. Sci. US 55,1504-1511. FRASER, R. S. S. (1973). The synthesis of tobacco mosaic virus RNA and ribosomal RNA in tobacco leaves. J. Gen. Vrol. 18, 267-279. IANDOLO, J. J. (1970). Device for slicing polyacrylamide gels. Anal. Biochem. 36, 6-10. IGLEWSKI, W. J., and FRANKLIN, R. M. (1967). Purifica-

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clease sensitive infectious material during biogenesis of tobacco mosaic virus. 2. Vererbungsl. 97, 166-185. SHIPP, W., and HASELKORN, R. (1964). Doublestranded RNA from tobacco leaves infected with TMV. hoc. Nat. Acad. Sci. US 52, 401-407. SIEGEL, A., ZAITLIN, M., and DUDA, C. T. (1973). Replication of tobacco mosaic virus. IV. Further characterization of viral related RNAs. Virology 58, 75-83. TAKEBE, I., OTSUKI, Y. (1969). Infection of tobacco mesophyll protoplasts by tobacco mosaic virus. hoc. Nut. Acad. Sci. US 64, 843-848. TAKEBE, I., OTSUKI, Y., and AOKI, S. (1971). Infection of isolated tobacco mesophyll protoplasts by tobacco mosaic virus. In “Les Cultures de Tissues de Plantes,” pp. 503-511. Centre National de la Recherche Scientifique, Paris. TAKEBE, I., AOKI, S., and SAKAI, F. (1975). Replication and expression of tobacco mosaic virus genome in isolated tobacco leaf protoplasts. In “Modification of the Information Content of Plant Cells” (R. Markham ed.). North-Holland Publishing Co., Amsterdam. WOLLUM, J. C., SHEARER, G. B., and COMMONER, R. (1967). The biosynthesis of tobacco mosaic virus RNA: Relationship to the biosynthesis of virusspecific ribonuclease-resistant RNA. Proc. Nat. Acad. Sci. US 58, 1197-1204.