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RNA-dependent RNA polymerases in uninfected and tobacco mosaic virus-infected tobacco leaves: Viral-induced stimulation of a host polymerase activity
VIIIOI.O(:Y
86,
241-253
(1978)
RNA-Dependent Virus-Infected
RNA Polymerases in Uninfected Tobacco Leaves: Viral-induced Polymerase Activity C. P. ROMAINE’
Department
of Plant
Pathology, Accepted
MILTON
AND
Cornell
University,
December
and Tobacco Mosaic Stimulation of a Host
ZAITLIN Ithaca,
New
York
14853
13, I977
The soluble RNA-dependent RNA polymerase activities in the 105,000-g supernatant fraction of homogenates prepared from uninfected and tobacco mosaic virus (TMV)-infected tobacco leaves have been compared. The enzymes were purified 40- to 50-fold from both mock-inoculated and TMV-infected leaves and were found to have identical behavior upon (NH&SO4 fractionation, Sephadex G-109 gel filtration, and DEAE-Bio-Gel and phosphocellulose chromatography. Although no qualitative differences in polymerase activity were detected, the specific activity of the polymerase from infected leaves was greater than that from mock-inoculated leaves at each step in the purification. Moreover, the most highly purified enzymes from both types of leaf tissue were indistinguishable with respect to a number of catalytic parameters, viz., requirements for RNA synthesis, kinetics of RNA synthesis, lack of template specificity, and character of the RNA products such as strandedness, T,,,, size, and complementarity to the TMV-RNA used as the template. SDS-polyacrylamide gel electrophoresis of the proteins in the most highly purified enzymes showed that both preparations contained 2 major and at least 13 minor corresponding polypeptides; none were unique to TMV infection. The evidence in toto suggests that the enhanced soluble RNA-dependent RNA polymerase activity following TMV infection is due to a stimulation of a host HNA-dependent RNA polymerase rather than to the genesis of a new viral-coded RNA polymerase. The possible relationships between the soluble host polymerase, the membrane-associated TMV replicase, and a 130,000-MW viral-coded polypeptide thought to be involved in TMV-RNA replication remain to be resolved. INTRODUCTION
pernatant fraction of clarified leaf homogenates (natively soluble enzyme) which is not virus specific (Duda et al., 1973; Brishammar and Juntti, 1974; Brishammar, 1975), and it still remains to be determined if this enzyme is obligately involved in viral RNA replication. The search for the viral replicase has been confused by the existence of a host RNA-dependent RNA polymerase in uninfected tobacco leaves (Duda et al., 1973; Bol et al., 1976; Fraenkel-Conrat, 1976). In all likelihood this enzyme is probably similar to the one reported in uninfected Chinese cabbage leaves (Astier-Manifacier and Cornuet, 1971). At the present time, the role of such enzymes in the nucleic acid metabolism of the host is obscure. Furthermore, the possible relationship between the host RNA polymerase and the membrane-
Infection of tobacco leaves by tobacco mosaic virus (TMV) results in a dramatic increase in RNA-dependent RNA polymerase activity. Basically two forms of such activity have been recognized in leaf extracts prepared from TMV-infected leaves. A virus-specific RNA-dependent RNA polymerase (replicase) associated with a membranous subcellular fraction (31,000-g pellet) has been shown to catalyze the synthesis of the replicative intermediate (RI) and replicative form (RF) of TMV-RNA (Bradley and Zaitlin, 1971; White and Murakishi, 1977). In addition, a RNA-dependent RNA polymerase is present in the su’ Present address: Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 168X2. 241
0042-6822/78/0861-0241aO2.(x)/O Copyright 0 1978 by Academic Press, In(.. All rights of reproduction in any form reserved
242
ROMAINE
AND ZAITLIN
associated replicase or natively soluble RNA-dependent RNA polymerase has not been convincingly determined. Although it has been recognized that a host enzyme system exists which has the potential to replicate the viral nucleic acid, infection of tobacco leaf cells by TMV elicits the formation of at least two proteins with molecular weights in the range of 130,000 to 165,000 (Zaitlin and Hariharasubramanian, 1972; Sakai and Takebe, 1974; Paterson and Knight, 1975; Bruening et al., 1976; Scalla et al., 1976). Both are viral-coded polypeptides because products of this approximate size are generated with TMV-RNA as the messenger in various in vitro protein-synthesizing systems (Roberts et cd., 1973; Roberts et aZ., 1974; Knowland, 1974; Knowland et aZ., 1975; Bruening et al., 1976), and cyanogen bromide peptide analysis of the in uiuo- and in uitro-generated 130,000-MW proteins have shown them to be similar if not identical (Romaine, 1977; Scalla, personal communication). Because the 130,000-dalton polypeptide is the predominant putative viral noncapsid polypeptide detected in infected tissues and corroborated in in vitro systems, it has been regarded as the prime candidate for the replicase (Sakai and Takebe, 1974; Hunter et al., 1976), although evidence for an enzymatic nature is lacking. The aim of the present work was to learn whether TMV induces a novel RNA-dependent RNA polymerase activity in infected tobacco cells. The approach undertaken was to purify the polymerases from uninfected and TMV-infected leaves with the hope of resolving a RNA polymerase unique to infection. In doing so, we have learned that the natively soluble RNA polymerase in homogenates of TMV-infected tobacco leaf cells has identical chromatographic and catalytic properties with the natively soluble host RNA polymerase extracted from uninfected cells. The inability to detect a new RNA polymerase in infected cells would indicate that the enhanced RNA polymerase activity following TMV infection is due to a stimulation of the preexisting host RNA-dependent RNA polymerase and not to the appearance of a new virus-coded enzyme.
MATERIALS
AND METHODS
Reagents. Unlabeled ribonucleoside 5’triphosphates (sodium salts: ATP, equine muscle; CTP, type III; GTP, type III), were purchased from Sigma Chemical Co. (St. Louis, Missouri); uridine 5’-[5-3H]triphosphate (tetralithium salt, sp act. 12-16 Ci/mmol) and ammonium sulfate (ultrapure) were from Schwarx/Mann (Orangeburg, New York); Sequanal grade of sodium dodecylsulfate (SDS) was from Pierce Chemical Co. (Rockford, Illinois); and electrophoretically purified ribonucleases A and T1 were from Worthington Biochemical Co. (Freehold, New Jersey). Buffers. Buffers were routinely autoclaved for 10 min or boiled prior to use and stored at -20”. Grinding buffer (GB). Grinding buffer was composed of 50 mbf Tris adjusted to pH 8.1 at 4’ with HCl, 0.4 1M sucrose, 10 mM KCl, 5 nut4 MgCh, 10% (v/v) glycerol, and 10 mM 2-mercaptoethanol (added just before use). Resuspending buffer (RB). Resuspending buffer was composed of 10 mM Tris adjusted to pH 8.1 at 4” with HCl, 10 mA4 KCl, 1 mM NazEDTA, 20% (v/v) glycerol, and 5 mM 2-mercaptoethanol (added just before use). Phosphate-EDTA buffer. PhosphateEDTA buffer was composed of 10 mM NazHP04 and 5 mM NazEDTA, pH 7.0, at room temperature. SSC buffer. SSC buffer was composed of 150 mM NaCl and 15 mM sodium citrate, pH 7.2, at room temperature. PKE buffer. PKE buffer was composed of 20 mM potassium phosphate, 10 mM KCl, 1 mM NazEDTA, 20% (v/v) glycerol, and 5 mAf 2-mercaptoethanol (added just before use), pH 7.5, at room temperature. Sample buffer. Sample buffer was composed of Tris-phosphoric acid, pH 6.7, at room temperature, 1% SDS, 1% mercaptoethanol, 10% glycerol, and 0.015% bromophenol blue (Maizel, 1971). Propagation of plants. Plants of tobacco, Nicotiana tabacum L. cv. Turkish Samsun, were grown in steam-sterilized soil in 6-in. pots in a greenhouse maintained at about 21’ except for the summer months
TOBACCO
RNA-DEPENDENT
when the temperature occasionally exceeded 30’. Virus purification and RNA preparation. The (VI) common strain of TMV was used exclusively in this study and was purified according to the method of Gooding and Hebert (1967). Tobacco leaf rRNAs were prepared by conventional phenol extraction and ethanol precipitation. Viral RNA (except TMV) and other nonviral nucleic acids were obtained from the frozen collection maintained in the laboratory. Inoculation. Generally, Turkish Samsun tobacco plants in the five- to six-leaf stage were used. Three partially expanded leaves of each plant were inoculated by rubbing both leaf surfaces with a brush which had been dipped in a solution of TMV (0.5 mg/ml) in 100 n&f sodium phosphate buffer, pH 7.0, containing a small amount of washed Celite. The leaves were rinsed with water immediately after inoculation and the plants placed in a controlled-environment chamber providing 25’ and 1500 fc of illumination at the leaf surface (16~hr photoperiod). At 3 days postinoculation, both directly inoculated leaves with chlorotic spots and small systemically infected vein-cleared leaves were used as a source of enzyme. Uninfected controls consisted of comparable leaves which were mock inoculated with a mixture of yeast RNA (50 pg/ml) and bovine serum albumin (0.45 mg/ml) in buffer with Celite. Partial purification of the soluble RNAdependent RNA polymerases. AU glassware was either acid-washed or heated. All purification steps were performed at 2 to 4” in the sequence given below: Preparation of crude extract. Seventy grams of fresh mock-inoculated or TMVinfected tobacco leaves with their midveins removed was placed in a polyethylene contamer immersed in an ice bath and chopped for several min in 1.25 ml of GB/g of tissue with an electric knife adapted to hold two razor blades. The brei was pressed through a single layer of washed Miracloth (Calbiochem Co.) and centrifuged at 1000 g for 5 min. The top 90% of the supernatant was recovered and centrifuged at 31,000 g for 20 mm. The supernatant was decanted and further clarified by centrifugation at
RNA
POLYMERASES
243
105,000 g for 1 to 1.5 hr at 4” in a Type 40 rotor in a Beckman L3-40 ultracentrifuge. (NH&SO4 fractionation. CrysWine (NH&SO4 was added slowly (l-2 min) to the supematant (100 ml) directly to 50% saturation with constant stirring on an icewater bath. Stirring was continued for an additional 15 min and the yellowish-white precipitate was collected by centrifugation at 20,000 g for 15 min and then dissolved with vortexing in 0.1 ml of cold RB/g of tissue (7 ml). The solution was incubated on ice for an additional 30 min and then clarified by centrifugation at 20,000 g for 10 min. Sephadex G-100 gel filtration. The (N&)$04-concentrated polymerase fraction was layered directly on top of a column (Pharmacia, 1.6~cm i.d. x 106 cm) of Sephadex G-100 equilibrated with 250 ml of RB. Fractions of 110 drops were collected at a flow rate of 12 ml&r by gravity flow. DEAE-Bio-Gel ion-exchange chromatography. The active fractions from the gel filtration step were pooled and applied to a 0.9-cm i.d. x 15-cm column (Pharmacia) of DEAE-Bio-Gel A, 100 to 200 mesh (BioRad Laboratories), that had been previously equilibrated with 25 ml of RB. The column was washed with a volume of RB equivalent to the sample volume to remove unadsorbed proteins and then developed with a linear gradient of 300 ml of RB also containing from 10 to 300 n&f KCl. Fractions of 196 drops were collected at a flow rate of 12 ml/hr using a Buchler peristaltic pump. The DEAE-Bio-Gel fractions containing enzyme activity were pooled and dialyzed against three changes of PKE buffer (total volume, 2 liters) overnight at 4O. Phosphocellulose ion-exchange chromatography. Phosphocellulose (Whatman P-11) was washed by decantation first with 0.1 N HCl in 50% ethanol and then with deionized water, followed by 0.1 N NaOH, and washed again with water (Wickner, 1973). The phosphocellulose was resuspended in PKE buffer and was packed into a 0.9-cm i.d. x 15-cm column (Pharmacia) under gravity. The column was equilibrated with 25 ml of PKE buffer and 34 ml of the dialyzed DEAE-Bio-Gel step enzyme was applied and washed on with 10 ml of PKE
244
ROMAINE
AND ZAITLIN
buffer. The column was then washed consecutively with 10 ml each of PKE buffer containing 0.05 M, 0.1 M, 0.2 M, and 0.4 M PO1, maintaining a flow rate of 8 ml/hr under gravity while 70-drop fractions were collected. Fractions containing high enzyme activity were pooled and constituted the final partially purified polymerases. Additional steps. Dilute enzyme preparations could be concentrated by dialysis against 20 vol of RB containing 30% PEG6000 at 4”. No appreciable loss of activity resulted using this procedure, providing dialysis did not proceed to the point where the enzyme was entirely dehydrated. RNA-dependent RNA polymerase assay. The standard reaction mixture (final volume, 50 $1, contained the following: 100 mM Tris (pH 8.0 for 33”), 10 mM MgC12, 0.025 pm01 each of ATP, CTP, and GTP, 7.5 mikf dithiothreitol, 20 mM (NH.&S04, 0.5 pg of actinomycin D, 1.25 @i of r3H]UTP, (ethanol removed by drying in uacuo just before use) 6 pg of TMV RNA, and 25 ~1 of enzyme, unless indicated otherwise. The enzyme reaction was initiated by adding the enzyme and was allowed to proceed for 1 hr at 33”, at which time the reaction was terminated by spotting the entire mixture onto a 2.3-cm circular 3-MM Whatman filter. The disks were processed and the acid-insoluble radioactivity was determined as described by Zaitlin et al. (1973). Data are expressed as net counts per minute, calculated by subtracting “0 times,” obtained by mixing enzyme with ice-cold reaction mixture and immediately terminating the reaction. Extraction of the polymerase reaction products. For analysis of the RNA products of the polymerase reaction, the reaction mixture was scaled up to 1 to 5 ml and after incubation for 4 to 5 hr, the RNA was phenol extracted and ethanol precipitated (Zaitlin et al., 1973). Polyacrylamide gel electrophoresis of RNA. Electrophoretic analysis of RNA was carried out in Plexiglas tubes (6-mm i.d.) containing g-cm long 1.8% polyacrylamide gels stabilized with 0.5% agarose (Jackson et al., 1971). The conditions for gel electrophoresis and fractionation have been described (Zaitlin et al., 1973.) RNA-RNA hybridization. The RNA
products were processed for hybridization as outlined previously (Zaitlin et al., 1973). The double-stranded RNAs were melted in a boiling water bath for 2.5 min followed by immediate cooling in an ice-water bath. Annealing was performed for 2 hr at 70” in SSC buffer (Zaitlin et al., 1973), and the extent of renaturation was determined by treating the sample with 10 ~1 of a mixture of ribonuclease A (100 pg/ml) and T1 (5 pg/ml) at 37’ for 30 min prior to determining TCA-insoluble radioactivity. Thermal denaturation of the pH]RNA products. The thermal denaturation kinetics of the in vitro-synthesized r3HJRNA was determined in a vessel containing water which was fashioned from a l-liter polypropylene bottle modified to hold six l-dram shell vials and a thermometer. Water was circulated around the vials and temperature was regulated (kO.5”) by a Haake bath. One-hundred-microliter aliquots of the [3H]RNA (1000-5000 cpm) in 0.1~ SSC were dispensed into Siliclad-treated vials, and duplicate samples were exposed for 5 min at the specified temperature, followed by immediate and continued cooling in an ice-water bath. After individual duplicate samples had been exposed at each of the designated temperatures, the vials were removed from the ice bath and centrifuged at 1000 g for 30 set to collect the solution. The solutions were adjusted to SSC by adding sufficient 10x SSC and TCA-insoluble ribonuclease-resistant counts were determined. Polyacrylamide gel electrophoresis of proteins. Proteins were precipitated from solution by adding cold 50% TCA to 10% and incubating for at least 2 hr at 4”. The precipitate was collected by centrifugation at 20,000 g for 15 min and washed twice with cold acetone. The final precipitate was recovered by centrifugation, air-dried at 4”, and then dissolved in sample buffer. Immediately proteins were denatured by heating for 4 hr at 55” and then again for 3 min at 100” just prior to electrophoresis. SDS-polyacrylamide gel electrophoresis was performed using the discontinuous buffer system of Maize1 (1971). Denatured proteins were separated on slab gels (0.05 x 14 x 10 cm) having a l-cm 5% stacking gel and a g-cm 10% running gel. The
TOBACCO
RNA-DEPENDENT
RNA
bisacrylamide ratio of the stacking gel was l.O35:30 and of the running gel was 0.8:30. Electrophoresis was done at room temperature at 50 V for 10 min and then at 100 V. The gels were stained in freshly prepared 0.2% Coomassie brilliant blue R-250 in methanol:water:glacial acetic acid (5:5:1, v/v/v) overnight at room temperature and destained in the same solvent without dye at 40° for 5 min and then in a 7% glacial acetic acid solution. Analytical methods. Protein concentration was determined by the method of Lowry et al. (1951) using crystalline BSA (Fraction V) as a standard. Conductivity measurements for determining salt gradients were made on a Yellowsprings Instrument Co. conductivity meter standardized with buffers of known ionic strength. RESULTS
Enzyme purification. The soluble RNAdependent RNA polymerase in mock-inoculated and TMV-infected tobacco leaves was partially purified with enzyme activity being monitored with exogenous TMVRNA as the template in the enzyme assay throughout the course of the purification. The purification scheme outlined was
adapted for 50 to 75 g of the deveined tobacco leaves and afforded a 40- to 50-fold purification with an approximate 5% yield with respect to the 105,000-g supernatant for both the mock-inoculated and TMVinfected leaf-soluble RNA polymerase (Table 1). When the enzyme in the high-speed supernatant from the crude homogenates was concentrated by (NH&S04 precipitation, the soluble polymerases in infected and uninfected leaves were observed to have similar “salting-out” properties. Approximately 70 to 85% of the activity precipitated in the range of 30 to 50% saturation (NH&S04 and greater than 90% from 0 to 50% saturation (Table 2). Higher concentrations of (NH&S04 precipitated considerable additional polymerase but with a low specific activity. Upon Sephadex G-100 gel filtration, polymerase activity from both infected and mock-inoculated leaves eluted in or just after the void volume (Figs. 1A and 1B) along with most of the protein, as determined by optical density measurements of the effluent at 280 nm. Generally, more than 100% of the initial polymerase activity in the 105,000-g supernatant is recovered at
TABLE PARTIAL
PURIFICATION Enzyme
1
OF THE SOLUBLE RNA-DEPENDENT RNA POLYMERAS= AND TMV-INFECTED TOBACCO LEAVES~ fraction
Total
protein (wdb
Total activity (units)’
.______ Mock-inoculated A. Soluble fraction B. 50% ammonium sulfate Sephadex G-100 E. DEAE-Bio-Gel G. Phosphocellulose TMV-infected B. Soluble fraction D. 50% ammonium dex G-100 F. DEAE-Bio-Gel H. Phosphocellulose
245
POLYMERASES
sulfate
pellet
and
138 76 38 0.14
pellet
Sepha-
144 72
Specific activity (units/mg of protein)
Yield
(%)
561 571
4.1 7.5
100 102
209 28
5.4 206
37 5
27 58
100 108
3899 4205
60 0.29
IN MOCK-INOCULATED
50 76 1026 8.--~~ _-~~--~ ~ supernatant) were prepared separately from 70 g of (deveined) at 3 days postinoculation as described under
a Soluble RNA polymerase crude extracts (105,000-g either mock-inoculated or TMV-infected tobacco leaves Materials and Methods. * Protein was determined by the method of Lowry et al. (1951). ’ One unit of enzyme activity is equivalent to the incorporation
2980 298
of 1 pmol
of [3H]UMP
at 33” for 1 hr.
246
ROMAINE
AND ZAITLIN TABLE
AMMONIUM ‘;f;;?f;
SULFATE Centrifugal
fraction
(S saturation)
O-20 20-30 30-40 40-50 w-60 60-70 >70
2
FRACTIONATION OF THE SOLUBLE RNA POLYMERASES UNINFECTED AND TMV-INFECTED TOBACCO LEAVES= Uninfected Total polymerase activity (cpm X 10e3)
Starting material Pellet Pellet Pellet Pellet Pellet Pellet Supernatant
411 9 24 145 182 14 3 0
PREPARED
FROM
TMV-infected Total protein (md
Total polymerase activity (cpm X 10m3)
Total protein (mid
12.65 0.40 0.78 1.01 7.63 2.44 1.33 0.20
LfJoo 20 132 573 380 45 4 30
18.00 0.17 0.78 1.60 10.64 2.10 1.37 0.14
a Soluble RNA polymerase extracts (105,000-g supematant) were prepared separately from 10 g of either uninfected or TMV-infected leaf tissue (deveined) as described under Materials and Methods. Sufficient solid (NH&SO4 was added slowly to the extracts to 20% saturation with constant stirring in an ice-water bath. Stirring was continued for an additional 15 min at which time the precipitate was removed by centrifugation at 20,609 g for 15 min and then dissolved in 1 ml of RB. The supematant was collected and the procedure was repeated for each of the (NH&SO4 concentrations indicated. All fractions were dialyzed batchwise against three changes of RB (total volume, 2 liters) for 20 br at 4” prior to polymerase assay. Protein was determined by the method of Lowry et al. (1951).
this step, presumably because of the removal of an inhibitor. Higher levels of polymerase activity were consistently obtained from TMV-infected leaves than from mock-inoculated leaves. After an initial experiment, it was found not to be necessary to assay all fractions since the polymerasecontaining fractions are yellow and elute from the column in the void volume. Polymerase activity from both tminfected and infected leaves eluted from DEAE-Bio-Gel as a single peak at 0.07 M KC1 just before the bulk of the protein as determined by optical density at 280 nm (Figs. 1C and 1D). Enzyme activity yields at this step ranged from 40 to 80% for both polymerases, the only difference being that a higher level of activity was obtained from infected leaves than from uninfected leaves. In a single experiment, a second minor peak of polymerase activity was detected which eluted at 0.11 to 0.13 M KC1 from DEAE-Bio-Gel (data not shown). This polymerase activity was present in extracts from both uninfected and infected leaves, which would discount the possibility that it was of viral origin. The polymerase from infected leaves could also be chromatographed on DEAE-Sephadex A-25 and eluted as a single peak of activity at ap-
proximately 0.09 A4 KCl. However, the yield of enzyme activity was usually 50% or less and the reproducibility of the method was low. The major purification of the RNA polymerases from other soluble proteins is accomplished by the phosphocellulose chromatography. As shown in Figs. 1E and lF, under the ionic conditions employed most of the protein (optical density at 280 nm) passes directly through the column, whereas most of the polymerase activity is adsorbed. Both mock-inoculated and infected leaf RNA polymerase activities eluted in the 0.95 M PO4 wash with the only difference again being quantitative. Polypeptides associated with enzyme preparations. The polypeptides in the various enzyme fractions at each step of the purification were analyzed by SDS-polyacrylamide gel electrophoresis to assess the purity of the enzyme preparations. This revealed that the most highly purified preparation, the phosphocellulose polymerase (Fig. 2G and 2H), is not homogeneous and still contains at least 15 different polypeptides detectable by staining with Coomassie brilliant blue (all are not apparent in Fig. 2), which range in apparent molecular weight from less than 25,000 to in excess of
TOBACCO
RNA-DEPENDENT
RNA
POLYMERASES
247
Ai
FIG. 1. Gel filtration and chromatography of the soluble RNA polymerases from mock-inoculated and TMV-infected tobacco leaves. A soluble subcellular fraction (105,000-g supernatant) was prepared from 70 g of either mock-inoculated or TMV-infected tobacco leaves at 3 days postinoculation and processed as described under Materials and Methods. (A) Sephadex G-100 gel filtration, polymerase activity from mockinoculated leaves. (B) Sephadex G-100 gel filtration, polymerase activity from TMV-infected leaves. Polymerase activity, w absorbance at 280 nm, M, void volume, VU. (C) DEAE-Bio-Gel chromatography, polymerase activity from mock-inoculated leaves. (D) DEAE-Bio-Gel chromatography, polymerase activity from TMV-infected leaves. Polymerase activity, W, absorbance at 280 nm, C----q KC1 concentration, -----. (E) Phosphocellulose chromatography, polymerase activity from mock-inoculated leaves. (F) Phosphocellulose chromatography polymerase activity from TMV-infected leaves. Polymerase activity, O-4; absorbance at 280 nm, O-----O; potassium phosphate concentration, -----.
130,000. The more highly purified polymerases prepared from uninfected and infected leaves had identical polypeptide compositions, and interestingly, the viralcoded 130,000-dalton polypeptide was not associated with the partially purified RNA polymerase activity from infected leaves. In three separate experiments this polypeptide was largely eliminated from the RNA-dependent RNA polymerase activity containing fractions following either DEAE-Bio-Gel or DEAE-Sephadex chromatography. The fate of the 130,000-dalton polypeptide at this step was not deter-
mined, however, since the yield of polymerase activity at each step of the purification and overaU was similar for both polymerases, and since no unique polymerase activity could be detected in infected leaves, it seems unlikely that a second polymerase activity, perhaps associated with this 130,000-dalton polypeptide, was lost at some step. Catalytic properties of the partiallypurified polymerase. The phosphocellulose enzymes from mock-inoculated and infected leaves are indistinguishable with respect to their requirements for RNA synthesis, and
248
ROMAINE
FIG. 2. Polyacrylamide gel analysis of the partial purification of the soluble RNA-dependent RNA polymerase in mock-inoculated and TMV-infected tobacco leaves. Proteins in aliquots of the polymerase fraction taken at each step in the purification designated in Fig. 1 and in Table 1 were electrophoresed on a 10% polyacrylamide slab gel in SDS (Maixel, 1971). Shown are the positions of the protein markers; /lgalactosidase (130,090 daltons), BSA (67,000 daltons), and ovalbumin (45,000 daltons). The arrowheads denote the position of the virus-coded 130,000-dalton polypeptide. Note that this polypeptide is not associated with the most highly purified polymerase preparations E through H. The soluble fraction (105,000-g supernatant): (A) mock-inoculated leaves and (B) TMV-infected leaves; the 50% (NH&S04 pellet and pooled polymerase fractions from the Sephadex G-100 gel filtration: (C) mock-inoculated leaves and (D) TMV-infected leaves; the pooled peak fractions from the DEAE-Bio-Gel column: (E) mock-inoculated leaves and (F) TMV-infected leaves; the pooled peak fractions from the phosphocellulose column: (G) mock-inoculated leaves and (H) TMV-infected leaves.
their properties are similar to previous reports of a detergent-solubilized enzyme from this laboratory (Zaitlin et al., 1973) and accordingly are not documented in detail here. Omission of the three ribonucleoside triphosphates resulted in a 97% reduction in enzyme activity. Mg2+ is required
AND ZAITLIN
for optimal activity which could be satisfied only to the extent of 5% by 1.25-n-&f Mn2+. Without exogenous TMV-RNA, incorporation was negligible and in several experiments ranged from 0 to 4% of the control which contained RNA. There is no detectable DNA-dependent RNA polymerase activity suggested by comparing [3H]UMP incorporation in the absence or presence of actinomycin D. Furthermore, addition of RNase (10 pg/ml) to the reaction mixture prior to adding the polymerase abolished activity, whereas DNase (10 pg/ml) had no observable deleterious effect, indicating that the template was RNA and not DNA. Under optimum conditions for enzyme activity, the kinetics of [3H]UMP incorporation by the partially purified polymerases were identical, being approximately linear for the first 3 hr of incubation and thereafter reaching a plateau. The RNA products synthesized by either of the polymerases at all degrees of purity were greater than 75% resistant to RNase digestion in high salt (2~ SSC), demonstrating that the RNAs were highly base-paired molecules. [3H]UMP incorporation by the phosphocellulose polymerases was measured using several viral and nonviral RNAs as templates at 180 pg/ml. Each of the RNAs tested could fulfill the template requirement with an efficiency that was similar for either of the polymerases. The most active templates were cowpea mosaic virus RNA and TMV-RNA. In general, viral RNAs seemed to be more effective templates than nonviral RNAs, a phenomenon which had been observed previously (Zaitlin et al., 1973). The RNA products synthesized by the soluble polymerases in the unfractionated 105,000-g supernatant were heterodispersed and of low molecular weight (Fig. 3A), whereas the more highly purified enzymes synthesized RNAs which were still heterodispersed in size but which were now of considerably higher molecular weight (Fig. 3B). The RNA products synthesized by the Sephadex G-100 and DEAE-Bio-Gel polymerases were analyzed similarly; an increase in the purity of the enzymes was accompanied by an increase in the average size of the RNA products (data not shown). As was observed with polymerases in crude leaf extracts, the polymerase from unin-
TOBACCO
RNA-DEPENDENT
t t
GEL
SLICE
NUMBER
FIG. 3. Radioactivity profile after polyacrylamide gel electrophoresis of the RNA products synthesized in vitro and catalyzed by the crude and partially purified soluble polymerases. Scaled-up standard reaction mixtures were incubated for 4 to 5 hr at 33”. The RNA products were phenol extracted and electrophoresed on g-cm 1.8% polyacrylamide-agarose gels at 5 mA/gel for 3 hr and 45 min. The gels were fractionated into 1.07-mm slices and radioactivity was determined. See Materials and Methods. The arrows denote the positions of the optical density marker, TMV RNA, and bromophenol blue dye. Migration is from left to right. (A) Crude polymerase fraction (105,000-g supernatant): from mock-inoculated leaves, G-0 (10,800 cpm applied); from TMV-infected leaves, 0-O (10,200 cpm applied). (B) Partially purified polymerase fraction (phosphocellulose): from mock-inoculated leaves, O-O (20,800 cpm applied); from TMV-infected leaves, @--O (20,300 cpm applied).
fected leaves synthesized a product of higher molecular weight than did the polymerase from infected leaves; however, this differential in molecular weight decreased with an increase in the purity of these enzymes. Nature of the enzyme products. To determine whether the phosphocellulose polymerases were synthesizing the complementary “minus” strand of the added TMV-RNA “plus” strand template or nascent plus strands, the products were melted
RNA
249
POLYMERASES
and then either self-annealed or reannealed in the presence of excess TMV-RNA plus strand or E. coli rRNAs. If reannealing is enhanced by the addition of excess TMVRNA to the reaction, then this would be an indication that the complementary minus strand was being synthesized. If on the other hand reannealing was decreased, then it could be concluded that the minus strand was being synthesized. Table 3 summarizes the results. Self-annealing of the RNA products followed by RNase treatment resulted in 6 to 9% of the radioactive RNA being converted to a RNase-resistant duplex (hybrid yield). When the products were melted and RNase-treated without reannealing, a significant reduction in hybrid yield was observed, which would discount the possibility that the self-annealing values were attributed to a RNase-resistant core in the RNA products. Reannealing was enhanced by adding 10 pg of TMV RNA to the reaction with hybrid yields on the order of 90%, indicating that the bulk of the RNA synthesized was complementary to the added TMV RNA template. This enhancement was specific for unique sequences in TABLE
3
HYBRIDIZATION OF THE RNA PRODUCTS SYNTHESIZED in Vitro BY THE PHOSPHOCEI.I.~:I,OSE POLYMERASES~ Reaction
condition
Hybrid Mock-inoculated
Self-anneal,
no
RNase No self-anneal, RNase Self-anneal, RNase Reanneal + 10 pg of TMV RNA, RNase Reanneal + 10 pg of E. coli rRNAs, RNase
100%~
(=1395cpm) 3.2
yield
(%I) TMV-infected
100% (=2073cpm) 0
6.3 85
9.1 93
6.5
8.8
n The phenol-extracted RNase-resistant RNA reaction products were melted by heating for 2.5 min in a boiling water bath and then quick cooled in an icewater bath. Where indicated, the melted RNAs were reannealed at 70” for 2 hr alone or with 10 /.rg of the specified RNA (reaction mixture volume of 135 ~1). Ten microliters of a mixture of ribonuclease A and T, (“RNase”) or 10 ~1 of water (“No RNase”) was added and the mixture was incubated for 30 min at 37” prior to determining TCA-insoluble radioactivity.
250
ROMAINE
AND
the TMV-RNA, since it was not observed when E. coli rRNAs were substituted for the TMV-RNA during reannealing. To assess the degree of complementary base pairs in the renatured hybrids, the thermal stability of the RNA hybrids synthesized in vitro by the phosphocellulosepurified polymerases was compared to that of the hybrid formed following denaturation and then reannealing with 100 pg/ml of TMV-RNA for 3 hr at 70”. The thermal stability of the hybrid synthesized in vitro by either of the polymerases had a melting temperature (T,) of 79 to 79.5” in 0.1X SSC which was in reasonably good agreement with the Tm’s of 80 to 81” obtained for the hybrids renatured with TMV-RNA (Fig. 4). The discrepancy of the T,,,‘s could not be reasonably explained; however, it could be concluded that the hybrid renatured in vitro and that synthesized in vitro were equally complementary to the TMVRNA. In all instances the transition breadth was approximately 25”, which is considered to be due to the heterodispersed size of the hybrid molecules (Gillespie et al., 1975). DISCUSSION
By all the chromatographic and catalytic criteria we have applied, the partially purified soluble enzyme from TMV-infected
ZAITLIN
tissues appears to be the same enzyme as that found in the soluble fraction of homogenates of uninfected tissues; however, the specific activity of the enzyme from infected tissue is considerably higher. A similar phenomenon has been observed with less purified enzymes in tobacco infected with either tobacco necrosis virus (Fraenkel-Conrat, 1976; Stussi-Garaud et al., 1977) or alfalfa mosaic virus (AMV) (Bol et al., 1976; LeRoy et al., 1977), although Bol et al. (1976) have apparently detected several RNA polymerases in a membranous fraction containing the AMV replicase. As has been observed earlier with TMV (Zaitlin et al., 1973) and a number of other RNA polymerases isolated from virus-infected tissues (Mouches et al., 1974; Weening and Bol, 1975; Stussi-Garaud et al., 1977; LeRoy et al., 1977), the soluble enzyme can only catalyze the synthesis of a RNA complementary to the RNA provided as the template. This enzyme is thus not the complete replicase, in that no nascent viral strands are produced. Perhaps the host enzyme is the “core” polymerase which lacks transcriptional control, and through its association with other viruscoded and host-coded proteins on the membrane it forms the fully-competent repli-
TEMPERATURE
FIG. 4. Thermal stabilities of the RNA hybrids synthesized in vitro by the phosphocellulose polymerases. Duplicate lOO-gl aliquots in 0.1~ SSC and the phenol-extracted RNase-resistant reaction products either in the native state or which had been melted and reannealed with 100 pgg/ml of TMV RNA for 3 hr at 70” were exposed for 5 min at the indicated temperature. The samples were adjusted to SSC and then scored for RNaseresistant radioactivity as described under Materials and Methods. RNA from: (A) mock-inoculated leaves, native hybrid, 0-O (2163 cpm = 100%); melted and renatured hybrid, m (1668 cpm = 100%); (B) TMVinfected leaves, native hybrid, @-O (4342 cpm = 100%); melted and renatured hybrid, C-O (3992 cpm = 100%).
TOBACCO
RNA-DEPENDENT
An equally plausible possibility is that the host polymerase has no role in viral RNA replication. The level of other enzymes has been shown to be stimulated following viral infection but without a role in replication. Leaf RNase is a good example of such a stimulation (Wyen et al., 1972). The kinetics for the appearance of both the soluble and membrane-associated polymerase activity following infection is similar, in that the rise in activity precedes the log phase in the virus growth curve but declines prior to the maximum in the production of virus (Romaine, 1977). We know that part of the stimulation of polymerase activity is a cellular response to injury from the inoculation (Duda, personal communication; Stein and Zaitlin, unpublished data), but as the control leaves were all mock-inoculated in our experiments, a significant proportion of the stimulated enzyme activity must be virally induced. There is no known role for a RNA-dependent RNA polymerase in healthy plants and only a hint that the alleged products of such an enzyme (i.e., dsRNA) exist in plants (Lewandowski et al., 1971). Enzymes with remarkedly similar properties exist in animal tissues (Downey et al, 1973) and suggestions that they might amplify mRNAs have been discounted (Boyd and Fitschen, 1977). Enzymes of this type must be widely distributed in plants, because in addition to tobacco and Chinese cabbage (Astier-Manifacier and Cornuet, 1971) we have found evidence for it in leaves of lettuce, beet, cowpea, corn, and pea (Romaine, 1977). Even though our studies and those of others (Fraenkel-Conrat, 1976; Stussi-Garaud et al., 1977; LeRoy et al., 1977) suggest that the host RNA-dependent RNA polymerase is stimulated, there are some suggestions of viral involvement of either natively soluble or solubilized polymerases. Some soluble polymerases show some tendency for an enhanced response to the RNA of the virus causing the infection (Hadidi and Fraenkel-Conrat, 1973; Sela and Hauschner, 1975), although specificity is usually not very great and could reflect the integrity of the various RNAs tested. Furthermore, White and Murakishi (1977) have provided evidence that the stimulation of the host polymerase may be related to cell
RNA
POLYMERASES
251
division. When TMV-infected tobacco cal1~scultures were grown on a mannitol medium to suppress cell division, the stimulation of the soluble host polymerase was not observed, but a rise in the membrane-associated TMV replicase activity was observed. Based on this evidence it was proposed that the host polymerase is not required in viral RNA synthesis. However, it should be recognized that a nonspecific stimulation of the host polymerase activity and a role in viral RNA replication are not mutually exclusive. That is, the stimulation of the host polymerase may be extraneous as it relates to viral RNA replication, but the host polymerase itself may be obligatory for that process. One question our study raises is the relationship of the 130,OOO-MW polypeptide found in TMV-infected tissues (Zaitlin and Hariharasubramanian, 1972; Scalla et al., 1976) to the soluble RNA-dependent RNA polymerase and to the membrane-associated TMV replicase. From our studies, this polypeptide obviously is not a constituent of the soluble polymerase, since as the enzyme is purified it disappears from the polymerase-containing fractions prepared from infected tissues at the DEAE-Bio-Gel step. This polypeptide apparently has no TMV-RNA-polymerizing activity by itself. However, several lines of evidence suggest it should be involved in TMV-RNA replication somehow and probably in an early event such as RNA replication: (1) In infected protoplasts it is an early translation product, appearing before viral coat protein (Sakai and Takebe, 1974; Paterson and Knight, 1975); (2) The gene for this polypeptide is on the 5’-end of TMV RNA (Hunter et al., 1976) and thus is most probably the earliest viral gene to be translated. Nothing presented here rules this protein out as a constituent of the TMV replicase, but it is certainly not a constituent of the viral-stimulated host RNA-dependent RNA polymerase. Furthermore, the relationship, if any, between the soluble host RNA polymerase and the membrane-associated TMV replicase remains to be resolved. ACKNOWLEDGMENTS This work was supported in part by NSF Grant BMS 75-09648. We thank Karen Weaber for her expert
252
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technical assistance and Roger Beachy and Alain Asselin for their advice and interest. REFERENCES ASTIER-MANIFACIER, S., and CORNUET, P. (1971). RNA-dependent polymerase in Chinese cabbage. Biochim. Biophys. Acta 232,464-493. BOL, J. F., CLERX-VAN HAASTER, C. M., and WEENING, c. J. (1976). Host and virus specific RNA polymerases in alfalfa mosaic virus infected tobacco. Ann. Microbial. (Inst. Pasteur) 127A, 183-192. BOYD, C. D., and FITSCHEN, W. (1977). Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication. Nucleic Acid Res. 4,461-471. BRADLEY, D. W., and ZAITLIN, M. (1971). Replication of tobacco mosaic virus: II, The in vitro synthesis of higher molecular weight virus-specific RNAs. Virology 45, 192-199. BRISHAMMAR, S., and JUNTTI, N. (1974). Partial purification and characterisation of soluble TMV replicase. Virology 59, 245-253. BRUENING, G., BEACHY, R. N., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in viva translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus. Virology 71,498-517. DOWNEY, K. M., BYRNES, J. J., JURMARK, B. S., and So, A. G. (1973). Reticulocyte RNA-dependent RNA polymerase. Proc. Nat. Acad. Sci. USA 70, 3400-3404. DUDA, C. T., ZAITLIN, M., and SIEGEL, A. (1973). In vitro synthesis of double-stranded RNA by an enzyme system isolated from tobacco leaves. Biochim. Biophys. Acta 319,62-71. FRAENKEL-CONRAT, H. (1976). RNA polymerase from tobacco necrosis virus infected and uninfected tobacco. Purification of the membrane-associated enzyme. Virology 72, 23-32. GILLESPIE, D., GILLESPIE, S., and WONG-STAAL, F. (1975). RNA-DNA hybridization applied to cancer research: Special reference to RNA tumor viruses. In “Methods in Cancer Research” (H. Busch, ed.), pp. 205-245. GOODING, G. V., and HEBERT, H. H. (1967). A simple technique for the purification of tobacco mosaic virus in large quantities. Phytopathology 67, 1285. HADIDI, A., and FRAENKEL-CONRAT, H. (1973). Characterization and template specificity of soluble RNA polymerase of brome mosaic virus. Virology 52, 363-372. HUNTER, T. R., HUNT, T., KNOWLAND, J., and ZIMMERN, D. (1976). Messenger RNA for the coat protein of tobacco mosaic virus. Nature (London) 260, 759-764. JACKSON, A. O., MITCHELL, D. M., and SIEGEL, A. (1971). Replication of tobacco mosaic virus: I, Isolation and characterization of double-stranded forms of ribonucleic acid. Virology 45, 182-191. KNOWLAND, J. S. (1974). Protein synthesis directed
by the RNA from plant virus in a normal animal cell. Genetics 78, 363-394. KNOWLAND, J., HUNTER, T., HUNT, T., and ZIMMERN, D. (1975). Translation of tobacco mosaic virus RNA and isolation of messenger for TMV coat protein. Colloq In&it Nat Sante Rech Med (INSERM) 47, 211-216. LEROY, C., STUSSI-GARAUD, C., and HIRTH, L. (1977). RNA-dependent RNA polymerases in uninfected and in alfalfa mosaic virus-infected tobacco plants. Virology 82,46-62. LEWANDOWSKI, L. J., KIMBALL, P. C., and KNIGHT, C. A. (1971). Separation of the infectious ribonucleic acid of potato spindle tuber virus from doublestranded ribonucleic acid of plant tissue extracts. J. Viral. 8, 809-812. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZEL, J. V. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and Koprowski, eds.), Vol. 5, pp. 179-246. Academic Press, New York. MOUCHES, C., Bovti, C., BARREAU, C., and Bovl, J. M. (1975). TYMV-RNA replicase. Colloq Instit Nat Sante Rech Med (INSERM) 47,109-120. PATERSON, R., and KNIGHT, C. A. (1975). Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus. Virology 64, 10-22. ROBERTS, B. E., and PATERSON, B. M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from commercial wheat germ. Proc. Nat. Acad. Sci. USA 70, 2330-2334. ROBERTS, B. E., PATERSON, B. M., and SPERLING, R. (1974). The cell-free synthesis and assembly of viral specific polypeptides into TMV particles. Virology 69,307-313. ROMAINE, C. P. (1977). RNA-dependent RNA polymerase activities in uninfected and tobacco mosaic virus-infected tobacco leaves; Stimulation of a host RNA-dependent RNA polymerase. Ph.D. thesis, Cornell University, Ithaca, N.Y. SAKAI, F., and TAKEBE, I. (1972). A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus. Mol. Gen. Genet. 118,93-96. SCALLA, R., BOUDON, E., and RIGAUD, J. (1976). SDS-polyacrylamide gel electrophoretic detection of two high molecular weight proteins associated with TMV infected tobacco. Virology 69, 339-345. SELA, I., and HAUSCHNER, A. (1975). Isolation and characterization of TMV RNA-dependent enzyme from TMV-infected tobacco leaves. Virology 64, 284-288. STUSSI-GARAUD, C., LEMIUS, J., and FRAENKEL-CONRAT, H. (1977). RNA-polymerases from tobacco necrosis virus infected and uninfected tobacco: II,
TOBACCO
RNA-DEPENDENT
Properties of the bound and soluble polymerases and the nature of their products. Virology 81, 224-236.
WEENING, C. J., and BOL, J. F. (1975).Viral RNA replication in extracts of alfalfa mosaic virus-infected Vicia faba. Virology 63, 77-83. WHITE, J. L., and MURAKISHI, H. H. (1977). In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: Solubilization of a membrane-bound replicase and partial purification. J. Viral. 21, 484-492. WICKNER, R. B. (1973). DNA polymerase II. In “Methods in Molecular Biology, Nucleic Acid Biosynthesis” (A. I. Laskin and J. A. Last, eds.), Vol. 4,
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pp. 157-166. Marcel Dekker, New York. WYEN, N. V., UDVARDY, J., ERDEI, S., and FARKAS, G. L. (1972). The level of a reratively purine-specific ribonuclease increases in virus-infected hypersensitive or mechanically injured tobacco leaves. Virology 48, 337-341. ZAITLIN, M., and HAHIHARASUBRAMANIAN, V. (1972). A gel electrophoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 47,296-305. ZAITLIN, M., DUDA, C. T., and PETTI, M. A. (1973). Replication of tobacco mosaic virus: V, Properties of the bound and solubilized replicase. Virology 53, 300-311.
86,
241-253
(1978)
RNA-Dependent Virus-Infected
RNA Polymerases in Uninfected Tobacco Leaves: Viral-induced Polymerase Activity C. P. ROMAINE’
Department
of Plant
Pathology, Accepted
MILTON
AND
Cornell
University,
December
and Tobacco Mosaic Stimulation of a Host
ZAITLIN Ithaca,
New
York
14853
13, I977
The soluble RNA-dependent RNA polymerase activities in the 105,000-g supernatant fraction of homogenates prepared from uninfected and tobacco mosaic virus (TMV)-infected tobacco leaves have been compared. The enzymes were purified 40- to 50-fold from both mock-inoculated and TMV-infected leaves and were found to have identical behavior upon (NH&SO4 fractionation, Sephadex G-109 gel filtration, and DEAE-Bio-Gel and phosphocellulose chromatography. Although no qualitative differences in polymerase activity were detected, the specific activity of the polymerase from infected leaves was greater than that from mock-inoculated leaves at each step in the purification. Moreover, the most highly purified enzymes from both types of leaf tissue were indistinguishable with respect to a number of catalytic parameters, viz., requirements for RNA synthesis, kinetics of RNA synthesis, lack of template specificity, and character of the RNA products such as strandedness, T,,,, size, and complementarity to the TMV-RNA used as the template. SDS-polyacrylamide gel electrophoresis of the proteins in the most highly purified enzymes showed that both preparations contained 2 major and at least 13 minor corresponding polypeptides; none were unique to TMV infection. The evidence in toto suggests that the enhanced soluble RNA-dependent RNA polymerase activity following TMV infection is due to a stimulation of a host HNA-dependent RNA polymerase rather than to the genesis of a new viral-coded RNA polymerase. The possible relationships between the soluble host polymerase, the membrane-associated TMV replicase, and a 130,000-MW viral-coded polypeptide thought to be involved in TMV-RNA replication remain to be resolved. INTRODUCTION
pernatant fraction of clarified leaf homogenates (natively soluble enzyme) which is not virus specific (Duda et al., 1973; Brishammar and Juntti, 1974; Brishammar, 1975), and it still remains to be determined if this enzyme is obligately involved in viral RNA replication. The search for the viral replicase has been confused by the existence of a host RNA-dependent RNA polymerase in uninfected tobacco leaves (Duda et al., 1973; Bol et al., 1976; Fraenkel-Conrat, 1976). In all likelihood this enzyme is probably similar to the one reported in uninfected Chinese cabbage leaves (Astier-Manifacier and Cornuet, 1971). At the present time, the role of such enzymes in the nucleic acid metabolism of the host is obscure. Furthermore, the possible relationship between the host RNA polymerase and the membrane-
Infection of tobacco leaves by tobacco mosaic virus (TMV) results in a dramatic increase in RNA-dependent RNA polymerase activity. Basically two forms of such activity have been recognized in leaf extracts prepared from TMV-infected leaves. A virus-specific RNA-dependent RNA polymerase (replicase) associated with a membranous subcellular fraction (31,000-g pellet) has been shown to catalyze the synthesis of the replicative intermediate (RI) and replicative form (RF) of TMV-RNA (Bradley and Zaitlin, 1971; White and Murakishi, 1977). In addition, a RNA-dependent RNA polymerase is present in the su’ Present address: Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 168X2. 241
0042-6822/78/0861-0241aO2.(x)/O Copyright 0 1978 by Academic Press, In(.. All rights of reproduction in any form reserved
242
ROMAINE
AND ZAITLIN
associated replicase or natively soluble RNA-dependent RNA polymerase has not been convincingly determined. Although it has been recognized that a host enzyme system exists which has the potential to replicate the viral nucleic acid, infection of tobacco leaf cells by TMV elicits the formation of at least two proteins with molecular weights in the range of 130,000 to 165,000 (Zaitlin and Hariharasubramanian, 1972; Sakai and Takebe, 1974; Paterson and Knight, 1975; Bruening et al., 1976; Scalla et al., 1976). Both are viral-coded polypeptides because products of this approximate size are generated with TMV-RNA as the messenger in various in vitro protein-synthesizing systems (Roberts et cd., 1973; Roberts et aZ., 1974; Knowland, 1974; Knowland et aZ., 1975; Bruening et al., 1976), and cyanogen bromide peptide analysis of the in uiuo- and in uitro-generated 130,000-MW proteins have shown them to be similar if not identical (Romaine, 1977; Scalla, personal communication). Because the 130,000-dalton polypeptide is the predominant putative viral noncapsid polypeptide detected in infected tissues and corroborated in in vitro systems, it has been regarded as the prime candidate for the replicase (Sakai and Takebe, 1974; Hunter et al., 1976), although evidence for an enzymatic nature is lacking. The aim of the present work was to learn whether TMV induces a novel RNA-dependent RNA polymerase activity in infected tobacco cells. The approach undertaken was to purify the polymerases from uninfected and TMV-infected leaves with the hope of resolving a RNA polymerase unique to infection. In doing so, we have learned that the natively soluble RNA polymerase in homogenates of TMV-infected tobacco leaf cells has identical chromatographic and catalytic properties with the natively soluble host RNA polymerase extracted from uninfected cells. The inability to detect a new RNA polymerase in infected cells would indicate that the enhanced RNA polymerase activity following TMV infection is due to a stimulation of the preexisting host RNA-dependent RNA polymerase and not to the appearance of a new virus-coded enzyme.
MATERIALS
AND METHODS
Reagents. Unlabeled ribonucleoside 5’triphosphates (sodium salts: ATP, equine muscle; CTP, type III; GTP, type III), were purchased from Sigma Chemical Co. (St. Louis, Missouri); uridine 5’-[5-3H]triphosphate (tetralithium salt, sp act. 12-16 Ci/mmol) and ammonium sulfate (ultrapure) were from Schwarx/Mann (Orangeburg, New York); Sequanal grade of sodium dodecylsulfate (SDS) was from Pierce Chemical Co. (Rockford, Illinois); and electrophoretically purified ribonucleases A and T1 were from Worthington Biochemical Co. (Freehold, New Jersey). Buffers. Buffers were routinely autoclaved for 10 min or boiled prior to use and stored at -20”. Grinding buffer (GB). Grinding buffer was composed of 50 mbf Tris adjusted to pH 8.1 at 4’ with HCl, 0.4 1M sucrose, 10 mM KCl, 5 nut4 MgCh, 10% (v/v) glycerol, and 10 mM 2-mercaptoethanol (added just before use). Resuspending buffer (RB). Resuspending buffer was composed of 10 mM Tris adjusted to pH 8.1 at 4” with HCl, 10 mA4 KCl, 1 mM NazEDTA, 20% (v/v) glycerol, and 5 mM 2-mercaptoethanol (added just before use). Phosphate-EDTA buffer. PhosphateEDTA buffer was composed of 10 mM NazHP04 and 5 mM NazEDTA, pH 7.0, at room temperature. SSC buffer. SSC buffer was composed of 150 mM NaCl and 15 mM sodium citrate, pH 7.2, at room temperature. PKE buffer. PKE buffer was composed of 20 mM potassium phosphate, 10 mM KCl, 1 mM NazEDTA, 20% (v/v) glycerol, and 5 mAf 2-mercaptoethanol (added just before use), pH 7.5, at room temperature. Sample buffer. Sample buffer was composed of Tris-phosphoric acid, pH 6.7, at room temperature, 1% SDS, 1% mercaptoethanol, 10% glycerol, and 0.015% bromophenol blue (Maizel, 1971). Propagation of plants. Plants of tobacco, Nicotiana tabacum L. cv. Turkish Samsun, were grown in steam-sterilized soil in 6-in. pots in a greenhouse maintained at about 21’ except for the summer months
TOBACCO
RNA-DEPENDENT
when the temperature occasionally exceeded 30’. Virus purification and RNA preparation. The (VI) common strain of TMV was used exclusively in this study and was purified according to the method of Gooding and Hebert (1967). Tobacco leaf rRNAs were prepared by conventional phenol extraction and ethanol precipitation. Viral RNA (except TMV) and other nonviral nucleic acids were obtained from the frozen collection maintained in the laboratory. Inoculation. Generally, Turkish Samsun tobacco plants in the five- to six-leaf stage were used. Three partially expanded leaves of each plant were inoculated by rubbing both leaf surfaces with a brush which had been dipped in a solution of TMV (0.5 mg/ml) in 100 n&f sodium phosphate buffer, pH 7.0, containing a small amount of washed Celite. The leaves were rinsed with water immediately after inoculation and the plants placed in a controlled-environment chamber providing 25’ and 1500 fc of illumination at the leaf surface (16~hr photoperiod). At 3 days postinoculation, both directly inoculated leaves with chlorotic spots and small systemically infected vein-cleared leaves were used as a source of enzyme. Uninfected controls consisted of comparable leaves which were mock inoculated with a mixture of yeast RNA (50 pg/ml) and bovine serum albumin (0.45 mg/ml) in buffer with Celite. Partial purification of the soluble RNAdependent RNA polymerases. AU glassware was either acid-washed or heated. All purification steps were performed at 2 to 4” in the sequence given below: Preparation of crude extract. Seventy grams of fresh mock-inoculated or TMVinfected tobacco leaves with their midveins removed was placed in a polyethylene contamer immersed in an ice bath and chopped for several min in 1.25 ml of GB/g of tissue with an electric knife adapted to hold two razor blades. The brei was pressed through a single layer of washed Miracloth (Calbiochem Co.) and centrifuged at 1000 g for 5 min. The top 90% of the supernatant was recovered and centrifuged at 31,000 g for 20 mm. The supernatant was decanted and further clarified by centrifugation at
RNA
POLYMERASES
243
105,000 g for 1 to 1.5 hr at 4” in a Type 40 rotor in a Beckman L3-40 ultracentrifuge. (NH&SO4 fractionation. CrysWine (NH&SO4 was added slowly (l-2 min) to the supematant (100 ml) directly to 50% saturation with constant stirring on an icewater bath. Stirring was continued for an additional 15 min and the yellowish-white precipitate was collected by centrifugation at 20,000 g for 15 min and then dissolved with vortexing in 0.1 ml of cold RB/g of tissue (7 ml). The solution was incubated on ice for an additional 30 min and then clarified by centrifugation at 20,000 g for 10 min. Sephadex G-100 gel filtration. The (N&)$04-concentrated polymerase fraction was layered directly on top of a column (Pharmacia, 1.6~cm i.d. x 106 cm) of Sephadex G-100 equilibrated with 250 ml of RB. Fractions of 110 drops were collected at a flow rate of 12 ml&r by gravity flow. DEAE-Bio-Gel ion-exchange chromatography. The active fractions from the gel filtration step were pooled and applied to a 0.9-cm i.d. x 15-cm column (Pharmacia) of DEAE-Bio-Gel A, 100 to 200 mesh (BioRad Laboratories), that had been previously equilibrated with 25 ml of RB. The column was washed with a volume of RB equivalent to the sample volume to remove unadsorbed proteins and then developed with a linear gradient of 300 ml of RB also containing from 10 to 300 n&f KCl. Fractions of 196 drops were collected at a flow rate of 12 ml/hr using a Buchler peristaltic pump. The DEAE-Bio-Gel fractions containing enzyme activity were pooled and dialyzed against three changes of PKE buffer (total volume, 2 liters) overnight at 4O. Phosphocellulose ion-exchange chromatography. Phosphocellulose (Whatman P-11) was washed by decantation first with 0.1 N HCl in 50% ethanol and then with deionized water, followed by 0.1 N NaOH, and washed again with water (Wickner, 1973). The phosphocellulose was resuspended in PKE buffer and was packed into a 0.9-cm i.d. x 15-cm column (Pharmacia) under gravity. The column was equilibrated with 25 ml of PKE buffer and 34 ml of the dialyzed DEAE-Bio-Gel step enzyme was applied and washed on with 10 ml of PKE
244
ROMAINE
AND ZAITLIN
buffer. The column was then washed consecutively with 10 ml each of PKE buffer containing 0.05 M, 0.1 M, 0.2 M, and 0.4 M PO1, maintaining a flow rate of 8 ml/hr under gravity while 70-drop fractions were collected. Fractions containing high enzyme activity were pooled and constituted the final partially purified polymerases. Additional steps. Dilute enzyme preparations could be concentrated by dialysis against 20 vol of RB containing 30% PEG6000 at 4”. No appreciable loss of activity resulted using this procedure, providing dialysis did not proceed to the point where the enzyme was entirely dehydrated. RNA-dependent RNA polymerase assay. The standard reaction mixture (final volume, 50 $1, contained the following: 100 mM Tris (pH 8.0 for 33”), 10 mM MgC12, 0.025 pm01 each of ATP, CTP, and GTP, 7.5 mikf dithiothreitol, 20 mM (NH.&S04, 0.5 pg of actinomycin D, 1.25 @i of r3H]UTP, (ethanol removed by drying in uacuo just before use) 6 pg of TMV RNA, and 25 ~1 of enzyme, unless indicated otherwise. The enzyme reaction was initiated by adding the enzyme and was allowed to proceed for 1 hr at 33”, at which time the reaction was terminated by spotting the entire mixture onto a 2.3-cm circular 3-MM Whatman filter. The disks were processed and the acid-insoluble radioactivity was determined as described by Zaitlin et al. (1973). Data are expressed as net counts per minute, calculated by subtracting “0 times,” obtained by mixing enzyme with ice-cold reaction mixture and immediately terminating the reaction. Extraction of the polymerase reaction products. For analysis of the RNA products of the polymerase reaction, the reaction mixture was scaled up to 1 to 5 ml and after incubation for 4 to 5 hr, the RNA was phenol extracted and ethanol precipitated (Zaitlin et al., 1973). Polyacrylamide gel electrophoresis of RNA. Electrophoretic analysis of RNA was carried out in Plexiglas tubes (6-mm i.d.) containing g-cm long 1.8% polyacrylamide gels stabilized with 0.5% agarose (Jackson et al., 1971). The conditions for gel electrophoresis and fractionation have been described (Zaitlin et al., 1973.) RNA-RNA hybridization. The RNA
products were processed for hybridization as outlined previously (Zaitlin et al., 1973). The double-stranded RNAs were melted in a boiling water bath for 2.5 min followed by immediate cooling in an ice-water bath. Annealing was performed for 2 hr at 70” in SSC buffer (Zaitlin et al., 1973), and the extent of renaturation was determined by treating the sample with 10 ~1 of a mixture of ribonuclease A (100 pg/ml) and T1 (5 pg/ml) at 37’ for 30 min prior to determining TCA-insoluble radioactivity. Thermal denaturation of the pH]RNA products. The thermal denaturation kinetics of the in vitro-synthesized r3HJRNA was determined in a vessel containing water which was fashioned from a l-liter polypropylene bottle modified to hold six l-dram shell vials and a thermometer. Water was circulated around the vials and temperature was regulated (kO.5”) by a Haake bath. One-hundred-microliter aliquots of the [3H]RNA (1000-5000 cpm) in 0.1~ SSC were dispensed into Siliclad-treated vials, and duplicate samples were exposed for 5 min at the specified temperature, followed by immediate and continued cooling in an ice-water bath. After individual duplicate samples had been exposed at each of the designated temperatures, the vials were removed from the ice bath and centrifuged at 1000 g for 30 set to collect the solution. The solutions were adjusted to SSC by adding sufficient 10x SSC and TCA-insoluble ribonuclease-resistant counts were determined. Polyacrylamide gel electrophoresis of proteins. Proteins were precipitated from solution by adding cold 50% TCA to 10% and incubating for at least 2 hr at 4”. The precipitate was collected by centrifugation at 20,000 g for 15 min and washed twice with cold acetone. The final precipitate was recovered by centrifugation, air-dried at 4”, and then dissolved in sample buffer. Immediately proteins were denatured by heating for 4 hr at 55” and then again for 3 min at 100” just prior to electrophoresis. SDS-polyacrylamide gel electrophoresis was performed using the discontinuous buffer system of Maize1 (1971). Denatured proteins were separated on slab gels (0.05 x 14 x 10 cm) having a l-cm 5% stacking gel and a g-cm 10% running gel. The
TOBACCO
RNA-DEPENDENT
RNA
bisacrylamide ratio of the stacking gel was l.O35:30 and of the running gel was 0.8:30. Electrophoresis was done at room temperature at 50 V for 10 min and then at 100 V. The gels were stained in freshly prepared 0.2% Coomassie brilliant blue R-250 in methanol:water:glacial acetic acid (5:5:1, v/v/v) overnight at room temperature and destained in the same solvent without dye at 40° for 5 min and then in a 7% glacial acetic acid solution. Analytical methods. Protein concentration was determined by the method of Lowry et al. (1951) using crystalline BSA (Fraction V) as a standard. Conductivity measurements for determining salt gradients were made on a Yellowsprings Instrument Co. conductivity meter standardized with buffers of known ionic strength. RESULTS
Enzyme purification. The soluble RNAdependent RNA polymerase in mock-inoculated and TMV-infected tobacco leaves was partially purified with enzyme activity being monitored with exogenous TMVRNA as the template in the enzyme assay throughout the course of the purification. The purification scheme outlined was
adapted for 50 to 75 g of the deveined tobacco leaves and afforded a 40- to 50-fold purification with an approximate 5% yield with respect to the 105,000-g supernatant for both the mock-inoculated and TMVinfected leaf-soluble RNA polymerase (Table 1). When the enzyme in the high-speed supernatant from the crude homogenates was concentrated by (NH&S04 precipitation, the soluble polymerases in infected and uninfected leaves were observed to have similar “salting-out” properties. Approximately 70 to 85% of the activity precipitated in the range of 30 to 50% saturation (NH&S04 and greater than 90% from 0 to 50% saturation (Table 2). Higher concentrations of (NH&S04 precipitated considerable additional polymerase but with a low specific activity. Upon Sephadex G-100 gel filtration, polymerase activity from both infected and mock-inoculated leaves eluted in or just after the void volume (Figs. 1A and 1B) along with most of the protein, as determined by optical density measurements of the effluent at 280 nm. Generally, more than 100% of the initial polymerase activity in the 105,000-g supernatant is recovered at
TABLE PARTIAL
PURIFICATION Enzyme
1
OF THE SOLUBLE RNA-DEPENDENT RNA POLYMERAS= AND TMV-INFECTED TOBACCO LEAVES~ fraction
Total
protein (wdb
Total activity (units)’
.______ Mock-inoculated A. Soluble fraction B. 50% ammonium sulfate Sephadex G-100 E. DEAE-Bio-Gel G. Phosphocellulose TMV-infected B. Soluble fraction D. 50% ammonium dex G-100 F. DEAE-Bio-Gel H. Phosphocellulose
245
POLYMERASES
sulfate
pellet
and
138 76 38 0.14
pellet
Sepha-
144 72
Specific activity (units/mg of protein)
Yield
(%)
561 571
4.1 7.5
100 102
209 28
5.4 206
37 5
27 58
100 108
3899 4205
60 0.29
IN MOCK-INOCULATED
50 76 1026 8.--~~ _-~~--~ ~ supernatant) were prepared separately from 70 g of (deveined) at 3 days postinoculation as described under
a Soluble RNA polymerase crude extracts (105,000-g either mock-inoculated or TMV-infected tobacco leaves Materials and Methods. * Protein was determined by the method of Lowry et al. (1951). ’ One unit of enzyme activity is equivalent to the incorporation
2980 298
of 1 pmol
of [3H]UMP
at 33” for 1 hr.
246
ROMAINE
AND ZAITLIN TABLE
AMMONIUM ‘;f;;?f;
SULFATE Centrifugal
fraction
(S saturation)
O-20 20-30 30-40 40-50 w-60 60-70 >70
2
FRACTIONATION OF THE SOLUBLE RNA POLYMERASES UNINFECTED AND TMV-INFECTED TOBACCO LEAVES= Uninfected Total polymerase activity (cpm X 10e3)
Starting material Pellet Pellet Pellet Pellet Pellet Pellet Supernatant
411 9 24 145 182 14 3 0
PREPARED
FROM
TMV-infected Total protein (md
Total polymerase activity (cpm X 10m3)
Total protein (mid
12.65 0.40 0.78 1.01 7.63 2.44 1.33 0.20
LfJoo 20 132 573 380 45 4 30
18.00 0.17 0.78 1.60 10.64 2.10 1.37 0.14
a Soluble RNA polymerase extracts (105,000-g supematant) were prepared separately from 10 g of either uninfected or TMV-infected leaf tissue (deveined) as described under Materials and Methods. Sufficient solid (NH&SO4 was added slowly to the extracts to 20% saturation with constant stirring in an ice-water bath. Stirring was continued for an additional 15 min at which time the precipitate was removed by centrifugation at 20,609 g for 15 min and then dissolved in 1 ml of RB. The supematant was collected and the procedure was repeated for each of the (NH&SO4 concentrations indicated. All fractions were dialyzed batchwise against three changes of RB (total volume, 2 liters) for 20 br at 4” prior to polymerase assay. Protein was determined by the method of Lowry et al. (1951).
this step, presumably because of the removal of an inhibitor. Higher levels of polymerase activity were consistently obtained from TMV-infected leaves than from mock-inoculated leaves. After an initial experiment, it was found not to be necessary to assay all fractions since the polymerasecontaining fractions are yellow and elute from the column in the void volume. Polymerase activity from both tminfected and infected leaves eluted from DEAE-Bio-Gel as a single peak at 0.07 M KC1 just before the bulk of the protein as determined by optical density at 280 nm (Figs. 1C and 1D). Enzyme activity yields at this step ranged from 40 to 80% for both polymerases, the only difference being that a higher level of activity was obtained from infected leaves than from uninfected leaves. In a single experiment, a second minor peak of polymerase activity was detected which eluted at 0.11 to 0.13 M KC1 from DEAE-Bio-Gel (data not shown). This polymerase activity was present in extracts from both uninfected and infected leaves, which would discount the possibility that it was of viral origin. The polymerase from infected leaves could also be chromatographed on DEAE-Sephadex A-25 and eluted as a single peak of activity at ap-
proximately 0.09 A4 KCl. However, the yield of enzyme activity was usually 50% or less and the reproducibility of the method was low. The major purification of the RNA polymerases from other soluble proteins is accomplished by the phosphocellulose chromatography. As shown in Figs. 1E and lF, under the ionic conditions employed most of the protein (optical density at 280 nm) passes directly through the column, whereas most of the polymerase activity is adsorbed. Both mock-inoculated and infected leaf RNA polymerase activities eluted in the 0.95 M PO4 wash with the only difference again being quantitative. Polypeptides associated with enzyme preparations. The polypeptides in the various enzyme fractions at each step of the purification were analyzed by SDS-polyacrylamide gel electrophoresis to assess the purity of the enzyme preparations. This revealed that the most highly purified preparation, the phosphocellulose polymerase (Fig. 2G and 2H), is not homogeneous and still contains at least 15 different polypeptides detectable by staining with Coomassie brilliant blue (all are not apparent in Fig. 2), which range in apparent molecular weight from less than 25,000 to in excess of
TOBACCO
RNA-DEPENDENT
RNA
POLYMERASES
247
Ai
FIG. 1. Gel filtration and chromatography of the soluble RNA polymerases from mock-inoculated and TMV-infected tobacco leaves. A soluble subcellular fraction (105,000-g supernatant) was prepared from 70 g of either mock-inoculated or TMV-infected tobacco leaves at 3 days postinoculation and processed as described under Materials and Methods. (A) Sephadex G-100 gel filtration, polymerase activity from mockinoculated leaves. (B) Sephadex G-100 gel filtration, polymerase activity from TMV-infected leaves. Polymerase activity, w absorbance at 280 nm, M, void volume, VU. (C) DEAE-Bio-Gel chromatography, polymerase activity from mock-inoculated leaves. (D) DEAE-Bio-Gel chromatography, polymerase activity from TMV-infected leaves. Polymerase activity, W, absorbance at 280 nm, C----q KC1 concentration, -----. (E) Phosphocellulose chromatography, polymerase activity from mock-inoculated leaves. (F) Phosphocellulose chromatography polymerase activity from TMV-infected leaves. Polymerase activity, O-4; absorbance at 280 nm, O-----O; potassium phosphate concentration, -----.
130,000. The more highly purified polymerases prepared from uninfected and infected leaves had identical polypeptide compositions, and interestingly, the viralcoded 130,000-dalton polypeptide was not associated with the partially purified RNA polymerase activity from infected leaves. In three separate experiments this polypeptide was largely eliminated from the RNA-dependent RNA polymerase activity containing fractions following either DEAE-Bio-Gel or DEAE-Sephadex chromatography. The fate of the 130,000-dalton polypeptide at this step was not deter-
mined, however, since the yield of polymerase activity at each step of the purification and overaU was similar for both polymerases, and since no unique polymerase activity could be detected in infected leaves, it seems unlikely that a second polymerase activity, perhaps associated with this 130,000-dalton polypeptide, was lost at some step. Catalytic properties of the partiallypurified polymerase. The phosphocellulose enzymes from mock-inoculated and infected leaves are indistinguishable with respect to their requirements for RNA synthesis, and
248
ROMAINE
FIG. 2. Polyacrylamide gel analysis of the partial purification of the soluble RNA-dependent RNA polymerase in mock-inoculated and TMV-infected tobacco leaves. Proteins in aliquots of the polymerase fraction taken at each step in the purification designated in Fig. 1 and in Table 1 were electrophoresed on a 10% polyacrylamide slab gel in SDS (Maixel, 1971). Shown are the positions of the protein markers; /lgalactosidase (130,090 daltons), BSA (67,000 daltons), and ovalbumin (45,000 daltons). The arrowheads denote the position of the virus-coded 130,000-dalton polypeptide. Note that this polypeptide is not associated with the most highly purified polymerase preparations E through H. The soluble fraction (105,000-g supernatant): (A) mock-inoculated leaves and (B) TMV-infected leaves; the 50% (NH&S04 pellet and pooled polymerase fractions from the Sephadex G-100 gel filtration: (C) mock-inoculated leaves and (D) TMV-infected leaves; the pooled peak fractions from the DEAE-Bio-Gel column: (E) mock-inoculated leaves and (F) TMV-infected leaves; the pooled peak fractions from the phosphocellulose column: (G) mock-inoculated leaves and (H) TMV-infected leaves.
their properties are similar to previous reports of a detergent-solubilized enzyme from this laboratory (Zaitlin et al., 1973) and accordingly are not documented in detail here. Omission of the three ribonucleoside triphosphates resulted in a 97% reduction in enzyme activity. Mg2+ is required
AND ZAITLIN
for optimal activity which could be satisfied only to the extent of 5% by 1.25-n-&f Mn2+. Without exogenous TMV-RNA, incorporation was negligible and in several experiments ranged from 0 to 4% of the control which contained RNA. There is no detectable DNA-dependent RNA polymerase activity suggested by comparing [3H]UMP incorporation in the absence or presence of actinomycin D. Furthermore, addition of RNase (10 pg/ml) to the reaction mixture prior to adding the polymerase abolished activity, whereas DNase (10 pg/ml) had no observable deleterious effect, indicating that the template was RNA and not DNA. Under optimum conditions for enzyme activity, the kinetics of [3H]UMP incorporation by the partially purified polymerases were identical, being approximately linear for the first 3 hr of incubation and thereafter reaching a plateau. The RNA products synthesized by either of the polymerases at all degrees of purity were greater than 75% resistant to RNase digestion in high salt (2~ SSC), demonstrating that the RNAs were highly base-paired molecules. [3H]UMP incorporation by the phosphocellulose polymerases was measured using several viral and nonviral RNAs as templates at 180 pg/ml. Each of the RNAs tested could fulfill the template requirement with an efficiency that was similar for either of the polymerases. The most active templates were cowpea mosaic virus RNA and TMV-RNA. In general, viral RNAs seemed to be more effective templates than nonviral RNAs, a phenomenon which had been observed previously (Zaitlin et al., 1973). The RNA products synthesized by the soluble polymerases in the unfractionated 105,000-g supernatant were heterodispersed and of low molecular weight (Fig. 3A), whereas the more highly purified enzymes synthesized RNAs which were still heterodispersed in size but which were now of considerably higher molecular weight (Fig. 3B). The RNA products synthesized by the Sephadex G-100 and DEAE-Bio-Gel polymerases were analyzed similarly; an increase in the purity of the enzymes was accompanied by an increase in the average size of the RNA products (data not shown). As was observed with polymerases in crude leaf extracts, the polymerase from unin-
TOBACCO
RNA-DEPENDENT
t t
GEL
SLICE
NUMBER
FIG. 3. Radioactivity profile after polyacrylamide gel electrophoresis of the RNA products synthesized in vitro and catalyzed by the crude and partially purified soluble polymerases. Scaled-up standard reaction mixtures were incubated for 4 to 5 hr at 33”. The RNA products were phenol extracted and electrophoresed on g-cm 1.8% polyacrylamide-agarose gels at 5 mA/gel for 3 hr and 45 min. The gels were fractionated into 1.07-mm slices and radioactivity was determined. See Materials and Methods. The arrows denote the positions of the optical density marker, TMV RNA, and bromophenol blue dye. Migration is from left to right. (A) Crude polymerase fraction (105,000-g supernatant): from mock-inoculated leaves, G-0 (10,800 cpm applied); from TMV-infected leaves, 0-O (10,200 cpm applied). (B) Partially purified polymerase fraction (phosphocellulose): from mock-inoculated leaves, O-O (20,800 cpm applied); from TMV-infected leaves, @--O (20,300 cpm applied).
fected leaves synthesized a product of higher molecular weight than did the polymerase from infected leaves; however, this differential in molecular weight decreased with an increase in the purity of these enzymes. Nature of the enzyme products. To determine whether the phosphocellulose polymerases were synthesizing the complementary “minus” strand of the added TMV-RNA “plus” strand template or nascent plus strands, the products were melted
RNA
249
POLYMERASES
and then either self-annealed or reannealed in the presence of excess TMV-RNA plus strand or E. coli rRNAs. If reannealing is enhanced by the addition of excess TMVRNA to the reaction, then this would be an indication that the complementary minus strand was being synthesized. If on the other hand reannealing was decreased, then it could be concluded that the minus strand was being synthesized. Table 3 summarizes the results. Self-annealing of the RNA products followed by RNase treatment resulted in 6 to 9% of the radioactive RNA being converted to a RNase-resistant duplex (hybrid yield). When the products were melted and RNase-treated without reannealing, a significant reduction in hybrid yield was observed, which would discount the possibility that the self-annealing values were attributed to a RNase-resistant core in the RNA products. Reannealing was enhanced by adding 10 pg of TMV RNA to the reaction with hybrid yields on the order of 90%, indicating that the bulk of the RNA synthesized was complementary to the added TMV RNA template. This enhancement was specific for unique sequences in TABLE
3
HYBRIDIZATION OF THE RNA PRODUCTS SYNTHESIZED in Vitro BY THE PHOSPHOCEI.I.~:I,OSE POLYMERASES~ Reaction
condition
Hybrid Mock-inoculated
Self-anneal,
no
RNase No self-anneal, RNase Self-anneal, RNase Reanneal + 10 pg of TMV RNA, RNase Reanneal + 10 pg of E. coli rRNAs, RNase
100%~
(=1395cpm) 3.2
yield
(%I) TMV-infected
100% (=2073cpm) 0
6.3 85
9.1 93
6.5
8.8
n The phenol-extracted RNase-resistant RNA reaction products were melted by heating for 2.5 min in a boiling water bath and then quick cooled in an icewater bath. Where indicated, the melted RNAs were reannealed at 70” for 2 hr alone or with 10 /.rg of the specified RNA (reaction mixture volume of 135 ~1). Ten microliters of a mixture of ribonuclease A and T, (“RNase”) or 10 ~1 of water (“No RNase”) was added and the mixture was incubated for 30 min at 37” prior to determining TCA-insoluble radioactivity.
250
ROMAINE
AND
the TMV-RNA, since it was not observed when E. coli rRNAs were substituted for the TMV-RNA during reannealing. To assess the degree of complementary base pairs in the renatured hybrids, the thermal stability of the RNA hybrids synthesized in vitro by the phosphocellulosepurified polymerases was compared to that of the hybrid formed following denaturation and then reannealing with 100 pg/ml of TMV-RNA for 3 hr at 70”. The thermal stability of the hybrid synthesized in vitro by either of the polymerases had a melting temperature (T,) of 79 to 79.5” in 0.1X SSC which was in reasonably good agreement with the Tm’s of 80 to 81” obtained for the hybrids renatured with TMV-RNA (Fig. 4). The discrepancy of the T,,,‘s could not be reasonably explained; however, it could be concluded that the hybrid renatured in vitro and that synthesized in vitro were equally complementary to the TMVRNA. In all instances the transition breadth was approximately 25”, which is considered to be due to the heterodispersed size of the hybrid molecules (Gillespie et al., 1975). DISCUSSION
By all the chromatographic and catalytic criteria we have applied, the partially purified soluble enzyme from TMV-infected
ZAITLIN
tissues appears to be the same enzyme as that found in the soluble fraction of homogenates of uninfected tissues; however, the specific activity of the enzyme from infected tissue is considerably higher. A similar phenomenon has been observed with less purified enzymes in tobacco infected with either tobacco necrosis virus (Fraenkel-Conrat, 1976; Stussi-Garaud et al., 1977) or alfalfa mosaic virus (AMV) (Bol et al., 1976; LeRoy et al., 1977), although Bol et al. (1976) have apparently detected several RNA polymerases in a membranous fraction containing the AMV replicase. As has been observed earlier with TMV (Zaitlin et al., 1973) and a number of other RNA polymerases isolated from virus-infected tissues (Mouches et al., 1974; Weening and Bol, 1975; Stussi-Garaud et al., 1977; LeRoy et al., 1977), the soluble enzyme can only catalyze the synthesis of a RNA complementary to the RNA provided as the template. This enzyme is thus not the complete replicase, in that no nascent viral strands are produced. Perhaps the host enzyme is the “core” polymerase which lacks transcriptional control, and through its association with other viruscoded and host-coded proteins on the membrane it forms the fully-competent repli-
TEMPERATURE
FIG. 4. Thermal stabilities of the RNA hybrids synthesized in vitro by the phosphocellulose polymerases. Duplicate lOO-gl aliquots in 0.1~ SSC and the phenol-extracted RNase-resistant reaction products either in the native state or which had been melted and reannealed with 100 pgg/ml of TMV RNA for 3 hr at 70” were exposed for 5 min at the indicated temperature. The samples were adjusted to SSC and then scored for RNaseresistant radioactivity as described under Materials and Methods. RNA from: (A) mock-inoculated leaves, native hybrid, 0-O (2163 cpm = 100%); melted and renatured hybrid, m (1668 cpm = 100%); (B) TMVinfected leaves, native hybrid, @-O (4342 cpm = 100%); melted and renatured hybrid, C-O (3992 cpm = 100%).
TOBACCO
RNA-DEPENDENT
An equally plausible possibility is that the host polymerase has no role in viral RNA replication. The level of other enzymes has been shown to be stimulated following viral infection but without a role in replication. Leaf RNase is a good example of such a stimulation (Wyen et al., 1972). The kinetics for the appearance of both the soluble and membrane-associated polymerase activity following infection is similar, in that the rise in activity precedes the log phase in the virus growth curve but declines prior to the maximum in the production of virus (Romaine, 1977). We know that part of the stimulation of polymerase activity is a cellular response to injury from the inoculation (Duda, personal communication; Stein and Zaitlin, unpublished data), but as the control leaves were all mock-inoculated in our experiments, a significant proportion of the stimulated enzyme activity must be virally induced. There is no known role for a RNA-dependent RNA polymerase in healthy plants and only a hint that the alleged products of such an enzyme (i.e., dsRNA) exist in plants (Lewandowski et al., 1971). Enzymes with remarkedly similar properties exist in animal tissues (Downey et al, 1973) and suggestions that they might amplify mRNAs have been discounted (Boyd and Fitschen, 1977). Enzymes of this type must be widely distributed in plants, because in addition to tobacco and Chinese cabbage (Astier-Manifacier and Cornuet, 1971) we have found evidence for it in leaves of lettuce, beet, cowpea, corn, and pea (Romaine, 1977). Even though our studies and those of others (Fraenkel-Conrat, 1976; Stussi-Garaud et al., 1977; LeRoy et al., 1977) suggest that the host RNA-dependent RNA polymerase is stimulated, there are some suggestions of viral involvement of either natively soluble or solubilized polymerases. Some soluble polymerases show some tendency for an enhanced response to the RNA of the virus causing the infection (Hadidi and Fraenkel-Conrat, 1973; Sela and Hauschner, 1975), although specificity is usually not very great and could reflect the integrity of the various RNAs tested. Furthermore, White and Murakishi (1977) have provided evidence that the stimulation of the host polymerase may be related to cell
RNA
POLYMERASES
251
division. When TMV-infected tobacco cal1~scultures were grown on a mannitol medium to suppress cell division, the stimulation of the soluble host polymerase was not observed, but a rise in the membrane-associated TMV replicase activity was observed. Based on this evidence it was proposed that the host polymerase is not required in viral RNA synthesis. However, it should be recognized that a nonspecific stimulation of the host polymerase activity and a role in viral RNA replication are not mutually exclusive. That is, the stimulation of the host polymerase may be extraneous as it relates to viral RNA replication, but the host polymerase itself may be obligatory for that process. One question our study raises is the relationship of the 130,OOO-MW polypeptide found in TMV-infected tissues (Zaitlin and Hariharasubramanian, 1972; Scalla et al., 1976) to the soluble RNA-dependent RNA polymerase and to the membrane-associated TMV replicase. From our studies, this polypeptide obviously is not a constituent of the soluble polymerase, since as the enzyme is purified it disappears from the polymerase-containing fractions prepared from infected tissues at the DEAE-Bio-Gel step. This polypeptide apparently has no TMV-RNA-polymerizing activity by itself. However, several lines of evidence suggest it should be involved in TMV-RNA replication somehow and probably in an early event such as RNA replication: (1) In infected protoplasts it is an early translation product, appearing before viral coat protein (Sakai and Takebe, 1974; Paterson and Knight, 1975); (2) The gene for this polypeptide is on the 5’-end of TMV RNA (Hunter et al., 1976) and thus is most probably the earliest viral gene to be translated. Nothing presented here rules this protein out as a constituent of the TMV replicase, but it is certainly not a constituent of the viral-stimulated host RNA-dependent RNA polymerase. Furthermore, the relationship, if any, between the soluble host RNA polymerase and the membrane-associated TMV replicase remains to be resolved. ACKNOWLEDGMENTS This work was supported in part by NSF Grant BMS 75-09648. We thank Karen Weaber for her expert
252
ROMAINE
AND ZAITLIN
technical assistance and Roger Beachy and Alain Asselin for their advice and interest. REFERENCES ASTIER-MANIFACIER, S., and CORNUET, P. (1971). RNA-dependent polymerase in Chinese cabbage. Biochim. Biophys. Acta 232,464-493. BOL, J. F., CLERX-VAN HAASTER, C. M., and WEENING, c. J. (1976). Host and virus specific RNA polymerases in alfalfa mosaic virus infected tobacco. Ann. Microbial. (Inst. Pasteur) 127A, 183-192. BOYD, C. D., and FITSCHEN, W. (1977). Direct evidence that ribosome bound RNA-dependent RNA polymerase does not play a role in globin messenger RNA replication. Nucleic Acid Res. 4,461-471. BRADLEY, D. W., and ZAITLIN, M. (1971). Replication of tobacco mosaic virus: II, The in vitro synthesis of higher molecular weight virus-specific RNAs. Virology 45, 192-199. BRISHAMMAR, S., and JUNTTI, N. (1974). Partial purification and characterisation of soluble TMV replicase. Virology 59, 245-253. BRUENING, G., BEACHY, R. N., SCALLA, R., and ZAITLIN, M. (1976). In vitro and in viva translation of the ribonucleic acids of a cowpea strain of tobacco mosaic virus. Virology 71,498-517. DOWNEY, K. M., BYRNES, J. J., JURMARK, B. S., and So, A. G. (1973). Reticulocyte RNA-dependent RNA polymerase. Proc. Nat. Acad. Sci. USA 70, 3400-3404. DUDA, C. T., ZAITLIN, M., and SIEGEL, A. (1973). In vitro synthesis of double-stranded RNA by an enzyme system isolated from tobacco leaves. Biochim. Biophys. Acta 319,62-71. FRAENKEL-CONRAT, H. (1976). RNA polymerase from tobacco necrosis virus infected and uninfected tobacco. Purification of the membrane-associated enzyme. Virology 72, 23-32. GILLESPIE, D., GILLESPIE, S., and WONG-STAAL, F. (1975). RNA-DNA hybridization applied to cancer research: Special reference to RNA tumor viruses. In “Methods in Cancer Research” (H. Busch, ed.), pp. 205-245. GOODING, G. V., and HEBERT, H. H. (1967). A simple technique for the purification of tobacco mosaic virus in large quantities. Phytopathology 67, 1285. HADIDI, A., and FRAENKEL-CONRAT, H. (1973). Characterization and template specificity of soluble RNA polymerase of brome mosaic virus. Virology 52, 363-372. HUNTER, T. R., HUNT, T., KNOWLAND, J., and ZIMMERN, D. (1976). Messenger RNA for the coat protein of tobacco mosaic virus. Nature (London) 260, 759-764. JACKSON, A. O., MITCHELL, D. M., and SIEGEL, A. (1971). Replication of tobacco mosaic virus: I, Isolation and characterization of double-stranded forms of ribonucleic acid. Virology 45, 182-191. KNOWLAND, J. S. (1974). Protein synthesis directed
by the RNA from plant virus in a normal animal cell. Genetics 78, 363-394. KNOWLAND, J., HUNTER, T., HUNT, T., and ZIMMERN, D. (1975). Translation of tobacco mosaic virus RNA and isolation of messenger for TMV coat protein. Colloq In&it Nat Sante Rech Med (INSERM) 47, 211-216. LEROY, C., STUSSI-GARAUD, C., and HIRTH, L. (1977). RNA-dependent RNA polymerases in uninfected and in alfalfa mosaic virus-infected tobacco plants. Virology 82,46-62. LEWANDOWSKI, L. J., KIMBALL, P. C., and KNIGHT, C. A. (1971). Separation of the infectious ribonucleic acid of potato spindle tuber virus from doublestranded ribonucleic acid of plant tissue extracts. J. Viral. 8, 809-812. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MAIZEL, J. V. (1971). Polyacrylamide gel electrophoresis of viral proteins. In “Methods in Virology” (K. Maramorosch and Koprowski, eds.), Vol. 5, pp. 179-246. Academic Press, New York. MOUCHES, C., Bovti, C., BARREAU, C., and Bovl, J. M. (1975). TYMV-RNA replicase. Colloq Instit Nat Sante Rech Med (INSERM) 47,109-120. PATERSON, R., and KNIGHT, C. A. (1975). Protein synthesis in tobacco protoplasts infected with tobacco mosaic virus. Virology 64, 10-22. ROBERTS, B. E., and PATERSON, B. M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9s RNA in a cell-free system from commercial wheat germ. Proc. Nat. Acad. Sci. USA 70, 2330-2334. ROBERTS, B. E., PATERSON, B. M., and SPERLING, R. (1974). The cell-free synthesis and assembly of viral specific polypeptides into TMV particles. Virology 69,307-313. ROMAINE, C. P. (1977). RNA-dependent RNA polymerase activities in uninfected and tobacco mosaic virus-infected tobacco leaves; Stimulation of a host RNA-dependent RNA polymerase. Ph.D. thesis, Cornell University, Ithaca, N.Y. SAKAI, F., and TAKEBE, I. (1972). A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus. Mol. Gen. Genet. 118,93-96. SCALLA, R., BOUDON, E., and RIGAUD, J. (1976). SDS-polyacrylamide gel electrophoretic detection of two high molecular weight proteins associated with TMV infected tobacco. Virology 69, 339-345. SELA, I., and HAUSCHNER, A. (1975). Isolation and characterization of TMV RNA-dependent enzyme from TMV-infected tobacco leaves. Virology 64, 284-288. STUSSI-GARAUD, C., LEMIUS, J., and FRAENKEL-CONRAT, H. (1977). RNA-polymerases from tobacco necrosis virus infected and uninfected tobacco: II,
TOBACCO
RNA-DEPENDENT
Properties of the bound and soluble polymerases and the nature of their products. Virology 81, 224-236.
WEENING, C. J., and BOL, J. F. (1975).Viral RNA replication in extracts of alfalfa mosaic virus-infected Vicia faba. Virology 63, 77-83. WHITE, J. L., and MURAKISHI, H. H. (1977). In vitro replication of tobacco mosaic virus RNA in tobacco callus cultures: Solubilization of a membrane-bound replicase and partial purification. J. Viral. 21, 484-492. WICKNER, R. B. (1973). DNA polymerase II. In “Methods in Molecular Biology, Nucleic Acid Biosynthesis” (A. I. Laskin and J. A. Last, eds.), Vol. 4,
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pp. 157-166. Marcel Dekker, New York. WYEN, N. V., UDVARDY, J., ERDEI, S., and FARKAS, G. L. (1972). The level of a reratively purine-specific ribonuclease increases in virus-infected hypersensitive or mechanically injured tobacco leaves. Virology 48, 337-341. ZAITLIN, M., and HAHIHARASUBRAMANIAN, V. (1972). A gel electrophoretic analysis of proteins from plants infected with tobacco mosaic and potato spindle tuber viruses. Virology 47,296-305. ZAITLIN, M., DUDA, C. T., and PETTI, M. A. (1973). Replication of tobacco mosaic virus: V, Properties of the bound and solubilized replicase. Virology 53, 300-311.