Cucumber mosaic virus-induced RNA polymerase: Partial purification and properties of the template-free enzyme

“,ROLcGY 62, 434-443 (19541

Cucumber Partial

Mosaic

Purification

Virus-Induced and Properties

RNA Polymerase: of the Template-Free

Enzyme GRAHAM

L. CLARK,

Department

KEITH

of Biochemistry,

W. C. PEDEN,’

Uniuersity Accepted

of Adelaide,

AND Adehde,

ROBERT

H. SYMONS

South Australia

5001

Augu.st 6. 197-J

The RNA-dependent RNA polymerase induced in cucumber cotyledons hy infection with cucumber mosaic virus (CMV) has been purified one-hundred-fold and its properties investigated. RNA polymerase activity was followed during the purification steps using CMV RNA and poly(C) as templates. The initial enzyme extract (Step 1) was partitioned in a dextranpolyethylene glycol liquid polymer phase system and the enzyme in the polyethylene glycol phase (Step 2) was further purified by adsorption onto and step-wise elution from DEAE-Sephadex: the enzyme (Step 3) was now free of nucleic acid templates. Stepwise elution from columns of phosphocellulose (Step 4) and then singlestranded DNA-agarose (Step 5) gave enzyme which was one-hundred-fold purified for CMV R&A and poly(C) as templates. Enzyme activity for these two templates copuril’ied through all steps; however, salt gradient elution from DNA-agarose gave separation 01’the two activities but the poly(C) copying activity was very lahile under the conditions used. The Step 5 enzyme was completely dependent on added template for activity. It copied poly(C) and was not specific for CMV RNA as it copied three other RNA species tested. It showed properties expected of an RNA-dependent RNA polymerase, e.g.. sensitivity to added pyrophosphate and ribonuclease. low activity with either single- or douhle-stranded DNA as template, and insensitivity to actinomycin D, rilampicin. n-amanitin. deoxyrihonuclease, and orthophosphate. The enzyme was unstable at 0” and 15”. hut has been stored in liquid nitrogen for 5 mo without loss of activity. When the same purification procedure was carried out using healthy plant material. negligible RNA replicase activity was found at all stages of the purification. Further purification steps will need to overcome the instahility of the enzyme which could be due to the protease activity in purified fractions and possihly also to a labile subunit structure of the enzyme. The possible structure of the enzyme has heen discussed in relation to the molecular properties of the phage Q$ and f’2 RNA replicases. INTRODUCTION

We have previously described the properties of a cucumber mosaic virus (CMV)induced RNA polymerase present in both soluble and particulate fractions of infected cucumber cotyledons (May and Symons, 1971; Peden et al., 1972; and references therein). Most of the work on the soluble RNA polymerase has been carried I Present address: M.R.C. Mammalian Unit. Department of Zoology, University burgh, Edinburgh. Scotland.

Genome of Edin-

out with an enzyme preparation which had only been purified severalfold and which still contained template nucleic acids. In order to show conclusively that the CMVinduced RNA polymerase contains a viralcoded function(s) and to fully describe its molecular and enzymatic properties, it is necessary to purify the enzyme to homogeneity. In this paper we describe the more extensive purification of the soluble CMVinduced RNA polymerase together with the properties of this enzyme which is com-

PLANT

VIRUS-INDUCED

pletely dependent on added template f’or a.ctivity. The purif’ication achieved so f’ar is appreciably greater than that achieved fhr any other soluble plant virus-induced RNA polymerase (e.g., see Hadidi and FraenkelConrat, 1973; Zaitlin et al., 1973). How much further this enzyme must be purified to reach homogeneity 1s unknown. Like the brome mosaic virus-induced RNA polymerase (Hadidi and Fraenkel-Conrat, 1973), the CMV-induced enzyme is very labile which makes any f’urther purif’ication dif’f’icult. MATEKIALS

AND

METHODS

Materials. In addition to those materials considered previously (May and Symons, 1971), dextran T500 and DEAE-Sephadex were obtained from Pharmacia, Sweden, polyethylene glycol 6000 from Union Carbide Australia Ltd., phosphocellulose (P-11) was a Whatman product, agarose (for electrophoresis) was obtained f’rom Sigma Chemical Co., and Lu-amanitin f’rom Calbiochem. Single-strand DNA-agarose was prepared as described by Schaller et al. (1972) and contained about 1.0 mg of DNA/ml of packed volume of’ 4%) agarose. Virus, viral RNA, and plants. Purif’ied CMV (Q strain) and CMV RNA were prepared as described by Peden and Symons (1973). Cucumber cotyledons were inf’ected with purified virus (0.2 mg/ml) and grown at 24-28” under fluorescent lighting f’or a 12-hr day. Plants were harvested for enzyme purification 7-13 days after infection. Assay of R.WA polymeruse activity. Enzyme assays were based on the rate of incorporation of’ [a-32P]GTP into acidinsoluble material and were carried out as described by May and Symons (1971) except that the assay volume was 0.17 ml and 1.0 mg of’ yeast RNA was added at the end of’the assay to aid the acid precipitation of the product. Templates used at 150 pg/ml were CMV RNA, yeast RNA, and poly(C); assays using poly(C) contained all f’our nucleoside triphosphates. Assays were linear for at least 60 min although they were usually t,erminated af’ter 30 min. Enzyme activity of’ various fractions was propor-

RNA POLYMERASE

435

tional to protein concentration as shown by assaying serial dilutions. However, increasing final concentrations of’ NH,Cl above 100 mM in the assay medium gave increasing inhibition of’ enzyme activity. A unit of RNA polymerase is defined as that amount which incorporated 1 pmole of [cY-~~P]GTP per minute into acid-insoluble material. Specif’ic activity is defined as units of enzyme activity per mg of’ protein. Protein estimation. During the early stages of’ enzyme purification. protein concentration was estimated by the method of Lowry et al. (1951) using bovine serum albumin as standard. However, during the later stages, a more sensitive method was required. As described below, protein samples were concentrated and fractionated by electrophoresis on polyacrylamide gels which were then stained and scanned to determine the total protein loading. Standard amounts of’ bovine serum albumin were run at the same time under the same conditions. Purification of CMV-induced RNA polymerase. A summary of the procedure used f’or 120 g of inf’ected plants is given in Table 1. All operations were performed at 4”. Step I. Preparation of’ the initial extract. This was prepared as described by May et al. (1969). Step 2. Liquid polymer phase separation. The initial extract was partitioned in a dextran-polyethylene glycol phase system, adapted f’rom that of Okazaki and Kornberg (1964). in order to separate most of the nucleic acids from the RNA polymerase activity. To 28 ml of’ initial extract. a solution of’ 9.3 ml of’ 20% (w/w) dextran 500 in water (final concentration 1.6’;; by weight). 26.0 ml of’:SO’!’ (w/w) polyethylene glycol in water (final concentration 6.4% by weight). 53 ml of’ 0.1 M NH,Cl, 0.09 M 2-mercaptoethanol, 0.05 M Tris+HCl buf’f’er, pH 8.5, and 27 g NaCl (final concentration 4 M) was slowly added dropwise and the mixture stirred at 0” for 2 hr. The mixture was then centrif’uged at 2000 rpm for 10 min at 0” and the resulting clear top polyethylene glycol phase recovered. The turbid green bottom dextran phase was

436

CLARK.

PEDEh’

Ah’11 SYMONS

TABLE SLiMMARt

Step no.

1 2 3 4 5

OF THE

Enzyme fraction

Initial extract Phase separation DEAE-Sephadex Phosphocellulose DNA-agarose

PLIHIFI(‘ATION

Volume (ml1

28 250 17 6 7

PROCEDURE

Tot al protein (mg)

64.4 -1’ 17 0.60 0.24

1

FOR THE

Specific activity .~.__poly(Cl 6.1 51 1010 1500

I’ All methods were as described in Materials and Methods. h The polyethylene glycol present in this fraction prevented

discarded. The NaCl was removed by dialysis overnight at 4” against several changes of 2 liters of 10% (v/v) glycerol, 20 mM TrisHCl, pH 8.5, 50 mM 2-mercaptoethanol, 1 mM EDTA. to give the Step 2 enzyme (250 ml). Step 3. Stepwise chromatography on DEAE-Sephadex. The dialyzed Step 2 enzyme was applied to a column of DEAESephadex (2.0 x 9 cm) equilibrated with 10%) glycerol, 50 mM NH,Cl, 20 mM TrisHCl, pH 8.5, 10 mM 2-mercaptoethanol. The column was washed with l-2 column volumes of the same buffer and the RNA polymerase activity eluted with the same buffer containing 0.35 M NH,Cl; the first 10 ml of eluate was discarded and the next 17 ml collected to give the Step 3 enzyme. This step ensured the complete removal of all template nucleic acids. Step 4. Stepwise chromatography on phosphocellulose. The Step 3 enzyme was diluted with Step 4 buffer without NH,Cl to a concentration of 0.2 M NH,Cl and the solution applied to a phosphocellulose column (1.0 x 9 cm) equilibrated with 30% glycerol, 0.2 M NH,Cl, 20 mM Tris-HCl, pH 8.5, 10 mM 2-mercaptoethanol (Step 4 buffer). The column was washed with l-2 column volumes of the same buffer and the RNA polymerase activity was eluted with the same buffer containing 0.5 M NH,Cl; the first 3 ml was discarded and the next 6 ml collected to give the Step 4 enzyme. Step 5. Stepwise chromatography on single-strand DNA-agarose. The Step 4 enzyme was diluted with Step 4 buffer

CMV-INDLICED

CM\ RNA 9.9 31.5 625 880

determination

RNA POI.YMEHUE” ~.__________~_ Total activit! Overall yield (units) (‘7 ) ~--__ CMV polypolyCM\ RNA RNA cc, (0 393 17:1 867 606 364

638 825 536 375 220

of the protein

100 44 220 154 92

100 130 85 59 33

concentration.

without NH,Cl to 0.1 M NH,Cl and the solution applied at 5 ml/hr to a column (2.0 x 5 cm) of DNA-agarose which had been packed slowly (5 ml/hr) in and well-washed with a solution of 30% glycerol, 0.1 M NH,Cl, 20 mM Tris-HCl, pH 8.5, 10 mM 2-mercaptoethanol. The column was washed with 1-2 column volumes of’ the same buffer and the RNA polymerase activity eluted with the same buffer containing 0.75 M NH,Cl at 10 ml/hr; the first 4 ml was discarded and the next 7 ml collected to give the Step 5 enzyme. When not used immediately for the following step, all enzyme fractions were stored in liquid nitrogen. Sodium dodec>rl sulfate (SDS)-polyacqflamide gel electrophoresis. Protein samples to be analyzed were dissociated by heating at 90” for 3 min in 1% SDS, 1% 2-mercaptoethanol, and were then fractionated by SDS-polyacrylamide gel electrophoresis (Weber and Osborn, 1969) on 10% acrylamide, 0.27% bis-acrylamide gels. Gels were stained with Coomassie Brilliant Blue according to the method of Fairbanks et al. (1971) and scanned with a Gilford gel scanner at 600 nm. Prior to electrophoresis, protein samples were concentrated by precipitation with cold trichloroacetic acid (final concentration 20%1, w/v) and the precipitate collected by centrifugation at 16,000 rpm for 10 min at 0”. The precipitate was then washed several times with cold acetone:ether (l:l, by volume) to remove the trichloroacetic acid.

PLANT

VIRUS-INDUCED

RESULTS

Purification Procedure for the Soluble CMV-Induced RNA Polymerase The results obtained for the five-step purification procedure starting with 120 g of infected cucumber plants harvested 7-13 days after infection are summarized in Table 1. The data are the averages of results for five separate, reproducible experiments. RNA polymerase activity at each step was followed using CMV RNA and poly(C) as templates. The salient features of each of the steps, which are described in detail in Materials and Methods, are as follows: Step 1. The preparation of the initial extract was carried out as described previously (May et al., 1969) and involved the grinding of infected plants with an extraction buffer which contained 50% saturated (NH,),SO,. Under these conditions, the RNA polymerase activity was insoluble. After washing the precipitated material once with the same buffer, the RNA polymerase activity was solubilized by suspension in the same buffer without This procedure, which re(NHMO,. moved essentially all ribonuclease activity (May et al., 1969), gave an g-fold protein purification relative to a crude soluble extract prepared by homogenizing plant material in the buffer without (NH,),SO, followed by centrifugation at 10,000 g for 10 min. The extent of purification of RNA polymerase activity in the initial extract of Step 1 above that in the crude soluble extract could not be determined because of low CMV RNA copying activity and the complete lack of detectable poly(C) copying activity in the crude extract (results not given). Steps 2 and 3. The Step 2 polyethylene glycol-dextran phase separation removed most of the template nucleic acid from the enzyme while the step-wise elution of the enzyme from DEAE-Sephadex (Step 3) was necessary to give an enzyme preparation which was completely dependent on added template for activity. Further, in contrast to stepwise elution from DEAESephadex (Step 3), which gave a 65%

RNA POLYMERASE

437

recovery from the previous step of RNA polymerase activity using CMV RNA as template (Table l), linear salt gradient elution gave much lower recoveries and was, therefore, not used in the purification procedure. In agreement with Schaller et al. (1972). the combination of phase separation and ion-exchange chromatography proved to be the most satisfactory procedure for the removal of template nucleic acids. Incomplete removal of nucleic acids was found by precipitation with protamine sulfate (up to 1 mg/ml) or streptomycin sulfate (l-10 mg/ml). The complete removal of template nucleic acids was essential for the unambiguous study of template requirements of the enzyme. In addition, the presence of residual nucleic acids resulted in frustratingly variable behavior of RNA polymerase activity during chromatography on DEAESephadex and phosphocellulose (results not given). Mouches et al. (1974) have also recently reported the use of a similar polyethylene glycol-dextran phase system to remove template nucleic acids from a partially purified turnip yellow mosaic virusinduced RNA replicase. Step 4. Although RNA polymerase activity with both CMV RNA and poly(C) as templates bound to DEAE-Sephadex (Step 3), it was completely adsorbed also to phosphocellulose. Stepwise elution from phosphocellulose with buffer containing 0.5 M NH,Cl gave a 20-fold purification of enzyme activity (Table 1). Gradient elution of the enzyme from phosphocellulose (results not given) gave no separation of polymerase activities with CMV RNA, poly(C), and yeast RNA as templates; under the conditions used, polymerase activity was eluted at 0.35 M NH,Cl with a recovery of 40-50s as compared with 70% recovery during stepwise elution (Table 1). Step li. The final step used here of affinity chromatography was the adsorption of polymerase activity on and stepwise elution from a column of single-strand DNA agarose with an approximately 1.5-fold increase in specific activity (results obtained on gradient elution are given be-

low). Single-strand DNA agarose was used here instead of RNA-agarose because of our previous report (May and Svmons. 1971) that the RNA polymerase binds to single-strand DNA but probably cannot use it as a template. In addition. the longer chains of DNA are less likely to elute from the agarose than the shorter chains of CM\ RNA which would then contaminate the enzyme preparation. The five-step purification procedure (Table 1) resulted in a go-fold purification of polymerase activity using CMV RNA as template with a yield of 33%)and a 250-fold purification using poly( C) as template with a yield of 92”‘rBof the activity in the initial extract. However, in the case of the poly(C) results, the activities measured after Steps 1 and 2 appear to be low; this was possibly due to the presence of some inhibitor which was removed at Step 3. The over-all purif’ication of the poly(C) activity in Table 1 is, therefore, probably an overestimate. When an initial enzyme extract was prepared from uninfected cucumber plants and taken through the same purification procedure to the end of Step 4, negligible RNA replicase activity was found at each step; this amounted to less than 0.2% of the activity found in corresponding fractions originating from infected plants and using either CMV RNA or poly(C) as templates. Properties of the Partially zyme (Step 5)

Purified

concentration was l:i mM magnesium using CMV RNA, poly(C), and yeast RNA as templates and was the same as that reported for the Step 1 enzyme (May and Symons. 1971). Template specificit>,. The effect of concentration of four types of RNA and of poly(C) on the polymerase activity with [32P]GTP as labeled substrate is shown in Fig. 1. Although CMV RNA was the best RNA template, appreciable activity was found with TMV RNA and with Escherichia coli ribosomal RNA and to a lesser extent with yeast RNA. In the four cases, maximum activity was reached with an RNA concentration of about 20 pug/assay (120 pglml). The high template activity of poly(C), previously reported for the Step 1 enzyme (May et al., 1969; May and Symons, 1971), was also found here with the Step 5 enzyme. Saturation of enzyme activity occurred at about 50 &assay (300 pg/ml) of poly(C) (Fig. 1). Step 5 enzyme showed polymerase activity with both native and heat-denatured salmon DNA which was about 12-159 of that found with the same concentration of CMV RNA (Table 2). This activity was not inhibited by 15 pglml of actinomycin D which indicates the absence of any DNA-

En-

Stability. Step 5 enzyme lost all activity with either CMV RNA or poly(C) as templates within 2-3 days when stored in buffer containing 30% glycerol (Step 5 elution buffer; Materials and Methods) at ~ 15” and within l-2 days at 0”. However, full enzyme activity was recovered after storage in liquid nitrogen for 5 mo in the same buffer. Limited dilution of the enzyme was possible provided the glycerol concentration remained at 30% (v/v). Dilution to 10% glycerol resulted in rapid and irreversible loss of activity. Essentially identical results were also obtained for the Step 4 enzyme. Magnesium optimum. Under the polymerase assay conditons used, the optimal

n-mTMvRNA 0

1 10 7.0 40 60 80 RNAor FULVMJCLEDTIDE AMED(&ISA”]

. 1 100

FK. 1. The effect of’template concentration on the activity of’ the CMV-induced RNA polymerase. Step 5 enzyme was assayed as described in Materials and Methods in the presence of’ varying amounts of’ RNA and polynucleotide templates as indicated.

PLANT

VIRUS-INDUCED

dependent RNA polymerase activity. Further, prior treatment of the DNA with 0.3 M NaOH at 37” for 2.0 hr in order to hydrolyze any contaminating RNA had no effect on its template activity (Table 2). These results illustrate further the lack of rigid template specificity of the viralinduced RNA polymerase, or alternatively, they could indicate the presence of some unusual contaminating RNA polymerase. Effect of nucleuses and inhibitors. Data on the effect of various additions to the normal polymerase assay using [32P]GTP as labeled substrate and with CMV RNA and poly(C) as templates are given in Table 2. RNA polymerase activity was markedly inhibited by the addition of pancreatic RNase (6 pg/ml) during the assay and of pyrophosphate (15 mM) but was little affected by the addition of pancreatic DNase, orthophosphate. actinomycin D, rifampicin, or tu-amanitin at the concentraTABLE PKOWKTIES

OF THE STEP 5 ENZYME-THE WITH CMV

System”

No added template CMV RNA ( 150 &ml) plus orthophosphate (15 mM) plus pyrophosphate (15 mM) plus actinomycin D (15 Kg/ml) plus rif’ampicin (30 &ml) plus tr-amanitin (25 &ml) plus pancreatic RNase (6 fig/ml) during assay plus pancreatic RNase (60 &ml) after assay’ plus deoxyrihonuclease (60 pgiml) Poly(C) (150&ml) plus orthophosphate (15 mM) plus pyrophosphate (15 mM)

tions given. These properties are those expected for an RNA-dependent RNA polymerase. The small effect of orthophosphate (15 mM) rules out any significant contribution of polynucleotide phosphorylase. As reported previously (May and Symons, 1971), the polymerization reaction was very sensitive to pancreatic RNase although once completed, the reaction product was 69% resistant to RNase digestion without any prior deproteinization step. Most of the product, therefore, existed in a double-stranded form. Separation of the CMV RNA Polymerase and Poly(C) Polymerase Activities on DNA-Agarose by Gradient Elution Although the polymerase activities with either CMV RNA or poly(C) as templates copurified through the five steps of Table 1, it was possible to separate these two activities by gradient elution from DNA2

EFFE~.T OF INHIBITORS

RNA,

PoLu(C).

Counts/ min

Relative activity (5)

50 16.250 13. 100 190 16,‘OO 14,450 15,000 520

0 100 80 1.’ 100 89 9’ 3.2

11,150

69

15.950

98

439

RNA POLYMERASE

AND NUCLEASES ON THE POLYMEHASE ACTIVITY

AND DNA

AS TEMPLATES

sL ystem”

plus Actinomycin D (15 &ml) plus rit’ampicin (30 &ml) plus tr-amanitin (25 ~g/ml) plus pancreatic RNase (6 &ml) during assay plus pancreatic RNase (60 &ml) after assay’ plus deoxyribonuclease (60 &nl) Native salmon DNA (150 &ml) plus actinomycin D (15 (.q$rnlI Denatured salmon DNA (150 pg/mlP plus actinomycin D (15 &ml) after alkaline treatment

Counts/ min

Rela tive activity (B )

62,300 59,700 61,800 1,160

91 87 90 1.7

39.900

58

67,400

98

1,970 2,050 1,980

-

68,500 100 2,250 58.600 86 150 0.2 1,940 ~___~ ” Step 5 enzyme was assayed for polymerase activity as described in Materials and Methods with additions of templates. nucleases, and inhibitors as indicated. Each assay contained 4 s 10” cpm of’ [w~~P]GTP. A zero-time background of 170 cpm has been subtracted from all results. ” Native salmon DNA in 5 mM EDTA. pH 8. was denatured by heating at 100” for 10 min followed by cooling in ice. For alkaline treatment to remove any contaminating RNA. denatured DNA was incubated in 0.3 M NaOH at :X0 Ior 2.0 hr and then neutralized before use. ’ At the end of’ the assay, pancreatic RNase was added to 60 fig/ml and incubation continued at 37” for a further 30 min before acid precipitation of the product.

440

(‘LARK,

PEDEN

FRBCTION

AND SJ.MOIi\s

NUMBER

FIG. 2. Gradient elution of’ the Step 1 enzyme from a column of single-stranded DNA-agarose. Step 4 enzyme (5 ml) was diluted with Step 1 buffer (see Materials and Methods) without NH,Cl to 0.1 M NH,Cl and which had been packed slowly (5 the solution applied at 5 ml/hr to a r3.5 - 2.0.cm column of’ DNA-agarose mlihr) in and well washed with 30”; glycerol. 0.1 M NH,Cl, 20 mM Tris-HCI, pH 8.n. 10 mM %-mercaptoethanol. The column was washed with I-2 column volumes of’ the same but’t’er bef’ore the linear gradient of’0.1 M-1.0 M NH,CI in the same but’ter was started; the flow rate was 12 ml/hr. Fractions of’ 1.9 ml were collected and assayed immediately for polymerase activity with CMY RNA, yeast RNA. and polytC) as templates. The concentrations of’ NH,CI were determined by rel’ractometry.

agarose (Fig. 2) even though this did not occur during stepwise elution from DNA-agarose (Step 5) nor during gradient elution from phosphocellulose (see above). The highly reproducible results (Fig. 2) showed that polymerase activity with either CMV RNA or yeast RNA was eluted with a peak at 0.3 M NH,Cl while the poly(C) copying activity was eluted at 0.45 M NH,Cl. Unfortunately, the recoveries of enzyme activity were low; about 35% of the original CMV RNA activity and only 5%)of the poly(C) activity. The significance of these observations is considered in the Discussion. Polyacrylamide Gel Electrophoresis of Enzyme Fractions Eluted from DNA Agarose In Fig. 3 are shown the SDSPpolyacrylamide gel patterns of the Step 5 enzyme (Fig. 3A) and of the CMV RNA copying activity (Fig. 3B) and poly(C) copying activity (Fig. 3C) obtained by gradient elution from DNA-agarose. The two greatest peaks with molecular weights of approx 60,000 and 55,000 were clearly visible on

stained gels but the lower molecularweight species (MW 20,000-40,000) were more diffuse, and definite bands were difficult to see clearly. In a separate set of experiments, the SDS-polyacrylamide gel pattern of the Step 4 enzyme was similar to that of the Step 5 enzyme shown in Fig. 3A (results not given). When healthy plant material was taken through to Step 4, a very similar gel pattern to that obtained with infected plants was also found. It was hoped that a comparison of the SDS-gel patterns of peak activity fractions obtained from DNA-agarose would provide data on the protein composition of the polymerase activities using CMV RNA and poly( C) as templates. Unfortunately, no definite conclusions can be drawn from the results of Fig. 3, presumably because the Step 5 enzyme is a long way from being homogeneous (see also Discussion). Two points can be noted however. The small 37,000 MW protein peak present in Fig. 3A and 3B is absent from Fig. 3C; the same result was obtained in three separate experiments using three different enzyme preparations. The differences in staining

PLANT

VIRUS-INDUCED

CMV RNA

L L

C POLVc

123b961*9 MIGRATION

(cm)

FIG. 3. SDSpolyacrylamide gel electrophoresis of Step 5 enzyme and of peak fractions of polymerase activity obtained by gradient elution from DNA-agarose. Protein samples were concentrated by acid precipitation and run on 10% SDS-polyacrylamide gels for 6.0 hr at 8 mA/gel; gels were then stained and scanned at 600 nm (Materials and Methods). The following proteins were used as molecular weight markers (Weber and Osborn, 1969); bovine serum albumin monomer (68,000), glutamate dehydrogenase (53,000), ovalbumin (43,000), pepsin (34,600), myoglobin (17,200), and cytochrome c (11,700). A. Step 5 enzyme (70 fig). R. Fractions 8-11 of Fig. 2 containing RNA polymerase activity. C. Fractions 17-19 of Fig. 2 containing poly(C) copying polymerase activity.

patterns for proteins with molecular weights of less than about 15,000 are probably not significant, as these patterns have varied from one experiment to another and probably reflect variable protease activity as considered below. The gel patterns of Fig. 3 are of protein samples of active enzyme preparations which had been placed at -15” as soon as they emerged from the DNA-agarose column and then stored in liquid nitrogen within 3-4 hr after the positions of the active fractions had been determined.

RNA POLYMERASE

441

Samples for SDS-gel electrophoresis were prepared by precipitation with trichloroacetic acid (see Materials and Methods) immediately after thawing. Gel patterns of protein samples which had been stored at 0” or -15” for l-2 wk or longer and which contained no RNA polymerase activity, showed the two large peaks clearly but there was a larger number of smaller peaks than those present in Fig. 3. These results suggested the presence of protease activity in the Step 5 enzyme preparation. Measurement of protease activity by the method of Rinderknecht et al. (1968) gave the following estimates in terms of trypsin activity as a standard; Step 3, 0.04 Fg/ml; Step 4, 0.02 pg/ml; Step 5, 0.02 pg/ml. Attempts to find an inhibitor for this protease activity were unsuccessful. For example, preincubation of the Step 3 enzyme for 30 min at 37” with 20 mM EDTA or with 50 pg/ml of phenylmethyl sulfonyl fluoride, tosyl-L-lysine chloroketone or methyl l-tosylamide2-phenylethyl-chloromethyl ketone had no effect on subsequent protease activity. DISCUSSION

The loo-fold purification of the CMVinduced RNA polymerase reported here represents the greatest purification yet achieved for any of the several plant virusinduced RNA polymerases studied so far. How much further this enzyme must be purified to reach homogeneity is unknown but it is possible that an overall purification of at least lO,OOO-fold will be needed. As a basis for speculation, the phage Q/3 RNA replicase was purified from 260- to 5000.fold to homogeneity (Eoyang and August, 1968; Kamen, 1972). E. cob DNA polymerase I, 2000-fold (Setlow, 1974) and E. coli DNA polymerase III, 40,000-fold (Otto et al., 1973). In the latter case, it was estimated that about 10 enzyme molecules were present per bacterial cell. Since the QP and f2 phage RNA replicases are the only RNA replicases which have been extensively purified and characterized so far, it is only natural that RNA replicases from other bacterial, plant, and animal sources will be compared with

them. These phage RNA replicases contain a heterogeneous subunit structure with part of the enzyme coded for by the phage and the rest being supplied by the host. Phage Q/3 and f% replicases appear similar in consisting of one viral-coded protein (II) and probably the same three host proteins (I, III, and IV) but they differ in the additional protein factors necessarv for the copying of homologous viral R%A plus strands (Kamen, 1972: Fedoroff and Zinder. 1973). Preliminary evidence indicates that the encephalomyocarditis virusinduced RNA replicase may have a similar subunit structure (Rosenberg et al., 1972). In contrast, the phage Rl’i RNA replicase can be separated into two components, one a viral coded protein and the other a host protein(s) which contains DNA-dependent RNA polymerase activity and which is sensitive to rifampicin (Igarashi, 1973). In the case of the CMV-induced RNA polymerase. a subunit structure is suggested by the lability of the enzyme on gradient elution from DEAE-Sephadex and the complete loss of activity of the Step 5 enzyme when diluted from 30% to 10% glycerol (see Results). More definitive evidence is given by the separation of the RNA and poly(C) copying activities by gradient elution from DNA-agarose (Fig. 2). Fedoroff and Zinder (1973) have reported the partial separation of the phage f’1 RNA and poly(C) copying activities of the partly purified f’L RNA replicase by gradient elution from DEAE-cellulose and by centrifugation through a glycerol gradient. They concluded that the poly(C) copying activity was part of the f’2 RNA replicase but that an extra protein was necessary to form the complete enzyme which copies RNA. Further, Kamen et al. (1972) have shown that Qp RNA replicase which lacks subunit I and contains only subunits II, III, and IV could copy poly(C) and the complementary viral RNA strand but not the plus RNA strand. It is possible that the separation of the RNA and poly(C) copying activities of the CMV-induced RNA polymerase may have a similar explanation. For example, if the complete enzyme consists of four subunits, A-D, then the poly(C) copying activity may consist of

subunits B, C. and D. This model require5 that the complete enzyme cannot copy poly(C~ since the RSA and JIO~~((‘) C-OJI~ing activities can be separated (Fig. 2). Any model must also consider the possibility of an associationdissociation equilibrium between the complete enzyme and its subunits as appears to occur with the Qp RNA replicase (Kamen, 19’72). A prominent feature of the CMVinduced RNA polymerase is its high activity with poly(C) as template when assayed with [32P]GTP in the presence or absence of the other three triphosphates (this work and May and Symons, 1971). The other polynucleotides, poly( A), poly( G) . and poly(U), showed low or negligible template activity when assayed with the appropriate labeled triphosphate (May and Symons, 1971): the poly(G)-dependent poly(G) polymerase activity reported by us was subsequently shown to be due to a very tight binding of [32P]GTP to poly(G) which was not removed during the work-up of the polymerase assays (unpublished results). As already considered, poly(C) copying activity is shown by the QP and f’% RNA replicases and also by the encephalomyocarditis virus-induced RNA polymerase (Rosenberg et al., 1972). However, the phage Rl? RNA replicase does not copy poly( C) (Igarashi and Bissonnette. 1971). The only other data available for plant virus RNA replicases are for the soluble brome mosaic virus-induced enzyme which copies poly(C) and poly(U:C) at a lower rate than with BMV RNA as template (Hadidi and Fraenkel-Conrat. 19X). An RNA-dependent RNA polymerase which shows very low activity with poly(C) and high activity with poly(U:C) as templates has been isolated from healthy Chinese cabbage plants; the activity of this enzyme increased several-fold after infection with turnip yellow mosaic virus (Astier-Manifatier and Cornuet, 1971). It is appreciated that we still have to provide concrete evidence that the enzyme considered here is involved in the in ~lico replication of viral RNA either as isolated or as part of a more complex enzyme structure. Although we have provided circumstantial evidence that the CMV-

PLANT

VIRUS-INDUCED

induced RNA polvmerase is involved in viral RNA replicatjon (Peden et al., 1972), more definitive evidence will only be provided by the complete purification and characterization of’ the enzyme. ACKNOWLEDGMENTS The authors thank the Rural Credits Development Fund of the Resewe Bank of’ Australia for financial support and Dr. R. 1. B. Francki for the provision ot glasshouse facilities. REFEKENCES ASTIEK-MANIFACIER. S., and COHNUET, P. (1971). RNA-dependent RNA polymerase in Chinese cabbage. Hiochim. Biophys. Acta 232, 484-493. EOYANC:.L., and ALI~LXT, .J. T. (1968). Phage Q@ RNA polymerase. Methods Enzymol. 12B, 530-540. FAIHBASKS, G.. STECK, T. L., and WALLACH, D. F. H. (1971). Electrophoretic analysis of’ the major peptides of’ the human erythrocyte membrane. Hiochemisty 10, 2606-2617. FEDOROFF, N. V., and ZIND~H. N. D. (197:I). Factor requirement of’ the bacteriophage f” replirase. Nature New Viol. 241, 105108. HAIX~I. A.. and FHAENKELCONKAT. H. (197:i). Characterization and specificity of soluble RNA polymerase of’ brome mosaic virus. Virology 52, 463-:17?. IC.ARASHI. S. J. (1973). Rl; RNA replicase. IV. Rifampicin sensitive component of the replicase. J. Biochem. 73, lZ&l:IO. ICARASHI, S. .J., and BISSOSNWI‘E, R. P. (1971). Effects of salts and antibiotics on the R17 replicase reaction. J. Hiochem. 70, 8:15-844. KAME~. R. (1972). A new method f’or the purification of’ Q/j RNA-dependent RNA polgmerase. Riochim. Hiophys. Acta 262, 88- 100. KAMEN. R., KONDO, M., ROMEH, W., and WEISSMANN. C. (1972). Reconstitution of’ Q6 replicase lacking subunit (Ywith protein-synthesis-interference t’acttrr i. Eur. J. Biochem. 31, 44.<51. LOLVR~. 0. H.. ROSEBROI,GH, N. .J., FARR. A. L., and RANDALL, R. .J. (1951). Protein measurements with the Folin phenol reagent. J. Rio/. Chem. 193, 265-275. MA\, ,J. T., GILLILAND. J. M.. and SYMONS. R. H. (1969). Plant virus-induced RNA polymerase:

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Properties of’ the enzyme partly purified from cucumber cotyledons infected with cucumber mosaic virus. Virology 39, 54-65. _.I 1 -., and SYMONS. R. H. (1971). Specificity ot lvl;‘~;‘~;~U,I,,,t-I , .I-~-- mosaic virus-induced RNA polymerase t’or RNA and polynucleotide templates, Virology 44, 517-526. MOLCHES, C.. and BOVE. J. M. (1974). Turnip yellow mosaic virus-RNA replicase: Partial purification of the enzyme from the solubilized enzyme-template complex. Viro/og> 58, 409-42X OKAZAKI, T.. and KORNBERC:. A. (1964). Enzymatic synthesis of’ deoxyribonucleic acid. XV. Purification and properties of a polymerase from Bacillus subtills. J. Hiol. Chem. 239, 259-268. OTTO, B., BONHOEFFER.F., and SCHALLER, H. (1973). Purification and properties of DNA polymerase III. Eur. J. Hiochem. 34, 440-447. PEDEN, K. W. C., MAY. J. T., and SYMONS. R. H. (1972). A comparison of’ two plant virus-induced RNA polymerases. Virology 47, 498-501. PEIIEN. K. W. C., and SYMONS. R. H. (197:1). Cucumber mosaic virus contains a functionally divided genome. Virology 53, 487-492. RINDERKNECHT. H.. GEOKAS. M. C., SILVERMAS. P.. and HAVERBACK. B. .J. (1968). A new ultrasensitive method f’or the determination of’ proteolytic activity. Clin. Chim. Acta 21, 197-20:(. ROSENBEH~. H., DISKIN. B.. ORON. L., and TRALIB. A. (197%). Isolation and subunit structure of polycytidylate-dependent RNA polymerase of encephalomyocarditis virus. Proc. Nat. Acad. Sci. USA 69, :I8153819. SCHALLER, H.. NLXSLEIN, C.. BONHOEFFER.F. J., KVRZ. C.. and NIETZSCHMANN. I. (1972). Affinity chromatography of DNA-binding enzymes on singlestranded DNA-agarose columns. Eur. J. Riochem. 26, 474-481. SETLOW. P. (1974). DNA Polymerase I f’rom Escherich& co/i. Methods Enzymol. 29, :I-12. WEBER. K., and OSBOKN. M. (1969). The reliability of molecular weight determinations by dodecyl sulphate-ptrlyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-4412. ZAITLIN, M., DUDS C. T., and PETTI, M. A. (1973). Replication of’ tobacco mosaic virus. V. Properties of the bound and solubilized replicase. Virology 53, :300-:I 1 1.