In vitro product of an RNA polymerase induced in broadbean by infection with broadbean mottle virus

VIROLOGY

49,

379-384

In:LVitro

(19%)

Product

of an RNA

by

Infection

Polymerase

Induced

Broadbean

Mottle

with

in

Broadbean

Virus

J. M. JACQUEMIN Laboraloire

de Pathologie

Vkgbtale,

Facultt! des Sciences Agronomiques, Accepted

May

6800 Gembloux,

Belgium

2, 1972

The RNA polymerase activity of a cell-free particulate fraction extracted from broadbean leaves infected with broadbean mottle virus was investigated. The RNA product labeled in vitro during a brief pulse of [3H]UTP in the presence of actinomycin D and exogenous RNA was shown by reannealing interference to have sequences homologous to parental viral RNA. The product was fractionated in 2 M LiCl and analysed by centrifugation in sucrose gradients. The major part of the acid-insoluble radioactivity did not precipitate in 2 M LiCl and was largely resistant to pancreatic ribonuclease in high salt. Some of the radioactivity associated with the LiCl-insoluble fraction of a 1.75~min pulse-labeled product was resistant to ribonuclease in high salt and had sedimentation properties expected for a replicative intermediate. After a 1-min chase, more of the LiCl-insoluble product became sensitive to ribonuclease and a portion of it sedimented in the region of viral RNA. INTRODUCTION

A particulate fraction from plants infected with broadbean mottle virus (BBMV) incorporated [3H] UTP into acidinsoluble products when incubated in the presence of actinomycin D and the necessary factors for RNA synthesis; little incorporating activity was detected in a comparable fraction from healthy plants (Semal, 1970). The RNA polymerase activity apparently was associated with template and addition of exogenous RNA was not required for RNA synthesis. Incorporation of labeled nucleotides was maxima1 at 4 days after inoculation and was dependent on the presence of the four ribonucleoside triphosphates (Romero and Jacquemin, 1971). The present communication describes some properties of the products which are synthesized in vitro by a crude RNA polymerasefraction from BBMV-infected leaves. MATERIALS plank and “Maxime”

var.

AND METHODS

virus. Broadbean were grown in

seedlings a green-

house. Very young plants, just emerging from soil, were inoculated by rubbing the lower epidermis of both unexpanded leaves of the first verticil with the juice of BBMVinfected broadbean. Inoculated plants were placed in a cabinet with continuous light and controlled (22-24’) temperature. Isolation of the crude RNA polymeruse. Two grams of leaves of 4-day infected broadbean were ground in a cold (4’) mortar in 40 ml of extraction buffer (0.05 M Tris-HCl, 0.01 M KCl, 0.008 M MgCh, pH 7.4, at room temperature) containing 0.001 M EDTA and 0.002 M mercaptoethanol. The extract was filtered through two layers of fine cloth and centrifuged for 5 min at 1OOOgat 4”. The supernatant fraction was adjusted to 20% glycerol and centrifuged at 10,OOOgfor 30 min. The resulting pellet-the crude enzyme preparation-was resuspended in 2.5 ml of polymerase buffer (0.05 M Tris-HCl, 0.01 n/r KCl; 0.008 M MgCh, pH S.S), containing 0.001 M EDTA and 0.002 M mercaptoethanol, and was used immediately.

379 Copyright All rights

@ 1972 by Academic Press, of reproduction in any form

Inc. re~erv

380

JACQUEMIN

Cell-free synthesis of RNA. Estimation of RNA polymerase activity was based on the incorporation of [3H]UTP into acid-insoluble material. The reaction mixture consisted of 0.5 ml of the crude RNA polymerase mixed with 0.1 ml of the necessary ingredients in polymerase buffer to make the following final concentrations: 500 pg/ml of each of unlabeled ATP, GTP, and CTP; 50 rCi/ml of [3H]UTP (l-2 Ci/mmole), 20 pg/ml of actinomycin D, 1.25 mg/ml of tricyclohexylamine salt of phosphoenolpyruvic acid; 20 pg/ml of pyruvate kinase, and 10 pg/ml of protective liver ribosomal RNA. The mixture was incubated at 30”, except when indicated. Each pair of results-RNA product treated or not with ribonuclease (RNase)-corresponds to 0.5 ml of the original crude RNA polymerase preparation, except for the experiments shown in Fig. 2, where the volume of t,he reaction mixture per individual result was reduced by half. Extraction of RNA. RNA was extracted by mixing the incubated mixture with 2 ml of water-saturated phenol and 4.5 ml of 0.5 % sodium dodecyl sulfate in 1 X SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0). The tubes were shaken for 5 min at room temperature, cooled, and centrifuged at 10,OOOgfor 10 min. Two-milliliter aliquots of the supernatant fluid were collected and the RNA was precipitated overnight at 4” by adding 2.5 vol of cold 95 % ethanol and 0.1 vol of 2 IM sodium acetate, pH 5.2. The RNA was pelleted at lO,OOOg,washed with cold 95 % ethanol, and resuspended in 2 X SSC. Fractionation with LiCl. RNA in 2 ml of 2 X SSC was adjusted to 2 M LiCl by mixing with an equal volume of 4 d4 LiCl, and kept overnight at 4”. The pellet of a 15min centrifugation at 10,OOOgwas resuspended in 0.02 M phosphate buffer, pH 7.2. After addition of 20 pg/ml of liver ribosomal RNA as carrier, the LiClsupernatant was treated with ethanol as above. Reannealing experiments. The procedure was described by Semal and Kummert (1971b). Double-stranded RNA was purified as follows: RNA extracted after 2 min

pulse-labeling was treated succcss~vely with RNase and Pronase and purified by passage through Sephadex G 200. The resultant labeled double-stranded RNA was heated at 100” for 10 min in 0.1 X SSC, and aliquots were mixed with increasing amounts of various RNAs. Reannealing experiments were carried out in 2 X SSC for 16 hr at 80”. Centrifugation in sucrose gradients. RNA in 0.3-0.4 ml was layered on top of a 22-ml column of a 5-20 % sucrose gradient in 0.1 M NaCl, 0.01 M acetate, pH 5.2, containing 1 pg/ml of sodium polyvinylsulfate. After centrifugation at 4” for 16 hr at 24,000 rpm in the 40,000 swing-out rotor of an MSE centrifuge, the tubes were pierced at the bottom and fractions of about 0.6 ml were collected after passagethrough an Uvicord flow-cell analyser. RNase resistance was tested by incubating the fractions in 2 X SSC with 5 rg/ml of pancreatic RNase for 30 min at 37”. Radioactivity measurements. RNA was precipitated in 5 % cold trichloroacetic acid containing 0.1% sodium pyrophosphate, in the presence of 18 pg/ml of carrier protein. The precipitates were collected on Millipore membranes (type HAWP) ; the filters were dried, placed in vials containing 10 ml of scintillation fluid consisting of 5 g PPO (2,5-diphenyloxazol) and 0.3 g POPOP (p-bis-2(5-phenyloxazol)-benzene) in 1.0 liter of toluene. The radioactivity was determined with a Nuclear Chicago scintillation counter, . with a counter et%ciency of 57 % for tritium. Chemicals. Actinomycin D was a gift from Merck, Sharp & Dohme Research Laboratories, Rahway, NJ. Unlabeled nucleotides were obtained from Schwarz, Mann. Pancreatic RNase, the tricyclohexylamine salt of phosphoenolpyruvic acid and pyruvate kinase were from Sigma Co. [5-3H]Uridine-5 triphosphate (l-2 Ci/ mmole) was obtained from the Radiochemical Center, Amersham. ’ PPO and POPOP were purchased from Packard Instrument Co., Common chemicals were from Merck Co. Ribosomal RNA from beef liver was from Schwartz/Mann.

PRODUCT

OF RNA POLYMERASE

RESULTS

TABLE

Characteristics of the crude RNA polymerase activity. The crude polymerase preparation was incubated for increasing time with [3H]UTP. The labeled RNA product was isolated and analysed for total and RNase-resistant acid-insoluble radioactivity. Figure 1 shows ,a rapid incorporation between 0 and 10 min. The product made during the first 2 min was 90-95 % resistant to RNase in 2 X SSC ; with longer incubations, the RNA product was partially sensitive to RNase. As shown in Table 1, incorporation was maximal at 30”. Very little labeling of RNA was obtained with leaf preparations from healthy plants. Reannealing of the early pulse-labeled product. The possible base sequence relationship between the in vitro RNA product and BBMV-RNA was investigated with hybridization experiments by measuring the interference of viral RNAs with the self-reannealing of the pulse-labeled doublestranded product synthesized in vitro. Self-reannealing was about 80-85 %. As shown in Fig. 2, addition of BBMV-RNA interfered with reannealing at low concentration, while turnip yellow mosaic virusRNA or liver ribosomal RNA had no effect. Thus, the product synthesized in vitro and BBMV-RNA have common sequences,

4

I

--

2

5

10

15

30

Ml”UteS FIG. 1. Kinetics of [“H]UTP incorporation by the crude RNA polymerase fraction incubated in the presence of actinomycin D and exogenous liver ribosomal RNA. Acid-insoluble radioacacid-insoluble radioactivity tivity (0-O); after RNase treatment of the product (5 rg/ml RNase for 30 min in 2 X SSC) (0-O).

381

IN BROADBEAN 1

EFFECT OF TEMPERATURE AND TIME OF INCUMTION ON THE RNA POLYMERME ACTIVITY OF A PARTICULATE CELL-FREE FRACTION FROM BROADBEAN LE.~VES~

Time of Healthy leaves, incuba- incubation tion at (min) 3o” 2 5 10

70 610 340

Leaves infected with broadbean mottle virus, incubation at 30”

37O

4o”

4180 5250 6670

2870 3870 5000

2350 2370 3530

a The crude RNA polymerase fraction was prepared from either healthy or infected leaves and incubated at different temperatures with [3H]UTP, actinomycin D, and the necessary ingredients for RNA synthesis. Aliquots were withdrawn at intervals; results are expressed as acidinsoluble cpm.

the product probably being a segment of the “plus” infecting strand of BBMV-RNA. Pulse chase analysis of the early product. To determine whether the pulse-labeled double-stranded RNA was a precursor of single-stranded viral RNA, labeling was performed for 2 min, and an excess of unlabeled UTP was then added to the reaction mixture. Total and RNase-resistant radioactivity precipitable by TCA were determined at intervals. As shown in Fig. 3, addition of UTP stopped incorporation of label and induced a rapid loss of RNase resistance of the product. The shift in RNase resistance was always obtained for polymerase preparations from symptomless plants but not for preparations from plants which showed beginning symptoms after 4 days of infection. RNA synthesized before and after the chase was analysed in 5-20 ‘;b sucrose gradients (Fig. 4). The bulk of RNA labeled during a 1.75~min pulse sedimented as a peak in the 14 S region, and was RNase resistant. The sedimentation pattern of the material extracted after a 2-min pulse followed by a 1-min chase showed an increased sensitivity to RNase, a reduction in the labeling of the 14 S peak, and an increased heterogeneity of sedimentation. Longer periods of pulse or chase gave similar results.

JACQUEMIN

382

EXOGENOUS (pglml)

RNA

2. Reannealing of the in vitro product of a pulse of [SHJUTP in the presence of exogenous liver ribosomal RNA. The purified doublestranded RNA product was heated in 0.1 X SSC for 10 min at 100”. Samples were then mixed with various unlabeled RNAs, and reannealing was performed in 2 X SSC final for 16 hr at 80’. Aliquots were then treated with RNaae. Results are expressed as RNase-resistant acid-insoluble radioactivity, in percentage of total acid-insoluble labeled material of each sample. (100% = about 2000 cpm). BBMV-RNA (O-O); TYMVRNA (e----0); liver ribosomal RNA (O--O). FIG. 2-rnin

-

MI ll”5,.

FIG. 3. Kinetics of [3H]UTP incorporation under “pulse-chase” conditions. The crude RNA polymerase was pulse-labeled for 2 min (see Fig. 1); a lOO-fold excess of unlabeled UTP was then added to an aliquot of the reaction mixture. Continuous labeling (O-O) ; continuous labeling, aliquots treated with RNase (@--a); chased sample (O---O); chased sample treated with RNase (LH).

FIG. 4. Sedimentation profile in a 5-20s sucrose gradient (sedimentation from right to left) of [3H] incorporated into RNA isolated from the RNA polymeraae preparation incubated with [ZH] UTP. Centrifugation was for 16 hr at 24,000 rpm. A: product after 1.75-min pulse; B: product after a 2-min pulse followed by a I-min chase in the presence of a 100-fold excess of unlabeled UTP. Total acid-insoluble radioactivity (O--O) ; acid-insoluble radioactivity after RNase treatment (5 fig/ml of RNase for 30 min at 37” in 2 X SSC) (@---0). Arrows refer to the sedimentation positions of heavy and light ribosomal RNAs from beef liver.

Fractionation with LiCl. As the pulsechased products were partially sensitive to RNase (Fig. 4), their solubility in 2 M LiCl was investigated in order to separate the RNAs according to single-strandedness and size. Totally or partially single-stranded RNAs of sufficient size are expected to be insoluble under such high salt conditions. Pulse-chase experiments were carried out, and samples were withdrawn at intervals. The RNA product was adjusted to 2 M LiCl and precipitated overnight at 4”. The pellet and the supernatant fraction were analysed for total and RNase-resistant acid-insoluble radioactivity. Table 2 shows that incorporation into LiCl-soluble material was largely unchanged after the chase, while sensitivity to RNase in the LiClinsoluble fraction was increased. Sucrose density-gradient analysis of the LiCl supernatant fraction and pellet before or after the chase appear in Figs. 5 and 6, respectively. The LiCl supernatant fraction (Fig. 5) sedimented as a single peak around 14 S

PRODUCT

FRACTIONATION

Aliquots withdrawn 0.15 min before the chase 1 min after the chase 2 min after the chase 3 min after the chase 4 min after the chase

OF

RNA

POLYMERASE

IN

383

BROADBEAN

TABLE 2 IN 2&f LiCl OF THE PRODUCTS OFTHE CRUDE RNA POLYMERASE FROM BRO~~DBEAN LEAVES INFECTED WITH BROADBEAN MOTTLE Total

RNA

LiCl-insoluble

RNA

fraction

FRACTION VIRUS"

LiCl-soluble

RNA

PREPARED

fraction

No RNase

RNaseb

Ko RNase

RNase*

No RNase

RNaseb

1520

1201

777

481

1100

1055

2217

1351

591

351

1285

1188

1777

1163

661

246

1011

843

2122

871

706

249

970

839

2089

894

680

229

1040

833

0 The crude RNA polymerase fraction was prepared from BBMV-infected leaves and pulse-labeled at 30” with [3H]UTP in the presence of actinomycin D and the necessary ingredients for RNA synthesis. At 2 min, an excess of unlabeled UTP was added and the fraction was further incubated; aliquots were withdrawn at intervals. RNA was extracted and fractionated in 2 M LiCl. b The RNA product was incubated for 30 min at 37” with 5 pg/ml of RNase in 2 X SSC. Results are expressed as acid-insoluble cpm.

FIG. 5. Sedimentation profile in a 5-20y0 sucrose gradient (sedimentation from right to left) of [3H] incorporation into the 2 M LiCl-soluble RNA isolated from the RNA polymerase preparation incubated with [$H] UTP. After a 2-min pulse, an excess of unlabeled UTP was added. A: product after a 1.75-min pulse; B: product after a 2-min pulse followed by a 1-min chase. Centrifugation was for 16 hr at 24,000 rpm.

and was unchanged after the chase. In contrast, the LiCl pellet before the chase was heterogeneous and partially sensitive to RNase, with a major peak at 14-16 S (Fig. 6). After the chase, there was a marked decrease in RNase resistance together with formation of new radioactive peaks, one of

FIG. 6. Sedimentation profile in a &20y0 sucrose gradient (sedimentation from right to left) of the 2 M LiCl-insoluble RNA isolated as above from the RNA polymerase preparation incubated with [‘H]UTP. After 2-min pulse, an excess of unlabeled UTP was added. A: product after 1.75min pulse B: product after 2-min pulse followed by a 1-min chase. Centrifugation was for 16 hr at 24,000 rpm. Total acid-insoluble radioactivity acid-insoluble radioactivity after (0-O); RNase treatment (5 pg/ml of RNaee for 30 min at 37” in 2 X SSC) (o-0). Arrows refer to the positions of heavy and light ribosomal RNAs from beef liver.

which (fraction 16) sedimented between both ribosomal RNAs at the place corresponding to the major OD peak of BBMVRNA.

384

JACQUEMIN DISCUSSION

The in vitro synthesis of RNA by cellfree particulate fractions prepared from virus-infected plants resulted generally in the labeling of polynucleotide chains complementary to endogenous RNA templates, the final products being associated with double-stranded RNA (Bove, 1967; Bradley and Zaitlin, 1971; May et al., 1970). With a crude RNA polymerase preparation from barley leaves infected with bromegrass mosaic virus (Semal and Kummert, 1971a) single-stranded RNA was chased from a double-stranded precursor, but the single-stranded products thus obtained were degraded by endogenous RNase. The labeled RNA in the double-stranded precursor was a segment of viral RNA (Semal and Kummert, 1971b). We used the same technique with the BBMV-broadbean system and got essentially similar results: single-stranded RNA was chased from double-stranded RNA precursor, but was destroyed upon further incubation of the system. Therefore, we added exogenous RNA in order to minimize the endogenous RNase effect, and we observed an increased duration of the synthesis and a stabilization of the single-stranded product. LiCl fractionation and sucrose gradient analysis revealed that the pulse-labeled RNA was made of two parts. A LiCIsoluble, RNase-resistant fraction was stable and did not turn over during the chase. A LiCl-insoluble, partially RNase-sensitive fraction generated single-stranded RNA during the chase, and displayed the general profile of replicative intermediates (Savage et d., 1971). Analysis of the total pulse-labeled product showed that the label in the RNaseresistant precursor was in the “plus” strand of BBMV RNA (Fig. 2) and indicated that exogenous ribosomal RNA added to the system did not participate as a template to RNA synthesis. The results also suggest that the single-stranded RNA generated during the chase is a segment of BBMV-RNA. Density-gradient analysis of the pulse-chased product showed an increase in RNase-sensitive material in the region of BBMV-RNA, thus suggesting the possible release of BBMV-RNA molecules from a 1416 S precursor during the

chase (Fig. 6). Part of t,he RNase-sensitive pulse-chased product has now be characterized as BBMV-RNA by analysis in polyacrylamide gel electrophoresis (Jacquemin, in preparation). Our results are in general agreement with those obtained by Kummert and Semal (1972), with the bromegrass mosaic virus-barley system. ACKNOWLEDGMENTS I thank Dr. A. Burny and Dr. J. Semal for discussions. I am indebted to the “Institut pour 1’Encouragement de la Recherche Scientifique dans 1’Industrie et 1’Agriculture” Brussels, for a predoctoral fellowship. This work was supported in part by a grant from the “Fonds National de la Recherche Scientifique” Brussels. I thank Mrs. Paulette Janssens for typing the manuscript. Thanks are expressed also to Dr. Ph. Dreze, Camira, Gembloux, for the use of a liquid-scintillation spectrometer. REFERENCES Bov15, J. M. (1967). Virus de la mosaique jaune du navet. Synthese asymetrique d’un segment de RNA viral. Ph D. Thesis, University of Paris. BRADLEY, D., and ZAITLIN, M. (1971). The in vitro synthesis of high molecular weight virusspecific RNAs. Virology 45, 192-199. KUMMERT, J., and SEMAL, J. (1972). Properties of single-stranded RNA synthesized by a crude RNA polymerase fraction from barley leaves infected with brome mosaic virus. J. Gen. Firol. In press. MAY, J., GILLILAND, J., and SYMONS, R. (1970). Properties of a plant virus-induced RNA polymerase in particulate fractions of cucumbers infected with cucumber mosaic virus. Virology 41, 653-664. ROMERO, J., and JACQUEMIN, J. M. (1971). Relation between virus-induced RNA polymerase activity and the synthesis of broadbean mottle virus in broadbean. Virology 45, 813-815. SAVAGE, Th., GRANBOULAN, N., and GIRARD, M. (1971). Architecture of the poliovirus replicative intermediate RNA. Biochimie 53, 533-543. SEMAL, J. (1970). Properties of the products of UTP incorporation by cell-free extracts of leaves infected with bromegrass mosaic virus or with broadbean mottle virus. Virology 46, 244-250. SEMAL, J., and KUMMERT, J. (1971a). Sequential synthesis of double-stranded and singlestranded RNA by cell-free extracts of barley leaves infected with brome mosaic virus. J. Gen. Vi’irol. 10, 79-89. SEMAL, J., and KIJMMERT, J. (1971b). In vitro synthesis of a segment of bromegrass mosaic virus ribonucleic acid. J. Gen. Fi’irol. 11, 189-192.