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RNA polymerase from tobacco necrosis virus-infected and uninfected tobacco
VIROLOGY
81,
224-236
(1977)
RNA Polymerase II. Properties
from Tobacco Necrosis Uninfected Tobacco
of the Bound
C. STUSSI-GARAUD, Department and Znstitut
of Molecular de Biologie
and Soluble Polymerases Products J. LEMIUS,
AND
Virus-Infected and the Nature
and of Their
H. FRAENKEL-CONRAT’
Biology and Virus Laboratory, University of California, Berkeley, California Moleculaire et Cellulaire, Laboratoire des Virus des Plantes, 15 rue Descartes, Strasbourg-cedex France Accepted
April
94720, 67084
15,1977
RNA polymerase activities in extracts of healthy and tobacco necrosis virus (TNV)infected tobacco plants were investigated. Considerable “bound” enzyme activity, associated with endogenous viral RNA template, was found in the 30,000 g pellet obtained from TNV-infected plants. This activity was usually not increased by addition of RNA. In the corresponding fraction from healthy plants a much lower activity was detected which was increased when RNA was added. The conditions favoring the enzyme activity and its general properties were investigated. The RNA synthesized by this enzyme on the endogenous template, be it in the particulate state or solubilized and ammonium sulfate-fractionated, was about 90% double-stranded and of high molecular weight. It corresponds to a viral plus strand. After melting, this RNA corn&rated with TNV-RNA. Thus it appears that the membrane-bound enzyme in TNV-infected plants represents a TNV replicase which is able to synthesize or finish full-length TNV-RNA on an endogenous template, the minus strand of TNV-RNA; this enzyme-template complex can be solubilized and fractionated without losing this ability. Nucleotide polymerizing activity was also detected in the 100,OOOg supernatant of healthy tobacco plants and this activity was significantly increased upon TNV infection. This activity which resembled in some respects that reported by Duda et al. (1973) was greatly stimulated by added RNA. The products synthesized by this “soluble” enzyme extracted from healthy and infected plants were of similarly low molecular weight; they consisted of both single- and doublestranded RNAs and behaved partly as expected for fragments of minus-strand viral RNA. INTrioDUCTION
The mechanisms of replication of viral RNA in eukaryotic host cells are largely unknown. Numerous studies have been undertaken on the replication of plant viral RNAs, and some properties of replicases were reported after infection with turnip yellow mosaic virus (TYMV) (Astier-Manifacier Cornuet, 1965; and Mouches et al., 1974); cucumber mosaic virus (CMV) (May et al., 1970; Clark et al., 1974); brome mosaic virus (BMV) (Semal and Kummert, 1971; Hadidi and Fraenkel’ Reprint Fraenkel-Conrat
requests should be addressed to Dr. at the University of California.
H.
Conrat, 1973); tobacco mosaic virus (TMV) (Zaitlin et al., 1973); alfalfa mosaic virus (AMV) (Weening and Bol, 1975; LeRoy et al., 1977); broadbean mottle virus (BBMV) (Romero, 19721; cowpea mosaic virus (Zabe1 et al., 1974, 1976); and others. In this paper, the tobacco necrosis virus (TNV) was used. This virus frequently occurs associated with a satellite virus, the RNA of which codes only for its own coat protein. The replication of this satellite depends entirely on the presence of TNV, presumably by using its replicase. A partial characterization of the bound replicase of the AC36 strain of TNV without satellite and of its products are presented here,
224 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
IN
VITRO
REPLICATION
as well as some data on soluble RNA polymerase in healthy and infected plants. The results of parallel studies on the solubilization and purification of the membrane-associated enzyme in healthy plants and upon infection with the Yarwood strain of TNV have alreadv been renorted (Fraenkel-Conrat, 1976). MATERIALS
AND
METHODS
Chemicals. Unlabeled triphosphates and actinomycin D were purchased from Sigma Chemical Co., St. Louis, MO. 15 3H]Uridine triphosphate (ammonium salt) in 50% ethanol (specific activity, 15 Cil mmol) and NCS solubilizer were purchased from the Radiochemical Centre, Amersham, England. Deoxyribonuclease, pancreatic ribonuclease, and T, ribonuclease were obtained from Worthington Biochemical Corporation, Freehold, N. J. Strain AC36 of tobacco necrosis virus (TNV) was kindly provided by Dr. R. J. Shepherd (University of CaliforniaDavis); and the Yarwood strain by Dr. C. E. Yarwood (University of CaliforniaBerkeley). Proteinase K was a generous gift from Boehringer Co., Mannheim, Germany. Buffers. The grinding buffer for most of the experiments reported here was 0.4 M sucrose; 10 mM KCl; 5 mM MgCl,; 50 mM Tris, pH 8.1 at 4”; 10% glycerol; and 10 mM mercaptoethanol. The resuspending and extraction buffer was 10 mM KCl; 25 mM NH&l; 10 mM Tris, pH 8.1 at 4”; 10% glycerol; and 5 mM 2-mercaptoethanol. This buffer was used in lieu of grinding buffer in an earlier study (Fraenkel-Conrat, 1976) and in some of the experiments reported here. The SSC buffer was 0.15 M NaCI, 0.015 M sodium citrate, pH 7.2. It was also used lo-fold diluted (0.1 x SSC) and 2-fold concentrated (2 x SSC). Plant and virus strains. Generally 2month-old Nicotiana tabacum plants, var. Xanthi nc., grown in a greenhouse, were inoculated with the AC36 or the Yarwood strain of TNV. The inoculated plants were then transferred to a temperature-controlled growth chamber (average temperature of 19”, 12-hr day under fluorescent lights). In some experiments tobacco
OF
TNV
225
plants var. Turkish Samsum and cowpea plants (Vignu sinensis) were used. Local lesions appeared 24-36 hr after infection. The leaves were generally harvested 2-3 days after inoculation for enzyme preparation and 7-8 days after infection for virus purification. Virus purification and TNV-RNA aration. Fresh or frozen infected
prep-
leaves were ground in the extraction buffer (100 ml for 100 g of leaves) in a Waring Blendor. The juice was squeezed through Miracloth (Calbiochem) and then centrifuged for 20 min at 30,000 g in a Sorvall centrifuge. The supernatant was kept at 4” overnight. After another centrifugation at 30,000 g it was ultracentrifuged for 3 hr at 105,000 g. The pellet was resuspended in deionized water. After clarification by centrifugation (20 min at 30,000 g) the virus was ultracentrifuged a second time and resuspended in deionized water. Concentration was estimated spectrophotometritally using A;&,* = 5 (Kassanis, 1970). If STNV was present, the two viruses were separated by sucrose gradient centrifugation (Fraenkel-Corn-at, 1976). TNV-RNA was extracted using the perchlorate method described by Wilcockson and Hull (1974). This method gave a higher yield of AC36 TNV-RNA than the phenol procedure used for Yarwood TNVRNA (Fraenkel-Conrat, 1976). The RNA was dissolved in sterilized deionized water and stored frozen at -25 or -70”. Preparation of replicase extracts. The method is similar to the one described by Zaitlin et al. (1973). Healthy and AC36infected leaves (2-3 days of infection) with their midrib removed were frozen at -70”. All the following operations were performed at 0”. The leaves were chopped in a cold mortar with a scalpel in the presence of 1.25 ml of grinding buffer per leaf, the average weight of which was about 1 g unless when extensively necrotized. After filtration through Miracloth the extracts were centrifuged for 5 min at 1000 g; the supernatants were centrifuged at 30,000 g for 20 min. The lOOO- and 30,000-g pellets were resuspended in 0.1 ml of resuspending buffer per number (or gram) of starting leaves and contained respectively, 10
226
STUSSI-GARAUD,
LEMIUS,
mg and 3.5 mg of protein/ml as estimated by the method of Lowry et al. (1951); the 30,000-g pellet constituted the particulate fraction which will be referred to as the “bound enzyme.” From the 30,000-g supernatant (about 1.2 mg of protein/ml) virus was usually removed at 105,000 g, and the proteins in the supernatant were precipitated with 50% saturated ammonium sulfate and resuspended in resuspending buffer. The resulting solution was adjusted to 15% saturation with ammonium sulfate and the precipitated proteins were eliminated by centrifugation at 30,000 g for 20 min. The solution was then adjusted to 35% saturation and centrifuged again, and the supernatant was brought to 50% ammonium sulfate saturation; the precipitated proteins were centrifuged as above, dissolved in resuspending buffer (0.1 ml/g of starting leaves) and termed “soluble enzyme.” Replicase assay. The enzyme activity tests were based on the incorporation of label supplied as [3H]UTP in the presence of the three other unlabeled nucleoside triphosphates into polynucleotides. A typical assay contained in 0.1 ml total volume: 50 n-&f Tris, pH 8.5 at 30”; 7.5 mM dithiothreitol; 25 n&f MgCl,; 2 20 mM (NH&SO,; 1 mM EDTA; 7pg of actinomycin D; 70 nmol each of ATP, GTP, and CTP; 13HlUTP, 5 &i in 1.33 nmol; and 30 ~1 of enzyme preparation; 20 pg of TNV- or TYMV-RNA was added when necessary. The reaction was started by addition of the enzyme. Thirty-microliter samples were removed at zero time and in duplicate after 60 min of incubation unless otherwise specified, and the reaction was stopped by applying to disks of DEAE 81 paper for radioactivity measurements according to the method of Litman (19681, as described in detail by Fraenkel-Conrat (1976). The incorporation data listed represent the differences between the zero-time counts (50-100 cpm) and the final counts. PHIRNA extraction. In order to isolate * It was recognized only later that the soluble enzyme was more active at lower Mg*+ concentrations (e.g., 5 mM), and thus most assays for this enzyme were performed at the level of Mg’+ most favorable for the bound enzyme.
AND
FRAElNKEL-CONRAT
the synthesized RNA in quantities suitable for further characterizations the standard polymerization assay was done on 2-4 ml of reaction medium. After 1 hr of incubation the solutions were adjusted to 1% SDS, treated with 50 pglml of proteinase K, and incubated 10 min at 37”. The RNA was then extracted by two treatments with one volume of water-saturated phenol. Phenol was eliminated from the aqueous phase by extractions with ether or by repeated alcohol precipitations in the presence of 0.025 M sodium acetate. The products of the solubilized and ammonium sulfate-fractionated enzyme were isolated after 20 min of incubation at 28” and worked up in a similar manner, frequently with added carrier TNV-RNA but without protease treatment; EDTA was added to 0.01 M. Reannealing experiments. Reannealing experiments were done with purified [3H]RNA after RNase treatment as follows: the purified L3H]RNA (5,000 to 10,000 cpm) was treated for 20 min at 37” with a mixture of pancreatic and T, RNases. The RNases were inactivated by incubation for 1 hr at room temperature in the presence of 0.1% diethylpyrocarbonate (DEPC). The RNA was then precipitated with two volumes of ethanol. After a 20-min centrifugation at 10,000 r-pm the pellet of RNA was dissolved in 0.1 x SSC buffer and divided into 25-~1 aliquots each containing 1000 cpm. Each aliquot was heated in a sealed capillary tube at 100” for 2 min. After quick cooling one sample was treated with the RNases to verify that thermal denaturation was complete. Reannealing experiments were done in duplicate on the other samples in 2x SSC buffer by incubating for 1 hr at 80” followed by slow cooling to 30” (2 hr) in the absence of added RNAs (self-reannealing) or in the presence of 20 pg of TNV-RNA or alfalfa mosaic virus (AMV) RNA as a homologous or heterologous competitor. In each case after reannealing, one of the duplicate samples was used as control and the other one was treated with the RNases. The radioactivity was then measured in each sample after applying the samples to Whatman disks and immediately washing in cold 5% TCA
IN VZTRO
REPLICATION
(Byfield and Sherbaum, 1966). Other methodological details are given in the footnotes to tables and in the figure legends. RESULTS
Demonstration of RNA ties in TNV-Infected bacco Plants
Replicase Activiand Healthy To-
The preparative procedure described in Materials and Methods gave three subcellular fractions: a 1000-g pellet, a 30,000-g pellet, and protein fractions obtained from the 105,000-g or, occasionally, 30,000-g supernatants. The incorporation of ribonucleotides was tested with these fractions in the presence or absence of added RNA from TNV or TYMV and of actinomycin D. The results (illustrated by a few examples in Table 1) showed that the extracts from infected plants had a much higher incorporating activity than those from healthy ones. The 1000-g pellet fraction was of rather low activity and was not further studied. The 30,000-g pellet fraction (“bound enzyme”) from healthy plants showed slight incorporation, only with added RNA, and this activity was not investigated further in this study.3 The nucleotide incorporation by bound enzyme from infected plants was considerable but, in contrast to that from healthy plants, not appreciably increased by addition of TNVRNA. The 30,000- or 105,000-g supernatant (S30, SlOO) from extracts of infected tissue, when tested directly in the presence or absence of TNV-RNA template, did not show significant activity (results not shown); but after a lo-fold concentration by ammonium sulfate precipitation (as described in Materials and Methods) significant incorporation of nucleotides occurred, which was greatly increased, particularly in the SlOO fraction, when RNA was added. Corresponding extracts from healthy plants also showed appreciable acB The activity characterized by current experiments that the physical similar to those soluble, enzyme
in uninfected plants was partly Fraenkel-Conrat (19761, and his further support his conclusion properties of this enzyme are very of the solubilized, and naturally from infected plants.
OF
TNV
227
tivity, particularly in the presence of added RNA, ranging from 30 to 80% of that found in the corresponding extract from infected plants. The fraction precipitated from SlOO fractions between 15 and 35% ammonium sulfate saturation showed highest RNA-dependent activity and was used as the source of “soluble enzyme”; it was generally tested in the presence of added RNA (Table 1). Table 2(A) shows that the incorporation of RNA precursors catalyzed by the bound enzyme was much higher in the absence of actinomycin D than under the standard assay conditions containing actinomycin D. This is probably due to a DNA-dependent RNA polymerase which is subject to actinomycin D inhibition. Pretreatment with DNase had the same effect as addition of actinomycin D. Freezing the leaves, which did not affect the actinomycin-resistant activity of infected plants (Table 2(B)) destroyed 90-100% of the DNA-dependent RNA polymerase activity. The activity of the soluble enzyme was not affected by omitting actinomycin D or by freezing (data not shown). Thus frozen leaves were generally used and assays performed in the presence of actinomycin. Table 3(A) shows that omission of one, two, or three nucleotides led to marked drops in incorporation by bound enzyme, but less so by soluble enzyme; the inhibition by adding pyrophosphate was marked for the bound enzyme but low for the soluble enzyme (Table 3(B)). The sum total of these results shows that the bound enzyme activity is largely an RNA-dependent RNA polymerase, which we shall refer to as an RNA replicase. This replicase was not dependent on and not stimulated by exogenous viral RNA but seemed to copy an endogenous viral template associated with it. In fact, pretreatment of the bound replicase with RNase (Table 3(C)) diminished the enzyme activity by 50%, presumably by partly destroying the endogenous template. The residual activity is probably due to a partial protection of the template within the membranes. Concerning the nature of the soluble enzyme, it appears to be heterogeneous, but according to new
228
STUSSI-GARAUD,
SYNTHESIS
OF RNA
LEMIUS,
CATALYZED
AND
TABLE 1 BY HEALTHY AND INFECTED L3H]Uridine Healthy -RNA
1000-g pellet Bound enzyme 30,000-g pellet Soluble enzyme 30,000-g supernatant (S30) proteins precipitated with ammonium sulfate O-15% saturation 15-35% saturation 35-50% saturation 15-35% saturationb 105,000-g supernatant (SlOO) X-35% saturationb LI Standard assay conditions using L3H]UTP, b By usual ammonium sulfate fractionation supernatant, the latter having been freed from
FRAENKEL-CONRAT
(cpm)
PLANT
incorporated
EXTRACT@ by fractions
plants
Infected
+ RNA
from plants
-RNA
+RNA
40
65
240
75
62
180
1170
1330
0 90 110 90
0 885 770 640
15
495
60 min of incubation; (first 50% saturation, virus particles.
data by Chifflot et al. (personal communication) it also contains a replicase-like activity, as is the case for the corresponding fraction from alfalfa mosaic virus-infected leaves, and to a lesser extent also from uninfected leaves. Time Course of Appearance of the Replicase Activity Each day over a period of 10 days after inoculation, nine healthy and nine infected leaves, randomly chosen, were harvested and frozen. Healthy and infected leaves were ground in buffer as usual. Tests for enzyme show a maximum of replicase activity on the fourth day after inoculation (Fig. 1). In other experiments, the maximum was being attained between the second and the fourth day after inoculation. The extract of healthy plants showed no significant activity (no RNA having been added in this experiment). Kinetics of Incorporation The kinetics of incorporation with the bound and soluble replicases were tested by performing large scale incubations: 30~1 samples were removed at different times of incubation for radioactivity measurements. The results are shown in Fig. 2.
0 315 140 530
55 1360 965 1875
65
1580
TNV-RNA was used. then 15-35%) of the
S30 or SlOO
The incorporation of RNA precursors catalyzed by the bound enzyme from infected plants increased rapidly over the first 2030 min but then reached a plateau for longer incubation times. The corresponding fraction from healthy plants did not show significant activity, no RNA being added. In contrast, with the soluble enzyme from infected plants the level of incorporation climbed rapidly for at least 4 hr (tested with added RNA) (Fig. 2B). With the healthy-plant extract, also tested in the presence of RNA, incorporation occurred during the first hour of incubation; during the following 3 hr the rate of incorporation decreased considerably but did not reach a plateau even after 4 hr of incubation. Optimal Conditions of Replicase Activity When tested under standard conditions in the presence of increasing amounts of magnesium, the activity of the bound enzyme increased sharply with magnesium concentration up to 10 mM and then leveled off up to 50 mM. On the other hand, the soluble enzyme was very sensitive to MgCl, with incorporation diminishing with increasing concentration (data not shown). The activity of the bound enzyme
IN VITRO
REPLICATION
OF
229
TNV
was similar over the range of pH from 7.25 Ribonuclease Resistance of the Products of to 8.9; the optimal temperature was 30” the Bound and Soluble Replicases and the optimal molarity of the Tris buffer After incubation under standard condiwas 50 mM. The enzyme activity was not tions, 30-~1 samples were withdrawn for affected by relatively high molarities of radioactivity measurements. Two other salt (0.2 M KC1 or (NH&SO,). samples were withdrawn at the same time and incubated for 20 min at 37”, one with TABLE 2 and the other without added RNase, before EFFECT OF ACTINOMYCIN D AND OF FREEZING THE counting the radioactivity. The products LEAVES ON RNA SYNTHESIS BY BOUND AND synthesized by both bound Yarwood TNV SOLUBLE ENZYME and AC36 TNV enzymes were 80-90% reRadioactivity insistant to RNase, showing the presence of corporated (cpm)” double-stranded RNA. After phenol exHealth Infected traction of products synthesized by YarY wood TNV enzyme the RNase resistance (A) Bound enzyme + AMD 85 585 was tested at 2x SW and about 0.1x SSC - DNase, fresh leaves (Table 4(A)). Here also, the product of the 1000 Bound enzyme AMD 800 bound enzyme was about 90% resistant to - DNase*, fresh leaves RNases in 2x SSC but was partly though Bound enzyme AMD 60 540 variably digested at low salt concentration. + DNaseb, fresh leaves Thus, regardless of the cause of this rela135 400 (B) Bound enzyme + AMD, tive stability at low salt, the bound enfresh leaves zyme synthesizes predominantly doubleBound enzyme + AMD, fro60 585 stranded RNA. The same is true for the zen (-20”) leaves Triton X-lOO-solubilized as well as for the a r3H]Uridine incorporation after 60 min of incuammonium sulfate-purified Yarwood TNV bation by standard assay. enzyme (Fraenkel-Conrat, 19761,both still b Samples held 20 min at 37” without DNase or representing an enzyme-template comwith added DNase (50 *g/ml) before incubation with RNA precursors. plex. The products of the soluble enzyme TABLE CHARACTERISTICS
3
OF POLYMER
FORMATION”
Bound enzyme Infected PFlni
(A)
(B)
(0
Control (all four triphosphates) -ATP - GTP -CTP - ATP, GTP -CTP, ATP -CTP, GTP -CTP, GTP, ATP Control +O.Ol M phosphate +O.Ol M pyrophosphate Control Preincubated 37”, 20 min Same + RNase A and T, (10 pg and 25 U/ml, respectively)
0 r3H]Uridine without added
425 80 185
55 50 450 270 90 425 320 210
Soluble Healthy _--------1 hr
988 491 502 424 318 309 261 272 890 760 635
incorporation (supplied as UTP) after 1 or 3 hr of incubation. RNA and of soluble enzyme with TNV-RNA.
plants 3 hr 1427 532 629 450 327 256 181 216
Tests
enzyme Infected ----------1 hr 2241 982 1374 864 688 563 452 641 3655 3765 2105
of bound
plants 3 hr 3072 1106 1475 808 583 439 275 370
enzyme
230 0 4
STUSSI-GARAUD,
LEMIUS,
AND
T
\. \
iu
.i .I
FRAENKEL-CONRAT
ble enzymes from healthy and infected plants synthesized L3H]RNA which was, respectively, 45 and 67% resistant to RNases in 2x SSC and totally digested by RNase in 0.1 x SSC buffer. Annealing
‘1. \. \.
0..
Experiments
Melting and annealing experiments on the products synthesized by the bound enzyme show that melting by heating provokes a complete disappearance of doublestranded structures (Table 5); after selfreannealing 72% of [3H]RNA was again engaged in double-stranded structures which are RNase resistant. However when reannealing was done in the presence of TNV-RNA only 4.5% of the labeled RNA was RNase resistant, while 84% was RNase resistant when heterologous RNA (AMV-RNA) was used as competitor. Thus the TNV-RNA displaced specifically the synthesized RNA, showing that the synthesized strands were plus strands and thus, by inference, the endogenous template was minus strand. For the products synthesized by the soluble enzyme the self-reannealing was about 30-50%. Addition of TNV-RNA did not displace the [3HlRNA. Thus the synthesized strand may be presumed to be complementary to the added TNV-RNA, although no template-product relationship has actually been demonstrated for the soluble enzyme in the present study.
/* ,fy*-tA \‘4I--&**&**,$--Lf,A.-.A.’ ,
I
I
1
3
5
I
I
,
9 7 &~ys of infection
FIG. 1. Time course of appearance of the bound replicase activity after inoculation of Xanthi nc. with TNV. Each day after infection nine healthy and nine infected leaves were harvested and frozen at -70”. They were ground in grinding buffer (1.25 ml/g of healthy leaves), using for the infected leaves the same volume as for the corresponding healthy leaves. Replicase assays were performed under standard conditions without added TNV-RNA. The points correspond to the radioactivity (in counts per minute) of 30-4 samples measured after 1 hr of incubation at 30”. Infected (0-O); healthy (A- - - -A,. *
from healthy or Yarwood TNV-infected plants usually showed only about 20% resistance to ribonuclease. When the same experiments were performed with the products synthesized by the bound enzyme of AC36 TNV-infected plants, we obtained 90% RNase resistance before phenol extraction. After purification by the proteinase K-SDS-phenol procedure described in Materials and Methods, the [3H]RNA was also 90% resistant to RNases in 2x SSC buffer and totally digested by the mixture of T, and pancreatic RNases in 0.1 x SSC (Table 4(B)); the solu-
Analysis of Products on Sucrose Gradients and on Polyacrylamide Gels Following phenol extraction, the RNA synthesized by enzymes of healthy and AC36 TNV-infected plants were centrifuged on sucrose gradients. The RNA produced by the bound enzyme from infected plants migrated as a peak of about 14 S and was almost totally RNase resistant (Fig. 3A). The small amount of RNA synthesized by the corresponding healthy extract migrated at 4-6 S. The RNA synthesized by the soluble enzyme of both healthy and infected extracts (Figs. 3B and C) migrated at about 4-6 S showing the presence of low molecular weight material which was about 50% RNase sensitive (Table 4).
IN VITRO
REPLICATION
OF
231
TNV
. 2-
. .
---A
/ 3-
/ %
2-.
_ e%--,-
_&-l-
,,*’ P
I
I
l 1
I’ 4’
I 1
,-‘~-------A
----
1;
------A
k
L--Ldd-dL
15
30
45
60
60 lncubotion
time
120
180
240
(in min)
FIG. 2. Kinetics of incorporation of nucleoside triphosphates catalyzed by the bound and the soluble enzymes extracted from healthy and infected plants. The replicase assays were performed in 300-4 total volume of assay medium containing 90 ~1 of healthy or infected enzyme fractions; the bound enzyme was used in the absence of TNV-RNA and the soluble enzyme in its presence. The points correspond to the radioactivity (in counts per minute) measured in 30-4 samples withdrawn after different incubation times. (A) Bound enzyme; (B) soluble enzyme; infected (O--O); healthy (A- - - -Al.
After phenol extraction and Sephadex gel filtration to eliminate unincorporated triphosphates, the synthesized products were chromatographed on polyacrylamide gels together with AMV-RNAs as markers, with results shown in Fig. 4. The double-stranded RNA synthesized by the bound enzyme (Fig. 4A) consisted of high molecular weight RNA molecules which migrated in three distinct and reproducible peaks. After melting of the double strands in 90% DMSO (Fig. 4B) most of the RNA migrated in the gel as two distinct peaks, the heavier comigrating with TNVRNA. The RNA synthesized by the soluble enzyme of the infected and healthy plants (Figs. 4C and D) consisted of low molecular weight RNA. No material which could correspond to TNV-RNA was detectable. These results are consistent with those obtained on sucrose gradients. Similar results were obtained with slightly different gel electrophoretic techniques (2% polyacrylamide, 0.5% agarose,
1 hr) for the products of the Yarwood TNV replicase, using bound, as well as Tritonsolubilized, and ammonium sulfate-fractionated enzyme fractions (data not shown). All yielded one very predominant product (about 80% of total) forming a sharp peak migrating between TNV-RNA and TMV-RNA and two lesser peaks between BMV-RNA components 1 and 4. The large component was not affected by RNase treatment but shifted to the position of the TNV marker upon heating or DMSO treatment. No detectable amounts of large products (>BMV-RNA 4) were produced by the soluble enzyme or by the bound enzyme from healthy plants, even when these were greatly stimulated in quantitative terms by the addition of a large RNA (TYMV-RNA). DISCUSSION
The results presented here show that bound and soluble RNA-dependent RNA polymerase activities can be found in
232
STUSSI-GARAUD,
LEMIUS,
AND
healthy and infected tobacco leaves. The bound enzyme activity detected in TNVinfected plants is not dependent on, and not stimulated by, TNV-RNA; the low bound enzyme activity in healthy plants is appreciable only when RNA is added. The bound enzyme extracted from infected plants is associated with an endogenous template which is the minus strand of TNV-RNA. This enzyme in vitro is able to make or finish double-stranded RNA resistant to RNases in which the “H-labeled strand is the plus strand and which comigrates with TNV-RNA after melting. The bound replicase present in TNV-infected plants shows some of the characteristics
generally observed for other plant virus replicases: The maximum of replicase activity is reached before the maximum of production of virus (Semal, 1970; Romero and Jacquemin, 1971; Hadidi and Fraenkel-Conrat, 1973; Zabel et al., 1974). The amount of incorporation of RNA precursors reaches a plateau after 5-30 min of incubation. Similar results were described by Semal and Hamilton (1968), Bradley and Zaitlin (19711, Jacquemin (1972), and Mouches et al. (1974). It was suggested that this phenomenon may be caused by the in vitro termination of nascent viral RNA chains. Low sensitivities to magnesium concentrations higher than 10 mM,
TABLE RIBONUCLEASE
Enzyme
fraction
RESISTANCE
and source
FRAENKEL-CONRAT
4 OF SYNTHESIZED
Added
Synthesized Total
(A)
(B)
Yarwood TNV-infected plants Bound enzyme, infected leaves Bound enzyme, healthy leaves Solubilized enzyme, infected leaves Solubilized enzyme, healthy leaves Ammonium sulfate-fractionated enzyme, infected leaves” Ammonium sulfate-fractionated enzyme, healthy leaves Soluble enzyme, infected leaves Soluble enzyme, infected leaves Soluble enzyme, healthy leaves Soluble enzyme, healthy leaves AC36 TNV-infected plants Bound enzyme, infected leaves Soluble enzyme, infected leaves Soluble enzvme, healthv leaves
RNA”
RNA
incorporation (cpm)
RNA
Remaining tion
--------------2x SSCb (%I
after in 0.1x
digesssc* (%)
None TYMV TYMV TYMV None
3075 185 3060 169 715
84 50 93 40 95
65’ 12 1.2 18 2.5
TYMV
220
28
19
TYMV None TYMV None
700 222 264 123
16 25 17 21
11 26 13 25
None TNV TNV
1000 1000 1000
88 67 45
0 0 0
u Data on phenol-extracted products obtained with various enzyme fractions (see Materials and Methods). Treatment of Yarwood TNV products with RNase directly in the incorporation mixture after addition of an equal volume of 4x SSC or water, respectively, indicated the same high resistance for all samples from infected leaves (except those made by the soluble enzyme) and low resistance (e.g., 20%) for the others. b Percentage of polymerized radioactivity that was RNase resistant. Pancreatic ribonuclease A (1 Kg for lo-50 Fg total RNA) was used for 30 min at 37” for set (A) and pancreatic + T, RNase for set (B) experiments. Equal aliquots (lo-30 ~1) of the RNA products were applied (see Materials and Methods) in set (A) to DEAE disks and in (B) to Whatman disks for counting the radioactivity directly or after RNase treatments. c This value represents an average of widely ranging data, high even upon digestion with RNases A + T,. d The product of an enzyme sample showing partial RNA dependence (incorporation of L3HlUTP, 100 and 310 cpm without and with added TYMV-RNA), made with added TYMV-RNA, was 45 and 17% resistant to digestion in 2x and 0.1 x SSC, respectively.
IN VZTRO REPLICATION TABLE REANNEALING 13HlRNA
EXPERIMENTS FROM synthesized
233
TNV
5
OF PRODUCTS SYNTHESIZED BY THE BOUND AND SOLUBLE HEALTHY AND AC36 TNV-INFECTED TOBACCO LEAVES RNase
by” Melting
Bound enzyme of infected plants” Soluble enzyme of healthv slants of infected plants *
OF
I
resistance
(%) of r3H]RNA
72
2.7 0
29 50
a 13HlRNAs were synthesized by bound enzyme without TNV-RNA. See Materials and Methods for experimental
added details.
in presence
of
TNV-RNA
AMV-RNA
4.5
84
61 73 RNA
EXTRACTED
after
Annealing
Self-reannealing
0
ENZYMES
and by soluble
62 58 enzymes
with
added
b cpm 0 1ooo-
24s 4
500-
d \ A’ %A9 e-e4e-@oo+-? . 15. .fractions . ** IO 5
FIG. 3. Sucrose gradient analysis of RNAs synthesized by the bound and soluble enzymes. Three milliliters of standard assay medium were extracted with phenol as described under Materials and Methods. The RNAs were dissolved in SSC buffer and loaded on a linear 5-20% sucrose gradient prepared in SSC. Fractions of 0.9 ml each were collected after a 16-hr centrifugation at 25,000 rpm in an SW 27 swinging bucket rotor. The points correspond to the radioactivity (in counts per minute) of 100 ~1 of each fraction. (A) Bound enzyme from infected plants; (B) soluble enzyme from healthy plants; (Cl soluble enzyme from infected plants; (O-O) before RNase treatment; (O--O) after RNase treatment. Arrows represent ribosomal and tRNA centrifuged as markers.
234
STUSSI-GARAUD,
LEMIUS,
AND FRAENKEL-CONRAT
FIG. 4. Polyacrylamide-gel electrophoresis of (A) 13H]RNA synthesized by the bound enzyme extracted from infected plants; (B) same RNA as (A) but after melting of the double strands in 90% DMSO; (0 r3HlRNA synthesized by the soluble enzyme of healthy plants; and (D) [3H]RNA synthesized by the soluble enzyme of infected plants. After phenol extraction and Sephadex G-75 filtration, the RNAs were loaded on 2.4% acrylamide gels (Loening, 1967); after 2.5 hr of electrophoresis at 5 mA/gel, the gels were cut in l-mm slices; each slice was digested with 90% NCS for 2 hr at 50”. The radioactivity was measured after adding 5 ml of scintillation fluid. Arrows correspond to migration of the four alfalfa mosaic virus RNA markers chromatographed under the same conditions; their molecular weights from left to right are: 0.3 x lo”, 0.7 x 106, 1 x 106, and 1.3 x 106. The dotted lines correspond to TNV-RNA marker (molecular weight, 1.5 x lo?.
to the pH of the reaction medium, and to high molarity of KC1 were also described by Zabel et al. (1974) for the enzyme found after CPMV infection. However, in the case of CPMV the enzyme was sensitive to ammonium sulfate concentrations higher than 50 mM while the TNV replicase seems to be more resistant to high molarities of salt (e.g., 0.2 M ammonium sulfate). The template-product-enzyme-
membrane complex is largely resistant to RNase. The earlier solubilization experiments (Fraenkel-Conrat, 1976) revealed that the TNV-induced enzyme-membrane complex is, like others, dissociated by nonionic detergent treatment. After detergent treatment the template generally remains attached to the enzyme, except in the case of BMV (Hadidi and Fraenkel-Conrat, 1973) where the solubilized enzyme is
IN
VITRO
REPLICATION
largely template dependent. The replicases tend to separate from the endogenous template to varying extents upon ammonium sulfate fractionation (FraenkelConrat, 19761, on density gradients (Zaitlin et al., 1973), by PEG-dextran twophase systems (Mouches et al., 1974; Clark et al., 1974; Fraenkel-Conrat, 19761, or by washing in a Mg2+-deficient buffer (Bol et al., 1976; Zabel et al., 1976). It is now shown that the TNV enzyme is able to make or finish TNV-RNA plus strands after solubilization and ammonium sulfate fractionation, as long as it is associated with endogenous template. Thus the membrane-bound state appears to play no role, at least in the elongation process. The soluble enzyme precipitated from the 105,000-g supernatant with ammonium sulfate is stimulated by added RNA and exists in healthy plants in a significant amount but increases greatly upon TNV infection. It may correspond to the enzyme described by Duda et al. (1973) except that the latter, probably due to the procedure used for its isolation, seems to carry endogenous plant RNA which it loses only upon sucrose gradient fractionation. Several other authors have isolated RNA polymerases from healthy plants which require addition of exogenous RNAs Astier-Manifacier and Cornuet [e.g., (1971) from Chinese cabbagge; Bol et al. (1976) and LeRoy et al. (1977) from tobacco, etc.]. These enzymes are stimulated by added RNAs, without showing any apparent specificity. Their action is favored by low magnesium concentrations and they are sensitive to high salt concentrations. The products of the soluble RNA polymerases appear to be only partially double stranded and of low molecular weight. Yet current experiments suggest that added RNAs can serve as templates, which would mean that healthy plant cells contain the enzymatic capability to replicate RNA. ACKNOWLEDGMENTS This investigation was supported by Research Grant No. PCM 7507854 from the National Science Foundation and by the “Commissariat a l’energie
OF
235
TNV
atomique” and the “Delegation cherche scientifique et technique”,
generale France.
de re-
REFERENCES ASTIER-MANIFACIER, S., and CORNUET, P. (1965). Isolation of the turnip yellow mosaic virus RNA replicase and asymmetrical synthesis of polynucleotides identical to TYMV-RNA. B&hem. Biophys. Res. Commun. 18, 283-287. ASTIER-MANIFACIER, S., and CORNUET, P. (1971). RNA-dependent RNA polymerase in Chinese cabbage. Biochim. Biophys. Acta 232, 484-493. BYFIELD, J. E., and SHERBAUM, 0. H. (1966). A rapid radioassay technique for cellular suspensions. Anal. Biochem. 17, 434-443. BOL, J. F., CLERX-VAN HAASTER, 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. BRADLEY, D. W., and ZAITLIN, M. (1971). Replication of tobacco mosaic virus. II. The in vitro synthesis of high molecular weight virus-specific RNAs. Virology 45, 192-199. CLARK, G. L., PEDEN, K. W. C., and SYMONS, R. H. (1974). Cucumber mosaic virus-induced RNA polymerase. Partial purification and properties of the template-free enzyme. Virology 62,434-443. 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. HADIDI, A., and FRAENKEL-CONRAT, H. (1973). Characterization and template specificity of soluble RNA polymerase of brome mosaic virus. Virology 52, 363-372. JACQUEMIN, J. M. (1972). In uitro product of an RNA polymerase induced in broadbean by infection with broadbean mosaic virus. Virology 49, 379384. KASSANIS, B. (1970). Tobacco necrosis virus. C.M.1.I A.A.B. Descriptions of Plant Viruses, Sheet No. 14 (B. D. Harrison and A. F. Murant, eds.). LEROY, C., STUSSI-GARAUD, C., and HIRTH, L. (1977). RNA dependent RNA polymerases stimulated upon alfalfa mosaic virus infection of tobacco plants. Virology, in press. LITMAN, R. M. (1968). A deoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acidcellulose. J. Biol. Chem. 243, 6222-6233. LOENING, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251257.
236
STUSSI-GARAUD,
LEMIUS,
LOWRY, 0. H., R~SEBROUCH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Ptotein measurement with the Folin phenol reagent. J. Bid. Chem. 193, 265275. MAY, J. T., GILLILAND, J. M., and SYMONS, R. H. (1970). Properties of a plant virus-induced RNA polymerase in particulate fractions of cucumbers infected with cucumber mosaic virus. Virology 41, 653-664. MOUCHES, C., BOVE, C., and BOVE, J. M. (1974). Turnip yellow mosaic virus-RNA replicase: Partial purification of the enzyme from the solubilized enzyme-template complex. Virology 58,409423. ROMERO, J. (1972). RNA synthesis in broadbean leaves infected with broadbean mottle virus. ViFOlOgy 48, 591-594. 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. 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 40, 244-250. SEMAL, J., and HAMILTON, R. I. (1968). RNA synthe-
AND
FRAENKEL-CONRAT
sis in cell-free extracts of barley leaves infected with bromegrass mosaic virus. Virology 36, 293302. SEMAL, J., and KUMMERT, J. (1971). In vitro synthesis of a segment of bromegrass mosaic virus ribonucleic acid. J. Gen. Viral. 11, 189-192. WEENING, C. J., and BOL, J. F. (1975). Viral RNA replication in extract of alfalfa mosaic virus infected Vicia faba. Virology 63, 77-83. WILCOCKSON, J., and HULL, R. (1974). The rapid isolation of plant virus RNAs using sodium perchlorate. J. Gen. Viral. 23, 107-111. ZABEL, P., WEENEN-SWAANS, H., and VAN KAMMEN, A. (1974). In vitro replication of cowpea mosaic virus RNA. I. Isolation and properties of the membrane-bound replicase. J. Viral. 14, 10491055. ZABEL, P., JONGEN-NEVEN, I., and VAN KAMMEN, A. (1976). In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme. J. Viral. 17, 679-685. 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.
81,
224-236
(1977)
RNA Polymerase II. Properties
from Tobacco Necrosis Uninfected Tobacco
of the Bound
C. STUSSI-GARAUD, Department and Znstitut
of Molecular de Biologie
and Soluble Polymerases Products J. LEMIUS,
AND
Virus-Infected and the Nature
and of Their
H. FRAENKEL-CONRAT’
Biology and Virus Laboratory, University of California, Berkeley, California Moleculaire et Cellulaire, Laboratoire des Virus des Plantes, 15 rue Descartes, Strasbourg-cedex France Accepted
April
94720, 67084
15,1977
RNA polymerase activities in extracts of healthy and tobacco necrosis virus (TNV)infected tobacco plants were investigated. Considerable “bound” enzyme activity, associated with endogenous viral RNA template, was found in the 30,000 g pellet obtained from TNV-infected plants. This activity was usually not increased by addition of RNA. In the corresponding fraction from healthy plants a much lower activity was detected which was increased when RNA was added. The conditions favoring the enzyme activity and its general properties were investigated. The RNA synthesized by this enzyme on the endogenous template, be it in the particulate state or solubilized and ammonium sulfate-fractionated, was about 90% double-stranded and of high molecular weight. It corresponds to a viral plus strand. After melting, this RNA corn&rated with TNV-RNA. Thus it appears that the membrane-bound enzyme in TNV-infected plants represents a TNV replicase which is able to synthesize or finish full-length TNV-RNA on an endogenous template, the minus strand of TNV-RNA; this enzyme-template complex can be solubilized and fractionated without losing this ability. Nucleotide polymerizing activity was also detected in the 100,OOOg supernatant of healthy tobacco plants and this activity was significantly increased upon TNV infection. This activity which resembled in some respects that reported by Duda et al. (1973) was greatly stimulated by added RNA. The products synthesized by this “soluble” enzyme extracted from healthy and infected plants were of similarly low molecular weight; they consisted of both single- and doublestranded RNAs and behaved partly as expected for fragments of minus-strand viral RNA. INTrioDUCTION
The mechanisms of replication of viral RNA in eukaryotic host cells are largely unknown. Numerous studies have been undertaken on the replication of plant viral RNAs, and some properties of replicases were reported after infection with turnip yellow mosaic virus (TYMV) (Astier-Manifacier Cornuet, 1965; and Mouches et al., 1974); cucumber mosaic virus (CMV) (May et al., 1970; Clark et al., 1974); brome mosaic virus (BMV) (Semal and Kummert, 1971; Hadidi and Fraenkel’ Reprint Fraenkel-Conrat
requests should be addressed to Dr. at the University of California.
H.
Conrat, 1973); tobacco mosaic virus (TMV) (Zaitlin et al., 1973); alfalfa mosaic virus (AMV) (Weening and Bol, 1975; LeRoy et al., 1977); broadbean mottle virus (BBMV) (Romero, 19721; cowpea mosaic virus (Zabe1 et al., 1974, 1976); and others. In this paper, the tobacco necrosis virus (TNV) was used. This virus frequently occurs associated with a satellite virus, the RNA of which codes only for its own coat protein. The replication of this satellite depends entirely on the presence of TNV, presumably by using its replicase. A partial characterization of the bound replicase of the AC36 strain of TNV without satellite and of its products are presented here,
224 Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
IN
VITRO
REPLICATION
as well as some data on soluble RNA polymerase in healthy and infected plants. The results of parallel studies on the solubilization and purification of the membrane-associated enzyme in healthy plants and upon infection with the Yarwood strain of TNV have alreadv been renorted (Fraenkel-Conrat, 1976). MATERIALS
AND
METHODS
Chemicals. Unlabeled triphosphates and actinomycin D were purchased from Sigma Chemical Co., St. Louis, MO. 15 3H]Uridine triphosphate (ammonium salt) in 50% ethanol (specific activity, 15 Cil mmol) and NCS solubilizer were purchased from the Radiochemical Centre, Amersham, England. Deoxyribonuclease, pancreatic ribonuclease, and T, ribonuclease were obtained from Worthington Biochemical Corporation, Freehold, N. J. Strain AC36 of tobacco necrosis virus (TNV) was kindly provided by Dr. R. J. Shepherd (University of CaliforniaDavis); and the Yarwood strain by Dr. C. E. Yarwood (University of CaliforniaBerkeley). Proteinase K was a generous gift from Boehringer Co., Mannheim, Germany. Buffers. The grinding buffer for most of the experiments reported here was 0.4 M sucrose; 10 mM KCl; 5 mM MgCl,; 50 mM Tris, pH 8.1 at 4”; 10% glycerol; and 10 mM mercaptoethanol. The resuspending and extraction buffer was 10 mM KCl; 25 mM NH&l; 10 mM Tris, pH 8.1 at 4”; 10% glycerol; and 5 mM 2-mercaptoethanol. This buffer was used in lieu of grinding buffer in an earlier study (Fraenkel-Conrat, 1976) and in some of the experiments reported here. The SSC buffer was 0.15 M NaCI, 0.015 M sodium citrate, pH 7.2. It was also used lo-fold diluted (0.1 x SSC) and 2-fold concentrated (2 x SSC). Plant and virus strains. Generally 2month-old Nicotiana tabacum plants, var. Xanthi nc., grown in a greenhouse, were inoculated with the AC36 or the Yarwood strain of TNV. The inoculated plants were then transferred to a temperature-controlled growth chamber (average temperature of 19”, 12-hr day under fluorescent lights). In some experiments tobacco
OF
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225
plants var. Turkish Samsum and cowpea plants (Vignu sinensis) were used. Local lesions appeared 24-36 hr after infection. The leaves were generally harvested 2-3 days after inoculation for enzyme preparation and 7-8 days after infection for virus purification. Virus purification and TNV-RNA aration. Fresh or frozen infected
prep-
leaves were ground in the extraction buffer (100 ml for 100 g of leaves) in a Waring Blendor. The juice was squeezed through Miracloth (Calbiochem) and then centrifuged for 20 min at 30,000 g in a Sorvall centrifuge. The supernatant was kept at 4” overnight. After another centrifugation at 30,000 g it was ultracentrifuged for 3 hr at 105,000 g. The pellet was resuspended in deionized water. After clarification by centrifugation (20 min at 30,000 g) the virus was ultracentrifuged a second time and resuspended in deionized water. Concentration was estimated spectrophotometritally using A;&,* = 5 (Kassanis, 1970). If STNV was present, the two viruses were separated by sucrose gradient centrifugation (Fraenkel-Corn-at, 1976). TNV-RNA was extracted using the perchlorate method described by Wilcockson and Hull (1974). This method gave a higher yield of AC36 TNV-RNA than the phenol procedure used for Yarwood TNVRNA (Fraenkel-Conrat, 1976). The RNA was dissolved in sterilized deionized water and stored frozen at -25 or -70”. Preparation of replicase extracts. The method is similar to the one described by Zaitlin et al. (1973). Healthy and AC36infected leaves (2-3 days of infection) with their midrib removed were frozen at -70”. All the following operations were performed at 0”. The leaves were chopped in a cold mortar with a scalpel in the presence of 1.25 ml of grinding buffer per leaf, the average weight of which was about 1 g unless when extensively necrotized. After filtration through Miracloth the extracts were centrifuged for 5 min at 1000 g; the supernatants were centrifuged at 30,000 g for 20 min. The lOOO- and 30,000-g pellets were resuspended in 0.1 ml of resuspending buffer per number (or gram) of starting leaves and contained respectively, 10
226
STUSSI-GARAUD,
LEMIUS,
mg and 3.5 mg of protein/ml as estimated by the method of Lowry et al. (1951); the 30,000-g pellet constituted the particulate fraction which will be referred to as the “bound enzyme.” From the 30,000-g supernatant (about 1.2 mg of protein/ml) virus was usually removed at 105,000 g, and the proteins in the supernatant were precipitated with 50% saturated ammonium sulfate and resuspended in resuspending buffer. The resulting solution was adjusted to 15% saturation with ammonium sulfate and the precipitated proteins were eliminated by centrifugation at 30,000 g for 20 min. The solution was then adjusted to 35% saturation and centrifuged again, and the supernatant was brought to 50% ammonium sulfate saturation; the precipitated proteins were centrifuged as above, dissolved in resuspending buffer (0.1 ml/g of starting leaves) and termed “soluble enzyme.” Replicase assay. The enzyme activity tests were based on the incorporation of label supplied as [3H]UTP in the presence of the three other unlabeled nucleoside triphosphates into polynucleotides. A typical assay contained in 0.1 ml total volume: 50 n-&f Tris, pH 8.5 at 30”; 7.5 mM dithiothreitol; 25 n&f MgCl,; 2 20 mM (NH&SO,; 1 mM EDTA; 7pg of actinomycin D; 70 nmol each of ATP, GTP, and CTP; 13HlUTP, 5 &i in 1.33 nmol; and 30 ~1 of enzyme preparation; 20 pg of TNV- or TYMV-RNA was added when necessary. The reaction was started by addition of the enzyme. Thirty-microliter samples were removed at zero time and in duplicate after 60 min of incubation unless otherwise specified, and the reaction was stopped by applying to disks of DEAE 81 paper for radioactivity measurements according to the method of Litman (19681, as described in detail by Fraenkel-Conrat (1976). The incorporation data listed represent the differences between the zero-time counts (50-100 cpm) and the final counts. PHIRNA extraction. In order to isolate * It was recognized only later that the soluble enzyme was more active at lower Mg*+ concentrations (e.g., 5 mM), and thus most assays for this enzyme were performed at the level of Mg’+ most favorable for the bound enzyme.
AND
FRAElNKEL-CONRAT
the synthesized RNA in quantities suitable for further characterizations the standard polymerization assay was done on 2-4 ml of reaction medium. After 1 hr of incubation the solutions were adjusted to 1% SDS, treated with 50 pglml of proteinase K, and incubated 10 min at 37”. The RNA was then extracted by two treatments with one volume of water-saturated phenol. Phenol was eliminated from the aqueous phase by extractions with ether or by repeated alcohol precipitations in the presence of 0.025 M sodium acetate. The products of the solubilized and ammonium sulfate-fractionated enzyme were isolated after 20 min of incubation at 28” and worked up in a similar manner, frequently with added carrier TNV-RNA but without protease treatment; EDTA was added to 0.01 M. Reannealing experiments. Reannealing experiments were done with purified [3H]RNA after RNase treatment as follows: the purified L3H]RNA (5,000 to 10,000 cpm) was treated for 20 min at 37” with a mixture of pancreatic and T, RNases. The RNases were inactivated by incubation for 1 hr at room temperature in the presence of 0.1% diethylpyrocarbonate (DEPC). The RNA was then precipitated with two volumes of ethanol. After a 20-min centrifugation at 10,000 r-pm the pellet of RNA was dissolved in 0.1 x SSC buffer and divided into 25-~1 aliquots each containing 1000 cpm. Each aliquot was heated in a sealed capillary tube at 100” for 2 min. After quick cooling one sample was treated with the RNases to verify that thermal denaturation was complete. Reannealing experiments were done in duplicate on the other samples in 2x SSC buffer by incubating for 1 hr at 80” followed by slow cooling to 30” (2 hr) in the absence of added RNAs (self-reannealing) or in the presence of 20 pg of TNV-RNA or alfalfa mosaic virus (AMV) RNA as a homologous or heterologous competitor. In each case after reannealing, one of the duplicate samples was used as control and the other one was treated with the RNases. The radioactivity was then measured in each sample after applying the samples to Whatman disks and immediately washing in cold 5% TCA
IN VZTRO
REPLICATION
(Byfield and Sherbaum, 1966). Other methodological details are given in the footnotes to tables and in the figure legends. RESULTS
Demonstration of RNA ties in TNV-Infected bacco Plants
Replicase Activiand Healthy To-
The preparative procedure described in Materials and Methods gave three subcellular fractions: a 1000-g pellet, a 30,000-g pellet, and protein fractions obtained from the 105,000-g or, occasionally, 30,000-g supernatants. The incorporation of ribonucleotides was tested with these fractions in the presence or absence of added RNA from TNV or TYMV and of actinomycin D. The results (illustrated by a few examples in Table 1) showed that the extracts from infected plants had a much higher incorporating activity than those from healthy ones. The 1000-g pellet fraction was of rather low activity and was not further studied. The 30,000-g pellet fraction (“bound enzyme”) from healthy plants showed slight incorporation, only with added RNA, and this activity was not investigated further in this study.3 The nucleotide incorporation by bound enzyme from infected plants was considerable but, in contrast to that from healthy plants, not appreciably increased by addition of TNVRNA. The 30,000- or 105,000-g supernatant (S30, SlOO) from extracts of infected tissue, when tested directly in the presence or absence of TNV-RNA template, did not show significant activity (results not shown); but after a lo-fold concentration by ammonium sulfate precipitation (as described in Materials and Methods) significant incorporation of nucleotides occurred, which was greatly increased, particularly in the SlOO fraction, when RNA was added. Corresponding extracts from healthy plants also showed appreciable acB The activity characterized by current experiments that the physical similar to those soluble, enzyme
in uninfected plants was partly Fraenkel-Conrat (19761, and his further support his conclusion properties of this enzyme are very of the solubilized, and naturally from infected plants.
OF
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227
tivity, particularly in the presence of added RNA, ranging from 30 to 80% of that found in the corresponding extract from infected plants. The fraction precipitated from SlOO fractions between 15 and 35% ammonium sulfate saturation showed highest RNA-dependent activity and was used as the source of “soluble enzyme”; it was generally tested in the presence of added RNA (Table 1). Table 2(A) shows that the incorporation of RNA precursors catalyzed by the bound enzyme was much higher in the absence of actinomycin D than under the standard assay conditions containing actinomycin D. This is probably due to a DNA-dependent RNA polymerase which is subject to actinomycin D inhibition. Pretreatment with DNase had the same effect as addition of actinomycin D. Freezing the leaves, which did not affect the actinomycin-resistant activity of infected plants (Table 2(B)) destroyed 90-100% of the DNA-dependent RNA polymerase activity. The activity of the soluble enzyme was not affected by omitting actinomycin D or by freezing (data not shown). Thus frozen leaves were generally used and assays performed in the presence of actinomycin. Table 3(A) shows that omission of one, two, or three nucleotides led to marked drops in incorporation by bound enzyme, but less so by soluble enzyme; the inhibition by adding pyrophosphate was marked for the bound enzyme but low for the soluble enzyme (Table 3(B)). The sum total of these results shows that the bound enzyme activity is largely an RNA-dependent RNA polymerase, which we shall refer to as an RNA replicase. This replicase was not dependent on and not stimulated by exogenous viral RNA but seemed to copy an endogenous viral template associated with it. In fact, pretreatment of the bound replicase with RNase (Table 3(C)) diminished the enzyme activity by 50%, presumably by partly destroying the endogenous template. The residual activity is probably due to a partial protection of the template within the membranes. Concerning the nature of the soluble enzyme, it appears to be heterogeneous, but according to new
228
STUSSI-GARAUD,
SYNTHESIS
OF RNA
LEMIUS,
CATALYZED
AND
TABLE 1 BY HEALTHY AND INFECTED L3H]Uridine Healthy -RNA
1000-g pellet Bound enzyme 30,000-g pellet Soluble enzyme 30,000-g supernatant (S30) proteins precipitated with ammonium sulfate O-15% saturation 15-35% saturation 35-50% saturation 15-35% saturationb 105,000-g supernatant (SlOO) X-35% saturationb LI Standard assay conditions using L3H]UTP, b By usual ammonium sulfate fractionation supernatant, the latter having been freed from
FRAENKEL-CONRAT
(cpm)
PLANT
incorporated
EXTRACT@ by fractions
plants
Infected
+ RNA
from plants
-RNA
+RNA
40
65
240
75
62
180
1170
1330
0 90 110 90
0 885 770 640
15
495
60 min of incubation; (first 50% saturation, virus particles.
data by Chifflot et al. (personal communication) it also contains a replicase-like activity, as is the case for the corresponding fraction from alfalfa mosaic virus-infected leaves, and to a lesser extent also from uninfected leaves. Time Course of Appearance of the Replicase Activity Each day over a period of 10 days after inoculation, nine healthy and nine infected leaves, randomly chosen, were harvested and frozen. Healthy and infected leaves were ground in buffer as usual. Tests for enzyme show a maximum of replicase activity on the fourth day after inoculation (Fig. 1). In other experiments, the maximum was being attained between the second and the fourth day after inoculation. The extract of healthy plants showed no significant activity (no RNA having been added in this experiment). Kinetics of Incorporation The kinetics of incorporation with the bound and soluble replicases were tested by performing large scale incubations: 30~1 samples were removed at different times of incubation for radioactivity measurements. The results are shown in Fig. 2.
0 315 140 530
55 1360 965 1875
65
1580
TNV-RNA was used. then 15-35%) of the
S30 or SlOO
The incorporation of RNA precursors catalyzed by the bound enzyme from infected plants increased rapidly over the first 2030 min but then reached a plateau for longer incubation times. The corresponding fraction from healthy plants did not show significant activity, no RNA being added. In contrast, with the soluble enzyme from infected plants the level of incorporation climbed rapidly for at least 4 hr (tested with added RNA) (Fig. 2B). With the healthy-plant extract, also tested in the presence of RNA, incorporation occurred during the first hour of incubation; during the following 3 hr the rate of incorporation decreased considerably but did not reach a plateau even after 4 hr of incubation. Optimal Conditions of Replicase Activity When tested under standard conditions in the presence of increasing amounts of magnesium, the activity of the bound enzyme increased sharply with magnesium concentration up to 10 mM and then leveled off up to 50 mM. On the other hand, the soluble enzyme was very sensitive to MgCl, with incorporation diminishing with increasing concentration (data not shown). The activity of the bound enzyme
IN VITRO
REPLICATION
OF
229
TNV
was similar over the range of pH from 7.25 Ribonuclease Resistance of the Products of to 8.9; the optimal temperature was 30” the Bound and Soluble Replicases and the optimal molarity of the Tris buffer After incubation under standard condiwas 50 mM. The enzyme activity was not tions, 30-~1 samples were withdrawn for affected by relatively high molarities of radioactivity measurements. Two other salt (0.2 M KC1 or (NH&SO,). samples were withdrawn at the same time and incubated for 20 min at 37”, one with TABLE 2 and the other without added RNase, before EFFECT OF ACTINOMYCIN D AND OF FREEZING THE counting the radioactivity. The products LEAVES ON RNA SYNTHESIS BY BOUND AND synthesized by both bound Yarwood TNV SOLUBLE ENZYME and AC36 TNV enzymes were 80-90% reRadioactivity insistant to RNase, showing the presence of corporated (cpm)” double-stranded RNA. After phenol exHealth Infected traction of products synthesized by YarY wood TNV enzyme the RNase resistance (A) Bound enzyme + AMD 85 585 was tested at 2x SW and about 0.1x SSC - DNase, fresh leaves (Table 4(A)). Here also, the product of the 1000 Bound enzyme AMD 800 bound enzyme was about 90% resistant to - DNase*, fresh leaves RNases in 2x SSC but was partly though Bound enzyme AMD 60 540 variably digested at low salt concentration. + DNaseb, fresh leaves Thus, regardless of the cause of this rela135 400 (B) Bound enzyme + AMD, tive stability at low salt, the bound enfresh leaves zyme synthesizes predominantly doubleBound enzyme + AMD, fro60 585 stranded RNA. The same is true for the zen (-20”) leaves Triton X-lOO-solubilized as well as for the a r3H]Uridine incorporation after 60 min of incuammonium sulfate-purified Yarwood TNV bation by standard assay. enzyme (Fraenkel-Conrat, 19761,both still b Samples held 20 min at 37” without DNase or representing an enzyme-template comwith added DNase (50 *g/ml) before incubation with RNA precursors. plex. The products of the soluble enzyme TABLE CHARACTERISTICS
3
OF POLYMER
FORMATION”
Bound enzyme Infected PFlni
(A)
(B)
(0
Control (all four triphosphates) -ATP - GTP -CTP - ATP, GTP -CTP, ATP -CTP, GTP -CTP, GTP, ATP Control +O.Ol M phosphate +O.Ol M pyrophosphate Control Preincubated 37”, 20 min Same + RNase A and T, (10 pg and 25 U/ml, respectively)
0 r3H]Uridine without added
425 80 185
55 50 450 270 90 425 320 210
Soluble Healthy _--------1 hr
988 491 502 424 318 309 261 272 890 760 635
incorporation (supplied as UTP) after 1 or 3 hr of incubation. RNA and of soluble enzyme with TNV-RNA.
plants 3 hr 1427 532 629 450 327 256 181 216
Tests
enzyme Infected ----------1 hr 2241 982 1374 864 688 563 452 641 3655 3765 2105
of bound
plants 3 hr 3072 1106 1475 808 583 439 275 370
enzyme
230 0 4
STUSSI-GARAUD,
LEMIUS,
AND
T
\. \
iu
.i .I
FRAENKEL-CONRAT
ble enzymes from healthy and infected plants synthesized L3H]RNA which was, respectively, 45 and 67% resistant to RNases in 2x SSC and totally digested by RNase in 0.1 x SSC buffer. Annealing
‘1. \. \.
0..
Experiments
Melting and annealing experiments on the products synthesized by the bound enzyme show that melting by heating provokes a complete disappearance of doublestranded structures (Table 5); after selfreannealing 72% of [3H]RNA was again engaged in double-stranded structures which are RNase resistant. However when reannealing was done in the presence of TNV-RNA only 4.5% of the labeled RNA was RNase resistant, while 84% was RNase resistant when heterologous RNA (AMV-RNA) was used as competitor. Thus the TNV-RNA displaced specifically the synthesized RNA, showing that the synthesized strands were plus strands and thus, by inference, the endogenous template was minus strand. For the products synthesized by the soluble enzyme the self-reannealing was about 30-50%. Addition of TNV-RNA did not displace the [3HlRNA. Thus the synthesized strand may be presumed to be complementary to the added TNV-RNA, although no template-product relationship has actually been demonstrated for the soluble enzyme in the present study.
/* ,fy*-tA \‘4I--&**&**,$--Lf,A.-.A.’ ,
I
I
1
3
5
I
I
,
9 7 &~ys of infection
FIG. 1. Time course of appearance of the bound replicase activity after inoculation of Xanthi nc. with TNV. Each day after infection nine healthy and nine infected leaves were harvested and frozen at -70”. They were ground in grinding buffer (1.25 ml/g of healthy leaves), using for the infected leaves the same volume as for the corresponding healthy leaves. Replicase assays were performed under standard conditions without added TNV-RNA. The points correspond to the radioactivity (in counts per minute) of 30-4 samples measured after 1 hr of incubation at 30”. Infected (0-O); healthy (A- - - -A,. *
from healthy or Yarwood TNV-infected plants usually showed only about 20% resistance to ribonuclease. When the same experiments were performed with the products synthesized by the bound enzyme of AC36 TNV-infected plants, we obtained 90% RNase resistance before phenol extraction. After purification by the proteinase K-SDS-phenol procedure described in Materials and Methods, the [3H]RNA was also 90% resistant to RNases in 2x SSC buffer and totally digested by the mixture of T, and pancreatic RNases in 0.1 x SSC (Table 4(B)); the solu-
Analysis of Products on Sucrose Gradients and on Polyacrylamide Gels Following phenol extraction, the RNA synthesized by enzymes of healthy and AC36 TNV-infected plants were centrifuged on sucrose gradients. The RNA produced by the bound enzyme from infected plants migrated as a peak of about 14 S and was almost totally RNase resistant (Fig. 3A). The small amount of RNA synthesized by the corresponding healthy extract migrated at 4-6 S. The RNA synthesized by the soluble enzyme of both healthy and infected extracts (Figs. 3B and C) migrated at about 4-6 S showing the presence of low molecular weight material which was about 50% RNase sensitive (Table 4).
IN VITRO
REPLICATION
OF
231
TNV
. 2-
. .
---A
/ 3-
/ %
2-.
_ e%--,-
_&-l-
,,*’ P
I
I
l 1
I’ 4’
I 1
,-‘~-------A
----
1;
------A
k
L--Ldd-dL
15
30
45
60
60 lncubotion
time
120
180
240
(in min)
FIG. 2. Kinetics of incorporation of nucleoside triphosphates catalyzed by the bound and the soluble enzymes extracted from healthy and infected plants. The replicase assays were performed in 300-4 total volume of assay medium containing 90 ~1 of healthy or infected enzyme fractions; the bound enzyme was used in the absence of TNV-RNA and the soluble enzyme in its presence. The points correspond to the radioactivity (in counts per minute) measured in 30-4 samples withdrawn after different incubation times. (A) Bound enzyme; (B) soluble enzyme; infected (O--O); healthy (A- - - -Al.
After phenol extraction and Sephadex gel filtration to eliminate unincorporated triphosphates, the synthesized products were chromatographed on polyacrylamide gels together with AMV-RNAs as markers, with results shown in Fig. 4. The double-stranded RNA synthesized by the bound enzyme (Fig. 4A) consisted of high molecular weight RNA molecules which migrated in three distinct and reproducible peaks. After melting of the double strands in 90% DMSO (Fig. 4B) most of the RNA migrated in the gel as two distinct peaks, the heavier comigrating with TNVRNA. The RNA synthesized by the soluble enzyme of the infected and healthy plants (Figs. 4C and D) consisted of low molecular weight RNA. No material which could correspond to TNV-RNA was detectable. These results are consistent with those obtained on sucrose gradients. Similar results were obtained with slightly different gel electrophoretic techniques (2% polyacrylamide, 0.5% agarose,
1 hr) for the products of the Yarwood TNV replicase, using bound, as well as Tritonsolubilized, and ammonium sulfate-fractionated enzyme fractions (data not shown). All yielded one very predominant product (about 80% of total) forming a sharp peak migrating between TNV-RNA and TMV-RNA and two lesser peaks between BMV-RNA components 1 and 4. The large component was not affected by RNase treatment but shifted to the position of the TNV marker upon heating or DMSO treatment. No detectable amounts of large products (>BMV-RNA 4) were produced by the soluble enzyme or by the bound enzyme from healthy plants, even when these were greatly stimulated in quantitative terms by the addition of a large RNA (TYMV-RNA). DISCUSSION
The results presented here show that bound and soluble RNA-dependent RNA polymerase activities can be found in
232
STUSSI-GARAUD,
LEMIUS,
AND
healthy and infected tobacco leaves. The bound enzyme activity detected in TNVinfected plants is not dependent on, and not stimulated by, TNV-RNA; the low bound enzyme activity in healthy plants is appreciable only when RNA is added. The bound enzyme extracted from infected plants is associated with an endogenous template which is the minus strand of TNV-RNA. This enzyme in vitro is able to make or finish double-stranded RNA resistant to RNases in which the “H-labeled strand is the plus strand and which comigrates with TNV-RNA after melting. The bound replicase present in TNV-infected plants shows some of the characteristics
generally observed for other plant virus replicases: The maximum of replicase activity is reached before the maximum of production of virus (Semal, 1970; Romero and Jacquemin, 1971; Hadidi and Fraenkel-Conrat, 1973; Zabel et al., 1974). The amount of incorporation of RNA precursors reaches a plateau after 5-30 min of incubation. Similar results were described by Semal and Hamilton (1968), Bradley and Zaitlin (19711, Jacquemin (1972), and Mouches et al. (1974). It was suggested that this phenomenon may be caused by the in vitro termination of nascent viral RNA chains. Low sensitivities to magnesium concentrations higher than 10 mM,
TABLE RIBONUCLEASE
Enzyme
fraction
RESISTANCE
and source
FRAENKEL-CONRAT
4 OF SYNTHESIZED
Added
Synthesized Total
(A)
(B)
Yarwood TNV-infected plants Bound enzyme, infected leaves Bound enzyme, healthy leaves Solubilized enzyme, infected leaves Solubilized enzyme, healthy leaves Ammonium sulfate-fractionated enzyme, infected leaves” Ammonium sulfate-fractionated enzyme, healthy leaves Soluble enzyme, infected leaves Soluble enzyme, infected leaves Soluble enzyme, healthy leaves Soluble enzyme, healthy leaves AC36 TNV-infected plants Bound enzyme, infected leaves Soluble enzyme, infected leaves Soluble enzvme, healthv leaves
RNA”
RNA
incorporation (cpm)
RNA
Remaining tion
--------------2x SSCb (%I
after in 0.1x
digesssc* (%)
None TYMV TYMV TYMV None
3075 185 3060 169 715
84 50 93 40 95
65’ 12 1.2 18 2.5
TYMV
220
28
19
TYMV None TYMV None
700 222 264 123
16 25 17 21
11 26 13 25
None TNV TNV
1000 1000 1000
88 67 45
0 0 0
u Data on phenol-extracted products obtained with various enzyme fractions (see Materials and Methods). Treatment of Yarwood TNV products with RNase directly in the incorporation mixture after addition of an equal volume of 4x SSC or water, respectively, indicated the same high resistance for all samples from infected leaves (except those made by the soluble enzyme) and low resistance (e.g., 20%) for the others. b Percentage of polymerized radioactivity that was RNase resistant. Pancreatic ribonuclease A (1 Kg for lo-50 Fg total RNA) was used for 30 min at 37” for set (A) and pancreatic + T, RNase for set (B) experiments. Equal aliquots (lo-30 ~1) of the RNA products were applied (see Materials and Methods) in set (A) to DEAE disks and in (B) to Whatman disks for counting the radioactivity directly or after RNase treatments. c This value represents an average of widely ranging data, high even upon digestion with RNases A + T,. d The product of an enzyme sample showing partial RNA dependence (incorporation of L3HlUTP, 100 and 310 cpm without and with added TYMV-RNA), made with added TYMV-RNA, was 45 and 17% resistant to digestion in 2x and 0.1 x SSC, respectively.
IN VZTRO REPLICATION TABLE REANNEALING 13HlRNA
EXPERIMENTS FROM synthesized
233
TNV
5
OF PRODUCTS SYNTHESIZED BY THE BOUND AND SOLUBLE HEALTHY AND AC36 TNV-INFECTED TOBACCO LEAVES RNase
by” Melting
Bound enzyme of infected plants” Soluble enzyme of healthv slants of infected plants *
OF
I
resistance
(%) of r3H]RNA
72
2.7 0
29 50
a 13HlRNAs were synthesized by bound enzyme without TNV-RNA. See Materials and Methods for experimental
added details.
in presence
of
TNV-RNA
AMV-RNA
4.5
84
61 73 RNA
EXTRACTED
after
Annealing
Self-reannealing
0
ENZYMES
and by soluble
62 58 enzymes
with
added
b cpm 0 1ooo-
24s 4
500-
d \ A’ %A9 e-e4e-@oo+-? . 15. .fractions . ** IO 5
FIG. 3. Sucrose gradient analysis of RNAs synthesized by the bound and soluble enzymes. Three milliliters of standard assay medium were extracted with phenol as described under Materials and Methods. The RNAs were dissolved in SSC buffer and loaded on a linear 5-20% sucrose gradient prepared in SSC. Fractions of 0.9 ml each were collected after a 16-hr centrifugation at 25,000 rpm in an SW 27 swinging bucket rotor. The points correspond to the radioactivity (in counts per minute) of 100 ~1 of each fraction. (A) Bound enzyme from infected plants; (B) soluble enzyme from healthy plants; (Cl soluble enzyme from infected plants; (O-O) before RNase treatment; (O--O) after RNase treatment. Arrows represent ribosomal and tRNA centrifuged as markers.
234
STUSSI-GARAUD,
LEMIUS,
AND FRAENKEL-CONRAT
FIG. 4. Polyacrylamide-gel electrophoresis of (A) 13H]RNA synthesized by the bound enzyme extracted from infected plants; (B) same RNA as (A) but after melting of the double strands in 90% DMSO; (0 r3HlRNA synthesized by the soluble enzyme of healthy plants; and (D) [3H]RNA synthesized by the soluble enzyme of infected plants. After phenol extraction and Sephadex G-75 filtration, the RNAs were loaded on 2.4% acrylamide gels (Loening, 1967); after 2.5 hr of electrophoresis at 5 mA/gel, the gels were cut in l-mm slices; each slice was digested with 90% NCS for 2 hr at 50”. The radioactivity was measured after adding 5 ml of scintillation fluid. Arrows correspond to migration of the four alfalfa mosaic virus RNA markers chromatographed under the same conditions; their molecular weights from left to right are: 0.3 x lo”, 0.7 x 106, 1 x 106, and 1.3 x 106. The dotted lines correspond to TNV-RNA marker (molecular weight, 1.5 x lo?.
to the pH of the reaction medium, and to high molarity of KC1 were also described by Zabel et al. (1974) for the enzyme found after CPMV infection. However, in the case of CPMV the enzyme was sensitive to ammonium sulfate concentrations higher than 50 mM while the TNV replicase seems to be more resistant to high molarities of salt (e.g., 0.2 M ammonium sulfate). The template-product-enzyme-
membrane complex is largely resistant to RNase. The earlier solubilization experiments (Fraenkel-Conrat, 1976) revealed that the TNV-induced enzyme-membrane complex is, like others, dissociated by nonionic detergent treatment. After detergent treatment the template generally remains attached to the enzyme, except in the case of BMV (Hadidi and Fraenkel-Conrat, 1973) where the solubilized enzyme is
IN
VITRO
REPLICATION
largely template dependent. The replicases tend to separate from the endogenous template to varying extents upon ammonium sulfate fractionation (FraenkelConrat, 19761, on density gradients (Zaitlin et al., 1973), by PEG-dextran twophase systems (Mouches et al., 1974; Clark et al., 1974; Fraenkel-Conrat, 19761, or by washing in a Mg2+-deficient buffer (Bol et al., 1976; Zabel et al., 1976). It is now shown that the TNV enzyme is able to make or finish TNV-RNA plus strands after solubilization and ammonium sulfate fractionation, as long as it is associated with endogenous template. Thus the membrane-bound state appears to play no role, at least in the elongation process. The soluble enzyme precipitated from the 105,000-g supernatant with ammonium sulfate is stimulated by added RNA and exists in healthy plants in a significant amount but increases greatly upon TNV infection. It may correspond to the enzyme described by Duda et al. (1973) except that the latter, probably due to the procedure used for its isolation, seems to carry endogenous plant RNA which it loses only upon sucrose gradient fractionation. Several other authors have isolated RNA polymerases from healthy plants which require addition of exogenous RNAs Astier-Manifacier and Cornuet [e.g., (1971) from Chinese cabbagge; Bol et al. (1976) and LeRoy et al. (1977) from tobacco, etc.]. These enzymes are stimulated by added RNAs, without showing any apparent specificity. Their action is favored by low magnesium concentrations and they are sensitive to high salt concentrations. The products of the soluble RNA polymerases appear to be only partially double stranded and of low molecular weight. Yet current experiments suggest that added RNAs can serve as templates, which would mean that healthy plant cells contain the enzymatic capability to replicate RNA. ACKNOWLEDGMENTS This investigation was supported by Research Grant No. PCM 7507854 from the National Science Foundation and by the “Commissariat a l’energie
OF
235
TNV
atomique” and the “Delegation cherche scientifique et technique”,
generale France.
de re-
REFERENCES ASTIER-MANIFACIER, S., and CORNUET, P. (1965). Isolation of the turnip yellow mosaic virus RNA replicase and asymmetrical synthesis of polynucleotides identical to TYMV-RNA. B&hem. Biophys. Res. Commun. 18, 283-287. ASTIER-MANIFACIER, S., and CORNUET, P. (1971). RNA-dependent RNA polymerase in Chinese cabbage. Biochim. Biophys. Acta 232, 484-493. BYFIELD, J. E., and SHERBAUM, 0. H. (1966). A rapid radioassay technique for cellular suspensions. Anal. Biochem. 17, 434-443. BOL, J. F., CLERX-VAN HAASTER, 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. BRADLEY, D. W., and ZAITLIN, M. (1971). Replication of tobacco mosaic virus. II. The in vitro synthesis of high molecular weight virus-specific RNAs. Virology 45, 192-199. CLARK, G. L., PEDEN, K. W. C., and SYMONS, R. H. (1974). Cucumber mosaic virus-induced RNA polymerase. Partial purification and properties of the template-free enzyme. Virology 62,434-443. 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. HADIDI, A., and FRAENKEL-CONRAT, H. (1973). Characterization and template specificity of soluble RNA polymerase of brome mosaic virus. Virology 52, 363-372. JACQUEMIN, J. M. (1972). In uitro product of an RNA polymerase induced in broadbean by infection with broadbean mosaic virus. Virology 49, 379384. KASSANIS, B. (1970). Tobacco necrosis virus. C.M.1.I A.A.B. Descriptions of Plant Viruses, Sheet No. 14 (B. D. Harrison and A. F. Murant, eds.). LEROY, C., STUSSI-GARAUD, C., and HIRTH, L. (1977). RNA dependent RNA polymerases stimulated upon alfalfa mosaic virus infection of tobacco plants. Virology, in press. LITMAN, R. M. (1968). A deoxyribonucleic acid polymerase from Micrococcus luteus (Micrococcus lysodeikticus) isolated on deoxyribonucleic acidcellulose. J. Biol. Chem. 243, 6222-6233. LOENING, U. E. (1967). The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel electrophoresis. Biochem. J. 102, 251257.
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LOWRY, 0. H., R~SEBROUCH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Ptotein measurement with the Folin phenol reagent. J. Bid. Chem. 193, 265275. MAY, J. T., GILLILAND, J. M., and SYMONS, R. H. (1970). Properties of a plant virus-induced RNA polymerase in particulate fractions of cucumbers infected with cucumber mosaic virus. Virology 41, 653-664. MOUCHES, C., BOVE, C., and BOVE, J. M. (1974). Turnip yellow mosaic virus-RNA replicase: Partial purification of the enzyme from the solubilized enzyme-template complex. Virology 58,409423. ROMERO, J. (1972). RNA synthesis in broadbean leaves infected with broadbean mottle virus. ViFOlOgy 48, 591-594. 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. 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 40, 244-250. SEMAL, J., and HAMILTON, R. I. (1968). RNA synthe-
AND
FRAENKEL-CONRAT
sis in cell-free extracts of barley leaves infected with bromegrass mosaic virus. Virology 36, 293302. SEMAL, J., and KUMMERT, J. (1971). In vitro synthesis of a segment of bromegrass mosaic virus ribonucleic acid. J. Gen. Viral. 11, 189-192. WEENING, C. J., and BOL, J. F. (1975). Viral RNA replication in extract of alfalfa mosaic virus infected Vicia faba. Virology 63, 77-83. WILCOCKSON, J., and HULL, R. (1974). The rapid isolation of plant virus RNAs using sodium perchlorate. J. Gen. Viral. 23, 107-111. ZABEL, P., WEENEN-SWAANS, H., and VAN KAMMEN, A. (1974). In vitro replication of cowpea mosaic virus RNA. I. Isolation and properties of the membrane-bound replicase. J. Viral. 14, 10491055. ZABEL, P., JONGEN-NEVEN, I., and VAN KAMMEN, A. (1976). In vitro replication of cowpea mosaic virus RNA. II. Solubilization of membrane-bound replicase and the partial purification of the solubilized enzyme. J. Viral. 17, 679-685. 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.