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Viral RNA replication in extracts of alfalfa mosaic virus-infected Vicia faba
VIROLOGY~~:
77-83(1975)
Viral RNA Replication
in Extracts
Infected
of Biochemistry,
State University, Accepted
Mosaic
Virus-
Vicia Faba
C.. J. WEENING Department
of Alfalfa
AND
J. F., BOL
Wassenaarseweg
64, Leiden,
The Netherlands
August 15, 1974
Extracts of alfalfa mosaic virus-infected broad bean leaves.were found to contain a membrane-bound replicative structure, consisting of an RNA polymerase associated with its template. In vitro this structure catalyzes the incorporation of [3H]CTP into an RNase-resistant structure, sedimenting after phenol extractionat about 15 S. Hybridization competition experiments revealed that in this 15-S structure the label was almost completely in viron type RNA. Under the experimental conditions there is no release of the newly synthesized RNA from the replicative structure.
A search for an RNA-dependent RNA polymerase activity in extracts of AMVinfected broad beans (Vicia f&a, local variety) was more successful. This paper describes the characterization of a membrane-bound enzyme-template complex, which can support the in vitro incorporation of RNA precursors into virion-type RNA.
INTRODUCTION
Preparations of alfalfa mosaic virus (AMV) are composed mainly of four different nucleoproteins: bottom component (B), middle component (M), top component b (Tb) and top component a (Ta), each containing a specific RNA molecule (Bol and Lak-Kaashoek, 1974). The genome of this virus is tripartite, and consists of B-RNA, M-RNA, and Tb-RNA (Van, Vloten-Doting et al., 1970). To become infectious, a mixture of these three RNA’s needs the addition of a small amount of coat protein or Ta-RNA (Bol et al., 1971). During the infection Ta-RNA is probably synthesized by a partial replication of TbRNA (Bol and Van Vloten-Doting, 1973; Mohier et al., 1974). To obtain further insight in the mechanism of the viral replication, and the possible role of the coat protein in this process, we initiated a study of the in vitro synthesis of viral RNA. In preliminary experiments attempts were made to obtain virus-specific RNA synthesis in extracts of AMV-infected tobacco (Samsum NN), prepared 1-5 days after infection according to the procedure described by Bradley and Zaitlin (1971). No significant differences with uninfected control plants were detectable in this way.
MATERIALS
METHODS
Plants and virus. Broad bean seedlings (Vicia f&a, local variety) were grown in a
greenhouse. The primary leaves of 7-dayold seedlings were infected by rubbing them with a purified AMV suspension (strain YSMV, 5 mg/ml). The infected seedlings were then grown under controlled temperature (23-24’) and continuous light. Eight days after infection chlorotic symptoms develop on the tertiary systemically infected leaves, becoming necrotic 10 days after infection. Leaves with chlorotic symptoms were taken for the preparation of the RNA-synthesizing leaf extract. The RNA-synthesizing activity was compared with that of similar leaves fr.om healthy plants. Preparation of the crude replicase extract. A crude replicase extract was pre77
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
78
WEENINGANDBOL
pared by the method of Jacquemin (1972) with some modifications. Four grams of the tertiary leaves were homogenized in a cold mortar in 35 ml of extraction buffer, containing 50 mM Tris-HCl, pH 7.4, 10 mM KCl, 1 mM EDTA, 8 mM MgC12, 8 mM 2-mercaptoethanol, and 50 pg/ml yeastRNA. The extract was filtered through nylon fine cloth and centrifuged for 5 min at 1000 g at 4”. The pellet was discarded. The supernatant was centrifuged (after addition of glycerol to a final concentration of 10%) at 10,000 g for 30 min. The resulting pellet (10,OOOP fraction) was resuspended in 1.5 ml of incubation buffer (the same buffer as extraction buffer, except the pH which was 8.6). Replicase assay. The 10,OOOPfraction of healthy and infected leaves were used for the assays. An amount of 0.5 ml of this fraction was mixed with 185 pg ATP, GTP, and UTP, 10 PCi [3H]CTP (sp act 28.3 Ci/mmole), 300 kg phosphoenol pyruvate and 10 kg pyruvate kinase. Unless stated otherwise the incubations were done in the presence of actinomycin D (AMD, 10 pg per incubation mixture). The mixture, with a total volume of 0.65 ml, was incubated for 20 min at 35” in a shaking waterbath. The reaction was terminated by extraction with phenol at room temperature. Extraction of incubation product. The in vitro-synthesized RNA was extracted by addition of 2.0 ml of 1.1% sodium dodecyl sulfate in 1 x SSC and 2.5 ml of watersaturated phenol to the incubated mixture. The tubes were shaken for 5 min at room temperature, cooled, and centrifuged for 5 min at 10,000 g. The water layer was collected, mixed with 2.5 vol of cold 96% ethanol and 0.3 ml 2 M sodium acetate, pH 5.2 and kept at 4” for 16 hr. The RNA precipitate was washed with cold 96% ethanol and resuspended in 2 x SSC (1 x SSC is 0.15 A4 NaCl, 0.015 A4 sodium citrate, PH 7.2), RNase treatment of the extracted incubation product. To determine the RNase resistance of the in uitro-synthesized phenol-extracted product, the RNA pellet was resuspended in 2 ml 2 x SSC and divided in two equal parts. One part was incubated
with RNase A (20 pg/ml) and RNase T, (60 units per ml) for 30 min at 37”; after incubation 0.1 mg of y-globulin was added, and the RNA was precipitated with an equal volume of a 10% TCA solution containing 2% pyrophosphate. The other part was precipitated directly with the TCA-pyrophosphate solution after the addition of y-globulin. The samples were collected on Whatman glass fiber disks (GF/A) and washed four times with 5ml portions of a 5% TCA/l% pyrophosphate solution, twice with 5 ml 96% ethanol. The dried filters were placed in tightly capped glass vials containing 10 ml of scintillation fluid consisting of 4 g PPO (2,5-diphenyloxazol) and 50 mg POPOP [p-bis-2(5phenyl-oxazol)-benzene] in 1.0 liter of toluene. The filters were counted in a Nuclear Chicago scintillation counter. For preparation of the RNase resistant in uitro-synthesized RNA, used for hybridization experiments, the RNase incubation was followed by a Pronase treatment (50 pg/ml, 30 min at 25”) and a reextraction with phenol. After ethanol precipitation the RNA was resuspended in 2 x SSC. Sucrose gradient analysis. Extracted RNA in 1 ml of 2 x SSC was layered on the top of a 5-20% linear sucrose gradient (made in 2 x SSC) and centrifuged for 22 hr at 25,000 rpm in the Spinco SW 27 rotor. Fractions of 0.5 ml were collected and alternately the total and RNase-resistant cpm were tested by the TCA-precipitation and GF/A-filtration methods. The sedimentation behavior of the product before phenol extraction was determined by layering of the total incubation mixture directly after incubation on the top of a discontinuous sucrose gradient (composed of equal volumes of lo%, 30%, and 50% sucrose in incubation buffer) and centrifuged for 30 min at 20,000 rpm in the Spinco SW 27 rotor. Reannealing experiments. Reannealing experiments were done with RNasetreated, in vitro-synthesized RNA, which had been sedimented in a 5-20% sucrose gradient. The fractions containing the double-stranded material were pooled, and the RNA was precipitated with ethanol. The double-stranded material was resus-
79
AMV RNA REPLICATION
solution from New England Nuclear. Actinomycin D was purchased from Calbiothem. RNase A (crystalline 5x) and yglobulin bovine (fraction 2) came from Nutritional Biochemicals Corporation; RNase T, (300,000 E/mg suspension) was obtained from Merck.
pended in 0.1 x SSC, melted at 100” for 10 min and reannealed in the presence or absence of unlabeled competitor RNA (50 c(g per sample). Reannealing was done in 2 x SSC at 80”. After 1 hr the temperature was allowed to decrease gradually to 37” in about 21/2hr. After reannealing the samples (0.5 ml) were diluted to 1 x SSC, and incubated with RNase A (100 &ml) for 15 min at 37”; radioactivity was measured after TCA precipitation and GF/A filtration. Reparation of viral 3H-labeled RNA. Two days after the infection of tobacco plants with AMV, leaves were cut in strips of 2 mm width. Ten grams of leaf material was incubated with 1 mCi [:H]uridine (25 Ci/mmole) in 5 ml 0.01 M phosphate buffer, pH 7.0, in a petri dish under continuous light of 2000 lux. Virus was harvested 5 days after infection (Van Vloten-Doting and Jaspars, 1972) and purified from contaminating plant m.aterial by gradient centrifugation (Verhagen and Bol, 1972). The labeled RNA was extracted with phenol and SDS as described by Bol and Van Vloten-Doting (1973). Chemicals. Unlabeled nucleotides (trisodium salt), phosphoenolpyruvate (tricyclohexylammonium salt), and pyruvate kinase were obtained from Boehringer Mannheim. The tetrasodium salt of (5-3H]cytidine 5’-triphosphate (sp act 28.3 Ci/ mmole) was received in a 50% ethanol
RESULTS
AMD-Resistant RNA Synthesis The 10,OOOP fractions, prepared 8, 9, and 10 days after the infection of broad bean seedlings with AMV, were assayed for polymerase activity. Comparable leaves of uninfected plants were used as a control. Table 1 shows that addition of AMD to healthy extracts results in a reduction of the 13H]CTP incorporation of about 90%. In extracts of infected leaves the insensitivity of the incorporation to AMD is significantly higher, reaching a maximum 9 days after infection. That the products synthesized in the extracts of infected and healthy leaves are of a different nature becomes apparent when the phenolextracted material, synthesized in the presence of AMD, is treated with RNase. In the 10,OOOPfraction of infected leaves the label is incorporated into a structure with a resistance to RNase of up to 88%, while the product synthesized by the healthy extract is almost completely sensitive to RNase. In the following experiments the RNA
TABLE INSENSITIVITY
1
TO ACTINOMYCIN D OF (3H]CTP INCORPORATION IN EXTRACTS OF INFECTED AND HEALTHY RIBONUCLEASE RESISTANCE OF THE SYNTHESIZED PRODWCT
[3H]CTP Incorporation
Days after infection” Infected
- AMD + AMD + AMD, + RNase” Resistance to AMD Resistance to RNase
(cpm)
(cpm) (cpm) (o/o) (%)
LEAVES.
Healthy
-
8
9
10
8
9
10
16,168
16,820 7,804 6,97 1 46 89
19,848 4,249 1,183 21 27
24,054 2,385 N.D.l 9 N.D.
23,510 3,120 236 13 7
20,474 2,743 369 13 13
3,875 1,688 24 44
a One half of a batch of 7-day-old broad bean seedhngs was infected with AMV, the other half was kept as a control. Comparable leaves of both groups of plants were assayed for polymerase activity 8,9, and 10 days after infection. b The phenol-extracted product, synthesized in the presence of AMI), was treated with RNase. Not determined.
80
WEENING
AND BOL
When the in vitro-synthesized RNA wa8 synthesis was studied in the presence of AMD, with extracts prepared 9 days after treated with RNase prior to gradient ten’trifugation, the sedimentation rate of the infection. RNase-resistant material was only slightljl Characterization of the in Vitro-Synthereduced (result not shown). This indicates sized RNA that possible (unlabeled) single-stranded That there is indeed a difference be- parts of the 15-S structure are relatively tween the RNA material synthesized by small. the 10,OOOPfractions of healthy and inBy hybridization experiments it was conb fected leaves becomes obvious when the firmed that the 15-S product is the result of phenol-extracted product is sedimented in virus-specific RNA synthesis. The RNasesucrose gradients. In extracts of healthy resistant product, purified by gradient cenleaves some label is incorporated in mate- trifugation, was melted and reannealed in rial sedimenting at 4 S (Fig. 1B). In addi- the presence or absence of competitor RNA tion to this 4-S material, the product of (Table 2). Without added RNA, about half infected leaves contains heterogeneous ma- the input counts were regained in an terial sedimenting at about 15 S (Fig. 1A). RNase-resistant structure after reannealTreatment of the gradient fractions with ing. When reannealing was done in the RNase revealed that the label in the 4-S presence of an excess of unlabeled AMVA material is completely sensitive to RNA, either from strain YSMV or 425, RNase. In the 15-S structure, however, almost all the label was rendered RNase the label is incorporated mainly into an sensitive. Addition of an excess of brome RNase-resistant form. mosaic virus RNA had no effect. This shows that in the 10,OOOPfraction label is incorporated into virion-type AMV-RNA. To demonstrate the presence of a com235 165 plementary template strand in the 15-S structure a 10,OOOPfraction was divided I I into two parts. One part was allowed to incorporate [3H]CTP, while: unlabeled RNA precursors were added to the other, part. Phenol-extracted RNA from both mixtures was sedimented in sucrose gradients, and the labeled product was used to TABLE
2
REANNEALINC OF in Vitro-LABELED DOUBLE-STRANDED 15-S RNA IN THE PRESENCE OF COMPETITOR RNA”
Competitor
None AMV-RNA AMV-RNA BMV-RNA FIG. 1. Sedimentation patterns of the phenolextracted products synthesized in the presence of AMD in extracts from infected (A) and healthy (B) plants. Fractions of 0.5 ml were collected and alternately the total (O--O) and RNase-resistant (O-----O) radioactivity was determined. Rihosomal RNA was used as a marker.
(strain YSMV) (strain 425)
RNase-resistant TCA-precipitable wm 1187 89 96 1170
” In uitro-synthesized RNA was treated with RNase and purified by gradient centrifugation as described in Materials and Methods. Samples containing 2396 cpm were used per reannealing mixture. One sample was melted and cooled directly in ice. After RNase treatment 95 acid-insoluble cpm were obtained. The presented figures are corrected for this value.
81
AMV RNA REPLICATION
identify the position of 15-S material. Unlabeled 15-S material was collected, and after melting it was reannealed in the presence of [3H]AMV-RNA (Table 3). The observation that some of the labeled AMVRNA is converted into a RNase-resistant structure demonstrates the presence of complementary RNA in the 15-S structure. Time Course of the [3H]CTP Incorporation
Figure 2 shows the time course of the [‘H]CTP incorporation. After about 5 min the total incorporation as well as the synthesis of RNase-resistant material reach a plateau. Variation of the pH of the incubation mixture from 7.2 to 8.6, or TABLE
3
ANNEALING OF MELTED UNLABELED 15-S RNA WITH [$H]AMV-RNA” 15-S RNA
+
13H]AMV-RNA
1520 99
la
-
RNase-resistant TCA-precipitable cpm after annealing
la
a The purification of unlabeled double-stranded 15-S RNA is described in the text. It was melted and reannealed in the presence of [aH]AMV-RNA of strain 425 (20,676 cpm/pg) as described in Materials and Methods.
variation of the temperature from 25” to 35” did not result in a prolonged incorporation. To see whether the synthesized plus strand material is released from the template, the pulse-chase experiment shown in Table 4 was performed. Label incorporated into RNase-resistant material during a 2-min pulse with [3H]CTP, is not converted into an RNase-sensitive form during a lo-min chase with an excess of unlabeled CTP. It was checked that RNA synthesis continues during the chase period by giving a 12-min pulse with [3H]CTP. These results (Fig. 2 and Table 4) indicate that, under the conditions used, the in vitro RNA synthesis is blocked in a certain stage, and that the synthesized plus strand material is not released from the template. Characterization of the Membrane-Bound Replicative Structure
Further insight into the nature of the replicative structure, present in the 10,OOOPfraction, was obtained by the following experiments: A 10,OOOPfraction of infected leaves was allowed to incorporate [3H]CTP, and was then sedimented in a discontinuous sucrose gradient for 30 min at 52,000 g. The acid-insoluble radioactivity of the fractions was determined before and after RNase treatment. The labeled product was found to cosediment with a green-colored band of membrane material, TABLE
4
EFFECT OF A lo-MIN CHASE ON THE RNase SENSITIVITYOF LABEL INCORPORATEDin Vitro DURING A 2-MIN PULSEa Sample number
“i
Minutes
TCA-precipitable cm
Pulse
Chase
Minus RNase
Plus RNase
2 2 12
10 -
1084 1067 2114
798 613 1311
20 time
(min.)
FIG. 2. Time course of the incorporation of [‘H]CTP into the 10,OOOPfraction of infected leaves. Incubation was done in the presence of AMD. At the indicated times the labeled product was extracted with phenol and divided into two parts. One half was directly precipitated with TCA (O--O), the other half was first incubated with RNase (A-A) and then precipitated with TCA.
1 2 3
‘The 10,OOOP fractions of samples 1 and 3 were incubated with [3H]CTP for 2 and 12 min, respectively. Sample 2 was incubated with 13H]CTP for 2 min and with 200 hg unlabeled CTP for an additional 10 min.
82
WEENING
and was completely resistant to RNase (Fig. 3 B). No such product was observed in a parallel experiment with healthy leaves (Fig. 3 A). Another 10,OOOP fraction was sedimented directly after isolation, and indicated fractions of the gradient (Fig. 3 C) were assayed for their capacity to incorpo-
,
J
7+--Y
Fraction
!O
number
FIG. 3. Analysis of the replicative complex present in the 10,OOOP fraction. The 10,OOOP fractions of healthy (A) and infected (B) leaves were incubated with [3H]CTP and then sedimented in discontinuous sucrose gradients for 30 min at 20,000 rpm in the Spinco SW 27 rotor. Radioactivity of the fractions (0.5 ml) was determined before (O---O) and after (O-----O) RNase treatment. Another 10,OOOPfraction from infected leaves (C) was sedimented directly after isolation, and indicated fractions of the gradient were tested for their capacity to incorporate [3H JCTP into an RNase-resistant structure.
AND BOL
rate [3H]CTP into a RNase-resistant structure. Most of the polymerase activity was found at the same position as the labeled RNA product. This demonstrates that in uitro-synthesized RNA remains associated with a membrane-bound enzyme-template complex. DISCUSSION
From the experiments reported here it can be concluded that the 10,OOOPfraction of AMV-infected broad bean leaves contains a membrane-bound replicative structure consisting of a minus-strand RNAtemplate (complementary to virion RNA) and an RNA polymerase. During the in vitro polymerase reaction label is incorpdrated into plus-strand RNA, probably by the elongation of nascent chains. The newly synthesized RNA remains associated to the replicative structure and is full) protected against RNase either by hydrogen bonding to the template or by associated proteins (8berg and Philipson, 1971). Upon phenol extraction, doublestranded RNA sedimenting at about 15 S is released from the replicative structure. It has been shown by Mohier et al. (1974’) that AMV-infected plants contain three replicative forms corresponding to B-RNA, M-RNA, and Tb-RNA, respectively. The possibility that our 15-S material is a mixture of these three replicative forms is presently being investigated by polyacrylamide gel electrophoresis. The choice of [3H]CTP as the labeled RNA precursor may be responsible for the incorporation of label into 4-S material in extracts from both healthy and infected leaves. Probably this is due to terminal incorporation into tRNA. This incorporaltion may have obscured the detection of viral RNA synthesis in our preliminary experiments with extracts of AMVIinfected tobacco. In the presence of AMD no difference was observed in the net incorporation of [3H]CTP in extracts of infected and healthy tobacco. When wk recently repeated these experiments we found that in extracts of infected leaves the label is incorporated mainly into an RNase-resistant 15-S structure, while in the healthy extracts only small RNase-sensitive RNA was labeled (unpublished re-
83
AMV RNA REPLICATION
sults). This indicates that extracts of AMV-infected tobacco contain a replicative structure similar to that induced in broad bean leaves. The isolation of a virus-specific particulate enzyme-template complex has been reported for a number of other viruses: turnip yellow mosaic virus (Bove et al., 1968), tobacco mosaic virus (TMV) (Bradley and Zaitlin, 1971), and bromegrass mosaic virus (BMV) and broad bean mottle virus (BBMV) (Semal, 1970). Like the AMV replicative structure, these enzyme preparations incorporate RNA precursors into virion-type RNA in an RNase-resistant form. In addition, Zaitlin et al. (1973) also observed some incorporation in the complement of TMV-RNA. Our hybridization competition experiment (Table 2) shows that at least 92% of the label is in plus-strand AMV-RNA. A sequential in vitro synthesis of doublestranded and single-stranded RNA by particulate enzyme preparations has been reported for BMV (Semal and Kummert, 1971) and BBMV (Jaquemin, 1972). The pulse-chase experiment of Table 4 demonstrates that the newly synthesized AMVRNA is not released from the template. The possibility that the reaction is arrested because some factor in the incubation mixture, e.g., the [3H]CTP concentration, is limiting, can be ruled out. Under the same reaction conditions a RNA-dependent RNA polymerase, solubilized by detergent from the 10,OOOPfraction of AMV-infected tobacco, catalyzed a linear incorporation of [3H]CTP for more than 30 min (C. J. Weening, C. M. Clerx-van Haaster, and J. F. Bol, to be published). Thus, the fact that the incorporation by the particular enzyme flattens out after 5 min (Fig. 2) must be due to a deficiency of the enzyme system. For a further understanding of the AMV replication a characterization of the solubiized RNA polymerase is required. Such xperiments are presently being perI ormed. ACKNOWLEDGMENTS Thanks are due to Drs. J. Semal and J. Kummert [Gembloux, Belgium) for helpful advice. The skillful
assistance of Mr. G. J. M. J. van den Aardweg is gratefully acknowledged. This work was sponsored by the Netherlands Foundation for Chemical Research (S.O.N.), with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.). REFERENCES BOL, J. F., VAN VLOTEN-DOTING, L., and JASPARS,E. M. J. (1971). A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus. Virology 46, 73-85. BOL, J. F., and VAN VLOTEN-DOTING, L. (1973). Function of top component a RNA in the initiation of infection by alfalfa mosaic virus. Virology 51, 102-108. BOL, J. F., and LAK-KAASHOEK, M. (1974). Composition of alfalfa mosaic virus nucleoproteins. Virology, 60, 476-484. BovB, J. M., Bovl, C., and MOCQUOT, B. (1968). Turnip Yellow Mosaic Virus-RNA synthesis in uitro: evidence for native doubie-stranded RNA. Biochem. Biophys. Res. Commun. 32, 480-486. 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. JACQUEMIN, J. M. (1972). In uitro product of an RNA polymerase induced in broadbean by infection with broadbean mottle virus. Virology 49, 379-384. MOHIER, E., PINCK, L.. and HIRTH, L. (1974). Replication of alfalfa mosaic virus RNAs. Virology 58,9-l& C)BERG, B., and PHILIPSON, L. (1971). Replicative structure of poliovirus RNA in uiuo. J. Mol. Biol. 58, 725-737. SEMAL, J. (1970). Properties of the product 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 KUMMERT, J. (1971). Sequential synthesis of double-stranded and single-stranded RNA by cell-free extracts of barley leaves infected with brome mosaic virus. J. Gen. Viral. 10, 79-89. VAN VLOTEN-DOTING, L., DINGJAN-VERSTEEGH, A. M., and JASPARS,E. M. J. (1970). Three nucleoprotein components of alfalfa mosaic virus necessary for infectivity. Virology 40, 419-430. VAN VLOTEN-DOTING, L., and JASPARS,E. M. J. (1972). The uncoating of alfalfa mosaic virus by its own RNA. Viro1og.v 48, 699-708. VERHAGEN, W., and BOL, J. F. (1972). Evidence for a pH-induced structural change of alfalfa mosaic virus. Virology 50, 431-439. 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.
77-83(1975)
Viral RNA Replication
in Extracts
Infected
of Biochemistry,
State University, Accepted
Mosaic
Virus-
Vicia Faba
C.. J. WEENING Department
of Alfalfa
AND
J. F., BOL
Wassenaarseweg
64, Leiden,
The Netherlands
August 15, 1974
Extracts of alfalfa mosaic virus-infected broad bean leaves.were found to contain a membrane-bound replicative structure, consisting of an RNA polymerase associated with its template. In vitro this structure catalyzes the incorporation of [3H]CTP into an RNase-resistant structure, sedimenting after phenol extractionat about 15 S. Hybridization competition experiments revealed that in this 15-S structure the label was almost completely in viron type RNA. Under the experimental conditions there is no release of the newly synthesized RNA from the replicative structure.
A search for an RNA-dependent RNA polymerase activity in extracts of AMVinfected broad beans (Vicia f&a, local variety) was more successful. This paper describes the characterization of a membrane-bound enzyme-template complex, which can support the in vitro incorporation of RNA precursors into virion-type RNA.
INTRODUCTION
Preparations of alfalfa mosaic virus (AMV) are composed mainly of four different nucleoproteins: bottom component (B), middle component (M), top component b (Tb) and top component a (Ta), each containing a specific RNA molecule (Bol and Lak-Kaashoek, 1974). The genome of this virus is tripartite, and consists of B-RNA, M-RNA, and Tb-RNA (Van, Vloten-Doting et al., 1970). To become infectious, a mixture of these three RNA’s needs the addition of a small amount of coat protein or Ta-RNA (Bol et al., 1971). During the infection Ta-RNA is probably synthesized by a partial replication of TbRNA (Bol and Van Vloten-Doting, 1973; Mohier et al., 1974). To obtain further insight in the mechanism of the viral replication, and the possible role of the coat protein in this process, we initiated a study of the in vitro synthesis of viral RNA. In preliminary experiments attempts were made to obtain virus-specific RNA synthesis in extracts of AMV-infected tobacco (Samsum NN), prepared 1-5 days after infection according to the procedure described by Bradley and Zaitlin (1971). No significant differences with uninfected control plants were detectable in this way.
MATERIALS
METHODS
Plants and virus. Broad bean seedlings (Vicia f&a, local variety) were grown in a
greenhouse. The primary leaves of 7-dayold seedlings were infected by rubbing them with a purified AMV suspension (strain YSMV, 5 mg/ml). The infected seedlings were then grown under controlled temperature (23-24’) and continuous light. Eight days after infection chlorotic symptoms develop on the tertiary systemically infected leaves, becoming necrotic 10 days after infection. Leaves with chlorotic symptoms were taken for the preparation of the RNA-synthesizing leaf extract. The RNA-synthesizing activity was compared with that of similar leaves fr.om healthy plants. Preparation of the crude replicase extract. A crude replicase extract was pre77
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
78
WEENINGANDBOL
pared by the method of Jacquemin (1972) with some modifications. Four grams of the tertiary leaves were homogenized in a cold mortar in 35 ml of extraction buffer, containing 50 mM Tris-HCl, pH 7.4, 10 mM KCl, 1 mM EDTA, 8 mM MgC12, 8 mM 2-mercaptoethanol, and 50 pg/ml yeastRNA. The extract was filtered through nylon fine cloth and centrifuged for 5 min at 1000 g at 4”. The pellet was discarded. The supernatant was centrifuged (after addition of glycerol to a final concentration of 10%) at 10,000 g for 30 min. The resulting pellet (10,OOOP fraction) was resuspended in 1.5 ml of incubation buffer (the same buffer as extraction buffer, except the pH which was 8.6). Replicase assay. The 10,OOOPfraction of healthy and infected leaves were used for the assays. An amount of 0.5 ml of this fraction was mixed with 185 pg ATP, GTP, and UTP, 10 PCi [3H]CTP (sp act 28.3 Ci/mmole), 300 kg phosphoenol pyruvate and 10 kg pyruvate kinase. Unless stated otherwise the incubations were done in the presence of actinomycin D (AMD, 10 pg per incubation mixture). The mixture, with a total volume of 0.65 ml, was incubated for 20 min at 35” in a shaking waterbath. The reaction was terminated by extraction with phenol at room temperature. Extraction of incubation product. The in vitro-synthesized RNA was extracted by addition of 2.0 ml of 1.1% sodium dodecyl sulfate in 1 x SSC and 2.5 ml of watersaturated phenol to the incubated mixture. The tubes were shaken for 5 min at room temperature, cooled, and centrifuged for 5 min at 10,000 g. The water layer was collected, mixed with 2.5 vol of cold 96% ethanol and 0.3 ml 2 M sodium acetate, pH 5.2 and kept at 4” for 16 hr. The RNA precipitate was washed with cold 96% ethanol and resuspended in 2 x SSC (1 x SSC is 0.15 A4 NaCl, 0.015 A4 sodium citrate, PH 7.2), RNase treatment of the extracted incubation product. To determine the RNase resistance of the in uitro-synthesized phenol-extracted product, the RNA pellet was resuspended in 2 ml 2 x SSC and divided in two equal parts. One part was incubated
with RNase A (20 pg/ml) and RNase T, (60 units per ml) for 30 min at 37”; after incubation 0.1 mg of y-globulin was added, and the RNA was precipitated with an equal volume of a 10% TCA solution containing 2% pyrophosphate. The other part was precipitated directly with the TCA-pyrophosphate solution after the addition of y-globulin. The samples were collected on Whatman glass fiber disks (GF/A) and washed four times with 5ml portions of a 5% TCA/l% pyrophosphate solution, twice with 5 ml 96% ethanol. The dried filters were placed in tightly capped glass vials containing 10 ml of scintillation fluid consisting of 4 g PPO (2,5-diphenyloxazol) and 50 mg POPOP [p-bis-2(5phenyl-oxazol)-benzene] in 1.0 liter of toluene. The filters were counted in a Nuclear Chicago scintillation counter. For preparation of the RNase resistant in uitro-synthesized RNA, used for hybridization experiments, the RNase incubation was followed by a Pronase treatment (50 pg/ml, 30 min at 25”) and a reextraction with phenol. After ethanol precipitation the RNA was resuspended in 2 x SSC. Sucrose gradient analysis. Extracted RNA in 1 ml of 2 x SSC was layered on the top of a 5-20% linear sucrose gradient (made in 2 x SSC) and centrifuged for 22 hr at 25,000 rpm in the Spinco SW 27 rotor. Fractions of 0.5 ml were collected and alternately the total and RNase-resistant cpm were tested by the TCA-precipitation and GF/A-filtration methods. The sedimentation behavior of the product before phenol extraction was determined by layering of the total incubation mixture directly after incubation on the top of a discontinuous sucrose gradient (composed of equal volumes of lo%, 30%, and 50% sucrose in incubation buffer) and centrifuged for 30 min at 20,000 rpm in the Spinco SW 27 rotor. Reannealing experiments. Reannealing experiments were done with RNasetreated, in vitro-synthesized RNA, which had been sedimented in a 5-20% sucrose gradient. The fractions containing the double-stranded material were pooled, and the RNA was precipitated with ethanol. The double-stranded material was resus-
79
AMV RNA REPLICATION
solution from New England Nuclear. Actinomycin D was purchased from Calbiothem. RNase A (crystalline 5x) and yglobulin bovine (fraction 2) came from Nutritional Biochemicals Corporation; RNase T, (300,000 E/mg suspension) was obtained from Merck.
pended in 0.1 x SSC, melted at 100” for 10 min and reannealed in the presence or absence of unlabeled competitor RNA (50 c(g per sample). Reannealing was done in 2 x SSC at 80”. After 1 hr the temperature was allowed to decrease gradually to 37” in about 21/2hr. After reannealing the samples (0.5 ml) were diluted to 1 x SSC, and incubated with RNase A (100 &ml) for 15 min at 37”; radioactivity was measured after TCA precipitation and GF/A filtration. Reparation of viral 3H-labeled RNA. Two days after the infection of tobacco plants with AMV, leaves were cut in strips of 2 mm width. Ten grams of leaf material was incubated with 1 mCi [:H]uridine (25 Ci/mmole) in 5 ml 0.01 M phosphate buffer, pH 7.0, in a petri dish under continuous light of 2000 lux. Virus was harvested 5 days after infection (Van Vloten-Doting and Jaspars, 1972) and purified from contaminating plant m.aterial by gradient centrifugation (Verhagen and Bol, 1972). The labeled RNA was extracted with phenol and SDS as described by Bol and Van Vloten-Doting (1973). Chemicals. Unlabeled nucleotides (trisodium salt), phosphoenolpyruvate (tricyclohexylammonium salt), and pyruvate kinase were obtained from Boehringer Mannheim. The tetrasodium salt of (5-3H]cytidine 5’-triphosphate (sp act 28.3 Ci/ mmole) was received in a 50% ethanol
RESULTS
AMD-Resistant RNA Synthesis The 10,OOOP fractions, prepared 8, 9, and 10 days after the infection of broad bean seedlings with AMV, were assayed for polymerase activity. Comparable leaves of uninfected plants were used as a control. Table 1 shows that addition of AMD to healthy extracts results in a reduction of the 13H]CTP incorporation of about 90%. In extracts of infected leaves the insensitivity of the incorporation to AMD is significantly higher, reaching a maximum 9 days after infection. That the products synthesized in the extracts of infected and healthy leaves are of a different nature becomes apparent when the phenolextracted material, synthesized in the presence of AMD, is treated with RNase. In the 10,OOOPfraction of infected leaves the label is incorporated into a structure with a resistance to RNase of up to 88%, while the product synthesized by the healthy extract is almost completely sensitive to RNase. In the following experiments the RNA
TABLE INSENSITIVITY
1
TO ACTINOMYCIN D OF (3H]CTP INCORPORATION IN EXTRACTS OF INFECTED AND HEALTHY RIBONUCLEASE RESISTANCE OF THE SYNTHESIZED PRODWCT
[3H]CTP Incorporation
Days after infection” Infected
- AMD + AMD + AMD, + RNase” Resistance to AMD Resistance to RNase
(cpm)
(cpm) (cpm) (o/o) (%)
LEAVES.
Healthy
-
8
9
10
8
9
10
16,168
16,820 7,804 6,97 1 46 89
19,848 4,249 1,183 21 27
24,054 2,385 N.D.l 9 N.D.
23,510 3,120 236 13 7
20,474 2,743 369 13 13
3,875 1,688 24 44
a One half of a batch of 7-day-old broad bean seedhngs was infected with AMV, the other half was kept as a control. Comparable leaves of both groups of plants were assayed for polymerase activity 8,9, and 10 days after infection. b The phenol-extracted product, synthesized in the presence of AMI), was treated with RNase. Not determined.
80
WEENING
AND BOL
When the in vitro-synthesized RNA wa8 synthesis was studied in the presence of AMD, with extracts prepared 9 days after treated with RNase prior to gradient ten’trifugation, the sedimentation rate of the infection. RNase-resistant material was only slightljl Characterization of the in Vitro-Synthereduced (result not shown). This indicates sized RNA that possible (unlabeled) single-stranded That there is indeed a difference be- parts of the 15-S structure are relatively tween the RNA material synthesized by small. the 10,OOOPfractions of healthy and inBy hybridization experiments it was conb fected leaves becomes obvious when the firmed that the 15-S product is the result of phenol-extracted product is sedimented in virus-specific RNA synthesis. The RNasesucrose gradients. In extracts of healthy resistant product, purified by gradient cenleaves some label is incorporated in mate- trifugation, was melted and reannealed in rial sedimenting at 4 S (Fig. 1B). In addi- the presence or absence of competitor RNA tion to this 4-S material, the product of (Table 2). Without added RNA, about half infected leaves contains heterogeneous ma- the input counts were regained in an terial sedimenting at about 15 S (Fig. 1A). RNase-resistant structure after reannealTreatment of the gradient fractions with ing. When reannealing was done in the RNase revealed that the label in the 4-S presence of an excess of unlabeled AMVA material is completely sensitive to RNA, either from strain YSMV or 425, RNase. In the 15-S structure, however, almost all the label was rendered RNase the label is incorporated mainly into an sensitive. Addition of an excess of brome RNase-resistant form. mosaic virus RNA had no effect. This shows that in the 10,OOOPfraction label is incorporated into virion-type AMV-RNA. To demonstrate the presence of a com235 165 plementary template strand in the 15-S structure a 10,OOOPfraction was divided I I into two parts. One part was allowed to incorporate [3H]CTP, while: unlabeled RNA precursors were added to the other, part. Phenol-extracted RNA from both mixtures was sedimented in sucrose gradients, and the labeled product was used to TABLE
2
REANNEALINC OF in Vitro-LABELED DOUBLE-STRANDED 15-S RNA IN THE PRESENCE OF COMPETITOR RNA”
Competitor
None AMV-RNA AMV-RNA BMV-RNA FIG. 1. Sedimentation patterns of the phenolextracted products synthesized in the presence of AMD in extracts from infected (A) and healthy (B) plants. Fractions of 0.5 ml were collected and alternately the total (O--O) and RNase-resistant (O-----O) radioactivity was determined. Rihosomal RNA was used as a marker.
(strain YSMV) (strain 425)
RNase-resistant TCA-precipitable wm 1187 89 96 1170
” In uitro-synthesized RNA was treated with RNase and purified by gradient centrifugation as described in Materials and Methods. Samples containing 2396 cpm were used per reannealing mixture. One sample was melted and cooled directly in ice. After RNase treatment 95 acid-insoluble cpm were obtained. The presented figures are corrected for this value.
81
AMV RNA REPLICATION
identify the position of 15-S material. Unlabeled 15-S material was collected, and after melting it was reannealed in the presence of [3H]AMV-RNA (Table 3). The observation that some of the labeled AMVRNA is converted into a RNase-resistant structure demonstrates the presence of complementary RNA in the 15-S structure. Time Course of the [3H]CTP Incorporation
Figure 2 shows the time course of the [‘H]CTP incorporation. After about 5 min the total incorporation as well as the synthesis of RNase-resistant material reach a plateau. Variation of the pH of the incubation mixture from 7.2 to 8.6, or TABLE
3
ANNEALING OF MELTED UNLABELED 15-S RNA WITH [$H]AMV-RNA” 15-S RNA
+
13H]AMV-RNA
1520 99
la
-
RNase-resistant TCA-precipitable cpm after annealing
la
a The purification of unlabeled double-stranded 15-S RNA is described in the text. It was melted and reannealed in the presence of [aH]AMV-RNA of strain 425 (20,676 cpm/pg) as described in Materials and Methods.
variation of the temperature from 25” to 35” did not result in a prolonged incorporation. To see whether the synthesized plus strand material is released from the template, the pulse-chase experiment shown in Table 4 was performed. Label incorporated into RNase-resistant material during a 2-min pulse with [3H]CTP, is not converted into an RNase-sensitive form during a lo-min chase with an excess of unlabeled CTP. It was checked that RNA synthesis continues during the chase period by giving a 12-min pulse with [3H]CTP. These results (Fig. 2 and Table 4) indicate that, under the conditions used, the in vitro RNA synthesis is blocked in a certain stage, and that the synthesized plus strand material is not released from the template. Characterization of the Membrane-Bound Replicative Structure
Further insight into the nature of the replicative structure, present in the 10,OOOPfraction, was obtained by the following experiments: A 10,OOOPfraction of infected leaves was allowed to incorporate [3H]CTP, and was then sedimented in a discontinuous sucrose gradient for 30 min at 52,000 g. The acid-insoluble radioactivity of the fractions was determined before and after RNase treatment. The labeled product was found to cosediment with a green-colored band of membrane material, TABLE
4
EFFECT OF A lo-MIN CHASE ON THE RNase SENSITIVITYOF LABEL INCORPORATEDin Vitro DURING A 2-MIN PULSEa Sample number
“i
Minutes
TCA-precipitable cm
Pulse
Chase
Minus RNase
Plus RNase
2 2 12
10 -
1084 1067 2114
798 613 1311
20 time
(min.)
FIG. 2. Time course of the incorporation of [‘H]CTP into the 10,OOOPfraction of infected leaves. Incubation was done in the presence of AMD. At the indicated times the labeled product was extracted with phenol and divided into two parts. One half was directly precipitated with TCA (O--O), the other half was first incubated with RNase (A-A) and then precipitated with TCA.
1 2 3
‘The 10,OOOP fractions of samples 1 and 3 were incubated with [3H]CTP for 2 and 12 min, respectively. Sample 2 was incubated with 13H]CTP for 2 min and with 200 hg unlabeled CTP for an additional 10 min.
82
WEENING
and was completely resistant to RNase (Fig. 3 B). No such product was observed in a parallel experiment with healthy leaves (Fig. 3 A). Another 10,OOOP fraction was sedimented directly after isolation, and indicated fractions of the gradient (Fig. 3 C) were assayed for their capacity to incorpo-
,
J
7+--Y
Fraction
!O
number
FIG. 3. Analysis of the replicative complex present in the 10,OOOP fraction. The 10,OOOP fractions of healthy (A) and infected (B) leaves were incubated with [3H]CTP and then sedimented in discontinuous sucrose gradients for 30 min at 20,000 rpm in the Spinco SW 27 rotor. Radioactivity of the fractions (0.5 ml) was determined before (O---O) and after (O-----O) RNase treatment. Another 10,OOOPfraction from infected leaves (C) was sedimented directly after isolation, and indicated fractions of the gradient were tested for their capacity to incorporate [3H JCTP into an RNase-resistant structure.
AND BOL
rate [3H]CTP into a RNase-resistant structure. Most of the polymerase activity was found at the same position as the labeled RNA product. This demonstrates that in uitro-synthesized RNA remains associated with a membrane-bound enzyme-template complex. DISCUSSION
From the experiments reported here it can be concluded that the 10,OOOPfraction of AMV-infected broad bean leaves contains a membrane-bound replicative structure consisting of a minus-strand RNAtemplate (complementary to virion RNA) and an RNA polymerase. During the in vitro polymerase reaction label is incorpdrated into plus-strand RNA, probably by the elongation of nascent chains. The newly synthesized RNA remains associated to the replicative structure and is full) protected against RNase either by hydrogen bonding to the template or by associated proteins (8berg and Philipson, 1971). Upon phenol extraction, doublestranded RNA sedimenting at about 15 S is released from the replicative structure. It has been shown by Mohier et al. (1974’) that AMV-infected plants contain three replicative forms corresponding to B-RNA, M-RNA, and Tb-RNA, respectively. The possibility that our 15-S material is a mixture of these three replicative forms is presently being investigated by polyacrylamide gel electrophoresis. The choice of [3H]CTP as the labeled RNA precursor may be responsible for the incorporation of label into 4-S material in extracts from both healthy and infected leaves. Probably this is due to terminal incorporation into tRNA. This incorporaltion may have obscured the detection of viral RNA synthesis in our preliminary experiments with extracts of AMVIinfected tobacco. In the presence of AMD no difference was observed in the net incorporation of [3H]CTP in extracts of infected and healthy tobacco. When wk recently repeated these experiments we found that in extracts of infected leaves the label is incorporated mainly into an RNase-resistant 15-S structure, while in the healthy extracts only small RNase-sensitive RNA was labeled (unpublished re-
83
AMV RNA REPLICATION
sults). This indicates that extracts of AMV-infected tobacco contain a replicative structure similar to that induced in broad bean leaves. The isolation of a virus-specific particulate enzyme-template complex has been reported for a number of other viruses: turnip yellow mosaic virus (Bove et al., 1968), tobacco mosaic virus (TMV) (Bradley and Zaitlin, 1971), and bromegrass mosaic virus (BMV) and broad bean mottle virus (BBMV) (Semal, 1970). Like the AMV replicative structure, these enzyme preparations incorporate RNA precursors into virion-type RNA in an RNase-resistant form. In addition, Zaitlin et al. (1973) also observed some incorporation in the complement of TMV-RNA. Our hybridization competition experiment (Table 2) shows that at least 92% of the label is in plus-strand AMV-RNA. A sequential in vitro synthesis of doublestranded and single-stranded RNA by particulate enzyme preparations has been reported for BMV (Semal and Kummert, 1971) and BBMV (Jaquemin, 1972). The pulse-chase experiment of Table 4 demonstrates that the newly synthesized AMVRNA is not released from the template. The possibility that the reaction is arrested because some factor in the incubation mixture, e.g., the [3H]CTP concentration, is limiting, can be ruled out. Under the same reaction conditions a RNA-dependent RNA polymerase, solubilized by detergent from the 10,OOOPfraction of AMV-infected tobacco, catalyzed a linear incorporation of [3H]CTP for more than 30 min (C. J. Weening, C. M. Clerx-van Haaster, and J. F. Bol, to be published). Thus, the fact that the incorporation by the particular enzyme flattens out after 5 min (Fig. 2) must be due to a deficiency of the enzyme system. For a further understanding of the AMV replication a characterization of the solubiized RNA polymerase is required. Such xperiments are presently being perI ormed. ACKNOWLEDGMENTS Thanks are due to Drs. J. Semal and J. Kummert [Gembloux, Belgium) for helpful advice. The skillful
assistance of Mr. G. J. M. J. van den Aardweg is gratefully acknowledged. This work was sponsored by the Netherlands Foundation for Chemical Research (S.O.N.), with financial aid from the Netherlands Organisation for the Advancement of Pure Research (Z.W.O.). REFERENCES BOL, J. F., VAN VLOTEN-DOTING, L., and JASPARS,E. M. J. (1971). A functional equivalence of top component a RNA and coat protein in the initiation of infection by alfalfa mosaic virus. Virology 46, 73-85. BOL, J. F., and VAN VLOTEN-DOTING, L. (1973). Function of top component a RNA in the initiation of infection by alfalfa mosaic virus. Virology 51, 102-108. BOL, J. F., and LAK-KAASHOEK, M. (1974). Composition of alfalfa mosaic virus nucleoproteins. Virology, 60, 476-484. BovB, J. M., Bovl, C., and MOCQUOT, B. (1968). Turnip Yellow Mosaic Virus-RNA synthesis in uitro: evidence for native doubie-stranded RNA. Biochem. Biophys. Res. Commun. 32, 480-486. 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. JACQUEMIN, J. M. (1972). In uitro product of an RNA polymerase induced in broadbean by infection with broadbean mottle virus. Virology 49, 379-384. MOHIER, E., PINCK, L.. and HIRTH, L. (1974). Replication of alfalfa mosaic virus RNAs. Virology 58,9-l& C)BERG, B., and PHILIPSON, L. (1971). Replicative structure of poliovirus RNA in uiuo. J. Mol. Biol. 58, 725-737. SEMAL, J. (1970). Properties of the product 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 KUMMERT, J. (1971). Sequential synthesis of double-stranded and single-stranded RNA by cell-free extracts of barley leaves infected with brome mosaic virus. J. Gen. Viral. 10, 79-89. VAN VLOTEN-DOTING, L., DINGJAN-VERSTEEGH, A. M., and JASPARS,E. M. J. (1970). Three nucleoprotein components of alfalfa mosaic virus necessary for infectivity. Virology 40, 419-430. VAN VLOTEN-DOTING, L., and JASPARS,E. M. J. (1972). The uncoating of alfalfa mosaic virus by its own RNA. Viro1og.v 48, 699-708. VERHAGEN, W., and BOL, J. F. (1972). Evidence for a pH-induced structural change of alfalfa mosaic virus. Virology 50, 431-439. 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.