Detection, isolation, and characterization of high molecular weight double-stranded RNAs in plants infected with velvet tobacco mottle virus

VIROLOGY126, 480-492 (1983)

Detection, Isolation, and Characterization of High Molecular Weight Double-Stranded RNAs in Plants Infected with Velvet Tobacco Mottle Virus P. W. G. C H U , R. I. B. F R A N C K I , 1 AND J. W . R A N D L E S Department of Plant Pathology, Waite Agricultural Research Institute, University of Adelaide, South Australi(~ Received August 19, 1982;accepted December 30, 1982

Two species of double-stranded (ds) RNA were detected in Nicotiana clevelandii infected with velvet tobacco mottle virus (VTMoV) which were not present in healthy plants. The ds-RNAs were first detected by polyacrylamide gel electrophoresis 2 to 4 and about 8 days after inoculation in the inoculated and systemically infected leaves respectively. Thereafter the concentrations of ds-RNA increased significantly. The VTMoV-specific ds-RNA was purified from plants 10 to 14 days after inoculation. The ds-RNA was electrophoresed on agarose gels, transferred to nitrocellulose paper and blot hybridized with 32P-labelled probes specific to nucleotide sequences of either the positive or negative sense strands of the various VTMoV RNA components: the linear single-stranded (ss) RNA 1 of M~ about 1.5 • 106 or RNAs 2 and 3, the circular and linear forms respectively, of viroid-like ss-RNA with Mr of about 1.2 • 105. One of the ds-RNA species, with Mr of about 2.8 • l0 G,was shown to contain base sequences specific to RNA 1 and the other, with Mr of about 3.6 • 106, specific to RNAs 2 and 3. The possible significance of the VTMoV-specific ds-RNAs to replication of the virus is discussed. INTRODUCTION Velvet tobacco mottle virus (VTMoV) has s m a l l i s o m e t r i c p a r t i c l e s of a b o u t 30 n m w h i c h e n c a p s i d a t e s i n g l e - s t r a n d e d (ss) R N A s o f a b o u t Mr 1.5 • 106 ( R N A 1) a n d h i g h l y b a s e p a i r e d , c i r c u l a r ( R N A 2) a n d l i n e a r ( R N A 3) s s - R N A s b o t h w i t h M r of a b o u t 1.2 X 105 ( R a n d l e s et al., 1981). H y bridization analyses using complementary D N A s ( c D N A s ) r e v e a l e d t h a t t h e b a s e seq u e n c e s of R N A 2 a n d 3 a r e i n d i s t i n g u i s h a b l e a n d h a v e no h o m o l o g y w i t h R N A 1 ( G o u l d , 1981). I n a d d i t i o n , t w o m i n o r R N A components were also isolated from V T M o V p a r t i c l e s , R N A s l a a n d l b w i t h Mr o f a b o u t 0.63 a n d 0.25 • l 0 s, r e s p e c t i v e l y ; b o t h w e r e s h o w n to h a v e b a s e s e q u e n c e h o m o l o g y w i t h R N A 1 ( G o u l d , 1981). I t h a s been demonstrated that both RNA 1 and 2 a r e e s s e n t i a l f o r i n f e c t i v i t y ( G o u l d et al., 1981b).

1To whom requests for reprints should be addressed. 0042-6822/83 $3.00 Copyright 9 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

In an endeavour to gain some unders t a n d i n g of V T M o V r e p l i c a t i o n w e h a v e compared the RNA contents of healthy and V T M o V - i n f e c t e d p l a n t s in s e a r c h o f R N A s t h a t m a y b e i n t e r m e d i a t e s in t h e s y n t h e s i s of t h o s e e n c a p s i d a t e d b y t h e v i r u s . I n t h i s p a p e r w e d e s c r i b e t h e d e t e c t i o n a n d isol a t i o n of t w o f r a c t i o n s o f h i g h m o l e c u l a r w e i g h t d o u b l e - s t r a n d e d (ds) R N A s specific to i n f e c t i o n . O n e of t h e s e c o n t a i n s b a s e s e q u e n c e s of R N A 1 a n d t h e o t h e r t h o s e o f R N A 2. MATERIALS AND METHODS Virus and Plant Materials

VTMoV was maintained and propagated in N i c o t i a n a clevelandii A. G r a y . a n d p u rified by clarification with chloroform and n-butanol followed by differential centrifugation as described for red clover nec r o t i c m o s a i c v i r u s ( G o u l d et al., 1981a). When synthesis of viral nucleic acids was to b e s t u d i e d , f o u r f u l l y e x p a n d e d l e a v e s of e a c h p l a n t w e r e i n o c u l a t e d e a c h w i t h 25 480

ds-RNAs IN VTMoV-INFECTED PLANTS ttl of purified VTMoV containing 1 m g/ m l virus and the plants were maintained at 25 ~ under continuous illumination of 10,000 lux.

Estimation of VTMo V in L e a f Tissues Virus concentrations of leaf extracts were determined by enzyme-linked immunosorbent assay (ELISA) as described by Chu and Francki (1982) using purified VTMoV preparations of known concentrations as standards.

Nucleic Acid Extraction and Purification RNA was isolated from purified VTMoV by sodium dodecylsulphate (SDS) and phenol extraction as described by Gould (1981). To isolate nucleic acids from leaf material, tissue was powdered in liquid nitrogen and extracted with SDS and phenol (Gould and Francki, 1981). To isolate virus-specific ds-RNA, nucleic acid preparations from leaf material harvested 10 to 14 days after infection were f u r t h e r purified by cetyltrimethylammonium bromide precipitation (Ralph and Bellamy, 1964) and then fractionated by adding LiC1 to 2 M. The ds-nucleic acids were precipitated with ethanol from the LiCIsoluble fraction and subjected to two cycles of chromatography on CF 11 cellulose columns (Jackson et al., 1971). The preparations were heated at 70 ~ for 3 min before loading onto the columns. Amounts of nucleic acids were estimated spectrophotometrically by assuming the ~260L'0"*~nmof 25 for ss-RNA and 20 for ds-RNA (Gould and Francki, 1981).

In Vivo Labelling of L e a f R N A Leaf strips were infiltrated and labelled as described by Rezaian et al. (1976). To each gram of leaf material was added 20 #Ci of [14C]uridine (529 mCi/mmol) which was then incubated at 25 ~ and 10,000 lux for 6 hr. The tissues were rinsed and the nucleic acids isolated as described above.

Enzymatic Digestion of Nucleic Acids T o digest RNA, nucleic acid preparations containing 2-5 tLg RNA in 200 #1 of

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0.01• SSC (SSC = 0.15 M NaC1 and 0.015 M sodium citrate, pH 7) were incubated with 1 #g/ml of pancreatic ribonuclease A (RNase) for 60 min at 37 ~ To digest ssRNA without affecting ds-RNA, the preparations were incubated as described above but in the presence of 2• SSC (H at t a and Francki, 1978). S1 nuclease (50 units/ml) in the presence of 0.3 M NaC1 was used for the digestion of all ss-nucleic acids (Gould, 1981) and deoxyribonuclease I (DNase) (10 ttg/ml) to digest DNA (Gould and Francki, 1981). Enzyme t r e a t m e n t was stopped by incubation with proteinase K (20 t~g/ml) at 37 ~ for 30 min followed by phenol extraction. Alkaline digestion of RNA was done by incubation in 0.3 M NaOH at 37 ~ for 60 min.

Gel Electrophoresis of R N A RNA preparations were electrophoresed either in 3% polyacrylamide gels in TBE buffer (10.8 g Tris, 5.5 g boric acid, and 0.93 g EDTA in 1 liter of water), pH 8.3, and 7 M urea (Gould, 1981) or in 1.5% agarose gels in TBE buffer (Mossop and Francki, 1977). When required, RNA preparations were heated at 80 ~ for 75 sec in a mixture of 50% formamide and 6% formaldehyde and electrophoresed in 3% polyacrylamide gels in 20 mM H E P E S - N a O H buffer, 1 mM EDTA, pH 7.8, containing 6% formaldehyde (Kiefer et al., 1982). Gels were stained with toluidine blue or when required with acridine orange for examination under ultraviolet light (McMaster and Carmichael, 1977). RNAs labelled with [14C]uridine were detected by fluorography (Bonner and Laskey, 1974). The molecular weights of ds-RNAs were estimated by comparing their electrophoretic mobilities with those of the 10 genomic ds-RNA segments of Fiji disease virus (FDV) (Reddy et al., 1975).

Preparation of s2P-Labelled, VTMo V R N A Specific Probes RNAs 1, 2, and 3 of VTMoV were purified by one cycle of sucrose density gradient centrifugation (Gould, 1981) and two cycles of polyacrylamide gel electrophoresis

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CHU, FRANCKI, AND RANDLES

in the presence of 7 M u r e a as described above. P r o b e s for detecting RNAs with nucleotide sequences c o m p l e m e n t a r y to VTMoV RNA 1 and to RNAs 2 and 3 (negative sense RNAs) were p r e p a r e d by in vitro labelling of RNA 1 and 2 f r a g m e n t s at t h e i r 5' ends with 32p essentially as described by Haseloff and Symons (1981). The RNA prepa r a t i o n s were f r a g m e n t e d by h e a t i n g at 90 ~ for 10 min in 45 m M NaHCO3, 5 m M Na2C03, and 0.05 m M EDTA, p H 9.0. The f r a g m e n t s were then incubated with 4 units of T4 polynucleotide kinase and 250 tLCi [~/32P]ATP in 20 m M Tris-HC1, 10 m M MgC12, 10 m M dithiothreitol, 1 m M spermidine, and 5% (v/v) glycerol, p H 9.0, at 37 ~ for 30 min, a f t e r which they were isolated by elution t h r o u g h a Sephadex G 50 column. P r o b e s for detecting RNAs with nucleotide sequences contained in VTMoV RNA 1 and RNAs 2 and 3 (positive sense RNAs) were p r e p a r e d by in vitro synthesis of c o m p l e m e n t a r y DNAs (cDNAs) to RNA 1 and 3 with avian myeloblastosis virus reverse transcriptase. Synthesis of RNA 1specific cDNA was done by the r a n d o m priming m e t h o d (Taylor et aL, 1976) as described by Gould and Symons (1977). F o r synthesis of cDNA specific to both RNA 2 and 3, p h o s p h a t a s e - t r e a t e d RNA 3 was polyadenylated, p r i m e d with oligo-(dT10), and then t r a n s c r i b e d (Palukaitis and Symons, 1978). F o r both procedures, [a~2P]dCTP was used as the labelled substrate.

Detection of VTMo V-Specific Nucleic Acids by Blot Hybridization Native nucleic acid samples (3-5 #g) were electrophoresed on 1.5% agarose gel slabs (1.5-mm thick) in TBE buffer and t h e n den a t u r e d with 50 m M N a O H for 20 min (A1wine et al., 1977; Zelcer et al., 1981). Nucleic acid samples d e n a t u r e d by h e a t i n g in f o r m a m i d e and f o r m a l d e h y d e were elect r o p h o r e s e d on 1.75% agarose gels in the presence of 6% f o r m a l d e h y d e as described above and the nucleic acids were t r a n s f e r r e d to nitrocellulose p a p e r (Thomas, 1980). Baking of the sheets, p r e h y b r i d i zation, hybridization, and w a s h i n g were as described by Kiefer et al. (1982).

Electron Microscopy ds-RNA was added to 100 m M T r i s - H C 1 , p H 8.5, containing 10 m M E D T A and 0.1 m g / m l of c y t o c h r o m e C (hyperphase) to a c o n c e n t r a t i o n of a b o u t 1 # g / m l and h e a t e d at 50 ~ for 3 min. T w e n t y - m i c r o l i t e r samples of the m i x t u r e were spread on a hypophase of 50 m M Tris-HC1, p H 8.5, containing 5 m M E D T A at 50 ~ The films were picked up on specimen grids, stained in u r a n y l acetate, r o t a r y shadowed, and exa m i n e d in a J E M CX electron microscope as described by Randles and H a t t a (1979).

Isolation and Ligation of VTMo V R N A 3 L i n e a r viroid-like RNA 3 of VTMoV was isolated by cutting out the corresponding band f r o m polyacrylamide gels electrophoresed in the presence of u r e a as described above. The pieces of polyacrylamide gel were embedded in m o l t e n 2% agarose buffered with 50 m M Tris, 33 m M sodium acetate, 2 m M EDTA, p H 8.3, and c o n t a i n i n g 1 ttg/ml ethidium bromide. The agarose gel was electrophoresed for 70 min at 60 mA and 70 V a t 4 ~ The RNA 3 band was detected by fluorescence in ultraviolet light, excised, and suspended in about 4 vol of 0 . 1 M sodium acetate, 1% SDS, and 1 m M EDTA. The agarose was m e l t e d rapidly in a microwave oven and e x t r a c t e d with an equal volume of w a t e r - s a t u r a t e d phenol. The m i x t u r e was agitated and kept at 60 ~ for 10 min, chilled to 0 ~ and centrifuged. The RNA was p r e c i p i t a t e d f r o m the buffer phase with 3 vol ethanol, washed in ethanol, dried u n d e r vacuum, and dissolved in water. Ligation of RNA 3 was done by the procedure of Snopek et al. (1976). Reaction m i x t u r e s of 10 #l each were incubated for 1 h r at 37 ~ T h e y contained 50 m M Tris, p H 7.4, 10 m M MgC12, 1 m M ATP; 20 m M dithiothreitol, 0.5 #g RNA, and 5 units of T4 RNA ligase (P-L Biochemicals Inc., Milwaukee, Wisc.). Reaction was stopped by adding 10 #1 10% SDS and h e a t i n g at 45 ~ for 10 min. Glycerol was added to 10% and the RNA was analysed by electrophoresis in polyacrylamide gels in the presence of urea.

ds-RNAs IN V T M o V - I N F E C T E D P L A N T S

RESULTS

Multiplication of VTMo V in Inoculated and Systemically Infected Leaves of N. clevelandii Leaves of N. clevelandii plants inoculated with VTMoV developed detectable lesions after about 4 days. Later, the lesions became necrotic, and 16 days after inoculation, the leaves dried up completely. Systemic symptoms were first observed about 6 days after inoculation. VTMoV was detected by ELISA in the inoculated leaves soon after the first appearance of lesions and increased in concentration for at least 9 days reaching a final concentrations of about 3 m g / g of fresh tissue (Fig. 1). The virus was first detected in the systemically infected leaves about 6 days after inoculation and the virus concentration increased for at least a f u r t h e r week reaching a final concentration of about 11 m g/ g of fresh tissue (Fig. 1). 12 w Go Go I--

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FIG. 1. Multiplication of VTMoV in inoculated (O) and systemically infected (O) leaves of Nicotiana clevelandii. Samples of leaf discs (5 m m in diameter) were collected f r o m each g r o u p of three p l a n t s at each time of s a m p l i n g and extracted in 20 vol of phosphate-buffered saline containing 0.05% Tween 20 and 2% polyvinylpyrrolidone. Virus content was determined by E L I S A on fivefold dilutions of the e x t r a c t using similar dilutions of purified virus as s t a n d a r d s (Chu and Francki, 1982).

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Accumulation of VTMo V-Specific R N A s in Infected Leaves N. clevelandii leaves inoculated with VTMoV were harvested at the time of inoculation and every 2 or 3 days t h e r e a f t e r for 16 days. Nucleic acid was extracted from the tissue samples and subjected to polyacrylamide gel electrophoresis in the presence of 7 M urea after incubation in 2• SSC only (Fig. 2A) and after incubation with RNase A in the presence of 2• SSC (Fig. 2B). Nucleic acid preparations from leaves harvested at the time of inoculation (healthy leaves) resolved into nine readily detectable bands (Fig. 2A, track a);//1 and //2 presumably being the 26 S and 18 S RNAs from cytoplasmic ribosomes; Ha and H4, the 23 S and 16 S RNAs from chloroplast ribosomes;//5, He, and/-/7, RNAs of unknown function; H8 the 5 S RNA; and H~ the 4 S tRNA. In nucleic acid preparations harvested as early as 2 days after inoculation when no viral antigen was detected by ELISA (Fig. 1), two virus-specific RNAs were already clearly present (V2 and V3 in Fig. 2A, track b). These two RNAs coelectrophoresed with the viroid-like RNA 2 and 3 isolated from preparations of VTMoV (Randles et al., 1981). Nucleic acid preparations from tissues harvested subsequently contained more virus-specific RNA 2 and 3 (Fig. 2A). Virus-specific RNA Vy was detected between 4 and 6 days after inoculation and the detection of RNAs Vx, and Vx,,was complicated by their electrophoretic mobilities being similar to t h a t of 18 S rRNA (//2 in Fig. 2A). However, when the ss-RNAs were digested from the nucleic acid preparations with RNase in the presence of 2• SSC, it can be seen t h a t the virus-specific RNA Vx,was detected in preparations from leaf tissues harvested as early as 2-4 days after inoculation. Later Vx- was also detected and both Vx,and Vxincreased in concentration t hereaft er (Fig. 2B). The resistance of RNAs Vx,and Vx-to RNase indicates that they are ds-RNAs. On the other hand, RNA Vy appears to be single-stranded because it was not detected in RNase-treated preparations (Fig. 2B). Subsequent experiments (data not

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FIG. 2. S y n t h e s i s of VTMoV-specific R N A s in inoculated leaves of Nicotiana clevelandii a t v a r i o u s t i m e s a f t e r inoculation. P l a n t s were s a m p l e d as described in Fig. 1 a n d t h e e x t r a c t e d nucleic acids were electrophoresed in 3% p o l y a c r y l a m i d e gels in t h e p r e s e n c e of 7 M u r e a a n d s t a i n e d in toluidine blue. P r i o r to electrophoresis, aliquots of t h e nucleic acids (each c o r r e s p o n d i n g to t h a t e x t r a c t e d f r o m 20 m g of leaf t i s s u e ) were i n c u b a t e d in 2• SSC at 37 ~ for 1 h r (A) a n d u n d e r s i m i l a r conditions b u t in t h e presence of 1 ttg/ml R N a s e (B). Nucleic acids f r o m h e a l t h y leaves were electrophoresed in t r a c k s a a n d t h o s e f r o m leaves infected w i t h VTMoV for 2, 4, 6, 8, 10, 13, a n d 16 d a y s in t r a c k s b - h , respectively. Host-specific R N A s a r e labelled w i t h t h e l e t t e r s H on t h e left a n d virus-specific R N A s labelled w i t h t h e l e t t e r s V on t h e r i g h t (see t e x t for details).

presented) established t h a t the electrophoretic mobility of RNA Vy is indistinguishable from t h a t of RNA lb, a minor component usually detected in VTMoV RNA preparations (Randles et al., 1981). RNAs Vx, and Vx,,were readily labelled with [ltC]uridine. They were stable to RNase in 2• SSC but not in 0.01• SSC. Furthermore, they were unaffected by incubation with $1 nuclease or DNase I but were digested with KOH (data not presented). These experiments confirmed t h a t Vx,and Vx,,are both ds-RNAs. With time after inoculation, a number of other changes in both host-specific and virus-specific RNA concentrations can be deduced from data presented in Fig. 2. They are as follows:

(1) A decrease in the concentration of ribosomal RNAs with time after inoculation, especially those of the chloroplast ribosomes (//3 a n d / / 4 in Fig. 2A). (2) A decrease in relative concentrations of the host-specific RNAs//5 a n d / / 6 with time after inoculation (Fig. 2A). As these RNAs decreased in concentration, the virus-specific RNAs Vx,, Vx,,, Vy, V2, and V3 all increased in concentration (Fig. 2A and B). (3) When the VTMoV viroid-like RNA 2 and 3 (V2 and V~ in Fig. 2A) were first detected two days after inoculation, the concentration of RNA 3 appears to have been higher than that of RNA 2. A similar ratio was maintained at 4 days after inoculation but at 6 days and thereafter, the

ds-RNAs IN VTMoV-INFECTED PLANTS RNA 2 concentration was higher than RNA 3. A similar experiment done using systemically infected N. clevelandii leaves gave essentially similar results. However, the virus-specific RNAs were detected a little later after inoculation, the RNAs 2 and 3 about 6 days after inoculation and RNAs Vx,, Vx,,, and Vy about 8 days after inoculation (data not presented).

Purification and Characterization VTMo V-Specific ds-RNA

of

VTMoV-specific ds-RNA Vx, and Vx,,purified by only one cycle of chromatography on CF 11 cellulose, yielded preparations which were still slightly contaminated by a number of other nucleic acids (Fig. 3A, track d). However, after a second chromatographic step, the ds-RNA was free of detectable amounts of other RNA species (Fig. 3B, track c). Electrophoresis in polyacrylamide gels in the presence of 7 M u r e a separated the ds-RNA into two closely migrating bands, Vx,usually contained at least five times as much material as Vx,.. These ds-RNAs had indistinguishable electrophoretic characteristics from those detected in nucleic acid preparations from VTMoV-infected N. clevelandii leaves (compare tracks a and d in Fig. 3A). A better separation of the two ds-RNA fractions was obtained by electrophoresis in agarose gels as shown in Fig. 3C (track b). Under these conditions, the ds-RNAs Vx, and Vx,.migrated as two separate diffuse zones behind the VTMoV ss-RNA 1 (V1 in Fig. 3C, track c). When electrophoresed with FDV dsRNA as marker molecules, the bands of Vx, and V• were much more diffuse than the ds-RNA components of FDV (data not presented). Using the molecular weights of the FDV RNA components (Reddy et al., 1975) as standards, it was calculated t h a t the Mr of Vx,iS about 3.6 • l0 Gand t h a t of V• about 2.8 • 106. Because the longest segment of FDV RNA has only Mr of 2.93 • 106 (Reddy et al., 1975), the size estimate of Vx,is only approximate. As expected, the purified ds-RNA fractions Vx,a n d V~,were unaffected when in-

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cubated with $1 nuclease, DNase, and RNase A in 2 • SSC but were digested when incubated in the RNase in 0,01 • SSC (data not presented). The ds-RNA (a mixture of Vx, and Vx,,) was found to have a melting curve characteristic of doublestranded nucleic acid with a Tm of about 82 ~ in 0,01• SSC (data not presented). Examination of the ds-RNA preparations by electron microscopy revealed the presence of only long linear molecules (data not presented). Whether untreated or suspended in a mixture of 50% formamide and 6% formaldehyde, the virus-specific ds-RNA (Vz and Vx~) migrated ahead of VTMoV RNA 1 when electrophoresed in polyacrylamide gels in the presence of 6% formaldehyde (data not presented). The ds-RNA bands fluoresced bright green whereas those of VTMoV RNA were red when stained with acridine orange and viewed under ultraviolet light. After heating at 80 ~ for 75 sec in the presence of formamide and formaldehyde, the electrophoretic and fluorescent properties of VTMoV RNA remained unchanged but the ds-RNA migrated as a diffuse band at about the same rate as VTMoV RNA 1 and fluoresced red (data not presented) indicating that it had been melted.

Hybridization Analysis of VTMo V-Specific RNAs Preparations of VTMoV RNA, purified virus-specific ds-RNA and nucleic acid preparations from healthy and VTMoVinfected N. clevelandii leaves were heated in formamide and formaldehyde and electrophoresed in agarose gels. The results presented in Fig. 4 show that none of the molecular probes detected any kind of material in nucleic acid preparations from healthy leaves (Fig. 4, tracks e, i, and m). This established that the probes were uncontaminated by any host-specific nucleic acids. As expected, VTMoV RNA (controls) did not hybridize with either probes specific for negative stranded RNAs (Fig. 4, tracks d and h). However, the cDNA 1 probe when hybridized to VTMoV RNA, detected a continuum of material with electrophoretic mobility ranging from that

CHU, FRANCKI, AND RANDLES

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FIG. 3. Purification and gel analysis of VTMoV-specific ds-RNA. (A) Nucleic acids subjected to one cycle of CF 11 cellulose column chromatography were electrophoresed on 3% polyacrylamide gel in the presence of 7 M urea showing the 2 M LiCl-soluble nucleic acid prior to chromatography (track a), that from the column eluting with 65% STE (50 mM Tris-HC1, 100 mM NaC1, 1 mM EDTA, pH 6.9)-35% ethanol (track b), 85% STE-15% ethanol (track c) and 100% STE (track d). Symbols for the various nucleic acid components are as in Fig. 2. V1 refers to VTMoV RNA 1 and the presence of DNA is also indicated. (B) Polyacrylamide gel electrophoresis (as described in A) of ds-RNA isolated by CF 11 cellulose chromatography (Panel A, track d) and rechromatographed under similar conditions. Marker VTMoV RNA isolated from purified virus was electrophoresed in track a; and the 85% STE-15% ethanol and 100% STE fractions of the ds-RNA preparation in tracks b and c, respectively. The five VTMoV RNA components are labelled V,, V,a, Vlb, V~, and V3 on the left. (C) Analysis of VTMoV-specific ds-RNA in agarose gel. RNA samples were electrophoresed in 1.5% agarose buffered in TBE. E. coli rRNA and VTMoV RNA were electrophoresed as markers in tracks a and c respectively and the ds-RNA in track b. Arrows marked 23 S and 16 S refer to the two species of E. coli rRNA; other symbols are as in other panels.

o f R N A 1 t o t h a t o f R N A 2 ( F i g . 4, t r a c k 1). W e c o n c l u d e t h a t t h i s m a t e r i a l c o n s i s t s o f i n t a c t V T M o V R N A 1, l a , a n d l b ( R a n d l e s e t al., 1981) a n d p a r t i a l l y d e g r a d e d molecules of these RNA species. The cDNA

3 probe detected at least five bands from p r e p a r a t i o n s o f V T M o V R N A ( F i g . 4, t r a c k p) w h i c h c o r r e s p o n d e d t o p o s i t i o n s e x pected for the monomers, dimers, trimers, tetramers, and pentamers of RNAs 2 and/

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FIG. 4. Blot-hybridization analysis of melted VTMoV RNA, VTMoV-specifieds-RNA, and nucleic acid preparations from healthy and VTMoV-infected Nicotiana clevelandii leaves. Nucleic acid samples (1.5 ttg of the ds-RNA and 2 #g of the others) were melted by heating in formamideformaldehyde and electrophoresed in 1.75% agarose gels in HEPES buffer containingformaldehyde. After electrophoresis, the nucleic acids were transferred to nitrocellulose paper which was autoradiographed after being blot hybridized with the following probes: 1 • l0 s cpm of ~2P-labelled VTMoV RNA 1 (tracks a-d); 1 • 10s cpm of 32P-labelled VTMoV RNA 2 (tracks e-h); 500,000 cpm of s2P-labelled cDNA 1 (tracks i-l); and 100,000 cpm of s2P-labelled cDNA 3 (tracks m-p). The nucleic acid preparations were loaded to the gels as follows: nucleic acids from healthy leaves in tracks a, e, i and m; that from VTMoV-infected leaves in tracks b, f, j, and n; ds-RNA in tracks c, g, k, and o; and VTMoV RNA in tracks d, h, l, and p. The electrophoretic mobilities of VTMoVRNA components (V1 and V2+s)are indicated as determined from a stained gel Arrowheads in track p point to the series of bands corresponding to polymers of VTMoV RNAs 2 and 3. Some of the higher polymers were only observed with difficulty and are not clearly visible in the photograph.

o r 3 a s i n d i c a t e d b y a p l o t of t h e m o b i l i t i e s v e r s u s t h e p o l y m e r i c s t a t e s of t h e s e R N A s ( g r a p h n o t p r e s e n t e d ) . T h e s i g n i f i c a n c e of t h i s p o l y m e r i c s e r i e s of R N A s w i t h b a s e s e q u e n c e s of V T M o V R N A 2 a n d 3 is a t p r e s e n t o b s c u r e . H o w e v e r , it is i n t e r e s t i n g t h a t a s i m i l a r p a t t e r n of R N A b a n d s h a s been detected by s i m i l a r techniques in p r e p a r a t i o n s of t o b a c c o r i n g s p o t v i r u s s a t e l l i t e R N A ( K i e f e r et al., 1982). All the probes detected material separated from the ds-RNA preparation. The RNA 1 and cDNA 1 probes detected bands of s i m i l a r m o b i l i t i e s (Fig. 4, t r a c k s c a n d k). T h e p o s i t i o n of t h e s t r o n g e s t b a n d s corr e s p o n d s to t h a t of V T M o V R N A 1 a n d t h e

w e a k e r o n e s a p p r o x i m a t e l y to v i r u s specific R N A Vy (Fig. 2A). H o w e v e r , t h e R N A 2 and cDNA 3 probes detected different p a t t e r n s of m a t e r i a l s i n t h e p r e p a r a t i o n s of d s - R N A (Fig. 4, t r a c k s g a n d o). W h e r e a s the RNA 2 probe detected mainly high mol e c u l a r w e i g h t m a t e r i a l (Fig. 4, t r a c k g), t h e c D N A 3 p r o b e d e t e c t e d , i n a d d i t i o n to a h i g h e r m o l e c u l a r w e i g h t f r a c t i o n , a series of l o w e r m o l e c u l a r w e i g h t f r a c t i o n s (Fig. 4, t r a c k o) c o r r e s p o n d i n g a p p r o x i m a t e l y to m o n o m e r s , d i m e r s , a n d t e t r a m e r s of V T M o V R N A 2 a n d / o r 3 (Fig. 4, t r a c k o). N u c l e i c acid p r e p a r a t i o n s f r o m V T M o V i n f e c t e d N. c l e v e l a n d i i l e a v e s e l e c t r o p h o -

488

CHU, FRANCKI, AND RANDLES

resed and h y b r i d i z e d w i t h the R N A 1 and c D N A 1 probes produced v e r y s i m i l a r p a t t e r n s except t h a t the one blotted w i t h the c D N A was m o r e intense (Fig. 4, t r a c k s b and j). These p a t t e r n s are also s i m i l a r to t h a t produced by VTMoV RNA p r o b e d with c D N A 1 (Fig. 4, t r a c k 1). This indicates t h a t the infected leaves c o n t a i n e d significant a m o u n t s of R N A 1 and its complem e n t a r y n e g a t i v e s t r a n d as well as s h o r t e r molecules c o n t a i n i n g sequences of R N A 1. W h e n the blots of nucleic acid p r e p a r a t i o n s f r o m VTMoV-infected leaves w e r e hybridized with R N A 2 and c D N A 3 probes, each p a t t e r n differed (Fig. 4, t r a c k s f and n). The R N A 2 p r o b e detected m o r e high m o l e c u l a r w e i g h t m a t e r i a l (Fig. 4, t r a c k f) t h a n the cDNA 3 (Fig. 4, t r a c k n). The large n u m b e r of poorly resolved b a n d s (Fig. 4, t r a c k s f and n) m a k e s i n t e r p r e t a t i o n of the r e s u l t s difficult. I t is especially puzzling w h y only a lightly labelled spot w a s detected in the position expected for R N A s 2 and 3 w h e n p r o b e d with c D N A 3 (Fig. 4, t r a c k n} a l t h o u g h b a n d s of R N A 2 and 3 w e r e readily detected in the nucleic acid p r e p a r a t i o n s when s i m i l a r gels were s t a i n e d with toluidine blue ( d a t a not presented~. H o w e v e r , d a t a in Fig. 4 ( t r a c k s f and n ~ do suggest t h a t in the R N A p r e p a r a t i o n f r o m infected leaves, the n e g a t i v e sense R N A 2-specific molecules a r e l a r g e r t h a n the positive sense ones, as observed also in the purified p r e p a r a t i o n of virusspecific d s - R N A (Fig. 4. t r a c k s g a n d ol. In a n o t h e r e x p e r i m e n t , VTMoV RNA and the virus-specific d s - R N A p r e p a r a t i o n were e l e c t r o p h o r e s e d in a g a r o s e gels without p r i o r melting. One p a r t of the gel was t r e a t e d with mild alkali (see M a t e r i a l s an d Methods l and the R N A was t r a n s f e r r e d to nitrocellulose p a p e r and probed as before (Fig. 5, t r a c k s a-h}. The o t h e r p a r t of the ge] c o n t a i n i n g s i m i l a r s a m p l e s of RNA w a s s t a i n e d w i t h toluidine blue (Fig. 5, t r a c k s i-j). S a m p l e s of VTMoV RNA p r e p a r a t i o n s (controls~ did not contain d e t e c t a b l e a m o u n t s of n e g a t i v e l y s t r a n d e d R N A s corr e s p o n d i n g to e i t h e r RNA 1 or R N A s 2 or 3 (Fig. 5, tracks b and d). H o w e v e r , VTMoV R N A I was detected with the c D N A 1 probe as expected (Fig. 5, t r a c k f), b u t c D N A 3

failed to detect VTMoV R N A s 2 and 3 (Fig. 5, t r a c k h). We suspect t h a t the alkali t r e a t m e n t step m a y h a v e over-digested these low m o l e c u l a r w e i g h t R N A s because the s a m e p r o b e d e t e c t e d R N A s 2 a n d 3 in u n t r e a t e d gels (Fig. 4). This could also have been due to a p r e f e r e n t i a l s e l f - a n n e a l i n g of those highly b a s e p a i r e d s t r u c t u r e s . Results p r e s e n t e d in Fig. 5 also show t h a t w h e n the d s - R N A s a m p l e s w e r e blotted w i t h each of the probes, the f a s t e r mig r a t i n g d s - R N A f r a c t i o n Vx,,(Fig. 5, t r a c k i) hybridized w i t h b o t h VTMoV R N A 1 and c D N A 1 (Fig. 5, t r a c k s a and e) b u t not w i t h the VTMoV R N A 2 or c D N A 3 p r o b e s (Fig. 5, t r a c k s c a n d g). This indicates t h a t Vx.contains b o t h positive and negative nucleotide sequences of VTMoV R N A 1. On the o t h e r hand, the slower m i g r a t i n g dsR N A b a n d Vx, (Fig. 5, t r a c k i) hybridized with both VTMoV R N A 2 and cDNA 3 (Fig. 5, t r a c k s c and g) b u t not w i t h the VTMoV RNA 1 or cDNA 1 p r o b e s (Fig. 5, t r a c k s a and e~. This indicates t h a t Vx,dS-RNA cont a i n s both positive a n d negative nucleotide sequences of VTMoV RNAs 2 and 3. Results p r e s e n t e d in Fig. 5 also indicate t h a t d s - R N A Vx ~with sequences of VTMoV R N A s 2 and 3 } is m o r e h e t e r o g e n e o u s t h a n d s - R N A Vx- ( c o m p a r e t r a c k s a and c. and e and g in Fig. 5}.

Ligation of VTMo V R N A 3 Two o b s e r v a t i o n s s u g g e s t t h a t R N A 3 m a y be a p r e c u r s o r of R N A 2. Firstly, no circular s t r u c t u r e s could be detected in association with virus specific d s - R N A p r e p a r a t i o n s f r o m infected leaves: and secondly, a t e a r l y s t a g e s of infection, the ratio of R N A 2 to RNA 3 was significantly lower t h a n l a t e r on (Fig. 2). F o r the linear, viroid-like R N A 3 to be a p r e c u r s o r of the covalently closed, c i r c u l a r R N A 2, an R N A ligation step would be required. This possibility w a s e s t a b l i s h e d by d e m o n s t r a t i n g t h a t T4 RNA ligase w a s capable of conv e r t i n g R N A 3 to R N A 2 (Fig. 6). DISCUSSION A n u m b e r of VTMoV-specific R N A s were detected in leaves of infected N. clevelandii

ds-RNAs IN VTMoV-INFECTED PLANTS a

b

c

d

e

f

g

h

489

i

j

..

Vx,

,9

Vx-

,9

V1

-----V2+

3

Fro. 5. Blot-hybridization analysis of native VTMoV RNA and VTMoV-specific ds-RNA. Five aliquots of a VTMoV RNA preparation (3 ~g each) and five of VTMoV-specific ds-RNA (2.5 ~g each) were electrophoresed in a slab of 1.5% agarose in TBE buffer. After electrophoresis, a portion of the gel with tracks containing one sample each of ds-RNA and VTMoV RNA was excised and stained with toluidine blue for use as markers (tracks i-j, respectively). The remaining gel was treated with NaOH to partially degrade the RNAs which were then transferred onto nitrocellulose paper (see Materials and Methods). The nitrocellulose was cut into four strips each containing a track of transferred ds-RNA (tracks a, c, e, and g) and one of VTMoV RNA (tracks, b, d, f, and h) and autoradiographed after blot hybridization with the following probes: 800,000 cpm each of 32p_ labelled VTMoV RNA 1 (tracks a and b) and 32p-labelled VTMoV RNA 2 (tracks c and d); 200,000 cpm of 32p-labelled cDNA 1 (tracks e and f); and 100,000 cpm of 32p-labelled cDNA 3 (tracks g and h).

b y e l e c t r o p h o r e s i s of t h e e x t r a c t e d n u c l e i c a c i d s in p o l y a c r y l a m i d e gels. T h i s w a s a c h i e v e d w i t h o u t r e c o u r s e to t h e i n h i b i t i o n of s y n t h e s i s of h o s t p l a n t R N A s b y i n h i b i t o r s s u c h a s a c t i n o m y c i n D. T h e h i g h l e v e l s to w h i c h V T M o V m u l t i p l i e s in t h e p l a n t ( F i g . 1) is p r o b a b l y r e s p o n s i b l e f o r this. T h r e e of t h e v i r u s - s p e c i f i c R N A s d e t e c t e d in l e a v e s w e r e s i m i l a r to t h r e e o f the RNAs isolated from VTMoV particles, t h e s e a r e R N A s l b , 2, a n d 3 ( R a n d l e s et al., 1981; G o u l d , 1981). T h e t w o o t h e r R N A s which became encapsidated (RNAs 1 and l a ) w e r e n o t d e t e c t e d in s t a i n e d o r fluor o g r a p h e d g e l s p r o b a b l y b e c a u s e t h e y do n o t r e a c h h i g h c o n c e n t r a t i o n s in i n f e c t e d

tissues and because they are sufficiently s i m i l a r in size to t h e l e a f r i b o s o m a l R N A s so a s n o t to b e r e s o l v e d f r o m t h e m . However, virus-specific RNAs of about t h e i r s i z e s w e r e d e t e c t e d in l e a f n u c l e i c acid preparations by blot hybridization ( F i g . 4). I n a d d i t i o n to R N A s w h i c h a r e e n c a p sidated by VTMoV, two VTMoV-specific dsR N A s p e c i e s w e r e d e t e c t e d in i n f e c t e d leaves. Blot-hybridization studies indicate t h a t one of t h e s e h a s b a s e s e q u e n c e s c h a r a c t e r i s t i c of R N A 1 ( R N A Vx0 a n d t h e o t h e r of R N A s 2 a n d 3 ( R N A Vx,) ( F i g . 5). R N A Vx- h a s a Mr o f a b o u t 2.8 • l 0 s w h i c h is c l o s e to a v a l u e e x p e c t e d f o r a d u p l e x o f R N A 1 w h i c h h a s a n e s t i m a t e d Mr of a b o u t

490

CHU, FRANCKI, AND RANDLES ab

RNA2 RNA3

FIG. 6. Conversion of linear VTMoV RNA3 to its circular form RNA2 by T4 RNA ligase. Adjacent tracks contain the RNA in reaction mixtures incubated at 37~ for 60 min with (a) and without (b) the RNA ligase. (See Materials and Methods for assay conditions).

1.5 X 106 (Randles et al., 1981; Gould, 1981). F u r t h e r m o r e , b l o t - h y b r i d i z a t i o n experim e n t s indicate t h a t on m e l t i n g R N A V~,, yielded two molecules s i m i l a r in size to R N A 1; one with sequences s i m i l a r to, a n d the o t h e r w i t h sequences c o m p l e m e n t a r y to those of R N A 1 (Fig. 5). R N A Vx, a p p e a r s to be m o r e h e t e r o geneous t h a n R N A Vx,, and h a s a Mr of a b o u t 3.6 X 106 (Fig. 5, t r a c k i). On melting, however, it a p p e a r s to b e h a v e differently f r o m R N A V~ in t h a t it yields high molecular w e i g h t R N A with sequences comp l e m e n t a r y to R N A s 2 and 3 and m u c h s m a l l e r molecules with sequences cont a i n e d in R N A s 2 and 3 (Fig. 4). I t s e e m s t h a t the Vx, d s - R N A molecule m a y consist of a single continuous negative R N A s t r a n d paired to a n u m b e r of positive s t r a n d s of various sizes. The functions of R N A Vx, a n d R N A Vx,, a r e u n k n o w n b u t t h e y m a y be i n t e r m e d i ates in the s y n t h e s i s of R N A s 2 and 3 and R N A 1 respectively. The p r o p e r t i e s of RNA V~, a r e s i m i l a r to those of the replicative f o r m (RF) R N A s of a n u m b e r of viruses w i t h s s - R N A g e n o m e s such as t h a t of tobacco m o s a i c virus (TMV) which a p p e a r s to be a p r e c u r s o r of v i r a l R N A (Jackson

et aL, 1972). Thus, R N A Vx~ m a y be the R F , of VTMoV R N A 1. I f R N A Vx, is a n i n t e r m e d i a t e in the replication of R N A s 2 a n d 3, the process m u s t be complex in t h a t a d s - R N A molecule, s o m e t h i n g like 15-times longer t h a n a duplex of R N A s 2 or 3, would be involved. I t is i n t e r e s t i n g to note t h a t the tobacco r i n g s p o t virus satellite R N A (TRSV s a t RNA) which is a s m a l l linear molecule s i m i l a r to t h a t of R N A 3, a p p e a r s to induce the s y n t h e s i s of a n u m b e r of high molecu l a r w e i g h t d s - R N A s in infected p l a n t s (Sogo and Schneider, 1982; K i e f e r et al., 1982). The l a r g e s t s t r u c t u r e is a b o u t 12times the expected size of a TRSV s a t - R N A duplex. An i n t r i c a t e m e c h a n i s m of its inv o l v e m e n t in TRSV s a t - R N A synthesis has been s u g g e s t e d (Bruening, 1981; Sogo and Schneider, 1982). T h e r e is also evidence recently r e p o r t e d b y B r a n c h et al. (1981) t h a t m u l t i m e r i c viroid replication i n t e r m e d i a t e s are p r e s e n t in p l a n t s infected with p o t a t o spindle t u b e r viroid (PSTV). With the l i m i t e d d a t a a v a i l a b l e at present, it is p r e m a t u r e to speculate in a n y detail on a possible m e c h a n i s m of VTMoV R N A s 2 a n d 3 synthesis. N e v e r t h e l e s s , it s e e m s likely t h a t the viroid-like R N A 3 of VTMoV r e p l i c a t e s via some f o r m of a rolling circle m e c h a n i s m to g e n e r a t e a high molecular w e i g h t negative sense RNA used as a t e m p l a t e for the s y n t h e s i s of the viroid-like RNA. This replicative i n t e r m e diate (RNA V~,) m a y then g e n e r a t e copies of RNA 3 which are in t u r n c o n v e r t e d to molecules of R N A 2. Such a m e c h a n i s m would require an R N A - d e p e n d e n t RNA p o l y m e r a s e a n d an R N A ligase. We h a v e recently detected an R N A - d e p e n d e n t R N A p o l y m e r a s e in VTMoV-infected leaves which is c a p a b l e of s y n t h e s i s i n g a ds-RNA w i t h p r o p e r t i e s of R N A Vx, (J. Rohozinski, P. W. G. Chu, a n d R. I. B. F r a n c k i unpublished results). A l t h o u g h t h e r e is no evidence for the p r e s e n c e of an R N A ligase in infected leaves, we have shown t h a t RNA 3 can be c o n v e r t e d to R N A 2 by T4 RNA ligase (Fig. 6). S i m i l a r ligase enzymes have been shown to be p r e s e n t in p l a n t tissues and are c a p a b l e of ligating s m a l l linear R N A molecules, including those of PSTV,

ds-RNAs IN VTMoV-INFECTED PLANTS

into covalently closed circles (Konarska et aL, 1981; Branch et al., 1982). ACKNOWLEDGMENTS We thank Dr. G. Bruening for introducing us to the blot-hybridization technique, for access to unpublished work, and for helpful discussions; Drs. R. H. Symons and J. Haseloff for generous gifts of enzymes and labelled nucleotide triphosphates and their helpful advice; Mrs. L. Wichman for art work; Mr. B. A. Palk for photography; and Mr. D. Talfourd for supply and maintenance of plants. This project was generously supported by a grant from the Australian Research Grants Committee. REFERENCES ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paperand hybridization with DNA probes. Proc. Nat. Acad. Sci. USA 74, 5350-5354. BONNER, W. M., and LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gets. Eur. J. Biochem. 46, 83-88. BRANCH, A. D., ROBERTSON, H. D., and DICKSON, E. (1981). Longer~than-unit-length viroid minus strands are present in RNA from infected plants. Proc. Nat. Acad Sci. USA 78, 6381-6385. BRANCH, A. D., ROBERTSON, H. D., GREER, C., GEGENHEIMER, P., PEEBLES, C., and ABELSON,T. (1982). Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ. Science 217,11471149. BRUENING, G. (1981). Biochemistry of plant viruses. In "Biochemistry of Plants" (P. K. Stumpf and E. E. Corm, eds.), Vol. 6, pp. 571-631. Academic Press, New York. CHU, P. W. G., and FRANCKI,R. I. B. (1982). Detection of lettuce necrotic yellows virus by an enzyme-linked immunosorbent assay in plant hosts and the insect vector. Ann. Appl. Biol. 100, 149-156. GOULD, A. R. (1981). Studies on encapsidated viroidlike RNA. II. Purification and characterization of a viroid-like RNA associated with velvet tobacco mottle virus (VTMoV). Virology 108, 123-133. GOULD, A. R., and FRANCKI, R. I. B. (1981). Immunochemical detection of ds-RNA in healthy and virus-infected plants and specific detection of viral ds-RNA by hybridization to labelled complementary DNA. ,]. Virol. Methods 2, 277-286. GOULD, A. R., FRANCKI,R. I. B., HATTA, T., and HOLLINGS, M. (1981a). The bipartite genome of red clover necrotic mosaic virus. Virology 108, 499-506. GOULD, A. R., FRANCKI, R. I. B., and RANDLES, J. W. (1981b). Studies on encapsidated viroid-like RNA.

491

IV. Requirement for infectivity and specificity of two RNA components from velvet tobacco mottle virus. Virology 110, 420-426. GOULD, A. R., and SYMONS, R. H. (1977). Determination of the sequence homology between the four RNA species of cucumber mosaic virus by hybridization analysis with complementary DNA. Nucleic Acids Res. 4, 3787-3802. HASELOFF, J., and SYMONS, R. H. (1981). Chrysanthemum stunt viroid: Primary sequence and secondary structure. Nucleic Acids Res. 9, 2741-2752. HATTA, T., and FRANCKI, R. I. B. (1978). Enzyme cytochemical identification of single-stranded and double-stranded RNAs in virus-infected plant and insect cells. Virology 88, 105-117. JACKSON,A. 0., MITCHELL,D. M., and SIEGEL,A. (1971). Replication of tobacco mosaic virus. I. Isolation and characterization of double-stranded forms of ribonucleic acid. Virology 45, 182-191. JACKSON,A. 0., ZAITLIN, M., SIEGEL, A., and FRANCKI, R. I. B. (1972). Replication of tobacco mosaic virus. III. Viral RNA metabolism in separated leaf cells. Virology 48, 655-665. KIEFER, M. C., DAUBERT, S. D., SCHNEIDER, I. R., and BRUENING, G. (1982). Multimeric forms of satellite of tobacco ringspot virus RNA. Virology 121, 262273. KONARSKA, M., FILIPOWICZ, W., DOMDEY, H., and GROSS, H. J. (1981). Formation of a 2'-phosphomonoester, 3',5'-phosphodiester linkage by a novel RNA ligase in wheat germ. Nature (London) 293, 112-116. MCMASTER, G. K., and CARMICHAEL, G. G. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Nat. Aca~ Sci. USA 74, 4835-4838. MossoP, D. W., and FRANCKI, R. I. B. (1977). Association of RNA 3 with aphid transmission of cucumber mosaic virus. Virology 81, 177-181. PALUKAITIS, P., and SYMONS, R. H. (1978). Synthesis and characterization of a complementary DNA probe for chrysanthemum stunt viroid. F E B S Letters 92, 268-272. RALPH, R. K., and BELLAMY, A. R. (1964). Isolation and purification of undegraded ribonucleic acids. Biochim. Biophya Acta 87, 9-16. RANDLES, J. W., DAVIES, C., HATTA, T., GOULD, A. R., and FRANCKI, R. I. B. (1981). Studies on encapsidated viroid-like RNA. I. Characterization of velvet tobacco mottle virus. Virology 108, 111-122. RANDLES, J. W., and HATTA, T. (1979). Circularity of the ribonucleic acids associated with cadang-cadang disease. Virology 96, 47-53. REDDY, D. V. R., BOCCARDO, G., OUTRIDGE, R., TEAKLE, D. S., and BLACK,L. M. (1975). Electrophoretic separation of dsRNA genome segments from Fiji

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disease and maize rough dwarf viruses. Virology 63, 287-291. REZAIAN, M. A., FRANCKI,R. I. B., CHU, P. W. G., and HATTA, T. (1976). Replication of tobacco ringspot virus. III. Site of virus synthesis in cucumber cotyledon cells. Virology 74, 481-488. SoGo, J. M., and SCHNEIDER, I. R. (1982). Electron microscopy of double-stranded nucleic acids found in tissue infected with satellite of tobacco ringspot virus. Virology ll7, 401-415.

SNOPEK,T. J., SUGINO, A., AGARWAL,K. L., and COZZARELLI, N. R. (1976). Catalysis of DNA joining by

bacteriophage T4 RNA ligase. Biochem. Biophys. Res. Commun. 68, 417-424. TAYLOR, J. M., ILLMENSEE,R., and SUMMERS,J. (1976). Efficient transcription of RNA into DNA by avian sarcoma virus polymerase. Biochim. Biophys. Acta 442, 324-330. THOMAS, P. S. (1980). Hybridization of denatured RNA and smaIl DNA fragments transferred to nitrocellulose. Proc. Nat. Acad. Sci. USA 77, 5201-5205. ZELCER, A., WEABER, K. F., BALAZS,E., and ZAITLIN, M. (1981). The detection and characterization of viral-related double-stranded RNAs in tobacco mosaic virus-infected plants. Virology 113, 417-427.