Low-molecular-weight RNAs associated with tobacco ringspot virus are satellites

VIROLOCY114,534-541(1981)

Low-Molecular-Weight

RNAs Associated with Tobacco Are Satellites’

M. A. REZAIAN*

AND

Ringspot Virus

A. 0. JACKSONY

*Department of Biology, Shiraz University, Shiraz, Iran, and tDepartment of Botany and Plant Pathology, Purdue University, W&t Lqfayette, Indiana 17207 Received May 15, 19sl; accepted June 24, 1981 The low-molecular-weight RNA associated with an isolate of tobacco ringspot virus [TRSV(F)] lacks polyadenylic acid [poly(A)] sequences. Complementary DNA (cDNA) specific to this RNA was made with an oligo(dT) primer after tailing the RNA with poly(A). However, a significant amount of cDNA was made only when the RNA was treated with alkaline phosphatase prior to tailing. Hybridization of RNA in solution and by a Northern blot technique showed that there was no detectable nucleotide sequence homology between TRSV(F) RNA and two of the low-molecular-weight RNAs associated with this virus. These associated RNAs are therefore, satellites of TRSV(F). The relationship of these satellites to a previously reported satellite of TRSV is discussed. INTRODUCTION

Tobacco ringspot virus (TRSV) is multicomponent and contains two species of RNA with molecular weights of about 1.4 x lo6 (RNA I) and 2.3 X lo6 daltons (RNA II) (Rezaian and Francki, 1973). Apart from the viral genome, low-molecular-weight RNA species have also been found in particles of this virus. A satellite RNA (sRNA) of 77,000 to 125,000 daltons which depends on TRSV for replication has been characterized (Schneider, 1976). Between 12 to 25 uniform strands of sRNA are packed in each satellite virion. More recently, two major and a minor species of RNA have been detected in another isolate of TRSV [TRSV(F)-Rezaian, 19801. These TRSV(F)-associated RNAs have molecular weights of approximately 114,000, 245,000, and 367,000 daltons. When analyzed by electrophoresis in 5% polyacrylamide, the 245,060-dalton component is resolved into three closely migrating species (Rezaian, 1980). i Journal paper No. 8553 of the Purdue University Agricultural Experiment Station. * To whom reprint requests should be addressed.

0042~6822/81/140534-08$02.00/0 Copyright All rights

Q 1981 by Academic Press, Inc. of reproduction in any form reserved.

Based on analogy with the defective interfering (DI) particles of animal viruses (Huang, 1977), Schneider (1976) has examined the properties of the sRNA of TRSV. This satellite resembles DI particles in having a protein capsid identical to the helper virus, but unlike DI particles, sRNA is not a deletion mutant of the virus genome (Schneider, 1976). As yet there is no conclusive evidence on the relationship between the nucleotide sequences of the sRNA and those of the helper TRSV (Schneider, 1976). The origin of TRSV(F)-associated RNA is also unknown (Rezaian, 1930). This study was designed to examine whether TRSV(F)-associated RNA species were derived from the viral genome. The results demonstrate that two of these RNAs have no detectable nucleotide sequences in common with the viral RNAs and hence are satellites of TRSV(F). MATERIALS

AND METHODS

Preparation of TRW and the viral RNA species. The culture line of TRSV referred to as TRSV(F) in a previous study (Rezaian, 1980), was propagated and purified 534

TRW

SATELLITE

as described before (Rezaian and Francki, 1973). RNA prepared from TRSV(F) (Rezaian, 1980) was suspended in distilled water and centrifuged through linear-log sucrose density gradients buffered in 100 mlM NaCl, 10 m&f Tris-HCl, pH 7.5 (Brakke and Van Pelt, 1970). Gradients containing 100 pg of RNA were centrifuged at 6” for 9 hr in a Beckman SW41 rotor at 40,000 rpm ‘and fractionated with an ISCO apparatus. Fractions containing either TRSV(F)associated RNA or TRSV(F) RNA were pooled separately. The separated RNA was precipitated by adding 1.1 vol of isopropanol, freezing at -70” in a polyallomer centrifuge tube, and then slowly thawing at room temperature. The precipitates were collected by ultracentrifugation for 1 hr at 40,000 rpm in the SW41 rotor, dried under a stream of nitrogen gas, and suspended in distilled water. Oligo(dT)-cellulosechromatography. Columns (6 mm in diameter) were packed with 0.1 g oligo(dT)-cellulose (P. L. Biochemicals) and used to separate polyadenylated RNA from nonpolyadenylated RNA as described by Mayo et al. (1979). The RNA in the fractions was precipitated by isopropanol and recovered as described above. Agarose gel electrophoresis. Horizontal slab gels contained 1.2% agarose and 5 mlM methylmercury hydroxide in electrophoresis buffer (50 mM boric acid, 5 mM Na2B407, 1 mM Naz EDTA, pH 8.2) as described by Bailey and Davidson (1976). Samples of RNA in electrophoresis buffer containing 10 mM methylmercury hydroxide and 10% glycerol were applied to sample slots and covered with paraffin melted at 60”. The entire surface of each gel was then covered with Saran wrap and electrophoresed at 0.7 V.cm-‘. The gels were subsequently soaked in 0.1% ethidium bromide in 0.5 M ammonium acetate and RNA bands were located by ultraviolet illumination (Bailey and Davidson, 1976). Treatment of RNA with alkaline phosphatase. Ribonuclease contamination in bacterial alkaline phosphatase (Sigma grade VII) was eliminated by treatment

RNAs

535

with diethylpyrocarbonate as outlined by Efstratiadis et al. (1977). Alkaline phosphatase activity was assayed optically using pnitrophenyl phosphate as a substrate (Worthington enzyme manual, 1972, p. 73). TRSV(F)-associated RNA (20 Bg) was incubated with 2 units of alkaline phosphatase in a volume of 100 ~1 containing 0.1 M Tris-HCl, pH 9, at 37”, for 30 min. After extraction with an equal volume of phenokchloroform (l:l), the RNA was recovered by isopropanol precipitation as described above and suspended in distilled water. Polyadenylation of TRSV(F)-associated RNA. TRSV(F)-associated RNA was polyadenylated at the 3’ end, using ATP:RNA adenyltransferase. The enzyme was a gift from Dr. Joel Milner, and was prepared by the method of Sipple (1973). The reaction mixture contained 50 mMTris-HCl, pH 7.9, at 37’, 10 n&f MgClz, 5 m&f MnClz, 300 mM NaCl, 500 Mg.ml-’ bovine serum albumin (BSA), 200 fl ATP, 100 pg.ml-’ RNA and 0.1 vol of enzyme. After incubation at 37” for 10 min the reaction was stopped by chloroform extraction and the RNA was precipitated with isopropanol. The precipitate was collected by ultracentrifugation, dried, and suspended in distilled water. Synthesis of cDNA. Complementary DNA was synthesized by a method similar to that described by Caton and Robertson (1979) and Pederson et al. (1980). Reaction mixtures contained 50 mM Tris-HCl, pH 8.3, at 42”, 70 mM KCl, 10 mM MgClz, 30 mM 2-mercaptoethanol, 500 &f each of dATP, dGTP, dTTP, 50 pjU 32P-labeled dCTP, 4 mM sodium pyrophosphate (Myers and Spiegelman, 1978), 80 pg.rnll’ RNA, either 20 pg.rnl-’ of oligo(dT)1z-18 or 360 pg. ml-’ calf thymus DNA fragments generated by DNase I digestion (Taylor et al., 1976) as a primer, and 10 units of avian myeloblastosis virus (AMV) reverse transcriptase per microgram of RNA. After incubation at 42” for 1 hr, the reaction was stopped by adding 2 vol of 0.3 M NaOH containing 0.5% sodium dodecyl sulfate (SDS) and incubated at 37” overnight. The mixture was neutralized with HCl and cDNA was purified by gel filtration through

536

REZAIAN

AND JACKSON

a column (0.6 X 25 cm) of Sephadex G-100 equilibrated with distilled water. RNA hybridization with cDNA. Liquid hybridization was performed as described by Pederson et al. (1980) except that the reaction was carried out in a single tube and 30-J aliquots were withdrawn with a micropipet at each time point. Hybrid formation was assayed by & nuclease purified from Aspergillus oryzae (a gift from Karl Pedersen of Dept. Botany, Purdue University). Digestions were carried out with 70 ~1 of enzyme. ml-’ at 45” and S1 resistant radioactivity was determined by trichloroacetic acid precipitation (Pedersen et al. 1980).

Northern blot and jilter hybridization techniques. RNAs separated by electrophoresis in agarose gels were transferred to DPT paper (Alwine et aZ., 1977) prepared as described by B. Seed of The California Institute of Technology (W. E. Timberlake, personal communication). The DPT paper was prepared as follows: Sheets of Whatman No. 540 filter paper weighing about 20 g were placed in a plastic bag to which 70 ml of 0.5 M NaOH containing 2 mg . ml-’ NaBH4 and 30 ml of 1,4-butanediol diglycidyl ether was added and the bag was sealed. After agitating overnight at room temperature, the bag was opened and the liquid was discarded. Then, 10 ml of 2-aminothiophenol in 40 ml of acetone was added, the bag was resealed, and agitated for 10 hr. The paper was transferred to a pan, washed twice with acetone, twice with 0.1 MHCl, five times with HzO, then air dried and stored at -20”. The resulting APT paper was diazotized, just before use, in a pan containing 500 ml of 1.2 M HCl and 13.5 ml of 1% NaNOz by incubating on ice for 30 min. The bright yellow DPT paper was then washed five times with cold Hz0 and twice with citrate-phosphate buffer (30.7 mM citric acid, 38.6 mM Na2HP04, pH 4.0). The procedure for transferring RNA to DPT paper after gel electrophoresis was essentially as described by Alwine et al. (1977) except that iodoacetate treatment was carried out in 0.2 M potassium phosphate buffer, pH 6.5, and subsequent

washing and blotting was done with citrate-phosphate buffer. Radioactive cDNA was tested for hybridization to RNA bound to the paper by a procedure similar to that used by Denhardt (1966) and Alwine et al. (1977) The paper strips containing RNA bands were placed in Sears Seal-A-Meal bags and prehybridized in 50% formamide, 1% glytine, 750 mMNaC1, 75 mMNa3 citrate, 25 mM NaHzP04, 25 mM NazHP04, 0.2% SDS, 0.1% BSA, 0.1% Ficoll, 0.1% polyvinylpyrollidone, 500 pg.ml-’ sheared calf thymus DNA, and 200 pg. ml-’ poly(rA), at 42”. After 12 hr, the liquid was removed from the bags and 6 ml of hybridization buffer containing 2 X lo6 cpm 3ZP-labeled cDNA probe was added and the bags were resealed. The buffer was the same as that used for prehybridization except that 0.02% each of BSA, PVP, and Ficoll were present and no glycine was added. Following 36 hr incubation at 42”, the paper strips were washed at 65” for 1 hr in 3X SSC (SCC is 150 mM NaCl, 15 mM Na citrate), and then for 15 min in each 2X, 1X, 0.5X, 0.25X, and 0.125X SSC containing 0.1% SDS. The papers were dried and exposed at -70” to Kodak X-ray film (X-Omat AR-5) supplemented with an intensifying screen (DuPont Cronex Quanta II). RESULTS

Purification of TRW(F)-Associated RNA b?d Oligo(dT)-Cellulose Chromatography Before synthesizing cDNA to TRSV(F)associated RNAs, it was essential to determine whether these RNAs contained tracts of poly(A). When a preparation of RNA isolated from TRSV(F) containing TRSV(F)-associated RNA was applied to oligo(dT)-cellulose in high-salt buffer, about one-third of the RNA bound to the column. This fraction was then eluted with the low-salt buffer. Analysis of the unbound and the bound RNA fractions by electrophoresis in agarose gels (Fig. 1) revealed that the unbound RNA consisted of TRSV(F)-associated RNAs contaminated with only trace amounts of TRSV(F) RNA (Fig. lb) and that the bound RNA

TRW SATELLITE

b

a

C

I FIG. 1. Electrophoresis of TRW(F) RNA and TRSV(F)-associated RNA, in agarose gels before and after fractionation over oligo(dT)-cellulose. Approximately 2 Fg of RNA was electrophoresed under denaturing conditions as outlined under Materials and Methods. (a) Unfractionated RNA. (b) Fraction eluted with high-salt buffer. (c) Fraction eluted with lowsalt buffer.

contained TRSV(F) RNA I and RNA II without detectable TRSV(F)-associated RNA (Fig. lc). This procedure revealed that TRSV(F)-associated RNA, unlike TRSV(F) RNA, lacked poly(A) tracts of sufficient length to bind to oligo(dT)-cellulose and also provided a convenient method for separating the two RNA classes. It is noteworthy that under the conditions of this study, the slower migrating TRSV(F)-associated RNA shown in Fig. 1 formed a smaller proportion of total RNA extracted from TRSV(F) than previously observed (Rezaian, 1980). The results were the same whether RNA was analyzed in sucrose density gradients or in denaturing agarose gels. A minor band reported previously (Rezaian, 1980) was not detectable in this study. Synthesis of DNA Complementary TRSV(F)-Associated RNA

to

The TRSV(F)-associated RNAs were used as substrates for polyadenylation

RNAs

537

with ATPRNA adenyltransferase. After the tailing reaction the RNAs were recovered as described under Methods, and used as templates in a cDNA reaction with oligo(dT)lz-18 as a primer. Results shown in Table 1 demonstrated that there was little incorporation of =P-labeled dCTP into acid insoluble material after such reactions. However, RNA preparations from brome mosaic virus (BMV), tailed and processed similarly in the same experiment, were efficient templates for cDNA synthesis (Table 1). TRSV(F)-associated RNAs were also efficiently copied if calf thymus DNA fragments were used as a primer (Table 1). It appeared, therefore, that TRSV(F)-associated RNA was not a suitable substrate for tailing by the above procedure. This conclusion was confirmed by the following experiment: Equimolar concentrations of BMV RNA, transfer RNA (tRNA), and TRSV(F)-associated RNA were used separately in poly(A) tailing reactions. The RNAs were then examined for binding to oligo(dT)cellulose. Although 51% of the BMV RNA and 36% of the tRNA bound to the column when applied in high-salt buffer, no significant amount of TRSV(F)-associated RNA was retained by oligo(dT)-cellulose under the same conditions. Neither BMV RNA or tRNA bound to the column prior to the tailing reaction. These results together with the lack of template activity of TRSV(F)-associated RNA after the tailing reaction (Table l), indicate that the 3’OH group at the end of the RNA may not be available for tailing. The possibility that these RNAs have phosphorylated 3’ ends was examined. In this experiment TRSV(F)-associated RNAs were treated with nuclease-free alkaline phosphatase, recovered by chloroform extraction and precipitated with isopropanol. When such RNA preparations were used in poly(A) tailing reactions and then used for cDNA synthesis, a significant amount of cDNA was made (Table 1). These findings indicate that the TRSV(F)associated RNAs may have phosphorylated 3’ ends which normally block the polyadenylation reaction. The cDNA prod-

REZAIAN

538

AND JACKSON TABLE

1

TEMPLATE ACTIVITY OF TRW(F)-ASSOCIATED

RNA FOR cDNA SYNTHESIS cpm [szPJdCTP incorporated cDNA’

Template RNA used in cDNA reaction

Experiment 2

Experiment 1

Primer

into

322

TRSV(F)-associated RNA after poly(A) tailing reaction but without alkaline phosphatase pretreatment

Oligo(dT)

343

BMV RNA after poly(A) tailing reaction

Oligo(dT)

4700

Not tested

TRSV(F)-associated RNA treated with alkaline phosphatase prior to poly(A) tailing reaction

Oligo(dT)

1285

1414

TRSV(F)-associated RNA without any pretreatment

Calf thymus fragments

DNA

5400

6479

No RNA

Calf thymus fragments

DNA

127

82

o 50-pl reactions were carried out in duplicate as specified under Materials and Methods. TCA insoluble radioactivity in 5-~1 aliquots was determined on GF/A filters (Whatman) after adding 200 ~1 thymus DNA (5 mg. ml-‘) as a carrier.

uct was polydisperse when analyzed by electrophoresis in agarose gels, but full copies of the fastest migrating TRSV(F)associated RNAs were also evident (data not shown). Hybridization of TRW(F)-Associated RNA to cDNA Probes In order to examine whether sequences of TRSV(F)-associated RNA were represented in the viral genome, cDNA to TRSV(F)-associated RNA was hybridized to excess quantities of homologous RNA and to the viral RNAs. The annealing reaction between TRSV(F)-associated RNA and its cDNA occurred over three decades of Rot (Fig. 2, curve a) which is broader than expected for a pseudo-first-order reaction involving

a single component (Fig. 2, curve a). Using a nonlinear least-squares program (Pearson et al., 19’77), the data were fitted to

looE80. :Q

2

60.

d vi z

40.

lFb *

1.

20

-5

-4

.

Jo

-3 Lop

-.

.

-2 E Rot

-1

-0

FIG. 2. Hybridization of RNA in excess with cDNA copied from TRSV(F)-associated RNA. Radioactive cDNA (6000 cpm per time point) with a specific activity of about 4 X 10’ cpm . pg-’ was hybridized with either TRSV(F)-associated RNA (curve a) or TRSV(F) RNA (curve b) present in more than 150-fold excess. Hybridization curves represent the best least-squares fit for pseudo-first-order reactions.

TRSV SATELLITE

a

b

c

TRW-RNA L

TRSVassociated RNA

+ +

FIG. 3. Northern hybridization of cDNA with the viral RNA. (a) RNA extracted from TRSV(F) RNA containing the virus-associated RNA, after electrophoresis in an agarose gel and staining with ethidium bromide. (b) Analyzed as in a, then transfered to DPT paper and hybridized with mP-labeled cDNA copied from TRSV(F) RNA purified by oligo(dT)-cellulose chromatography. RNA bands hybridizing to the radioactive cDNAs were detected by autoradiography. (c) Same as b but hybridized with cDNA copied from the TRSV(F)-associated RNAs purified by removal of TRSV RNA by oligo(dT)-cellulose chromatography.

pseudo-first-order kinetics expected for either one or two abundance classes. The root mean square (RMS) errors were 0.0454 and 0.0238 for one and two abundance classes, respectively. These results indicate that the TRSV(F)-associated RNA used in this experiment consists of at least two hybridizing components. Analysis of the two TRSV(F)-associated RNAs used in this study by sucrose density gradient centrifugation at 60,000 rpm for 7 hr resolved the two RNAs from one another (data not shown). From the area under the peaks, the proportions of the slower sedimenting and the faster sedimenting RNAs were 96 and 4%, respectively. Using these figures &tljz values of 7.4 X 10e4 and 1.8 X lo-’ mol. sec. liter-’ were calculated for the two RNAs, respectively. The ratio of the two R.&,z values is close to the ratio of the size of these two RNAs. When TRSV(F) RNA was separated

RNAs

539

from TRSV(F)-associated RNA, by sucrose density gradient centrifugation followed by oligo(dT)-cellulose chromatography, and then used to hybridize with cDNA to TRSV(F)-associated RNA, the reaction occurred very slowly with a R&j2 of approximately 0.12 mol. sec. liter-’ (Fig. 2, curve b). This hybridization rate was slower than expected if nucleotide sequences of the TRSV(F)-associated RNA had sequences in common with the viral RNA and was most likely due to contamination of TRSV(F) RNA with about 0.1% of the TRSV(F)-associated RNA. It is noteworthy that even though the TRSV(F)-associated RNA was well resolved from TRSV(F) RNA in sucrose density gradient columns, TRSV(F) RNA prepared by this method was still significantly contaminated with TRSV(F)-associated RNA because RNA in the TRSV(F) fraction of the’gradient hybridized very efficiently with cDNA to the TRSV(F)-associated RNA (results not shown). These results indicated that some TRSV(F)-associated RNA aggregates with TRSV RNA. It was therefore necessary to dissociate these RNAs under denaturing conditions before conducting hybridization experiments involving gel electrophoresis. This was achieved by analyzing total RNA extracted from TRSV(F) in agarose gels containing 5 m&f methyl mercury hydroxide followed by the Northern hybridization procedure. The RNA bands were transferred to DPT paper and pieces of paper containing immobilized RNA from different lanes in the gel were used to hybridize separately with q-labeled cDNA to either TRSV(F) RNA or TRSV(F)-associated RNA. Results shown in (Fig. 3) show that these two classes of RNA each hybridized to their respective cDNA and there was no detectable cross-hybridization between TRSV(F) RNAs and TRSV(F)-associated RNAs. DISCUSSION

Results of the kinetic and Northern hybridization experiments described above clearly demonstrate that the nucleotide

540

REZAIAN

AND JACKSON

sequences of TRSV(F)-associated RNA are distinct from those of TRSV RNA. Therefore by the definition of Mossop and Francki (19’78), TRSV(F)-associated RNA species are satellites and not defective interfering nucleic acids. Further analysis of the kinetic hybridization results indicate that the two TRSV(F)-associated RNAs have unique nucleotide sequences. Hybridization of excess TRSV(F)-associated RNA to its cDNA in liquid occurred over three decades of Rot which is consistent with the presence of more than a single abundance class of RNA (Davidson, 1976). When the data were fitted to pseudofirst-order kinetics with two components, a smaller RMS error was obtained than expected of a single component suggesting that the two RNA species have distinct sequences, at least in part. This conclusion, however, needs to be verified by hybridization experiments in which the individual RNAs are used to prepare radioactive cDNA probes. TRSV(F)-associated RNA lacks sufficient poly(A) sequences to bind to oligo(dT)-cellulose and fails to serve as a template for synthesis of cDNA when oligo(dT)12-18is used as a primer for reverse transcriptase. Under the same conditions, TRSV RNA which is polyadenylated (Mayo et al., 19’79) readily binds to oligo(dT)-cellulose and is efficiently used as a template for cDNA synthesis. The TRSV(F)-associated RNA also becomes an efficient template for cDNA synthesis if oligo(dT) is substituted with fragments of calf thymus DNA (Taylor, 1976) in the reaction mixture (Table 1). Several experiments indirectly indicate that the 3’ end of TRSV(F)-associated RNA is phosphorylated. First, TRSV(F)associated RNA is an inefficient substrate for poly(A) polymerase under conditions where BMV RNA and tRNA are tailed. Second, after phosphatase treatment TRSV(F)-associated RNA improves as a PO&~) polymerase substrate, and third, sufficient poly(A) is added to the RNA to enable synthesis of cDNA in the presence of oligo(dT) and reverse transcriptase.

The relationship of TRSV(F)-associated RNAs to the satellite (sTRSV) reported earlier by Schneider (1976) is at present unknown. However, the following parallels may be drawn: (a) The molecular weight of the smallest TRSV(F)-associated RNA (Rezaian, 1980) falls within the range of molecular weights given for sTRSV RNA (Schneider, 1976), (b) virions encapsidating sTRSV RNA or TRSV(F)associated RNAs are heterogeneous and sediment between middle and bottom components of TRSV (Rezaian, 1980; Schneider; 1976), (c) both satellites apparently have arisen during virus replication for experimental purposes (Schneider, 1976; Rezaian, 1980). However, it has not been determined whether sTRSV RNA has any sequence homology with the viral RNA. ACKNOWLEDGMENTS

We thank Drs. Brian A. Larkins, Joel J. Mimer, and Karl Pedersen for helpful discussions and assistance with methods. This work was supported by a grant from the National Science Foundation (PCM 79-20505). AMV reverse transcriptase was a gift from J. W. Beard, Life Sciences Inc.

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TRSV SATELLITE H., CHACONAS,G., DOUGALL,D. K., and KAFATOS, C. K. (1977). End labeling of enzymatically decapped mRNA. Nucleic Acids. Res. 4,4165-4174. HUANG, A. S. (1977). Viral pathogenesis and molecular biology. Biochem. Rev. 41.811-821. MAYO, M. A., BARKER, H., and HARRISON, B. D. (1979). Polyadenylate in the RNA of five nepoviruses. J. Gen. Viral 43.603-610. MOSSOP,D. W., and FRANCKI,R. I. B. (1978). Survival of a satellite RNA in tivo and its dependence on cucumber virus replication. Virology 36, 562-566. MYERS, J. C., and SPIEGELMAN, S. (1978). Sodium pyrophosphate inhibition of RNA-DNA hybrid degradation by reverse transcriptase. Proc. Nat. Acad. Sci. USA 75,5329-53X3. PEARSON, W. R., DAVIDSON, E. H., and BRI’ITEN, R. J. (1977). A program for least square analysis of reassociation and hybridization data. Nucleic Acids Res. 4.1727-1737. PEDERSEN,K., BLOOM,K. S., ANDERSON,J. N., GLOVER, D. V., and LARKINS, B. A. (1980). Analysis of the

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complexity and frequency of zein genome. Biochemistry 19,1644-1650. REZAIAN, M. A. (1980). Three low molecular weight RNA species detected in tobacco ringspot virus. virozog?g 166,400-407. REZAIAN, M. A., and FRANCKI, R. I. B. (1973). Replication of tobacco ringspot virus. I. Detection of a low molecular weight double-stranded RNA from infected plants. Virologyd 56,238-249. SCHNEIDER,I. R. (1976). Defective plant viruses. In “Virology in Agriculture” (J. A. Romberger, J. D. Anderson, and Rex L. Powell, eds.), pp. 201-219. Allanheld, Osmun, Monclair, N. J. SIPPLE, A. E. (1973). Purification and characterization of adenosine triphosphate:ribonucleic acid adenyltransferase from Escherichia coli. Eur. J. Biochem. 37,34-40. TAYLOR, J. M., ILLMENSEE, R., and SUMMERS,J. (1976). Efficient transcription of RNA into DNA avian sarcoma virus polymerase. Biochim. Biophye. Acta 442,324-3.30.