RNA-dependent RNA polymerase associated with subviral particles of Fiji disease virus

VIROLOGY

70, 29%300 (1976)

RNA-Dependent

RNA Polymerase Associated Fiji Disease Virus MASATO

Department

of Plant Pathology,

IKEGAMI

AND

with Subviral

Particles

of

R. I. B. FRANCKI

Waite Agricultural

Research Institute, South Australia

University

of Adelaide,

Accepted October 21, 1975

RNA-dependent RNA polymerase activity was detected in concentrated extracts of leaf gall tissue from Fiji disease virus (FDV)-infected sugarcane leaves but not in similar extracts from healthy leaf tissue. The polymerase activity was correlated with FDV antigen and some polymerase activity was also detected in preparations of FDV subviviral particles. Optimal polymerase activity occurred at about 35”, at pH between 8.5 and 9.0, and in the presence of 8 mM MgCl, and 200 m&f NH,Cl. The polymerase product was single-stranded RNA, over 80% of which annealed to FDV-RNA. Similarities of the FDV associated enzyme to those of reovirus and structurally similar viruses are discussed. INTRODUCTION

Fiji disease virus (FDV), like some other plant viruses, including wound tumor virus (WTV), rice dwarf virus (RDV), and maize rough dwarf virus (MRDV), is structurally similar to reovirus and related viruses infecting mammals and to cytoplasmic polyhydrosis virus (CPV) of insects (Hutchinson and Francki, 1973; Ikegami and Francki, 1974, 1975; Reddy et al., 1975). Fenner et al. (1974) have included all these viruses in the family Reoviridae. It has been demonstrated that virions of reovirus (Borsa and Graham, 1968; Shatkin and Sipe, 19681, CPV (Lewandowski et al., 19691, WTV (Black and Knight, 1970), bluetongue virus (BTV; Verwoerd and Huismans, 1972; Martin and Zweerink, 19721, and RDV (Kodama and Suzuki, 1973) all contain a transcriptase. In this paper we report evidence that FDV subviral particles possess a similar enzyme. MATERIALS

AND METHODS

FDV-infected plant material. Infected sugarcane plants (var. NCO-310) grown in field plots or in a glasshouse were used. Virus-induced galls were excised from the leaves for use as starting material, from which FDV subviral particles were puri-

fied and enzymatically active extracts prepared. Preparation of enzymatically active extracts. All extracts were prepared at 4” and kept in an ice bath until assayed. Gall tissue was ground in a pestle and mortar with some acid-washed. sand by two methods: Method 1. Each gram of fresh tissue was extracted in 2.5 ml of 50 mM TrisHCl, 100 n-&f NH&l, and 90 m&f 2-mercaptoethanol, pH 8.4 (TAM buffer), and strained through cheesecloth. The strained liquid was centrifuged for 1 min at 500 g to remove cell debris and the supernatant was centrifuged at 200,000 g for 1 hr. The pellet was suspended in 200 ~1 of TAM buffer unless otherwise stated. Method 2. Each g of tissue was extracted in 2.5 ml of 0.1 M glycine, 5 mM EDTA, pH 8.5 (GE buffer), and strained through cheesecloth. The extract was centrifuged for 1 min at 500g and Nonidet P40 was added to the supernatant to a concentration of 1%. After 10 min at 4” the extract was centrifuged at 5000 g for 10 min, layered over 2 ml of 10% sucrose in GE buffer, and centrifuged at 200,000 g for 1 hr. The pellet was suspended in either 0.1 M NaCl, 0.05 M Tris-HCI, 5 mM EDTA, pH 7.5 (STE buffer), or in TAM buffer. 292

Copyright All rights

0 1976 by Academic Press, Inc. of rewoduction in anv form wanrwrl

RNA

POLYMERASE

Purification of FDV subviral particles and isolation of FDV-RNA. FDV subviral particles were purified as described by Ikegami and Francki (1974). FDV-RNA was prepared by the pronase-SDS method (Ikegami and Francki, 1975) followed by extraction with 90% aqueous phenol containing 0.1% hydroxyquinoline. The RNA was precipitated with 3 vol of ethanol, was washed three times with ethanol and once with acetone, and dried under vacuum. The precipitate was suspended in 0.01 M phosphate buffer, pH 8.2, containing 0.3 M NaCl (Shatkin and Sipe, 1968). Isolation of RNA from sugarcane leaf tissue and leaf extracts. Total leaf RNA from sugarcane leaf tissue and RNA from enzymatically active extracts were prepared by the phenol-SDS method as described by Francki and Jackson (1972) and were further purified by precipitation with cetyltrimethylammonium bromide as described by Ralph and Bellamy (1964). Assay of RNA-dependent RNA polymerase activity. Fifty-microliter samples of preparations to be assayed were added to 200 ~1 of assay medium. Unless otherwise stated, the medium contained 25 pmoles Tris-HCl, pH 8.5; 2 Fmoles MgCl,; 1 pmole phospho(enol)pyruvate (PEP); 25 pg (6 units) pyruvate kinase; 0.5 pmole (ATP); 0.2 adenosine-5’ triphosphate pmole uridine-5’-triphosphate (UTP); 0.2 pmole cytosine-5’-triphosphate (CTP); 0.02 pmole guanosine-5’-triphosphate (GTP) containing approximately 1 X 10” cpm of Iw~*P]GTP; and 4 M Actinomycin D (AMD). Details of incubation conditions and the means of assays done in duplicate are given in the figures and tables. The reactions were terminated by chilling, the RNA was precipitated with trichloroacetic acid (TCA), and the precipitates were assayed for radioactivity as described by Francki and Randles (1972). RNA-RNA hybridization technique. ““P-labelled RNA samples from polymerase active extracts were mixed with samples of test RNA preparations in 0.01 M phosphate buffer, pH 8.2, containing 0.3 M NaCl. The mixtures were heated at 100 for 10 min and cooled slowly as recommended by Schonberg et al. (1971). When cooled, the samples were incubated with

293

OF FDV

10 pglpl RNAse at 37” for 30 min. Yeast RNA was added (200 pglsample) and the mixtures were precipitated with TCA and assayed for radioactivity as described above. Serological assays. All serological tests were done by the immunodiffusion technique as described by Francki and Habili (1972). An antiserum to FDV particles (Ikegami and Francki, 1974) was used to assay FDV-specific protein and antiserum to poly[Il: poly[Cl (Francki and Jackson, 1972) to assay ds-RNA. Sources of chemicals. Tris(hydroxymethyl) aminomethane (Tris; Sigma 7-91, ribonuclease (RNase), deoxyribonuclease (DNase), phospho(eno1) pyruvate, pyruvate kinase, and unlabelled nucleoside triphosphates were obtained from Sigma Chemical Co., St. Louis, Missouri; Nonidet P40 was obtained from the Shell Co.; 32P-labelled GTP was kindly supplied by Dr. R. H. Symons, Department of Biochemistry, University of Adelaide; Actinomycin D (Dactinomycin, pure substance) was a gift from Merck, Sharpe, and Dohme. All other chemicals were commonly available reagent grade preparations. RESULTS

Preliminary

Experiments

Initially we were unable to detect significant RNA-dependent RNA polymerase activity in purified preparations of FDV subviral particles. However, enzymatic activity was readily detected in crude extracts of leaf galls from FDV-infected sugarcane containing subviral particles (Ikegami and Francki, 19741, whereas similar extracts from healthy leaf tissue had negligible activity. After fractionating active extracts by differential centrifugation between 500 and 17,000 g, significant polymerase activity was detected in all the fractions. Distribution of FDV antigen among these fractions was similar to that of the enzyme activity. Subsequent experiments demonstrated that more than 90% of the polymerase activity was sedimented into pellets after centrifugation at 200,000 g for 1 hr. Moreover, the proportion of the polymerase activity sedimenting under these

294

IKEGAMI

AND

conditions was not different if the nonionic detergent, Nonidet P40, was added to the extract to a concentration of l%, a -restment designed to solubilize cytoplasmic membranes. Properties

of the Polymerase

The kinetics of polymerase activity in concentrated extracts of FDV-induced galls are shown in Fig. 1. The rate of incorporation of+ label f&m [32PlGTP into a TCA-insoluble product at 30” increased rapidly during the first 20 min of incubation but did not increase significantly thereafter. Under the same conditions, a similarly prepared extract from healthy sugarcane leaf tissue failed to incorporate detectable amounts of label (Fig. 1). Incorporation of [32PlGTP by the polymerase was dependent on the presence of the other three nucleoside triphosphates but not on the addition of PEP and PEP kinase (Table 1). Insensitivity of the system to DNAse and AMD (Table 1) indicates that the enzyme activity is independent of DNA. However, [32P]GTP incorporation was almost completely inhibited by RNAse (Table 1). The polymerase activity was dependent on Mg2+, the optimum concentration being about 8 m&f (Fig. 2). The enzyme was also greatly stimulated by NH,+ but not by Na+, the optimum NH,+ concentration being about 200 & (Fig. 3). TABLE PROPERTIES OF THE RNA Experiment

Reaction

FRANCKI

The optimum temperature for enzyme activity is around 35” (Fig. 4) and the optimum pH is between 8.5 and 9.0 (Fig. 5). Borsa and Graham (1968) and Shatkin and Sipe (1968) demonstrated that heat treatment or digestion with a-chymotrypsin, respectively, activated the RNA-dependent RNA polymerase of purified reovirus preparations. Similar treatments of concentrated extracts of galls from FDV100

r ‘t

b

n--

riu. 1. Rate of increase of TCA-insoluble 32P-labelled product in the complete assay medium incubated at 30” containing extracts from FDV-induced galls (0-a) and from healthy sugarcane leaf tissue (A-A). The extracts were prepared by Method 2 and suspended in TAM buffer as described in Materials and Methods. 1

POLYMERASE ASSOCIATED WITH FDV INFECTION

system

[%PlGMP

incorporation

wm

Percentage of complete system

1

Complete” - ATP - CTP - UTP - ATP, - CTP, - UTP - gall extract

1655 111 479 76 31 0

100 6.7 29 4.6 1.9 0.0

2

Complete” - PEP, - PEP Kinase - AMD + DNase (25 pg/ml) + RNase (20 pg/ml)

1803 1772 1561 1716 55

100 98 87 95 3.1

fl Extracts from FDV-induced galls were prepared by Method 1 and polymerase activity described in Materials and Methods. The enzyme activity was assayed at 30” for 40 min.

assayed as

RNA

infected leaves slightly decreased matic activity (Table 2). Correlation of Polymerase FDV Antigen

Activity

POLYMERASE

295

OF FDV

enzy-

0

Expt.

1

3

Expt.

2

X

Expt.

3

NHdCl NaCl

with

Since nearly all the polymerase activity in extracts from FDV-induced gall tissue sedimented after centrifugation for 60 min at 200,000 g, even after treatment with detergent, it would appear that the enzyme must be associated with a relatively large macromolecular structure. The sedimentation rate of this structure was compared to that of FDV antigen by subjecting an enzymatically active preparation to centrifugation in a sucrose density-gradient. Each fraction from the gradient was assayed for the presence of FDV antigen and for polymerase activity. Results of the experiment (Fig. 6) indicate a good correlation between the presence of FDV antigen and polymerase activity, suggesting that the enzyme is virion associated. In a subsequent experiment, the antigen content and polymerase activity of a purified preparation of FDV subviral particles was com-

I

/ 100

0

1 200 SALT

I 300

(mM)

FIG. 3. Relationship

between RNA polymerase activity in extracts from FDV-induced galls and monovalent cation concentration. The extracts were prepared by Method 1 as described in Methods and Materials; however, extracts used in Experiments 1 and 2 were suspended in buffer containing various amounts of NH&l. Reaction mixtures were incubated at 30” for 40 min (Experiments 1 and 3) and 60 min (Experiment 2). Radioactivity of the samples in these experiments ranged from 290 to 4978 cpmi assay.

pared to those of a concentrated gall tissue extract. The results (Table 3) demonstrate that both preparations incorporated [32PlGTP into a TCA-insoluble precipitate and that both contained FDV antigen. However, on the basis of antigen content, the purified subviral particles were only about 10% as active in incorporating [32PlGTP as the concentrated gall extract. Properties

FIG. 2. Relationship between RNA polymerase activity in extracts from FDV-induced galls and MgCl, concentration. The extracts were prepared by Method 1 and assayed at 30” for 40 min as described in Materials and Methods.

of the Polymerase

Product

The product of the polymerase is almost entirely a single-stranded polyribonucleotide since over 90% of it was readily digested by RNAse (Table 4). Self-annealing and annealing after the addition of RNA from healthy sugarcane leaf tissue, rendered about half the radioactive product resistant to RNAse digestion whereas annealing in the presence of added FDVRNA increased this to 80% (Table 4).

296

IKEGAMI

AND

These observations indicate that most of the single-stranded RNA product has base sequences complementary to FDV-RNA. When a concentrated gall extract incu-

./

FRANCKI

bated in the polymerase assay medium with f3*PlGTP for 60 min at 30” was subjetted to sucrose density-gradient centrifugation, nearly all the TCA-insoluble radioactive material sedimented at the same 1 rate as FDV antigen (Fig. 7). Under these

.

.r ‘A

r.

,--

FIG. 4. Relationship of RNA polymerase activity in extracts from FDV-induced galls and temperature. The extracts were prepared by Method 1 and assayed as described in Materials and Methods except that MgCl, concentration was 4 n&f throughout. Incubation was for 40 min. TABLE

OH FIG. 5. Relationship of RNA polymerase activity in extracts from FDV-induced galls and pH. Preparation of extracts and assay conditions as described in Fig. 4 except that all samples were incubated at 30”. 2

EFFECTS OF HEAT AND WCHYMOTRYPNN TREATMENTS ON RNA POLYMERASE ACTIVITY IN EXTRACTS FROM FDV-INDUCED GALL TISSUE Experiment

Treatment

of gall extract

[3zP1GMP incorporation cpm

1

2

Untreateda Heated 40” Heated 50” Heated 60” Heated 60”

for for for for

20 20 10 20

se@ set set set

Untreated” + 50 pg/ml a-chymotrypsin’ + 100 pg/ml a-chymotrypsin + 200 fig/ml o-chymotrypsin + 400 pg/ml a-chymotrypsin

932 782 833 829 648

100 84 89 89 70

1813 1433 1450 1437 968

100 79 80 79 53

n Extracts of FDV-induced galls were prepared by Method 1 and enzyme activity in Materials and Methods. Incubation was at 30” for 40 min. b Heat treatment was applied before assaying polymerase activity. r a-Chymotrypsin was included in the assay medium.

was assayed as described

RNA

POLYMERASE

incubation conditions, synthesis of polymerase product would have been completed within the first 15 min of incubation (Fig. 1). In a subsequent experiment, a similar . 700

1

f.00

i

TOP

FRACT1ON

NUMBCR

BOTTOM

FIG. 6. Correlation of RNA polymerase activity with FDV antigen in extracts from FDV-induced gall tissue. An extract was prepared by Method 2 and suspended in STE buffer as described in Materials and Methods. A sample (500 ~1) of the extract was layered on a 30-60% linear sucrose densitygradient buffered with STE and centrifuged at 40,000 rpm for 30 min in a Spinco SW50 rotor. Fractions were collected by puncturing the bottom of the tube and each fraction was subdivided for assay of polymerase activity in duplicate and FDV antigen. Enzyme activity was assayed at 30” for 40 min as described in Materials and Methods except that the assay medium contained 175 n&f NH&l and 90 m&f 2-mercaptoethanol. FDV-antigen was assayed by immuno-diffusion as described in Materials and Methods.

297

OF FDV

preparation incubated for only 20 min was deproteinized by phenol-SDS extraction and subjected to sucrose density-gradient centrifugation. The results, summarized in Fig. 8, indicate that most of the product remained at the top of the gradient whereas FDV ds-RNA (assayed by immunodiffusion against poly[I]: poly[C]antiserum) sedimented well into the gradient. Electrophoresis of similar preparations of deproteinized polymerase product in polyacrylamide gels (Loening, 19681 of varying pore sizes (2.43%) failed to resolve any clearly distinct peaks of radioactivity. Most of the labeled material migrated ahead of the 18 S leaf rRNA and a large proportion of it migrated between 18 and 5 S rRNA. These observations indicate that the single-stranded RNA transcribed from FDV-RNA was of low molecular weight and heterogeneous and was not released from subviral particles following synthesis, but was separated from FDV ds-RNA on deproteinization. DISCUSSION

The RNA-dependent RNA polymerase in extracts of FDV-induced galls appears to be a transcriptase which transcribes single-stranded RNA from FDV ds-RNA. Furthermore, correlation of the transcriptase activity with FDV antigens suggests that the enzyme is an integral part of subviral particles as found in other Reoviridae including reovirus, CPV, WTV, BTV, and

TABLE 3 COMPARISON OF THE RNA POLYMERA~E ACTIVITY IN EXTRACTS FROM FDV-INDUCED PURIFIED FDV SUBVIRAL PARTICLES Preparation

Gall extract’ Undiluted Diluted i/z Diluted i/4 Diluted ‘ia FDV subviral

particles”

Polymerase activity (cpm)”

4109 2584 1842 938 1228

Antigen dilution end-point”

GALL TISSUE AND Ratio (polymerase activityiantigen dilution end-point) .-___

8 4 2 1 16

” Assays done as described in Materials and Methods. Incubation was at 30” for 40 min. b Antigen dilution end-points are expressed as reciprocals of highest dilutions of antigen visible precipitation line in immunodiffusion tests against anti-FDV serum. o Extracts prepared by Method 1 described in Materials and Methods. ’ Preparations purified as described by Ikegami and Francki (1974).

514 646 921 938 77 ___-producing

a

298

IKEGAMI

AND FRANCKI TABLE 4

ANNEALING OF THE RNA

POLYMERABE PRODUCT WITH FDV-RNA

Addition to reaction mixture

Treatment

RNAse resistance cm

Nil Nil RNA from healthy sugarcane leaves (11 pg/assay) FDV-RNA (3 pg/assayl

Percentage of product added

Unheated* Heated and cooled Heated and cooled

53 382 451

6.3 46 54

Heated and cooled

701

84

(1Polymerase product was extracted with phenol-SDS and purified as described in Materials and Methods. Each reaction mixture contained 839 cpm of the polymerase product. * Polymerase product was incubated with 10 pg/ml RNase at 37” for 30 min without any prior treatment and then precipitated with TCA as described in Materials and Methods. c Polymerase product was heated and cooled slowly as described in Materials and Methods before RNAse treatment and TCA precipitation.

:: : 5007 E( *y/E;,, T II g 0. .-..d,’

i

16d 2I i “47 i4 i 4

+ ;i”’ ,i‘,,,, \ ‘4..^L-.-0, .. .....-.........I...?~~~:~:~:.< I: i B 9 1031121314IS 16(7 181’1 i23456’ I TOP BOTTOM FRACTION NUMBER

FIG. 7. Cosedimentation of polymerase product RNA and FDV-antigen in sucrose density-gradients. An extract from FDV-induced gall tissue was prepared by Method 2 and suspended in STE buffer and was incubated in the standard polymerase assay medium at 30” for 60 min as described in Materials and Methods. The incubated mixture (500 ~1) was fractionated by sucrose density-gradient centrifugation as described in Fig. 6. The fractions were assayed for TCA-insoluble radioactivity and FDV-antigen as described in Materials and Methods.

RDV (Borsa and Graham, 1968; Shatkin and Sipe, 1968; Lewandowski et al., 1969; Black and Knight, 1970; Verwoerd and Huismans, 1972; Martin and Zweerink, 1972; Kodama and Suzuki, 1973). Transcriptase of all Reoviridae are remarkably similar in their requirement for Mg2+ at a concentration of about 8 mM for maximum activity. This requirement contrasts with transcriptase-containing viruses belonging to other virus groups; viruslike particles with ds-RNA isolated from Penicillium sp. require only 5 mM Mg*+ for maximum activity (Alaoui et al., 1974; Chater and Morgan, 19’741, Rhabdoviruses about 4-6 mM Mg*+ (Baltimore et al., 1970; Francki and Randles, 1972) and Myxoviruses are dependent on Mn*+ and

not Mg2+ (Penhoet et al., 1971; Chow and Simpson, 1971). The FDV transcriptase was also stimulated by NH++, about 200 mM being optimum. Apparently little attention has been paid to the effect of monovalent cations on transcriptases of other Reoviridae. However, Levin et al. (1970) reported that both K+ and NH,+ were able to stimulate the polymerase of reovirus. Van Etten et al. (1973) have also shown that the RNA polymerase of the ds-RNA bacteriophage 46 can be stimulated by the addition of NH4+, the optimum concentration being 75-100 n&f. The FDV transcriptase differs from those of other Reoviridae in that it maintained activity for only 15-20 min at 30” (Fig. l), whereas enzymes of other Reoviri-

RNA

T’OP

POLYMERASE

FRICTION

NUMBtR

299

OF FDV

il,lrr””

FIG. 8. Sedimentation of RNA isolated from an extract of FDV-induced gall tissue incubated with lS’P]GTP. The extract was prepared by Method 2, suspended in TAM buffer and incubated at 30” for 60 min in the standard polymerase assay medium as described in Materials and Methods. The incubated mixture was deproteinised by the phenol-SDS procedure and subjected to centrifugation in a IO-30% linear sucrose density-gradient in SSC buffer (0.15 M NaCl and 0.015 M Na acetate, pH 7) for 4.5 hr. The gradients were fractionated as described in Fig. 6 and each fraction was assayed for TCA-insoluble radioactivity and dsRNA antigen as described in Materials and Methods. Arrows indicate the position to which marker cucumber mosaic virus RNAs sedimented in a sister tube.

dae have been observed to incorporate labelled nucleoside triphosphates over periods of hours. With reovirus, such incorporation continued at a constant rate for at least 10 hr (Levin et al., 1970). Unlike some Reoviridae, but like CPV (Lewandowski et al., 1969) and WTV (Black and Knight, 1970), FDV does not require chymotrypsin or heat treatment to activate its polymerase. These treatments appear to remove the outer layer of the virions to expose enzymatically active cores or subviral particles (Shatkin and Sipe, 1968; Banerjee and Shatkin, 1970; Shatkin and Lafiandra, 1972). This exposure of reovirus cores appears to be a reversible reaction and the enzyme activity is masked in the reassembled virions (Astell et al., 1972). FDV requires no activation, probably because the outer layer of its particles is easily lost during extraction from plant tissues; indeed, we have been unable to prevent this loss during purification (Ikegami and Francki, 1974). All these observations point to the conclusion that FDV is very much less stable than most of the other Reoviridae studied. Although the FDV polymerase appears to transcribe FDV-RNA with a high degree of base sequence fidelity (Table 41, it does not appear to release the transcripts from subviral particles (Fig. 7). The size

heterogeneity of the polymerase product may be due either to incomplete transcription of the genome segments or to activity of endogenous RNAse in the gall extracts. ACKNOWLEDGMENTS We thank Dr. R. H. Symons for gifts of :r”P-labelled GTP; Dr. P. B. Hutchinson for supplies of FDV-infected sugarcane material; Drs. R. G. Milne and G. Boccardo for sending us manuscripts prior to publication; Mrs. L. Wichman for the drawings; and Mr. K. W. Jones for maintaining plants in the glasshouse. The first author was supported by an Adelaide University Research Grant Scholarship and the project was supported in part by grants from the Colonial Sugar Refining Company and the Australian Research Grants Committee. REFERENCES ALAOUI, R., JACQUEMIN, J. M., and BOCCARDO, G. (19741. Ribonucleic acid polymerase activity associated with virus-like particles isolated fromPenicillium stoloniferum ATCC 14586. Acta Viral. 18, 193-202. S. C., LEVIN, D. H., and ASTELL, C., SILVERSTEIN, ACS, G. (1972). Regulation of the reovirus RNA transcriptase by a viral capsomere protein. Virology 48, 648-654. BALTIMORE, D., HUANG, A. S., and STAMPFER, M. (1970). Ribonucleic acid synthesis of vesicular stomatitis virus II. An RNA polymerase in the virion. Proc. Nat. Acad. Sci. USA 66, 572-576. BANERJEE, A. K., and SHATKIN, A. J. (1970). Transcription in vitro by reovirus-associated ribonucleic acid-dependent polymerase. J. Viral. 6, l-11.

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AND

BLACK, D. R., and KNIGHT, C. A. (1970). Ribonucleic acid transcriptase activity in purified wound tumor virus. J. Virol. 6, 194-198. BORSA, J., and GRAHAM, A. F. (1968). Reovirus: RNA polymerase activity in purified virions. Biohem. Biophys. Res. Commun. 33, 895-901. CHATER, K. F., and MORGAN, D. H. (19741. Ribonucleic acid synthesis by isolated viruses of Penicillium stoloniferum. J. Gen. Viral. 24, 307-317. CHOW, N., and SIMPSON, R. W. (1971). RNA-dependent RNA polymerase activity associated with virions and subviral particles of myxoviruses. Proc. Nat. Acad. Sci. USA 68, 752-756. FENNER, F., PEREIRA, H. G., PORTERFIELD, J. S., JOKLIK, W. K., and DOWNIE, A. W. (1974). Family and generic names for viruses approved by the International Committee on Taxonomy of Viruses, June 1974. Invervirology 3, 193-198. FRANCKI, R. I. B., and HABILI, N. (1972). Stabilization of capsid structure and enhancement of immunogenicity of cucumber mosaic virus (Q strain) by formaldehyde. Virology 48, 309-315. FRANCKI, R. I. B., and JACKSON, A. 0. (1972). Immunochemical detection of double-stranded ribonucleic acid in leaves of sugar cane infected with Fiji disease virus. Virology 48, 275-277. FRANCKI, R. I. B., and RANDLES, J. W. (1972). RNAdependent RNA polymerase associated with particles of lettuce necrotic yellows virus. Virology 47, 270-275. HUTCHINSON, P. B., and FRANCKI, R. I. B. (1973). Sugarcane Fiji disease virus. In “Description of Plant Viruses 119.” Commonwealth Mycological Institute/Association of Applied Biologists. IKEGAMI, M., and FRANCKI, R. I. B. (19741. Purification and serology of virus-like particles from Fiji disease virus-infected sugarcane. Virology 61, 327-333. IKEGAMI, M., and FRANCKI, R. I. B. (1975). Some properties of RNA from Fiji disease subviral particles. Virology 64, 464-470. KODAMA, T., and SUZUKI, N. (1973). RNA polymerase activity in purified rice dwarf virus. Ann. Phytopath. Sot. Japan. 39, 251-258. LEVIN, D. H., MENDEL~OHN, N., SCHONBERG, M., KLETT, H., SILVERSTEIN, S., KAPULER, A. M., and

FRANCKI Acs, G. (19701. Properties of RNA transcriptase in reovirus subviral particles. Proc. Nat. Acad. Sci. USA 66, 890-897. LEWANDOWSKI, L. J., KALMAKOFF, J., and TANADA, Y. (1969). Characterization of a ribonucleic acid polymerase activity associated with purified cytoplasmic polyhydrosis virus of the silk-worm Bombyx mori. J. Virol. 4, 857-865. LOENING, U. E. (1968). The fractionation of high molecular weight RNA. In “Chromatographic and Electrophoretic Techniques” (I. Smith, ed.) Vol. 2, pp. 437-442. W. Heinemann (Medical Books) Ltd., London. MARTIN, S. A., and ZWEERINK, H. J. (1972). Isolation and characterization of two types of blue tongue virus particles. Virology 50, 495-506. PENHOET, E., MILLER, H., DOYLE, M., and BLATTI, S. (1971). RNA-dependent RNA polymerase activity in influenza virions. Proc. Nat. Acad. Sci. USA 68, 1369-1371. RALPH, R. K., and BELLAMY, A. R. (19641. Isolation and purification of undegraded ribonucleic acids. Biochem. Biophys. Acta 87, 9-16. REDDY, D. V. R., BOCCARDO, G., OUTRIDGE, R., TEAKLE, D. S., and BLACK, L. M. (1975). Electrophoretie separation of da-RNA genome segments from Fiji disease and maize rough dwarf viruses. Virology 63, 287-291. SCHONBERG, G., SILVERSTEIN, S. C., LEVIN, D. H., and Acs, G. (1971). Asynchronous synthesis of the complementary strands of the reovirus genome. Proc. Nat. Acad. Sci. USA 68, 505-508. SHATKIN, A. J., and LAFIANDRA, A. J. (19721. Transcription by infectious subviral particles of reovirus. J. Viral. 10, 698-706. SHATKIN, A. J., and SIPE, J. D. (1968). RNA polymerase activity in purified reovirus. Proc. Nat. Acad. Sci. USA 61, 1462-1469. VAN ETTEN, J. L., VIDAVER, A. K., KOSKI, R. K., and SEMANCIK, J. S. (1973). RNA polymerase activity associated with bacteriophage $6. J. Virol. 12, 464-471. VERWOERD, D. W., and HUISMANS, H. (1972). Studies on the in vitro and the in vivo transcription of the blue tongue virus genome. Ondersteopoort J. Vet. Res. 39, 185-192.