Studies on encapsidated viroid-like RNA I. Characterization of velvet tobacco mottle virus

VIROLOGY

108,

Ill-122

(1981)

Studies on Encapsidated Viroid-like RNA I. Characterization

of Velvet Tobacco

Mottle

Virus

J. W. RANDLES,’ C. DAVIES, T. HATTA, A. R. GOULD,’ AND R. I. R. FRANCKI Department

of’Plant

Pathology,

Waite

Agricultural Research South Australia

Accepted

July

II,

Institute,

University

of Adelaide,

1.980

Velvet tobacco mottle virus (VTMoV) isolated from Nicotiana velutina growing wild in arid Central Australia was transmitted by inoculation to a limited number of plant species of which N. clevelandii was the most convenient experimental host. The virus was also transmitted from field-grown plants to N. velutina and N. clevelandii by the mirid Cyrtopeltis nicotianae. VTMoV preparations purified by clarification with organic solvents and differential centrifugation contained polyhedral particles about 30 nm in diameter sedimenting as a single component at about 116 S. The particles were shown to be located in the nucleus, cytoplasm, and vacuoles of infected plant cells. Virus dissociated in the presence of mercaptoethanol and sodium dodecyl sulfate (SDS) separated into one major and two minor polypeptides with estimated molecular weights of 33,000, 36,600 and 31,000, respectively. Single-stranded RNA isolated from VTMoV by extraction with phenol was separated into five components with apparent molecular weights of 1.5 x 10fi, 0.63 x 108, 0.25 x lo”, 0.16 x 106, and 0.12 x lo6 referred to as RNAs 1, la, lb, 2, and 3, respectively. It appears that RNAs la and lb are breakdown products of RNA 1, as shown elsewhere, and electron microscopic examination of the other species showed that whereas RNAs 1 and 3 are linear molecules, RNA 2 is circular. The similarity of RNAs 2 and 3 to the RNA of viroids is discussed. VTMoV has been compared with several RNA plant viruses with small polyhedral particles. Only solar-mm nodiflorum mottle virus appears to share some of its unique features and the two have been shown to be antigenically related.

plants. Someof its properties are described in this and the accompanyingpaper, with Nicotiana velutina (Wheeler), a native particular emphasis on its unusual RNA tobacco common to Central Australia (Black, 1957)and sometimesreferred to as complement. velvet’tobaccobecauseof its characteristic MATERIALS AND METHODS leaf texture (Willis, 1972),has been preViruses. VTMoV isolatedfrom N. velutina. viously reported as a host of nicotiana velutina mosaic virus (NVMV; Randles et in northeasternSouth Australia was mainal., 1976). More recently plants of this tained in a greenhouse in Nic&ana species with prominent virus-like symp- clevelandii A. Gray by mechanicalinoeulaive toms unlike those of NVMV have beenob- tion. Other viruses used for corn served in two areasof northeastern South studies(Table 1)were simiI&y ~~~~~. Purijicattin. of tiracscss.VTMoV is a very Australia. A previously undescribedvirus, wtih we havenamedvelvet tobaccomottle stablevirus reachinghigh ~n~ntr~~~~ in virus (VTMoV), was isolated from these the leaves of infe&ed N. clev&rPdii. It was purified by several proceduresinvolving extraction in phosphatebuBers, cl&’ To whom reprint requests should be addressed. fieation with organic solvents, and dif* Present address: Department of Biochemistry, University of Adelaide, South Australia. ferential centrifugation.The methodusually INTRODUCTION

111

~2-as22w1/010111-12$02.~/0 Copyright 0 1981 by Academic Press, Inc. All rights of reprctduetion in any form reserved.

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RANDLES

ET AL.

TABLE VIRUSES

Virus

USED

1

FOR COMPARATIVE

Source of isolate

PURPOSES

Experimental

host

Reference

From Solanum nodijorum Jacquin. isolated by R. S. Greber in Queensland

Nicotiana clevelandii A. Gray

Greber (1973)

Southern bean mosaic virus (SBMV)

From the Waite Agricultural Research Institute Collection

Phaseolus vulgaris L. cv. Hawkesbury Wonder

Shepherd (1971)

Sowbane mosaic virus

From Chenopodium trigonon Roem. & Schult. isolated by D. J. Teakle in Queensland

Chenopodium Willd.

Teakle (1968)

Tomato bushy stunt virus (TBSV)

From Glasshouse Crops Research Institute (U. K.) Collection (Type isolate)

Nicotiana clevelandii A. Gray

Martelli et al. (1971)

Carnation mottle virus (CaMV)

From Victorian Plant Research Institute Collection

Chenopodium Willd.

Hollings and Stone (1970)

Galinsoga mosaic virus

From Galinsoga parvijlara Cav. isolated by G. M. Behncken

Phaseolus vulgaris L. cv. Hawkesbury Wonder

Behncken (1970)

Kennedya yellow mosaic virus (KYMV)

From Kennedya rubicunda (Schneev) isolated by J. Dale in New South Wales

Pi-sum satiwum L. cv. Greenfeast

Dale and Gibbs (1976)

Cucumber mosaic virus (CMV)

Q strain from the Waite Agricultural Research Institute Collection

Nicotiana L.

Francki et al. (197913)

Tobacco ringspot virus (TRSV)

From the Waite Agricultural Research Institute Collection

Cucumis sativum L.

Randles and Fran&i (1965)

Echtes Ackerbohnemosaik virus (EAMV)

From Viciafaba L. isolated by J. W. Randles

Vicia faba L. cv. Leviathan Long Pod

Randles and Dub6 (1977)

Solanum nodiflorum virus (SNMV)

mottle

(SMV)

(GMV)

used to purify the virus and which resulted in yields of about 1 mg of virus/g of freshly harvested leaf material was as follows. The material was extracted with 70 mM phosphate buffer, pH 7, containing 3 mM EDTA and 0.1% thioglycollic acid (2 ml/g of leaf material). The slurry was strained through cheesecloth, n-butanol was added to a final concentration of 9% while stirring, and the extract was left at 4” for 20 min. After clarification by centrifugation at 10,000 g for 10 min, the supernatant fluid was centrifuged at 78,000 g for

quinoa

quinoa

glutinosa

4 hr and the pellets were resuspended in ‘70 mM phosphate buffer, pH 7. After clarification by centrifugation at 10,000 g for 10 min, the supernatant fluid was centrifuged at 200,000 g for 75 min, the pellets were again resuspended in 70 mM phosphate buffer, pH 7, and then emulsified with an equal volume of chloroform for 5 min at 4”. The emulsion was broken by centrifugation at 27,000 g for 10 min, the aqueous layer was recovered and subjected to another cycle of differential centrifugation. The final virus pellets were re-

VELVET

TOBACCO

MOTTLE

VIRUS

1 1:;

arations were denatured in buffered suspended in the required buffer. When formamide, spread under den&&g condi&IS dhightv purity was required, the preptions, stained, and rotary shadowed as dearations were subjected to centrifugation in 5-258 sucrose (in 20 mM phosphate scribed by Randles and Hatta (1979). All specimens were examined in a JEM 100CX buffer, pH 7.4) density-gradients either in electron microscope. a Spineo SW 41 rotor at 40,000 rpm for 1 hr or a SW 27 rotor at 26,090 rpm for 2 hr. Nucleic acid and protein analysis. DeThe single virus band was localized and tails of nucleic acid isolation and electrorecovered using an ISCO Model 640 den- phoretic separation of components are desity-gradient fractionator and ultraviolet scribed in detail in an accompanying paper monitor, concentrated by centrifugation at (Gould, 1981). For protein analysis, virus preparations were dissociated at 100” for 3 300,000 g for 2 hr, and resuspended in buffer. min either in 50 m&f Tris-HCl, pH 6.8, SMMV was purified as above, SBMV, containing 2% sodium dadecyl sulfate (SDS) SMV, CaMV, TBSV, and GMV (Table 1) and 1% 2-mercaptoethanol (ME) or in unessentially as described by Harrison and buffered 8 nlr urea eontaming 1% SDS and Nixon (1960); CMV as described by Francki 1% ME. Electrophoresis was in 13% polyet al. (1979b); KYMV as by Gibbs (1978); acrylamide gels as described by Laemmli EAMV as by Gibbs and Paul (1970); (1970). TRSV as by Rezaian and Fran&i, 1973. Serology. Antisera were prepared in rabbits injected subcutaneously or intramuscuRESULTS larly with virus preparations emulsified with equal volumes of Freunds complete Field Incidence and Transmission oj’ adjuvant. All serological tests were done VTMoV by double diffusion in 0.75% agar gels prepared with 10 nul4 phosphate buffer, Numerous N. velutina plants with dispH 7, containing 0.02% sodium azide ease symptoms growing in the wild were observed during a survey in May 1979 at (Francki and Habili, 1972). Sedimentation and buoyant density meas- Cobblers Sandhill (29”46’; 139*57’) and near Innamincka (27”46’; 140”42’). The leaves of uwments. Ultracentrifugal analyses were done in a Spinco Model E using plain and diseased plants had a yellow mosaic with wedge window cells in an AnD rotor. prominent blistering (Fig. 1) from which Buoyant density measurements were done virus was readily transmitted by meehanical inoculation to N. clevelundii (Fig. 2). either in the analytical ultracentrifuge Similarly infected N. velutina plants were (Chervenka, 1969) or in a preparative ultraobserved during a subsequent visit to Cobcentrifuge (Francki et al., 1980). Estimation of virus concentration. The blers Sandhill in November 1979 and the EB&‘&,, for VTMoV was assumed to be 5 on area was examined in more detail. Here the the basis of its approximate RNA con- plants were growing in deep sand in an tent (see Results) and values for other vi- ecological association with Nicotiumz glauca ruses used were those quoted in the referGrah. and Phyllanthus lacunarius von ences presented in Table 1. Mueller. A’ 600-m line transect survey disElectron microscopy. Virus preparations closed that symptoms were associated with were negatively stained with 3% uranyl N. velutina to the extremity of its distriacetate (Francki et al., 1979a). Leaf tis- bution. This suggested that the virus sue was fixed, embedded, and sectioned as spreads rapidly since the plant is an annual described by Hatta and Francki (1978)- and we have been unable to detect any Ribonuclease (RNase) treatment of tissues seed transmission of VTMoV in N, v&&ma ~ to digest the RNA of ribosomes was done Without reference to symptoms, VTMoV as described by Hatta and Fran&i (1979). was detected serologically in 9 of 20 Immune electron microscopic tests were randomly sampled N. velutina plants but done using the antibody decoration method not in any of 12 N. glauca or ten P. (Mime and Luisoni, 1977). Nucleic acid prep- lacunarius testedI

RANDLESETAL.

114

FIG. 1. Leaf symptoms of VTMoV infection on N. velutina in the field. plant showing necrotic FIG. 2. Symptoms of VTMoV on an experimentally infected N. clewelandii local lesions on the inoculated leaves and mosaic and leaf crinkling on systemically infected leaves.

During the November survey at Cobblers Sandhill, N. velutina plants were infested with larvae and adults of Cyrtopeltis nicotianae (Konigsberger) (Hemiptera; Miridae). Insects collected in the field and caged for 5 days on healthy N. velutina seedlings in groups of 5 per plant, transmitted virus to 7 of 10 plants. Transmission of VTMoV by C. nicotianae was confirmed in another experiment in which groups of 6 to 20 insects transmitted the virus to all of the seven N. clevelandii test plants used. Experimental Host tomatology

Range

and

Symp-

VTMoV infection by mechanical inoculation induced systemic vein clearing and later, mosaic in N. velutina and N. clevelandii (Fig. 2) whereas a milder mosaic was induced in Nicotianu glutinosa L and N. clevelandii x N. glutinosa hybrid plants. The inoculated leaves of N. clevelandii developed necrotic lesions (Fig. 2). Lesions were also produced on the inoculated leaves of N. glauca without development of systemic symptoms. Petunia hybrida Vilm. was shown to be infected by

VTMoV without exhibiting. symptoms and the virus was recovered from the inoculated leaves of Nicotiana tabacum L. cv. White Burley and Xanthi n.c., Datura strumonium L. and Gomphrena globosa L. with no discernible symptoms. VTMoV failed to infect Solanum nodijlorum Jacquin, S. melongena L., Physalis jloridana Rydb., Capsicum annuum L., and Lycopersicon esculentum L. in the family Solanaceae; Phaseolus vulgar-is L., Pisum sativum L., Vigna sinensis Savi, Glycine max (L.) Merrill, and Vicia fuba L. in the Leguminosae; Cucumis sativus L. in the Cucurbitaceae; Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd. in the Chenopodiaceae; Brussica pekinensis (Lour.) Rupr. in the Cruciferae; and Zinnia elegans Jacq. in the Compositae. Properties tions

of Puri)ied

VTMoV

Prepara-

Negatively stained preparations of highly infectious VTMoV contained homogeneous populations of polyhedral particles about 30 nm in diameter (Fig. 3). The particles were readily distinguished from those of

VELVET

TOBACCO MOTTLE

FIG. 3. Electron micrograph of a purified preparation of VTMoV negatively stained with uranyl acetate. (Bar represent 100 nm.)

VIRUS

11’5

resolving power of the analytical ultracentrifuge, showed that the bulk of the material had a buoyant density of 1.37 with g*cmW3, but a minor component slightly higher density was also resolved (Fig. 5). Nucleoprotein with a buoyant density of 1.37g. cmm3 should contain about 22% nucleic acid (Gibbs and Harrison, 1976). Purified preparations of VTMoV had ultraviolet absorption spectra characteristic of nucleoprotein with A,,dA 2%.ratios of about 1.54 indicating a nucleic acid content of approximately 18% (Gibbs and Harrison, 1976). From these data we conclude that the particles of VTMoV contain about 20% nucleic acid. Serology

CMV (cucumovirus group), KYMV (tymovirus group), EAMV (comovirus group), TRSV (nepovirus group), and TBSV (tombusvirus group) as well as those of the as yet unclassified CaMV and GMV. However, the particles were indistinguishable from those of SBMV which has recently been designated as the type member of the southern bean mosaic virus group, and SMV which has tentatively been assigned to the group (Matthews, 1979). Particles of SNMV were also indistinguishable in their appearance from those of VTMoV. Examination of VTMoV preparations in the analytical ultracentrifuge established the presence of a single macromolecular species, cosedimenting with particles of SBMV (Fig, 4) which have a sedimentation coefficient of 115 S at infinite dilution (Shepherd, 1971). When VTMoV was centrifuged to equilibrium in CsCl, some material banded in a narrow zone with buoyant density 1.37 g *erne3 and from this, intact virus particles were recovered. However, some material also remained near the top of the gradients. On the other hand, virus preparations which had been fixed with glutaraldehyde

An antiserum with homologous titer of 111024 was produced in one rabbit after two subcutaneous and one intramuscular

FIG. 4. Schlieren diagram of a preparation containing 2 mg/ml of purified VTMoV (above) and a mixture of 2 mg/ml VTMoV and 2 mg/ml SBMV (belowj. Photograph was taken at a bar angle of W’, 12 min after the AnD rotor had reached a speed of 33,450 rpm. Sedimentation is from left to right. FIG. 5. Equilibrium density gradient centrifugation in C&l of approximately 66 /Ig of VTMoV purified by the standard technique (see Materials and Methods) including a sucrose density-gradient centrifugatian step. The initial density of the CsCl was approximately 1.36 g.cm-3. Photograph was taken at a bar angle of 50” after centrifugation for 21 hr at 42,040 rpm. (In this experiment the cell was overloaded to demonstrate the presence of the minor component).

116

RANDLESETAL.

FIG. 6. Homologous and heterologous serological reactions in gel-diffusion tests between antisera prepared against VTMoV (v) and SNMV (s) and purified preparations of VTMoV (V) and SNMV (S). Homologous titers of anti-VTMoV and SNMV sera were l/512 and l/256, respectively.

injection over a period of 27 days. Tests done by Drs. M. Hollings (Glasshouse Crops Research Institute, Littlehampton, Surrey, U. K.) and G. M. Behncken (Plant Pathology Branch, Department of Primary Industries, Indooroopilly, Queensland) using this serum and its homologous antigen failed to detect any serological relationships between VTMoV and the following viruses with small polyhedral particles sedimenting as a single component in the ultracentrifuge: broad bean mottle, carnation mottle (CaMV), carnation ringspot, cymbidium ringspot, galinsoga mosaic (GMV), glycine mottle, narcissus tip necrosis, red clover necrotic mosaic, saguaro cactus, southern bean mosaic (SBMV), several strains of tomato bushy stunt (TBSV), and turnip crinkle viruses. However, a relationship was established between VTMoV and SNMV, a virus isolated from S. nodi&wum in Queensland (Greber, 1973; Hollings et al., 1979). Tests done in our laboratory confirmed that VTMoV and SNMV are serologically related but not identical. With an antiVTMoV serum, homologous and heterologous titers were found to be l/1024 and l/256, respectively; and with an anti-SNMV serum, l/256 and l/64, respectively. Spur formation in heterologous immunodiffusion tests was observed when the antisera were tested against preparations of the two viruses placed in adjacent wells (Fig. 6). The close serological relationship between the two viruses was confirmed by using antisera to the two viruses, collected from rabbits 10 days after immunization with a single injection of 1 mg virus per animal;

the homologous and heterologous titers were, respectively, l/64 and l/16 with the anti-VTMoV and l/123 and l/32 with the anti-SNMV serum. Isolates of VTMoV collected at the Cobblers Hill and Innamincka sites showed no distinguishable serological differences. Despite the similarities in the morphology of VTMoV and SBMV particles and their sedimentation properties, we confirmed that there was no detectable serological reaction between the two viruses. Using both immunodiffusion, and the more sensitive immunoelectron microscopy tests (Boccardo et al., 1980), no positive reactions were observed with antisera which had homologous titers of l/1024. Similar tests also failed to disclose any relationship between VTMoV and SMV, a tentative member of the southern bean mosaic virus group (Matthews, 1979). Properties

of Nucleic Acid from VTMoV

Nucleic acids prepared from purified VTMoV, either by the phenol-SDS or the Pronase-SDS techniques (Randles et al., 1977), were both infectious, indicating that no covalently linked protein is needed for biological activity as recently reported for SBMV (Veerisetty and Sehgal, 1979). When subjected to electrophoresis in polyacrylamide gels under nondenaturing conditions (Peden and Symons, 1973), four components were resolved and the one with the fastest mobility was present in the largest amount (Fig. 7A). Under denaturing conditions (Air et al., 1976), three major nucleic acid species (1, 2, and 3) and two minor ones (la and lb) were resolved (Fig. 7B); all were sensitive to RNase in buffer containing 0.18 M salt indicating that they are single stranded (ss RNA@. RNA 1 has an electrophoretic mobility very similar to that of SBMV RNA (Fig. 7C) indicating a molecular weight of about 1.5 x log (Shepherd, 1971). The mobilities of RNAs la, lb, 2, and 3 indicate apparent molecular weights of about 0.63 x 106, 0.25 x 106, 0.16 x 10s, and 0.12 x 106, respectively, when compared with the RNAs of CMV and its satellite RNA (Fig. 7D; Fran&i et al. (1979b)). The relative proportions of each of

VELVET

TOBACCO MOTTLE

B

C

VIRUS

D

E

F

FIG. 7. Polyacrylamide gel electrophoretic analyses of RNA preparations. (A) RNA isolated from purified VTMoV electrophoresed under nondenaturing conditions. (B-D) Electrophoresis in the same gel system under denaturing conditions in 7 M urea (see Materials and Methods); (B) similar preparation to that in A showing separation of viroid-like RNA band upon denaturation; (C) RNA isolated from purified SBMV; (D) RNA isolated from purified cucumber mosaic virus containing satellite RNA (five RNA fractions were resolved with molecular weights of about 1.35 x lo’, 1.13 x lOti, 0.82 x lo”, 0.35 x 106, and 0.10 x 106; however, on photographic reproduction the two largest species appear as a single band). (E, F) Electrophoresis in the same denaturing gel as above; total leaves, respectively. RNA isolated from VTMoV-infected (E) and healthy (F) N. elevelandii

the encapsidated VTMoV-RNA species, determined by densitometry of stained polyacrylamide gels, were 7, 4, 7, 47, and 35% in decreasing order of molecular weight. The same RNA components were detected in VTMoV purified from N. velutina plants collected in the field, showing that the composition of encapsidated RNA was unchanged by experimental passage of virus through N. clevelandii. In preparations of RNA extracted by the phenol-SDS procedure from VTMoVinfected N. clevelandii leaves, RNAs 2 and 3 were readily detected by polyacrylamide gel electrophoresis and accounted for between 36 and 56% of the total RNAs present in the leaves (Figs. ‘7E and F). Evidence that RNAs la and lb are degradation products of RNA 1 is presented in an accompanying paper (Gould, 1981).

Electron microscopic examination of denatured RNA 1 and 3 preparations revealed the presence of long and short linear molecules, respectively (Figs. 8A and C). However, in preparations of RNA 2, small circular molecules of about the same length as those of linear RNA 3 were observed almost exlusively (Fig. 8B). The molecular weights of the two RNAs are therefore similar, and the different molecular weight estimates for RNA 2 and 3 from gel electrophoretic analysis are presumably due to the cirda&y of RNA 2. We assume that the value obtained for the linear RNA 3 (0.12 x lo”) is also that for RNA 2. Properties

of VTMoV

Coat Protein

Polypeptides isolated from preparations of purified VTMoV migrated as one major

118

RANDLES

ET AL.

FIG. 8. Electron microscopy of RNA fractions isolated from purified VTMoV. (A) RNA 1 showing long linear molecules. (B) RNA 2 showing small circular molecules. (C) RNA 3 showing short linear molecules. (Bar represents 500 nm.)

and two minor bands when subjected to electrophoresis in polyacrylamide gels under denaturing conditions (Fig. 9B). The mobility of the major band corresponds to a molecular weight of about 33,000 when compared with a series of marker proteins (Figs. 9C-E) whereas the minor bands were estimated to have molecular weights of 36,000 and 31,000. All these proteins are larger than that of SBMV coat protein (Fig. 9A) which has an estimated molecular weight, determined by polyacrylamide gel electrophoresis, of about 27,000 (Hill and Shepherd, 1972). Although only a single polypeptide has been reported in purified preparations of SBMV (Shepherd, 1971; Hill and Shepherd, 1972), a second minor band can be observed in Fig. 9A, the significance of which has not been investigated. Detection and Distribution of VTMoV Particles in Cells of Diseased Plants

Thin sections of leaf tissue from VTMoVinfected N. velutina and N. clevelandii with mosaic or vein-clearing symptoms showed

the presence of numerous electron-dense particles about 22 nm in diameter. These particles were similar in appearance to ribosomes but were often distinguishable by their clearer rounded outlines (Fig. 10). The viral nature of the particles was confirmed by two types of experiments. First, their appearance was unchanged by RNase treatment of tissues after aldehyde fixation prior to embedding which results in degradation of RNA of ribosomes, reducing their ability to take up stain (Hatta and Francki, 1979). Second, their appearance was indistinguishable from that of particles in sections of purified virus preparations, pelleted by ultracentrifugation and processed for electron microscopy by the same method as used for leaf tissues. RNase-resistant VTMoV particles were detected in cells of all leaf tissue types. They were observed in the nuclei, cytoplasm, and vacuoles of infected cells, being most numerous in the cytoplasm (Fig. 10). Vesicles of various sizes containing electron-dense strands were observed in leaf

VELVET

TOBACCO

cells from VTMoV-infected plants (large arrows in Fig. 10) but not in those from healthy plants. The vesicles are seen inside the endoplasmic reticulum and the nuclear envelope; they resemble those observed in leaf cells infected by SBMV (Weintraub and Ragetli, 1970). Electrondense strands were also observed in the cytoplasm (small arrows in inset to Fig. 10) and appear similar to those observed in cells infected with SBMV (de Zoeten and Gaard, 1969) and SMV (Milne, 1967).

MOTTLE

A

VIRUS

BC

119

DE

DISCUSSION

VTMoV has a number of properties which suggest that it is a representative of a previously unrecognized virus group. Three of these properties in particular appear to be unique. First, the isolation of three protein components with molecular weights of 31,000, 33,000, and 36,000 is not known for any other virus, except perhaps for the serologically related SNMV (Greber, 1973), which has been reported as having a major polypeptide (MW 30,200) and sometimes two minor components (MW 27,000 and 27,750) (Hollings et al., 1979). The significance of these observations requires further investigation. Second, and probably the most significant characteristics of VTMoV, is the presence of an encapsidated circular single-stranded RNA component. The structural properties of this RNA (Gould, 1981) are characteristic of those of viroids (Diener, 1979) and its nucleotide composition is unrelated to that of the 1.5 x lo6 MW ss linear RNA also encapsidated in the particle (Gould, 1980). We have examined the RNA composition of a number of ss RNA viruses with small polyhedral particles (unpublished results), and SNMV is the only other virus with a similar viroid-like RNA component. The viroid-like RNAs of the two viruses are compared in a forthcoming paper (Gould and Hatta, in press). Third, to our knowledge, VTMoV is the only virus shown to be transmitted by a mirid bug. Previous reports appear to be unconfirmed (Harris, 1979, and personal communication).

FIG. 9. Polyacrylamide gel eleetrophoretic analyses of protein preparations. (A) SBMV protein. (B) VTMoV protein. (C) Ovalbumin (marker protein of molecular weight 43,000). (D) y-Globulin (marker protein, molecular weight of heavy and l@ht chains, 50,000 and 23,500, respectively). (E) Bovine serum albumin (marker protein, molecular weight 68,000; Weber and Osborn (1969)).

We have named VTMoV using the traditional method of employing the vernacular name of the host in which it is first found, together with a description of the disease it causes. Although a close serological relationship exists between VTMoV and SNMV, we decided not to use the same vernacular name as the previously described virus beeause our isolate does not infect Solarium nodi$mm in tests conducted both in our laboratory and in Queensland by Mr. R. S. Greber (personal communication). Nevertheless, the viruses must be considered as serotypes and the degree of relationship between the nucleotide sequence of their 1.5 x lo6 MW RNAs is considered by Gould and Hatta (in press). This taxonomic paradox illustrates a serious deficiency in the vernacular nomenclature used in plant virology. On the basis of criteria such as gross particle morphology and the distribution of

120

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ET AL.

leaf cell infected with VTMoV. Numerous virus FIG. 10. Thin section of N. clewelandii particles can be seen in both cytoplasm and nucleus (N). Large arrows point to virus-specific vesicles and small arrows to strands (st) observed in the cytoplasm of infected cells. Inset is a section from tissue treated with RNase to remove ribosomal RNA as an aid to virus particle identification (Hatta and Francki, 1979). Endoplasmic reticulum (ER) was more frequently observed than in healthy tissue. (Bars represent 500 nm.)

particles within the host, VTMoV resembles SBMV, the type member of the southern bean mosaic virus group (Matthews, 1979). Particles of VTMoV and SBMV cosediment, have similar buoyant densities, and identical morphologies in negatively stained preparations. Both viruses are distributed throughout all types of tissue and induce the same cytopathic changes. SBMV also contains a ss RNA of

molecular weight 1.5 x lo6 (Shepherd, 19’71) but is reported to have only one polypeptide (Shepherd, 1971; Hill and Shepherd, 1972) which we have confirmed is smaller than all three found in VTMoV. The differences in protein and RNA composition lead us to conclude that VTMoV is not a member of the SBMV group. Because VTMoV displays no significant

VELVET

TOBACCO

heterogeneity in the sedimentation and buoyant density of its particles, it seems likely that all the particles contain the same molar complement of RNA, made up either as a single 1.5 x lo6 RNA, or as approximately 12 viroid-like circular or linear molecules (each with an estimated MW of 120,000). (The great excess of the viroidlike RNA over that of the 1.5 x lo6 RNA precludes the possibility that each particle contains a uniform mixture of the RNAs.) Such a pattern of encapsidation could explain the similar gross physical properties of SBMV and VTMoV, despite their different compositions. We have no evidence that the viroidlike RNA is a plant viroid. The infectivity of RNA 1 and the viroid-like RNA (RNAs 2 and 3) has been tested in preliminary experiments. Neither appears to replicate independently of the other (manuscript in preparation) suggesting that infection by VTMoV depends on the replication of both components, and that both comprise the viral genome. Whether this is the case, or whether the viroid-like RNA exerts a regulatory function on virus replication in the manner proposed for viroids in plants (Diener, 1979), will be the subject of further investigations. The detection of the viroid-like RNA in association with a virus raises a number of questions; did viroids originate in association with helper viruses (Diener, 1974); does circularity of RNA have some functional significance; can the viroid-like RNA be considered as a viroid of a virus? ACKNOWLEDGMENTS We thank Drs. M. Hollings and G. M. Behncken for some serological tests; Mr. R. S. Greber, Drs. G. M. Behncken, A. J. Gibbs, M. Hollings, and P. R. Smith for virus cultures; Mr. R. S. Greber for access to unpublished data; Mr. G. F. Gross of the South Australian Museum for the insect identification; and Mr. D. Talfourd for supply and maintenance of plants. One of us (T. H.) acknowledges financial support from a Commonwealth Special Research Grant of the Department of Primary Industry, and another (A.R.G.) from the Commonwealth Scientific and Industrial Research Organisation. The work was also supported by grants from the Australian Research Grants Committee.

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