The bipartite genome of red clover necrotic mosaic virus

VIROLOGY

108, 499-506 (1981)

The Bipartite Genome of Red Clover Necrotic Mosaic Virus A. R . GOULD,*' R . I . B . FRANCKI,* T . HATTA,*

AND

M . HOLLINGSt

*Department of Plant Pathology, Waite Agricultural Research, University of Adelaide, Glen Osne nd, South Australia and tGlasshouse Crops Research Institute, Littlehampton, Sussex, England Accepted August 28,

1980

Purified preparations of red clover necrotic mosaic virus isolated in Australia have been shown to contain three RNA components whose electrophoretic mobilities in polyacrylamide gel electrophoresis indicate molecular weights of 1 .5 x 106 (RNA 1), 0 .5 x 10 1 (RNA 2), and 0.14 x 10 6 (RNA 3) . Comparisons of the RNAs by hybridization analysis with 1 H-labeled complementary DNAs synthesized in vitro have established that RNAs 1 and 2 are unique RNA molecular species with little or no sequence homology between them . However, RNA 3 appears to be a complex mixture of breakdown fragments of both RNA 1 and RNA 2 . Infectivity experiments with highly purified preparations of RNAs 1 and 2 have demonstrated that both molecules are essential for infectivity .

INTRODUCTION

A virus with small polyhedral particles was isolated from white clover and alfalfa in Victoria, Australia in 1965 but its identity was not established (P. R . Smith and R . H . Taylor, private communication) . We have now identified this virus as a strain of red clover necrotic mosaic virus (RCNMV) and have studied its genomic RNA which is the subject of this paper . MATERIALS AND METHODS

Virus maintenance and assay . The virus isolated from white clover by Drs . P . R . Smith and R . H . Taylor was maintained in a glasshouse by mechanical inoculation to Nicotiana clevelandii A . Gray and assayed on primary leaves of Vigna sinensis Savi . cv . Blackeye. Virus purification . Virus was purified from systemically infected leaves of N . clevelandii . Each 100 g of leaf material was ground in 200 ml of 0 .1 M phosphate buffer, pH 7 .4, containing 0 .1% thioglycollic acid and strained through several layers of cheesecloth . The juice was emulsified with an equal volume of 1 :1 mixture of ' Present address : Department of Biochemistry, University of Adelaide, South Australia . 499

chloroform and butanol and then centrifuged at 10,000 g for 10 min . The supernatant was centrifuged at 78,000 g for 60 min and the pellets were resuspended in 20 mM phosphate buffer, pH 7 .4 . The differential centrifugation was repeated twice and the final virus pellets were suspended in 20 mM phosphate buffer, pH 7.4 . Up to 50 mg virus was obtained from 100 g of leaf tissue . When virus of higher purity was required, the preparations were subjected to centrifugation in 5-25% sucrose (in 20 mM phosphate buffer, pH 7 .4) density gradients in a Spinco SW27 rotor at 26,000 rpm for 120 min . The single virus band was localized and recovered using an ISCO Model 640 density gradient fractionator and ultraviolet monitor, concentrated by centrifugation at 300,000 g for 2 hr, and resuspended in the phosphate buffer . Southern bean mosaic virus (SBMV) was purified as already described (Randles et al ., 1980) . Serology . Antisera to the Australian isolate of the virus were prepared in rabbits injected subcutaneously and serological tests were done by double diffusion in 0 .75% agar gels prepared with 10 mM phosphate buffer, pH 7, containing 0 .02% sodium azide (Francki and Habili, 1972) . Antisera to 0042-0822/81/020499-05$02 .00/0 Copyright c 1981 by Academic Prees, Inc . All rights of reproduction in any form reserved .

500

GOULD ET AL .

other strains were prepared by one intravenous and two intramuscular injections (with Freund's complete adjuvant) (Hotlings and Stone, 197Th) . Purified virus preparations were used in all tests . Sedimentation and buoyant density measurements . Ultracentrifugal analyses were done with Schlieren optics in a Spinco Model E using plain and wedge window cells in an An D rotor and sedimentation coefficients determined by the protractor method (Markham, 1960) . Estimation of virus concentration . The EP'u 250pni for the virus was assumed to be 5 on the basis of its approximate RNA content (Hollings and Stone, 1977b) . Electron microscopy . Purified virus preparations diluted in water were stained with 2% phosphotungstic acid, (PTA) pH 6.8, or 3% uranyl acetate and examined in a JEM 1000X electron microscope (Francki et al ., 1979) . Isolation and purification of viral RNA . RNA was isolated from virus preparations and the components separated by electrophoresis in polyacrylamide gels under denaturing conditions (Gould, 1980) . Preparation of complementary DiVA transcripts and RNA -cDNA hybridization procedures . Complementary DNA (cDNA) to the individually purified viral RNAs was prepared as described by Gould and Symons (1977) except that [3H]dCTP was used instead of [a- 32 P]dATP as the radioactive precursor . RNA-cDNA hybridizations and S, nuclease assays were done as described by Gould and Symons (1977) . RESULTS

Identification of the Australian Isolate of RCNMV

The previously undescribed virus isolated from white clover and alfalfa in Victoria was purified and when negatively stained with uranyl acetate, polyhedral particles about 34 nm in diameter were observed in the electron microscope (Fig . 1) . When stained with PTA, particles from the same preparations measured only about 31 nm ; however, their outlines were not as clearly defined . The particles sedimented in the analytical ultracentrifuge as a single component of about 135 S at infinite dilution

(Fig . 2) . Isopycnic ultracentrifugation of highly purified virus disclosed that the bulk of the material had a buoyant density of 1.37 g em' in CsCl, although a small component reached equilibrium at a slightly higher density (Fig . 3) . However, under preparative conditions the two components could not be resolved . Purified virus preparations were tested against antisei a to a number of viruses with small polyhedral particles . No positive reactions were observed with antisera to several members of the tombusvirus group, including the PLCV-456, PLCV-232, and type strains of tomato bushy stunt virus, carnation Italian ringspot virus, and artichoke mottled crinkle virus (Hollings and Stone, 1975) ; to the clover and cymbidium strains of cymbidium ringspot virus (Hollings and Stone, 1977a) and to the type strain of carnation ringspot virus (Hollings and Stone, 1970) . Similar tests using preparations of the above viruses failed to react with antisera to the Australian virus isolate (having homologous titers between 1)256 and 1 .12048) . However, serological tests with five strains of red clover necrotic mosaic virus (RCNMV) from Europe (Hollings and Stone, 1977b) and their antisera demonstrated that the Australian virus was antigenically related to four of the European RCNMV strains (Table 1) . From these data we conclude that the Australian virus is a strain of RCNMV (RCNMV-Aus .) but there is known to be considerable antigenic variation among the strains (Hollings and Stone, 1977b) . RCNMV-Aus . infected a number of plant species producing symptoms characteristic of RCNMV (Hollings and Stone, 1977b), including N . clevelandii, N . tabacum ev . White Burley, N . glutinosa, Cucumis sativus, Ocimum basilicum, Pha.seolus vulgaris, and Vigna sinesis . On V . sinesis, the symptoms of RCNMV-Aus . were similar to those described for the Swedish strain of the virus, necrotic lesions on the inoculated leaves followed by systemic mottle and apical necrosis (Hollings and Stone, 1977h) . Properties of RNA from RCNMV-Aus .

RNA preparations from purified RCNMVAus . separated into three components when

RED CLOVER NECROTIC MOSAIC VIRUS RNA

501

FIG . 1 . Electron micrograph of a purified preparation of RCNMV-Aus . stained with uranyl acetate (bar represents 100 nm) . FIG . 2 . Schlieren diagram of a preparation containing about 2 .4 mg/ml RCNMV-Aus . (above) and a mixture of about 2 .4 mg/ml RCNMV-Aus . and about 2 mg/ml southern bean mosaic virus (below) . Photographs were taken at a bar angle of 50°, 7 min after the An D rotor had reached a speed of 33,450 rpm . Sedimentation is from left to right . FIG . 3 . Equilibrium density gradient centrifugation in CsCI of approximately 15 µg of RCNMV-Aus . The initial density of the CsCI was about 1 .38 g cm - ' . The photograph was taken at a bar angle of 50° after centrifugation for 19 .5 hr at 44,700 rpm .

electrophoresed in 3% polyacrylamide gels under denaturing conditions (Air et al ., 1976) . The components designated 1, 2, and 3 (Fig . 4, track B) were estimated to have molecular weights of about 1 .5 x 106, 0.5 x 106, and 0 .14 x 10 6 respectively, when their electrophoretic mobilities were compared to those of cucumber mosaic virus

(CMV) RNAs and its satellite RNA (sat RNA) (Fig . 4, track A) . The RNA components were separated by repeated polyacrylamide gel electrophoresis (Gould, 1980) and shown to be relatively pure (Fig . 4, tracks C-E) . While RNA 3 was usually detectable, its amount was variable but always low .

GOULD ET AL .

502 TABLE I

and 500 nucleotides, respectively (Fig . 5) . It has been shown that cDNA transcripts prepared by this method are representative of the entire RNA template without enrichment of any particular regions (Gould and Symons, 1977) . We have assumed that the eDNA transcripts from RNAs 1 and 2 have similar properties. The hybridization kinetics of ['H]cDNAs to a 40 molar excess of unlabeled RNAs are presented in Fig . 6 . The homologous hybridization curves of RNAs 1 and 2 have sharp transitions with R pt,l, values of 1 .0 x 10-2 and 2 .89 x 10-' mol -see liter - ', respectively, consistent with nucleotide complexities expected for RNAs of their molecular weights (Table 2) . No rapidly annealing fractions were detected in either of the hybridizations indicating that no reiterated sequences were present in either of the RNAs . Heterologous hybridizations also showed sharp transitions but with higher Rat values (Fig . 6, Table 2) . Calculations from the data indicate that RNA 1 was contaminated by 2 .3% [(2 .89 x 10-'/ 1.26 x 10-') x 100] by weight of RNA 2 and RNA 2 by 5 .1% [(1 .0 x 10--2/1.95 x 10 - ') x 100] of nucleotide sequences derived from RNA 1 . However, when the RNA 1 preparation was subjected to fur-

SEROLOGICAL RELATIONSHIPS BETWEEN THE AUSTRALIAN AND EUROPEAN STRAINS OF

RCNMV"

Anti-RCNMV-Aus . sera (dilated 14) Virus preparatinn RCNMV-A . . RCNMV-H RCNMV-S RCNMV-Sw RCNMV-TpM-34 RCNMV-TpM-4A

Source of virus Australia England Suuland

Hnmolegeus titer 1'2,043

Homologcms liter 1266 i'-

Sweden Czechoslovakia Czechoslovakia

Antiserum (diluted 1/4)

4 . RCNMV-An, . preparation

RCNMV-Aus. (Homologous Liter 11266) RCNMV-Aus (Homelegeus Liter 1/2,043) RCNMV-H RCNMV-S RCNMV-SW RCNMV-rpM-34

-++

++

RCNMV-TpM-48

^ AU teats were done by immonodiffusion in age, gels as described in Materials and Methods . Formation of strong to weak precipitin lines to +, respectively, and the absence of preupithi are indicated by 1 1 lines is indicated by - . 'All viruspreparatiuNprnducedstrongprecipitinline, (+-to -i) when tested against homologous antisera . .All antisera pmluced strong pmeipitin lines (++ m + + i) wherr tested against homologous antigen preparations .

cDNAs to RNAs 1 and 2 were prepared as described by Taylor et at. (1976) and shown to have average chain lengths of 800 A

B

C

D

E

B

RNA1

RNA 2

RNA 3

FIG . 4 . Electrophoresis of viral RNA preparations in 3% polyacrylamide gels in the presence of 7 M urea. (A) CMV (Q strain) RNAs 1-4 and its satellite RNA; (B) unfractionated RCNMV-Aus . -RNA ; (C) purified RNA 1 from RCNMV-Aus . ; (D) purified RNA 2 from RCNMV-Aus . ; (E) purified RNA 3 from RCNMV-Aus .



Ea U

GEL

SLICE

NUMBER

FIG . 5 . Size distribution of [3H]eDNAs as determined by electrophoresis in 3% polyacrylamide gels in the presence of 7 M urea . Radioactivity was determined from gel slices digested with NCS tissue solubilizer by liquid scintillation spectrophotometry . The arrows marked 1 and 2 indicate the average molecular weights transcribed from RNA 1 (• - •) and RNA 2 (0 0), respectively . The positions of the RCNMV-Aus .-RNAs in the gels are marked by arrow RNA 1 and RNA 2 . RNAs of cucumber mosaic virus and its satellite RNA were also used as markers (not shown) .

100

80

60

40

20

0

-5

-4

-2

-3 Log Rot (mol sec

1_t

0

I

)

FIG . 6. Hybridization kinetics of RCNMV-Aus. RNAs with cDNAs transcribed from them . Homologous reactions between RNA 1 and eDNA 1 (9 - •) and between RNA 2 and cDNA 2

(x-x) ; heterologous reactions between RNA 1 and cDNA 2 (0 - 0 in Expt. 1 and A - A in Expt . 2) and between RNA 2 and cDNA 1 Arrows indicate R4 1, 2 values

which, with other data, are summarized in Table 2 . 503



504

GOULD ET AL .

HYBRIDIZATION OF

RCNMV-Aus RNA component (MW)

TABLE 2 RCNMV-ALS. RNAS AND [ 3 H]CDNA Number of residues

TRANSCRIPTS

RNA species from which [3 HIcDNA was transcribed

Percentage hybridization

RNA 1 (1 .5 x 10 6 )

1 2 (Expt . 1) 2 (Expt . 2)

0-100 0-100 0-100

RNA 2 (0 .5 x 10 6 )

1 2

0-100 0-100

1470

1 .95 x 10 - ' 2 .89 x 10-,

RNA 3 (0 .14 x 106 )

1 2

0-86° 6-69^

4414° 1470`

-1 .0 x 10 -2 -1 .0 x 10 - '

4411

r„t,,, (moLsec liter - ') Determined

Expected"

1 .0 x 10 . 2 126 x 10 -' 4 .57

0 .8 x 10

Contamination (w/w) by other RNA (%)

2

2 .2 0 .06 5 .1 2 .6 x 10 -3 -100 -160

° Calculated using CMV-RNA 4 as a molecular weight standard (0-355 x 106 ) and a R o t,,, of 1 .8 x 10 -3 mot -see liter - ' (Peden and Symons, 1973 ; Gould and Symons, 1977; Gould et al ., 1978) . ° Hybridization taken to a Rot„, of 1 .0 mot sec liter - ' . Displayed complex hybridization kinetics with a broad transition which did not go to completion over 3-4 Log R o t units .

ther purification by reelectrophoresis in polyacrylamide gel, its contamination by RNA 2 was reduced to 0 .063% (2 .89

x 10 -3/4 .57 x 100) by weight (Fig . 3 and Table 2) . The thermal denaturation of the cDNA-

100o o

0

80

I z

60

0 F

I

N O

Tm 88°M

x

40

f z w U C . 0 d a 20

I I I 50 70 90

a -0 100

TEMPERATURE ("C)

FIG . 7. Thermal stability of RNA-DNA hybrids of RCNMV-Aus . RNA I-cDNA 1 (O-O) and RNA 2-cDNA 2 (• - •) . The arrow indicates the T m of both hybrids (88°) .



RED CLOVER NECROTIC MOSAIC VIRUS RNA TABLE INFECTIVITY OF

3

RCNMV-Aus . RNAs

1 AND 2

AND THEIR MIXTURES

Experiment number 1

InoculumRNA 1 (130 gg/ml) RNA 1 (65 gg/ml) + RNA 2 (80 gg/ml) RNA 2 (160 gg/ml) RNA 1 (65 gg/nil) + RNA 2 (80 jig/ml) RNA 1 (26 gg/ml) RNA 1 (13 jig/ml) + RNA 2 (16 gg/ml) RNA 2 (32 gg/ml) RNA 1 (13 gg/ml) + RNA 2 (16 gg/ml)

2

RNA 1 (50 gg/mq RNA 1 (25 gg/ml) + RNA 2 (25 gg/ml) RNA 2 (50 gg/ml) RNA 1 (25 gg/ml) + RNA 2 (25 pg/ml)

Lesions on 10 half-leaves' 19 104

0l 98 5 18

0 28 1 360

0 300

RNA 1 (25 gg/ml) RNA 1 (12.5 jig/ml) + RNA 2 (12 .5 gg/mp

76

RNA 2 (25 gg/ml) RNA 1 (12.5 gg/ml ) + RNA 2 (12 .1 gg/ml)

29"

505

hybridization kinetics were observed (data not shown), indicating that this low-molecular-weight RNA was largely a heterogeneous mixture of nucleotide sequences derived from both RNA 1 and RNA 2 (Table 2) . The results of tests on the infectivity of RNAs 1 and 2 alone and in combinations are presented in Table 3 . RNA 2, although contaminated with 5 .1% by weight of nucleotide sequences derived from RNA 1, failed to produce any lesions (Table 3, Expts . 1 and 2) which indicates that the sequences were noninfectious RNA I fragments . On the other hand, RNA 1 contaminated by only 2 .3% by weight of RNA 2 produced significant numbers of lesions (Table 3, Expt . 1) ; probably due to the contaminating RNA 2 being aggregated intact molecules . However, tests with RNA 1 which contained only 0 .06% contamination by weight of RNA 2 leave no doubt that RCNMV-Aus . has a functionally divided genome requiring both RNAs 1 and 2 to initiate infection (Table 3 . Expt . 2) . DISCUSSION

0I

I

a Hybridization analysis with eDNA's to RNAs 1 and 2 revealed that in Experiment 1 RNA 1 was contaminated by 2 .3% of RNA 2 and RNA 2 by 5 .1% of RNA 1 . In Experiment 2 the same preparation of RNA 2 was used but RNA 1 was further purified by another cycle of polyacrylamide gel electrophoresis and the contamination of RNA 1 by RNA 2 was reduced to 0 .06% by weight . " Assays done on opposite half-leaves of primary cowpea leaves . Lesion numbers joined by brackets indicate that assays were done on the opposite halves of the same leaves .

RNA hybrids showed that under our experimental conditions, hybrid formation was highly specific with no significant mismatching. The hybrids melted with sharp transitions and high T u,'s (Fig . 7) . When RNA 3 was hybridized to cDNAs transcribed from RNAs 1 and 2, complex

Data on the physical, antigenic, and biological properties of RCNMV-Aus . described in this paper leave no doubts that it is closely related to the European strains of the virus (Hollings and Stone, 1977b) . Detailed studies of nucleic acid isolated from RCNMV-Aus. demonstrate that it has a bipartite genome consisting of two unique ssRNA molecules with molecular weights of about 0 .5 x 108 and 1 .5 x 106 . These RNAs are encapsidated in such a way that no particle heterogeneity is evident from their appearance in the electron microscope or from their sedimentation behavior . Several taxonomic groups of plant viruses with small polyhedral particles have been established by the International Committee on Taxonomy of Viruses, three of which contain viruses with bipartite ssRNA genomes, the comovirus, nepovirus, and pea enation mosaic virus groups (Matthews, 1979) . However, RCNMV does not appear to fit into any of these groups by virtue of its particle homogeneity . In this respect it is more like carnation ringspot virus, the particles of which sediment as a single



GOULD ET AL .

506

component (Hollings and Stone, 1970) which contain two functional ssRNAs with molecular weights of 0.5 x 106 and 1 .5 x 106 (Dodds et al ., 1977), and a single coat protein of molecular weight about 40,000 as determined by polyacrylamide gel electrophoresis (R . J . Barton and M . Hollings, unpublished data) . ACKNOWLEDGMENTS We thank Mrs . F. Leggat for supplying us with a culture of the virus ; Dr . P . R. Smith for access to his unpublished data ; Mr . P . T. Atkey for some electron microscopic work ; Drs . R . H . Symons and P . Palukaitis for gifts of avian myeloblastosis virus reverse transcriptase and S, nuclease ; Mr . C . J . Grivell for able technical assistance ; Mrs . L . Wichman fur fine drawings ; and Mr . D . Talfourd for supply and maintenance of plants . One of us (A .R .G .) acknowledges financial support from the Commonwealth Scientific and Industrial Research Organization, and another (T,H,) from a Commonwealth Special Research Grant of the Department of Primary Industry . The work was also supported by a grant from the Australian Research Grants Committee . REFERENCES and COULSON, A . R . (1976) . Nucleotide and amino acid sequences of gene G of OX174 . J . Mob . Biol . 108, 519-533. DODDS, J . A., TREMAINE, J. H ., and RONALD, W. P . (1977) . Some properties of carnation ringspot virus single- and double-stranded ribonucleic acid . Virology 83, 222-328 . 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 ., HATTA, T ., GRYLLS, N . E ., and GRIVELL, C . J . (1979) . The particle morphology and some other properties of chloris striate mosaic virus . Ann . Appl . Biol . 91, 51-59. AIR, G . M ., SANGER, F .,

A . R . (1980). Studies on encapsidated viroidlike RNA . II . Purification and characterization of a viroid-like RNA associated with velvet tobacco mottle virus . Virology, 108, 123-133 . GOULD, A . R ., PALUKAITIS, P., SYMONS . R . H ., and Mossop . D . W. (1978) . Characterization of a satellite RNA associated with cucumber mosaic virus . Virology 84, 443-455 . COULD, 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. HOLLINGS, M ., and STONE, 0 . M . (1970) . Carnation ringspot virus . CMIIAAB Descriptions of Plant Viruses No . 21 . HOLLINGS, M., and STONE, 0 . M . (1975) . Serological and immunoelectrophoretie relationships among viruses in the tombusvirus group . Ann . Appl. Biol . 80,37-48 . HOLT INGS, M ., and STONE, 0 . M . (1977a). Cymbidium ringspot virus . CMIIAAR Descriptions of Plant Viruses No. 178 . HOLLINGS, M ., and STONE, 0 . M. (1977b) . Red clover necrotic mosaic virus . CMIIAAB Descriptions of Plant Viruses No. 181 . MARKHAM, R . (1960) . A graphical method for the rapid determination of sedimentation coefficientsBiochem . J. 77, 516-519. MATTHE WS, R . E . F. (1979). Classification and Nomenclature of Viruses . Third report of the International Committee on Taxonomy of Viruses . Intervirology 12, 132-296 . PEDEN, K . W . C ., and SYMONS, R . H . (1973) . Cucumber mosaic virus contains a functionally divided genome. Virology 53, 487-492 . GOULD,

RANDLES, J . W., DAVIES, C ., HATTA, T ., GOULD, A . R ., and FRANCKI, R . I . B . (1980) . Studies on

encapsidated viroid-like RNA . I . Characterization of velvet tobacco mottle virus . Virology, 108, 111122 . TAYLOR, J . M ., ILLMENSEE, R ., and SUMMERS, J . (1976) . Efficient transcription of RNA into DNA by avian sarcoma virus polymerase . Biochim . Biophys . Acda 442, 324-330 .