Some properties of Tulare apple mosaic and ILAR viruses suggesting grouping with tobacco streak virus

VIROLOGY

70,

440-450 (1976)

Some Properties

of Tulare Apple Mosaic and ILAR Viruses Suggesting Grouping with Tobacco Streak Virus1 R. M. LISTER2

Department

of Botany

and Plant Pathology,

AND

K. N. SAKSENA3

Purdue

Accepted November

University,

West Lafayette,

Indiana

47907

4,1975

Particle sedimentation and size in Tulare apple mosaic and some isometric labile ringspot (ILAR) viruses fulfill criteria previously suggested for grouping plant viruses with tobacco streak virus; namely, a heterogeneity in particle size but homogeneity in particle density. Such viruses apparently comprise different classes of nucleic acid with distinctive sizes and functions, encapsidated in corresponding classes of particles. On the basis of these and other criteria, recent evidence also suggests the inclusion of other quasi-isometric viruses and alfalfa mosaic virus in the group.

tially isometric particles differentiable as Tulare apple mosaic virus (TAMV) has differently sedimenting particle classes. been isolated only once in nature, from an Moreover it has been suggested (Lister et apple tree with mosaic symptoms in Tu- al., 1972) that such viruses might be inlare County, California (Yarwood, 1955; cluded in a group, typified by TSV, in Fulton, 1971a). Though serologically unre- which a primary common feature would be lated, it shares several distinctive proper- that the particle classes are differentiated ties with tobacco streak virus (TSV), by size, not density. Here we present data supporting this which is widespread (Fulton, 1971b), and criterion for group membership, especially with several common viruses of perennial rosaceous plants, called ILAR (isometric for TAMV, but also for representative labile ringspot) viruses by Fulton (1968). ILAR viruses. An abstract of some of these The ILAR viruses comprise various sero- results appeared earlier (Lister and Saklogically interrelated strains of prunus ne- sena, 1975). We suggest also that results crotic ringspot virus (Fulton, 197Oa),apple published while this manuscript was in mosaic virus (sensu Fulton, 19721, and preparation (Gonsalves and Garnsey, 1975; rose mosaic virus, together with prune Van Vloten-Doting, 19751, showing dependence on structural protein for nucleic dwarf virus. The distinctive properties shared by acid infectivity in TSV, citrus leaf rugose TAMV, TSV, and ILAR viruses include virus, and alfalfa mosaic virus, tend both to confirm and extend such a grouping. sensitivity to oxidized plant polyphenols, infectivity dilution curves suggesting parMATERIALS AND METHODS ticle interaction in infection, and essenINTRODUCTION

1 Journal Paper No. 5969 of the Purdue University Agricultural Experiment Station. Supported in part by the National Science Foundation (GB37764). * Portions of this work were done while RML was at the East Malling Research Station, near Maidstone, England. 3 Postdoctoral Associate. Present address, the Carnegie-Mellon University, Pittsburgh, Pennsylvania. 440 Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

Virus

culture

and

purification.

The

sources and origins of the viruses mainly used are listed in Table 1. Most work was done with TAMV transmitted from apple cuttings supplied by Dr. G. Nyland. Symptoms were typical of TAMV (Fulton, 1971a1, and purified preparations reacted specifically with TAMV antisera supplied by Dr. R. Fulton. The ILAR viruses re-

TAMV

AND

ILAR

VIRUSES TABLE

GROUP

WITH

441

TSV

1

SOURCES OF VIRUSES USED FOR ANALYSIS Designation Apple mosaic virus

(AMV-Pl

Apple mosaic virus (AMV-Sl European plum line pattern (EPLP) Prunus necrotic ringspot (NRSV-Cl061 Prunus necrotic ringspot (NRSV-Gl Tobacco streak virus (TSVM(H)) Tulare apple mosaic virus (TAMV)

Source

Supplier

“Paradise” apple, Calif. “Lord Lambourne” apple, England Plum, England

R. W. Fulton, Madison, Wise. R. Cropley, East Mailing, England R. Cropley, East Malling, England R. Cropley, East Malling, England

Cherry (necrotic line pattern disease), England Cherry, Wise. Tobacco, Wise. Apple,

Calif.

acted serologically as described under Results. TAMV was usually cultured for purification in Christie’s Nicotianu hybrid (Christie, 19691, systemically infected for lo-12 days. The ILAR viruses were cultured in cotyledons of cucumber (Cucumis satiuus L., cvs. Chicago Pickling or Ohio). Except where noted, plants were grown in greenhouses with temperatures fluctuating around 26-28” with seasonal variations in light and day length, but with auxiliary tungsten lighting in the winter months to improve vegetative growth. TAMV was purified essentially by methods developed for TSV (Clark and Lister, 1971; Lister et al., 1972). All processing was at about 4”. Fresh, chilled leaf was extracted at 1:2, w/v, in 0.02 M disodium phosphate containing 0.02 M sodium diethyldithiocarbamate (DIECA) and 0.02 M sodium thioglycollate. The extract (pH about 7.2) was squeezed through cheesecloth and adjusted to and maintained at pH 4.8-5.0 during 2-hr storage. After centrifugation (10 min at 10,000 g) the supernatant fluid was then adjusted to pH 6.5, and made 10 and 1% (w/v), respectively, with polyethylene glycol (MW 6000 = PEG) and NaCl. The precipitate obtained by centrifugation was suspended overnight in 0.01 M pH 7 phosphate buffer, when the suspension was again clarified by low-speed centrifugation, after adjust-

R. W. F&on, Madison, Wise. R. W. Fulton, Madison, Wise. G. Nyland, Davis, Calif.

Reference F&on,

1965

De Sequeira,

1967

Seneviratne and Posnette, 1970 Posnette and Cropley, 1956 Fulton,

1968

Lister, Ghabrial, Saksena, 1972 Yarwood, 1955

and

ment to pH 5. Subsequent concentration and purification was by one or two cycles of differential ultracentrifugation, resuspending pellets in 0.05 M pH 5 acetate buffer. When frozen leaf was used, it was first crushed and infiltrated in uucuo with the cold reducing solution or with phosphate buffer at pH 7 containing 0.01 M DIECA (Mink et al., 1963) before proceeding as above. Yields varied between 2-6 mg/lOO g of leaf assuming E!@ = 6 (Fulton, 1967). The ILAR viruses were purified by a modification of Fulton’s procedure (1967), using hydrated calcium phosphate (HCP). Fresh, chilled tissue was extracted as above, but in buffer containing 1% PEG (w/v). After clarification with HCP, virus was concentrated by precipitation with 10% PEG and 0.8-l% NaCl (w/v), resuspending precipitates in 0.02 M phosphate buffer at pH 7.0. Further purification was by one or two cycles of differential ultracentrifugation followed by rate-zonal density-gradient centrifugation (Brakke, 1963). Yields were not determined accurately but were much lower than for TAMV. Analysis of preparations. Analysis by ulspectrophotometry, density-gradient tracentrifugation, free boundary electrophoresis, electron microscopy, and serological testing were as described for TSV (Lister et al., 1972). Similarly, antisera to

442

LISTER

AND

TAMV were made by injecting purified preparations into rabbits, first im (with Freund’s incomplete adjuvant) and then, after 2 weeks, iv at weekly intervals over a 4-week period. For infectivity tests with TAMV, the different particle classes were separated as for TSV (Lister et al., 1972), by three to five successive cycles of rate-zonal sucrose density-gradient centrifugation using first the Spinco SW 27 rotor, and then the SW 41 rotor; but ribonuclease-free sucrose was used, because infectivity was drastically reduced by using the technical grade. The local lesion assay host was Phaseolus vulguris cv. Bountiful.

SAKSENA

the analytical ultracentrifuge for TV, MV and BV in various dilutions of unfractionated virus, are plotted in Fig. 2. Extrapolation to infinite dilution, assuming linearity, gave values for TV, MV, and BV of 93, 106, and 114 S, respectively. Comparisons of uv-absorbance spectra for the separated components and for unfractionated preparations indicated the same protein-nucleic I

1

RESULTS

TAMV Rate-zonal and anulyticat ultrucentrifugations. TAMV, prepared as described

or by the butanol-clarification procedure of Mink et al. (19631,resolved into three major and one minor sedimenting components when analyzed by analytical ultracentrifugation or rate-zonal sucrose density-gradient centrifugation. Figures 1, 4, and 7, illustrating typical uv-absorbance profiles from density-gradient analyses, show the proportions of the major components obtained, corresponding to top, middle, and bottom (TV, MV, and BV), by the convention used previously for TSV (Clark and Lister, 1971; Lister et al., 1972). The minor component was usually resolved as a small shoulder preceding the TV component, in a position corresponding with that of the “super-top” @TV) component seen in some preparations of TSV (Lister et al., 1972). By analogy with other viruses of this type, including citrus rugose leaf virus (see Discussion), we take BV, MV, TV, and STV to be equivalent, respectively, to the nucleoprotein components in TSV named NPl, NP2, NP3, and NP4, by Van Vloten-Doting (1975); but for the present, we retain our nomenclature for simplicity of comparison with earlier papers. Components sedimented to the same level in rate-zonal density gradients at pH 5 (acetate) or pH 7 (phosphate). Sedimentation coefficients (szoJ as determined in

I

1 RELNTIVE DEPTH

FIG. 1. UV-absorbance profiles of a formalinized preparation of TAMV analyzed by: BC, rate-zonal density-gradient centrifugation on a 5-20% sucrose gradient (Spine0 SW 41 rotor, 90 min at 39,000 rpml before treatment with CsCl; C, equilibrium zonal density-gradient centrifugation in 38% CsCl dissolved in 0.01 M pH 5 acetate buffer (24 hr at 45,000 rpm in Spinco SW 65 rotor at 25”). AC is the profile of the peak material recovered from the CsCl gradient, re-analyzed by rate-zonal density-gradient centrifugation.

$Pu

12345676 CONC. (mg/ml)

FIG. 2. Plot of variation tion, for TAMV.

of sZ+ with concentra-

TAMV

AND

ILAR

VIRUSES

acid ratio for each, with A280,26o values of 0.73 for preparations with negligible scattering at 320 run. These values are similar to those of Bamett and Fulton (1969). With TSV, both the yield of viral nucleoprotein and component ratio vary significantly with host, and under certain conditions, substantial proportions of the STV component are produced (Lister et al., 1972; Saksena and Lister, unpublished). With TAMV, viral nucleoprotein yields from Christie’s Nicotiana hybrid were about double those from N. &veZandii, 510 times those from N. tabacun or cucumber, and more than 10 times those from Chenopodium quinoa, but component ratios were essentially the same. Moreover, component ratios were also similar for preparations made from Christie’s hybrid and C. quinoa raised at 15, 22, or 30”, although yields at 30” were negligible. Thus, in all the TAMV preparations made, STV was an almost undetectable component. Equilibrium-zonal density-gradient centrifugations. TAMV was unstable in 40%

CsCl at pH 5 or 7, and at 4 or 25”, but it was stabilized by making solutions 9% with formalin for about 30 min at room temperature and then removing excess formalin by dialysis overnight at 4”. When so treated, the virus sedimented in equilibrium-zonal density-gradient centrifugations as a single band, at a density of 1.37 (25”) in solutions of CsCl at pH 5 or 7 (0.1 M acetate or 0.05 M phosphate). Recycling

4.0

c

p” -0-l.O-2.0

WITH

TSV

443

this band of viral material on rate-zonal density gradients after recovery by dilution, pelleting by ultracentrifugation, and resuspension in buffer, again resolved it into approximately the usual proportions of TV, MV, and BV components (Fig. 1). This result parallels the behavior of TSV (Lister et al., 19721,and indicates heterogeneity in particle size, not density. Antigenicity of the separated components. TV, MV, and BV separated by four

successive cycles of rate-zonal density-gradient centrifugation, reacted with undiluted antiserum to TAMV (titer 1:128) to form single precipitin lines, which coalesced without spur formation, indicating antigenic identity. Electrophoretic analysis. When subjected to free-boundary electrophoretic analysis as previously applied with TSV (Lister et al., 19721, TAMV preparations migrated as a single peak at pH 7 and 6, indicating electrophoretic homogeneity. Preparations showed the same centrifugal heterogeneity before and after electrophoresis at pH 7. An additional small electrophoretic peak migrating more slowly than the major peak appeared at pH 5 and 4, but virus dialyzed to pH 3 or 2 precipitated completely, suggesting that this secondary peak was due to aggregation of part of the viral nucleoprotein near the isoelectric point, which was about pH 3.74 (Fig. 3). Electrophoretic peak ratios did not coincide with the centrifugal heterogeneity of the preparations used (cf. Fig. 4). More-

---

x0; P.O8,.0-

GROUP

Bp ! 2.0 ‘4.0 5.0 ’ 3.0mvm ” 6.07.0 ” a0 9.0 I PH -

-

FIG. 3. Plot of variation of electrophoretic mobility with pH for TAMV (major peak). Dashed lines are presumed extrapolations. Lower, middle, and upper schlieren patterns are for ascending limb boundaries after 240, 260, and 280 min at 10 mA at pH 4, 5, and 6 (0.1 Fm), respectively (electrophoretic migration from left to right).

444

LISTER AND SAKSENA

I

I

RELATIVE

DEPM

FIG. 4. UV-absorbance profiles of: above, ratezonal density-gradient centrifugation analysis of a preparation of TAMV, below, analysis of nucleic acid extracted from same preparation and electrophoresed on 2.5% polyacrylamide/0.5% agarose composite gel. Electrophoresis was in Loening’s buffer + 0.2% SLS for 2 hr at 6 mA/gel. Positions of components corresponding to nucleoprotein components TV, MV, and BV are marked T, M, and B, respectively.

over, the secondary electrophoretic peak was larger at pH 4 than at pH 5, consistent with increased aggregation at the lower PH. Vim1 protein and nucleic acid. SDSpolyacrylamide-gel electrophoresis of unfractionated virus (Ghabrial and Lister, 1974) gave a single band, indicating a single structural protein. Viral nucleic acid prepared by the SDSphenol procedure of Clark and Lister (19711, using bentonite to reduce nuclease activity, was resolved by polyacrylamidegel electrophoresis into three peaks, in ratios approximately corresponding to those of the nucleoprotein components (Fig. 4). Their molecular weights, estimated by comparing their mobilities with that of Escherichia coli ribosomal RNA, and with cowpea chlorotic mottle virus RNA and tobacco mosaic virus RNA (using the values of Fowlks and Young, 1970), were 0.74, 0.92, and 1.Ol x 10” for the nucleic acid of TV, MV, and BV, respectively. In the

same series of experiments, molecular weights for the RNA’s from TSV strain M were estimated at 0.78, 0.98, and 1.04 for the nucleic acids of TV, MV, and BV, respectively. These values are in reasonable agreement with previous estimates (Clark and Lister, 1971; Ghabrial and Lister, 1974). Electron microscopy. Electron microscopy of the separated TV, MV, and BV components of TAMV suggested that they corresponded to particle size classes (Fig. 5). This was confirmed by plots of measurements of about 200 particles from each component (Fig. 6). Mean diameters for the particle classes were 27.5, 29.5, and 31 nm for TV, MV, and BV, respectively. Although particles were extensively degraded in phosphotungstate at pH 7, most appeared well preserved in uranyl acetate at pH 4.7, but many were oval in outline, rather than circular. In the same series of experiments, mean diameters of particles of the STV, TV, and MV components of TSV-M were 24.6, 27.5, and 29.5 nm, respectively. The values for the TV and MV components agree closely with those for TSV-GV published earlier (Lister et al., 1972). Infectivity

of the separated components.

We attempted to resolve the question of component infectivity in two ways, first by comparing the infectivities of droplet fractions from rate-zonal density gradients, and second by comparing the infectivity of separated components remixed in various combinations. In three experiments using the first approach, peak infectivities were at (Fig. 71, or just below the MV position. In other experiments, BV as separated by successive cycles of rate-zonal density-gradient centrifugation was usually more infective than MV, but the infectivity of TV was always very low. Mixing the separated components at concentrations at which they were individually noninfective, or nearly so, gave infectivity (Table 2). Mixtures of TV and BV were moderately infective, mixtures of MV and BV more so, and mixtures of all three components were highly infective. In these experiments, the TV used remained noninfective even when used-concentrated ten-

TAMV

AND

ILAR

VIRUSES

GROUP

WITH

TSV

445

FIG. 5. Particles of TAMV (left) and NRSV-G (right) from separated TV (top row), MV (middle row), and BV (bottom row) fractions, negatively stained with uranyl acetate. Magnification approx 170,000 for TAMV and 150.000 for NRSV-G.

fold (0.15 AZ&, and although the BV com- sibility of interaction of mutually compleponents concentrated similarly were quite mentary particles within classes, as proinfective, this could have been due to posed by Fulton (1970b1,is not excluded. traces of MV and TV particles contaminating this fraction. Considering the diffi- ILAR Viruses culty of particle separation, the simplest Serotype grouping. ILAR viruses can be interpretation of our results is that infec- grouped into serotypes by their reactions tivity requires the interaction of particles with appropriate antisera (Fulton, 1968). from each particle class, although the pos- Two of these are exemplified by the apple

446

LISTER AND SAKSENA TAW (TV,

30 2

NRSV-G 1MV I

Dr. R. W. Fulton) and to the Hop-A-NRSV and Hop-C-NRSV strains (Bock, 1967) grouped the ILAR viruses we investigated into these two serotypes. Typical results showing different reaction intensities are illustrated in Fig. 8. European plum line pattern (EPLP), Sequeira’s AMV-S, Fulton’s AMV-P, and a virus isolate from Fuggle hop (AMV-F) were of the AMV type, and NRSV-Cl06 and NRSV-G were of the NRSV type. These results are in accord with those of others summarized by Fulton (1968). Rate-zonal analysis. Preparations of the ILAR viruses resolved into several distinct components when centrifuged on ratezonal density gradients at pH 7. The proportions of components were somewhat characteristic, but varied more than with TAMV, especially after repeated subculturing. The numbers of components clearly resolved also varied. However, resolution was improved when virus was treated with formalin (as described for TAMV), and the results of many centrifugations suggested that all the viruses typically comprised four resolvable components (Fig. 9). In Fig. 9, the codes STV, TV, MV, and BV can be assigned to components by analogy with TAMV and TSV. Assuming this analogy, with the AMVtype viruses EPLP, AMV-S, and AMV-P, components STV and BV predominated, whereas with NRSV-Cl06 and NRSV-G the STV component resolved only as a small shoulder. Except with AMV-F, the components of the apple mosaic group viruses sedimented faster than those of the necrotic ringspot group (Fig. 9). Equilibrium density-gradient centrifugation. Several analyses were made of

FIG. 6. Histograms of particle classes TV, MV, and BV for (above) TAMV (1 mm = 6.6 A) and (below) NFtSV-G (1 mm = 7.4 A).

mosaic virus,

AMV-P, and the necrotic ringspot virus, NRSV-G, of Fulton (Table 1). In gel diffusion tests, antisera to the AMV-P and NRSV-G strains (supplied by

NRSV-G, NRSV-C106, and AMV-P, prepared by rate-zonal density-gradient centrifugation, formalinized, and then subjected to prolonged centrifugation (at 4 or 25” for 24-48 hr) after mixing with CsCl (final concentration 36% at pH 7). The results (Fig. 10) always suggested slight density heterogeneity, to the extent that preparations sedimented as two barely resolvable species. The “light” and “heavy” spe-

cies were too close to separate, but ratezonal analysis of fractions containing predominantly one or other species (Fig. 10)

TAMV

FIG. 7. Infectivities of selected TAMV in relation to UV-absorbance TABLE INFECTIVITIES

ILAR

VIRUSES

GROUP

WITH

TSV

droplet fractions from a rate-zonal density-gradient centrifugation profile. Centrifugation 90 min at 39,000 rpm in a Spinco SW 41 tube.

447

of

2

OF TAMV NUCLEOPR~TEIN COMPONENTS

Fraction”

Lesions totals on 10 halfleaves Expt.

TV MV BV TV + BV TV + MV MV + BV TV + MV + BV Unfractionated (I Each fraction of 0.015 A,,,,.

AND

0 2 1 26 3 38 84 14

1

Expt. 2

Expt. 3

1 0 3 51 21 149 464 -

0 1 0 27 7 121 210 -

was used at a final concentration

indicated that most of the typical viral nucleoprotein was in the “light” species. Conceivably the “heavy” species is a host contaminant, sedimenting along with virus. If it represents a proportion of denser virions in the preparations, these do not appear to be specifically from one particular component. Electron microscopy. De Sequeira (1966) differentiated two serologically identical sedimenting species in AMV-S, quoting modal particle diameters as 25.4-26.3 and 28.7-29.6 nm. Our measurements of particles in the separated TV, MV, and BV components from a preparation of NRSVG (Fig. 6) indicated mean diameters of 26.6, 30, and 32.5 nm, respectively. The particles were degraded in phosphotungstate but were preserved in uranyl acetate. Many appeared oval rather than circular,

FIG. 8. Representative gel double-diffusion reactions between ILAR viruses and antisera. Center wells contain undiluted antisera to: A, a Hop ANRSV type isolate (Bock, 1967); B, EPLP; C, AMVP, D, NRSV-G. Outer wells contained partially purified samples of: 1, plum decline virus (= NRSV; Seneviratne and Posnette, 1970); 2, “Ontario” ringspot virus (= ORSV = NRSV, lot. cit.); 3, NRSVC106; 4, AMV-S; 5, plum decline virus; 6, “Ontario” ringspot virus. Homologous antiserum titers as in Bock (1967).

and sausage-shaped or bacilliform particles were common in MV and BV (Fig. 5), but we measured only essentially isometric particles for the histograms in Fig. 6. DISCUSSION

On the basis of sedimentation and particle measurements, indicating size heterogeneity, but density homogeneity, TAMV fulfills the criteria suggested (Lister et al.,

LISTJZR AND SAKSENA

RELt’Tl”E DEPTH FIG. 9. Two typical sets of UV-absorption profiles of rate-zonal density-gradient analyses of six different preparations of AMV-S, AMV-F, EPLP, and NBSV-C106, selected to show that each has at least four components. Centrifugation 100 min (top) or 120 min (bottom) at 39,000 r-pm in Spinco SW 41 tubes. Each set run in sister tubes. Absorbing peaks on left are residual host contaminants.

1972) for grouping viruses with TSV. As with TSV also, electrophoretic analysis of intact virus and viral protein, and the antigenic behavior of separated particle classes, indicate homogeneity of the structural protein. Nucleic acid/protein ratio was the same for each particle class, and although the possibility of heterogeneity in the nucleic acid contained in specific particles is not excluded, the number and proportion of nucleic acid species observed conformed approximately with the particle classes. Similarly, despite the indications of some density heterogeneity, the evidence from centrifugal analysis and electron mi-

croscopy strongly suggests that the ILAR viruses investigated group with TSV and TAMV. Evidence of the same type also suggests inclusion of elm mottle virus in the group (Jones and Mayo, 1973), and new evidence of serological relationship with TAMV (Lister, Gonsalves, and Garnsey, unpublished) suggests including the citrus rugose leaf and citrus variegation viruses (Garnsey, 1975). The particles of all such viruses appear to result from the encapsidation of different classes of nucleic acid, with distinctive sizes and functions, in corresponding classes of virions comprising different numbers of identical protein subunits. The occurrence of four classes of virions, . in varying proportions, appears to be the rule. The reasons for the variations in proportions, which often appear to be characteristic of virus, strain, or culture conditions (e.g., Lister et al., 1972), is unknown. Though most particles appear essentially isometric in electron micrographs, ovoid and bacciliform shapes are common, and with TSV, previous work (Lister et al., 1972; Ghabrial and Lister, 1974) showed that particle sizes and construction were not consistent with theoretical values for a series of icosahedral particles comprising identical subunits.

FIG. 10. UV-absorbance profiles of: left, CsCl equilibrium density-gradient analysis of A&IV-P and NBSV-C106; right, rate-zonal density-gradient analyses (I-A-Cs and II-A-Cs) of samples I and II (delimited by arrows) collected from the gradients, compared with analyses of the same preparations not centrifuged in C&l (V-B-G).

TAMV

AND

ILAR

VIRU ‘SES GROUP

That particle size is predicated on nucleic acid content implies that the nucleic acid has a structural role in maintaining particle integrity. This is supported by the absence of empty protein shells, by observations with TSV of RNAse sensitivity (Clark and Lister, 1971), and by observations with TSV, TAMV, AMV-P, and NRSV-Cl06 that particles are substantially degraded by incubation of preparations in 0.04-0.08% SDS at pH 7 for 30 min at room temperature (Lister and Hsiao, unpublished; see Acknowledgments). These properties distinguish viruses stabilized by protein-RNA rather than proteinprotein interactions (Boatman and Kaper, 1972; Kaper, 1973). Interestingly, the above criteria also fit alfalfa mosaic, a virus with particles that at first sight do not suggest grouping with TSV and that has an aphid vector. Classification of alfalfa mosaic virus with viruses considered here is also strongly supported by recent evidence that coat protein is required for infectivity of alfalfa mosaic, citrus rugose leaf, citrus variegation, and tobacco streak viruses, and that the coat proteins are interchangeable for this purpose (Van Vloten-Doting, 1975; Gonsalves and Garnsey, 1975; and personal communication). It would be of considerable interest to know if this phenomenon applies also with ILAR viruses. Meanwhile we suggest that, in the classification proposed by Harrison et al. (1971), it would be appropriate, for the present, to group alfalfa mosaic virus, TSV, TAMV, the ILAR viruses, elm mottle virus, and similar viruses, on the basis of having what appear to be important physical and biological properties in common. ACKNOWLEDGMENTS Besides those listed as supplying virus isolates and antisera, we wish to thank Dr. Charles E. Bracker for electron microscopy, Mrs. Margaret AnnYoung and Mr. Robert Kennedy for technical assistance, and Miss Nina Hsiao who assisted as an N.S.F. High School Trainee in investigating the effects of SDS on virion integrity. Use of laboratory facilities at East Malling Research Station by one of us (R.M.L.1 during a sabbatical leave, is gratefully acknowledged.

WITH

TSV

449

REFERENCES BARNETT, 0. W., and FULTON, R. W. (1969). Some chemical properties of Prunus necrotic ringspot and Tulare apple mosaic viruses. Virology 39,556561. BOATMAN, S., and KAPER, J. M. (1972). Forces responsible for the generation of virus structure: The use of SDS to probe protein-RNA interactions. Proceedings, 1st John Innes Symposium, Norwich, England, pp. 123-124. North Holland, Amsterdam. BOCK, K. R. (1967). Strains of Prunus necrotic ringspot in hop (Hum&us lupulus L.1. Ann. App. Biol.

59, 437-446. BRAKKE, M. K. (1963). Photometric scanning of centrifuged density gradient columns. Anal. Biothem. 5, 271-283. CHRISTIE, S. R. (1969). Nicotiana hybrid developed as a host for plant viruses. Plant Dis. Rep. 53,939941. CLARK, M. F. and LISTER, R. M. (19711. Preparation and some properties of the nucleic acid of tobacco streak virus. Virology 45, 61-74. DE SEQUEIRA, 0. A. (1966). Purification and serology of an apple mosaic virus. Virology 31, 314-322. FOWLKS, E., and YOUNG, R. J. (1970). Detection of heterogeneity in plant viral RNA by polyacrylVirology 42, 548-550. amide gel electrophoresis. FULTON, R. W. (1965). A comparison of two viruses associated with plum line pattern and apple mosaic. Zast. Bilja 16, 427-430. FULTON, R. W. (1967). Purification and some properties of tobacco streak and Tulare apple mosaic viruses. Virology 32, 153-162. FULTON, R. W. (1968). Relationships among the ringspot viruses of Prunus. Deut. A&d. Landwirtsch. Wiss. Berlin Tagundsber. 97, 123-138. FULTON, R. W. (1970a). Prunus necrotic ringspot virus. “Descriptions of Plant Viruses.” No. 5, Commonw. Mycol. Inst./Assoc. Appl. Biol. FULTON, R. W. (1970b). The role ofparticle heterogeneity in infection by tobacco streak virus. Virology 41, 288-294. FULTON, R. W. (1971a). Tulare apple mosaic virus. “Descriptions of Plant Viruses.” No. 42, Commonw. Mycol. Inst./Assoc. Appl. Biol. FULTON, R. W. (1971b). Tobacco streak virus. “Descriptions of Plant Viruses.” No. 44, Commonw. Mycol. Inst./Assoc. Appl. Biol. FULTON, R. W. (1972). Apple mosaic virus. “Descriptions of Plant Viruses.” No. 83, Commonw. Mycol. Inst./Assoc. Appl. Biol. GARNSEY, S. M. (1975). Purification and serology of the Florida isolate of citrus variegation virus. In “Proceedings of the Sixth Conference of the International Organization of Citrus Virologists,” pp. 169-175. University of California, Division of Agricultural Sciences, Berkeley.

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GHABRIAL, S. A., and LISTER, R. M. (1974). Chemical

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and physiochemical properties of two strains of tobacco streak virus. Virology 57, l-10. GONSALVES, D., and GARNSEY, S. M. (1975). Functional equivalence of an RNA component and coat protein for infectivity of citrus leaf rugose virus. Virology 64, 23-31. HARRISON, B. D., FINCH, J. T., GIBBS, A. J., HOLLINGS, M., SHEPHERD, R. J., VALENTA, V., and WETTER, C. (1971). Sixteen groups of plant vi-

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