Towards a system for the identification and classification of potyviruses

VIROLOGY

‘73, 350-362 (1976)

Towards

a System for the Identification Potyviruses

I. Serology

and Amino Acid Composition S. M. MOGHAL’

Department

of Plant Pathology,

AND

and Classification of Six Distinct

Viruses

R. I. B. FRANCKI”

Waite Agricultural Research Institute, South Australia Accepted April

of

University

of Adelaide,

20,1976

Antigenic relationships of six distinct potyviruses were studied by immunodiffusion tests using highly purified sonicated virus preparations and anti-intact virus sera devoid of detectable antibodies to host-plant antigens. Three variants of bean yellow mosaic virus (BYMV) including BYMV sensu strict0 and two variants of pea mosaic virus (PMV and SPMV) were shown to be antigenically very similar and also relatively closely related to lettuce mosaic virus (LMV). Distant antigenic relationships were detected between the BYMV variants and bean common mosaic virus (BCMV); between BCMV and passionfruit woodiness virus (PWV); and between PWV and potato virus Y (PVY). No antigenic relationships were detected between any of these viruses and sugarcane mosaic virus (SCMV). Antibodies in anti-viral sera were very poor in recognizing coat proteins dissociated with LiCl from homologous viruses and failed altogether to recognise those dissociated with pyrrolidine. Attempts to prepare antisera in mice against isolated viral coat proteins dissociated with either LiCl or pyrrolidine were unsuccessful due to poor immunogenicity of the preparations. Electrophoretic mobilities of the viral coat proteins relative to marker proteins in the presence of sodium dodecyl sulphate suggest that the protein subunits of all the viruses studied have molecular weights of about 33,000. However, the coat proteins were prone to partial degradation. The amino acid compositions of the antigenically closely related viruses were very similar, but similarities of those distantly related were no greater than those of the apparently unrelated viruses. The problems in the use of serological and amino acid composition data obtainable with currently available techniques for the classification of potyviruses are discussed. INTRODUCTION

of their biological properties and particle morphology. It seems that some of these viruses may prove to be sufficiently similar to be considered synonymous. For example, it has already been suggested that bean yellow mosaic virus (BYMV) and pea mosaic virus (PMV) should be considered as strains of the same virus (Taylor and Smith, 1968; Bos, 1970; Bos et al., 1974). If the taxonomy of potyviruses is to be orderly, much more work will have to be done on the detailed characterization of the individual viruses. The properties of viruses already named should be checked for similarities sufficient to identify those

Nearly 100 apparently distinct viruses have been listed as possible members of the potyvirus group by Edwardson (19741, and thus it must be the largest of the plant virus groups so far defined (Harrison et al., 1971; Shepherd et al., 1976). Most of the viruses listed by Edwardson (1974) have not been characterized in any detail and many have been described on the basis ’ Present address: Department of Plant Pathology, A.R.I., Tandojam, Sind, Pakistan. * Author to whom requests for reprints should be addressed. 350 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

CLASSIFICATION

which may be synonymous. Every effort must also be made to avoid description of new members of the group without adequate justification. Since the potyvirus group is already large, it would also be desirable to divide its members into smaller groups. In order to achieve these aims, clearly defined criteria are required for the identification and classification of potyviruses. With this in mind we have investigated some characteristics of a range of potyviruses in an endeavour to determine which characteristics are likely to prove useful in identifying and classifying members of the group. Gibbs (1969) and Edwardson (1974) have already made suggestions as to how the potyviruses may be subdivided. Antigenic properties of viruses and their amino acid compositions are commonly used criteria in classification (Gibbs, 1969). In this paper we report serological and amino acid composition data on six distinct potyviruses isolated in Australia. BYMV (three variants), lettuce mosaic virus (LMV), bean common mosaic virus (BCMV), and passionfruit woodiness virus (PMV), all of which commonly infect leguminous plants, are compared with potato virus Y (PVY), which infects dicotyledons but has few hosts in the Leguminosae, and sugarcane mosaic virus (SCMV), which is largely confined to graminaceous hosts (Edwardson, 1974). MATERIALS

AND

METHODS

Virus Isolates Virus isolates are listed in Table 1 together with their origins. The cultures were maintained in an insect-proof glasshouse in the plants indicated in Table 1. Special precautions were taken to avoid any of the viruses becoming contaminated with broad bean stain virus when propagated in Vicia faba which sometimes carried this virus in its seed (Moghal and Francki, 1974). All transmissions were done by sap inoculation. Virus Purification Attempts to purify the viruses by methods described in the literature were unsatisfactory in our hands. However, satisfac-

OF POTYVIRUSES

351

tory preparations were obtained by using the following two methods. Method 1. (For BYMV isolates, LMV, PWV, and PVY): Systemically infected leaves were harvested 14-16 days after inoculation, cooled to 4” and homogenized in a Waring blender with 2 vol (w/v) of 0.5 M borate buffer, pH 8, containing 0.15% thioglycollic acid and one-half vol (w/v) each of chloroform and carbon tetrachloride. The emulsion was broken by centrifugation at 8000 g for 10 min and the buffer layer was recovered and filtered through filter paper (Whatman No. 4). Polyethylene glycol 6000 (PEG) and NaCl were added to final concentrations of 4 and 1.75% (w/v), respectively; the mixture was stirred for 15 min and left to stand at 4” for 1 hr. Precipitated virus was recovered by centrifugation at 8OOOgfor 10 min and was resuspended in one-fifth the original vol of 0.5 M borate buffer, pH 8, by gentle agitation for 2 hr. Insoluble material was removed by centrifugation at 8000 g for 10 min and the virus was sedimented from the supernatant by centrifugation at 78,000 g for 75 min (Spinco 30 rotor). Pellets were resuspended by gentle agitation overnight in 0.05 M borate buffer, pH 8, containing 0.005 M EDTA. The differential centrifugation step was repeated and virus pellets were suspended in 0.05 M borate buffer, pH 8 (1 ml/50 g of original material). The virus was further purified by rate-zonal centrifugation in lo-40% sucrose density gradients buffered with 0.05 M borate buffer, pH 8.0, for 3 hr at 24,000 rpm in a Spinco SW25 rotor. The virus zones were located with an ISCO density gradient fractionator and ultraviolet scanner. The recovered virus was dialyzed overnight against 0.05 M borate buffer, pH 8, and concentrated by centrifugation at 78,000 g for 90 min. Method 2. (For BCMV and SCMV): Extraction and initial clarification was done as in Method 1. However, instead of precipitation with PEG, Triton X-100 was added to the slow-speed supernatant fluid to 5% (v/v) and the mixture was stirred at 4” for 30 min. After clarification at 8000 g for 10 min, virus was isolated by three cycles of differential centrifugation fol-

352

MOGHAL

AND

FRANCKI

TABLE LIST

Details

Isolate

1

OF POTYVIRUSES

USED

of isolation

Experimental

propagation

hosts

mosaic virus

From Canna sp. isolated by D. S. Teakle in Queensland (Accession No. 191)

Phuseolus vulguris L. cv. Windsor Long Pod or Tweed Wonder; Viciu fubu L. cv. Leviathan Long Pod

Bean yellow mosaic virus isolate [pea mosaic (PMWI”

From Pisum sativum L. described by Taylor and Smith (1968) as PMV I

Viciu fabu L. cv. Leviathan Long Pod; Pisum sutivum L. cv. Green Feast; Pisum urvense L.

Bean yellow mosaic virus [sweet pea mosaic isolate (SPMV)p

From Lathyrus odor&us L. isolated by the authors in South Australia

Vicia fubu L. cv. Leviathan Pod; Pisum urvense L.

Lettuce (LMV)”

From Luctuca sutiuu L. isolated by J. K. McGechan in New South Wales

Luctuca sutiva L. cv. Climax; Pisum sutivum L. cv. Green Feast or Victory Freezer; Pisum arvense L.

Bean yellow (BYMV)”

mosaic

virus

Long

Bean common rus (BCMV)

mosaic vi-

From Phuseolus vulguris L. (Navy bean old selection 49) isolated by G. M. Behncken in Queensland

Phuseolus vulguris bury Wonder

Passionfruit virus (PWV)

woodiness

From Pussifloru edulis L. isolated by G. M. Behncken in Queensland.

Phaseolw vulguris L. cv. Bountiful; Nicotiana clevelundii Gray

From Nicotiana tubucum L. isolated by J. Finley in Queensland

Nicotianu glutinosu clevelundii Grey

From Sorghum halpense (L.) Pers. isolated by D. Persley in Queensland

Zeu muys L. cv. Iochief

Potato virus Y (PVY)

Sugarcane (SCMV)

mosaic

virus

L. cv. Hawkes-

L.; Nicotiunu

0 These virus isolates all reacted with antisera prepared against BYMV and PMV obtained from Dr. M. Hollings (Glasshouse Crops Research Institute, Littlehampton, Sussex, England) and with an antiserum prepared against LMV obtained from Dr. J. Tomlinson (National Vegetable Research Station, Wellesbourne, Warwick, England).

lowed by sucrose density-gradient centrifugation as described for Method 1. Preparation

of Viral Proteins

Protein was isolated by the LiCl method similar to that described by Francki and McLean (1968). Purified virus (4-5 mg/ml) was mixed with an equal volume of 4 M LiCl and kept for 24-48 hr at -15”. After thawing at 4”, the precipitated RNA was removed by centrifugation at 2500 g for 15 min and the supernatant fluid was centrifuged at 105,000 g to remove any undegraded virus and the protein was precipi-

tated from the supernatant fluid by adding 2.5 vol of saturated ammonium sulphate. The precipitate was isolated by centrifugation and resuspended in 8 M urea. The salt and urea were removed by exhaustive dialysis against several changes of deionized water and the protein was freeze-dried. Some virus proteins were also prepared by the pyrrolidine dissociation method as described by Shepard (1972). Serological

Techniques

Antisera were prepared in rabbits by three subcutaneous injections adminis-

CLASSIFICATION

tered at lo-day intervals. On each occasion, 1 mg of purified virus emulsified with an equal volume of Freund’s complete adjuvant was injected. Antiserum was also prepared against host plant antigens by immunization with a suspension of material obtained from healthy plants extracted by the method used for virus purification and concentrated by centrifugation at 78,000 g for 90 min. Antisera to some viruses were also prepared in mice immunized by administering 50-pg doses of the viruses intraperitoneally (Ikegami and Francki, 1974). All serological tests were done by two-dimensional immunodiffusion tests as described by Francki and Habili (1972), except that all virus preparations were sonicated for 20 min at 0” in a Bronsonic unit to break up the particles for easier diffusion in the agar gels (Tomlinson and Walkely, 1967). This method is less sensitive than the precipitin tube method but avoids the appearance of nonspecific precipitates. For example, the same anti-PVY serum had a homologous titre of l/2056 when tested by the tube precipitin test but only l/128 by the immunodiffusion assay. Polyacrylamide-Gel Proteins

Electrophoresis

umns. Tryptophan was determined spectrophotometrically as described by Beaven and Holiday (1952). Analytical

Acid Analysis

About l-2 mg of lyophilized protein was dissolved in 3.2 ml of 6 N HCl, and one drop of 5% aqueous phenol was added to prevent the degradation of tyrosine (Delange et al ., 1969). The mixture was hydrolysed in evacuated and sealed tubes at 110” for 24,48, and 72 hr, evaporated to dryness in uacuo, resuspended in 12.5% sucrose in 0.1 N HCl, and applied to a Beckman Model 120C amino acid analyzer with Chromatronix type LC-9MV analysis col-

Techniques

Virus preparations were examined in a Unicam SP1800 spectrophotometer in quartz cuvettes with a path length of 10 mm. Virus concentrations were estimated by using the extinction coefficient E!j$,,,,, = 3 (Brunt, 1970). Approximate sedimentation coefficients were estimated by cocentrifugation in sucrose density gradients with preparations of tobacco mosaic virus (sZo, = 190; Wildy, 19711, and the three components of tobacco ringspot virus (sZozL’= 53, 91, and 126; State-Smith, 1970). Electron Microscopy Crude leaf extracts (Hitchborne and Hills, 1965) and purified virus preparations were negatively stained with 2% phosphotungstic acid adjusted with KOH to pH 6.8 and examined in a Philips 1OOC electron microscope. Some purified virus preparations were examined after shadowing with platinum-carbon. RESULTS

of

The molecular weights of viral proteins were determined in 10 or 7.5% polyacrylamide gels as described by Weber,and Osborn (1969). Both viral and marker proteins were treated as described by Agrawal and Tremaine (1972) prior to electrophoresis. Cytochrome c (mw = 11,700) was present in all gels to calculate relative mobilities. Amino

353

OF POTYVIRUSES

Properties

of Purified

Viruses

Purified preparations of all the viruses were colourless and opalescent and devoid of noticeable amounts of contaminating materials as judged by electron microscopy. Furthermore, no host plant antigens were detected in the preparations by immunodiffusion tests with antisera to host plant antigens and no antibodies to host plant antigens were detected in antisera prepared against the purified virus preparations. For acceptable yields of the viruses (at least 20 mg/kg of leaf material) to be obtained, particular attention to detail was required, and even minor departures from the described purification schedules resulted in low virus yields and/or impure preparations. In sucrose density-gradients, particles of the viruses sedimented mainly as single components of about 154 S. Infectivity was confined to fractions containing ultraviolet absorbing material.

354

MOGHAL

AND

FRANCKI

ships between five of the six viruses studied; the sixth (SCMV) appears to be unrelated to any of the others (Table 2). Of the two viruses showing close relationship (BYMV isolates and LMV), BYMV also appears to be distantly related to BCMV but no relationship between BCMV and LMV was observed. BCMV, in turn, appears to be related to PWV and PWV to PVY, but no relationship between BCMV and PVY could be demonstrated (Table 2). A positive reaction was detected between anti-PVY serum and antigen preparations of LMV. However, this apparent relationship is not clear since PVY antigen did not react with antisera to LMV (Table 2 and Fig. 11, one of which had a higher homologous titre than that of the anti-PVY serum (Table 2).

Ultraviolet spectra of preparations of all the viruses were similar and characteristic of nucleoprotein with minima at about 247 nm and maxima at about 260 nm. The 260/ 280 nm ratio varied between 1.20 and 1.37 before correction for light scattering, and the viruses were calculated to have an RNA content of 5.5-6.0% (Layne, 1957). The particle length distributions in purified virus preparations were not significantly different from those in crude leaf extracts, indicating that the purification procedures caused neither significant particle breakage nor end-to-end aggregation. Antigenic Comparison of the Six Distinct Viruses Results of homologous and heterologous immunodiffusion tests with preparations of the six viruses and their antisera are summarised in Table 2. The antigenic relationship between BYMV and PMV appears to be relatively close confirming the results reported by Taylor and Smith (1968) and Bos et al. (19741, whereas the other positive heterologous reactions indicate more remote relationships. The data indicate a continuous range of relation-

Closeness of the Antigenic BYMV and LMV

2

RELATIONSHIPS

Antiserum to:

of

Immunodiffusion tests with BYMV, PMV, and SPMV and their homologous antisera placed in adjacent wells, produced a confluent immunoprecipitin line without detectable spur formation (Fig. 2). However, in similar tests with LMV and PMV

TABLE SEROLOGICAL

Relationship

AMONG

SIX

POTYVIRUSES

Test antigen” BYMV

PMV

SPMV

BYMP PMV’ SPMV”

512” 128 128

128 512 64

128 128 256

LMV”’ LMW’

32 16

64 32

BCMV

2

PWVb

PWV

PVY

16 32 32

2 1 1

0 0 0

0 0 0

0 0 0

32 32

256 128

0 0

0 0

0 0

0 0

2

2

0

64

2

0

0

0

0

0

0

2

128

4

0

PvY*

0

0

0

2

0

8

128

0

SCMV’

0

0

0

0

0

0

0

LMV

BCMV

SCMV

128

” All antigen concentrations were adjusted to 500 pg/ml, and 15-4 samples were placed in each well. h Antisera produced in rabbits. c Antisera produced in mice (plasma fraction used). d Reciprocal of maximum antiserum dilution producing a visible immunoprecipitin line (figures in italics refer to homologous reactions). (’ Viral immunogen isolated from infected P. aruense. ’ Viral immunogen isolated from L. satiua.

CLASSIFICATION

355

OF POTYVIRUSES

differences between the BYMV isolates, the homologous and heterologous titres of unabsorbed antisera indicate that small antigenic differences may be present (Table 2). It is well known that individual

FIG. 1. Double immunodiffusion in agar gel between purified preparations of LMV (L) and PVY (Y) and their respective antisera (1 and y). Homologous titres of 1 and y were l/256 and l/128, respectively, and antigen concentrations were adjusted to 500 pglml.

FIG. 3. Double immunodiffusion in agar gel betwen purified preparations of LMV (L) and PMV (P) and their respective antisera (1 and p). Homologous titres of 1 and p were l/256 and l/512, respectively, and antigen concentrations were adjusted to 500 Kg/ ml. TABLE SEROLOGICAL

3

RELATIONSHIP

BETWEEN

BYMV

AND

LMV Antiserum to?

FIG. 2. Double immunodiffusion in agar gel between purified preparations of BYMV (B), PMV (P), and SPMV (S), and their respective antisera (b, p, and s). Homologous titres ofb, p, and s were l/512, l/ 512, and l/256, respectively, and all antigen concentrations were adjusted to 500 pg/ml.

a spur was detected (Fig. 3). Furthermore, the intragel absorption tests summarized in Table 3 indicate that whereas the antigenie properties of BYMV and PMV could not be differentiated from one another, they could be differentiated from those of LMV. Attempts to Distinguish the BYMV Isolates Serologically Although the immunodiffusion tests (Fig. 2) and the intragel absorption tests (Table 3) failed to detect any antigenic

Test antigene

Absorbin& antigen BYMV

PMV

LMV

BYMV

BYMV PMV LMV

256d 0 0 16

128 0 0 8

16 0 0 0

PMV

BYMV PMV LMV

128 0 0 4

512 0 0 4

32 0 0 0

LMV

BYMV PMV LMV

16 0 0 0

32 0 0 0

256 8 8 0

a Antisera produced in rabbits. b Antiserum wells were charged with 15 ~1 of antigen (500 @g/ml) 24 hr before charging with antiserum. c All antigen concentrations were adjusted to 500 pg/ml, and 15-~1 samples were placed in each well. d Reciprocal of maximum antiserum dilution producing a visible immunoprecipitin line (figures in italics refer to homologous reactions).

356

MOGHAL

AND

FRANCKI

antisera to a given antigen vary considerably. Hence, it was decided to determine if the three isolates of BYMV could be distinguished by examining antisera from several animals at various stages of immunization; to this end the following experiment was done. Groups of four mice were immunized with preparations of each of the three BYMV isolates, each animal receiving 50 /*g of antigen at the beginning of the experiment and 1, 3, and 5 weeks thereafter. The mice were bled at 1-2 week intervals and the serum from each bleeding was titrated against antigen preparations of all three virus isolates. Results of the experiment, summarised in Fig. 4, demonstrate that antisera to each virus reacted with both homologous and heterologous antigens, but the homologous titres were always higher than the heterologous. Statistical analysis of the results by the Kruskal-Wallis technique (Siegel, 1956) showed that whereas the antigenic properties of SPMV were not significantly different from those of PMV or BYMV, those of BYMV were significantly different from those of PMV (P < 0.01). I

Immunogenicity Proteins

and Antigenicity

of Viral

It would appear from the work of Shepard et al. (1974) that distant antigenic relationships among potyviruses may be more easily traced by using antisera prepared against degraded viruses. To assess the virtues of this approach, attempts were made to prepare antibodies in the ascitic fluid of mice inoculated with ascites tumors (Ikegami and Francki, 1974). The following antigens were used: (1) undegraded virus preparations of BYMV, PVY and BCMV; (2) preparations of BYMV, PVY, BCMV, and PWV degraded by the LiCl method; and (3) pyrrolidine-dissociated viral protein preparations from BYMV and PVY. Each antigen preparation was administered to five individual mice and the harvested ascitic fluid from each batch of animals was pooled. Results summarised in Table 4 demonstrate that of the antigens used, only preparations of the intact viruses were sufficiently immunogenic to yield fluids with

0

I 2

I

TIME

I 4

,

I 6

I

I 8

I

(weeks)

FIG. 4. Antibody production in mice immunized with purified preparations of BYMV (a), PMV (b), and SPMV (cl. Each serum sample was titrated against purified preparations of BYMV (O-O), PMV (O- - -O), and SPMV (r- - -7) adjusted to a concentration of 400 fig/ml. Animals were immunized at the beginning of the experiment and 2 and 5 weeks thereafter. The results are expressed as mean reciprocal titres (four animals per treatment).

detectable antibody under our experimental conditions. The data in Table 4 also confirm the clear antigenic relationship between BYMV and LMV as well as the distant relationships between BYMV and BCMV and between PWV and PVY. Furthermore, the data demonstrate that antibodies raised against intact BYMV, PVY, and BCMV failed to recognize the proteins from their homologous viruses isolated by the pyrrolidine dissociation method. The same antibodies did recognize proteins from their homologous viruses isolated by

CLASSIFICATION

LiCl dissociation; however, very much less efficiently than the intact viral antigens. Subunit

Sizes of Viral

Proteins

Proteins prepared from BYMV (all three isolates) and LMV migrated as two bands when electrophoresed in polyacrylamidegels in the presence of sodium dodecyl sulphate (SDS). Coelectrophoresis of proteins from the different virus isolates failed to detect significant differences in their mobilities. By coelectrophoresis with marker proteins (Fig. 5), it was calculated that the slower migrating polypeptide (accounting for over 90% of the total protein) has a molecular weight of around 33,000 and the faster migrating polypeptide of about 28,000. These results are similar to those reported by Huttinga and Mosch (1974) except that some of their protein preparations had higher proportions of the faster migrating polypeptide and that they obtained a value of 34,000 for the molecular weight of their slower migrating polypeptide. TABLE SEROLOGICAL Antibodies

ACTIVITIES

OF INTACT

357

OF POTYVIRUSES

POTYVIRUS

to:”

When electrophoresed under similar conditions to those reported in Fig. 5, BCMV, PWV, and SCMV protein preparations all yielded a major polypeptide with a molecular weight of 33,000, indistinguishable in electrophoretic mobility from those of the BYMV isolates and LMV. However, BCMV protein contained no detectable polypeptide of 28,000 daltons but an even faster component was observed with an estimated molecular weight of 26,000. On the other hand, SCMV protein yielded only a single polypeptide (33,000 daltons). Contrary to the results reported by Hiebert and McDonald (1973) and by Huttinga and Mosch (1974), we did not detect significant amounts of a polypeptide with molecular weight 33,000 in preparations of PVY protein and only a single polypeptide of 28,000 daltons was observed. Although the types of polypeptides detected in protein preparations of the viruses studied were consistent, the proportion of material in each did vary from experiment to experiment. For example, we 4 PARTICLES

AND THEIR

c BYMV

PVY

BCM

-

0

0 0 0

P-P 0 0 0

L-P 0 0 0

0 0 0

1 0 0

0 0 0

0 0 0

0 0

0 0

1 0

0 0

1L-P

'P

L-P

0 0 0

2 0 0

Virus P-P L-P

0 0 0

0 0 0

BCMV

Virus L-P

0 0

0 0

0 0

0 0

PWV

L-P

0

0

0

0

ViNsc

BYMV

PVY

Virus’ P-P L-P

64O 0 0

Virus 1‘-P 0 0 f 0 64 0 0

PWV

-

-

0

0

64 0

-0

-

-

0

I

DISSOCIATED

Test antigen*

P-P -

0 0 0 0

-

0

-

1L-P 0 0 0

i

PROTEINS

LMV T

-

IP-P L-P

-

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0

0 0

0 0

0

0

0

a Antibodies were produced in ascitic fluid of mice. Each animal was injected intraperitoneally with 50 pg of antigen with an equal volume of Freund’s complete adjuvant (total volume of 200 ~1) at weekly intervals for 3 weeks. A booster injection was given after a further 2 weeks and immediately afterwards each mouse was injected intraperitoneally with 1 ml of a fresh suspension of a Krebs Ascites tumor cell suspension. Ascitic fluid was collected 6-8 days later as described by Ikegami and Francki (1974). * All antigen concentrations were adjusted to 500 Fg/ml, and 15-~1 samples were placed in each well. L’ Purified virus preparations sonicated as described in Materials and Methods. d Viral protein prepared by pyrrolidine dissociation. c Viral protein prepared by LiCl dissociation. ’ Purified virus preparations without any further treatment. 0 Reciprocal of maximum antiserum dilution producing a visible immunoprecipitin line (figures in italics refer to homologous reactions).

358

MOGHAL

AND

observed that protein from a BYMV preparation which had been purified as quickly as possible contained much less of the polypeptide of 28,000 daltons than that from virus purified by the standard procedure. It would appear that the polypeptides with molecular weights lower than 33,000 may be the results of degradation by proteolytic enzymes as suggested by Huttinga and Mosch (1974). It would also seem likely from our observations that the tendency of viral proteins to degrade may vary with the virus and perhaps also with the host from which they are purified. Amino Acid Composition

of Viral Proteins

The amino acid analyses of the viral proteins are summarized in Table 5. The numbers of residues of each amino acid per protein subunit were calculated using the results of the polyacrylamide-gel electrophoretic data as a guide. The results indicate that the protein subunits of the viruses studied contain about 290 amino acid residues, a value well above those previously reported for other potyviruses (Damirdagh and Shepherd, 1970; State-Smith and Tremaine, 1970; Miki and Oshima, 1972; Hill and Shepherd, 1972; Hill et al., 1973; Makkouk and Gumpf, 1975). However, the amino acid molar ratios of the viruses show similarities. Pearson’s correlation coefficients (Snedecor and Cochran, 1967) of the amino acid data reported in Table 5 and those reported for other potyviruses by other authors are presented in Table 6. The highest correlation coefficients (0.987-0.994) were those between the three isolates of BYMV which could not be distinguished serologitally (Fig. 2, Table 3). They were also very high (0.937-0.945) between LMV and the BYMV isolates which have been shown to be serologically closely related. However, the correlation coefficients between the other viruses did not reflect their apparent distant serological relationships (Table 2). DISCUSSION

Methods used here achieved the purification of several potyviruses in adequate yields, high degree of purity, and without significant changes in the particle size dis-

FRANCKI

tribution However, we are not satisfied that the virus coat proteins were recovered in their native condition. Hiebert and McDonald (1973), McDonald and Hiebert (1975), and Michelin-Lausarat and Papa (1975) have already reported that coat protein of potyviruses can undergo partial degradation in vitro. Our results support this conclusion, but at present we cannot exclude the possibility that the coat proteins can also undergo some degradation in ho. Several different values for the subunit size of potyvirus coat proteins have been reported in the literature. Based on amino acid analysis and the number of cyanogen

abcde -1

-

_

4-;llrr5+b 61)rc +

I

.-_

&

FIG. 5. Electrophoresis of potyvirus coat proteins in 10% polyacrylamide-gels in the presence of SDS as described in Materials and Methods. (a) Mixture of BYMV coat protein with the following marker proteins: 1 = bovine serum albumin (MW = 68,000), 2 = glutamate dehydrogenase (53,000), 3 = ovalbumin (43,000), 4 = cucumber mosaic virus coat protein (26,300), 5 = tobacco mosaic virus coat protein (17,000). (b) PMV coat protein. (c) SPMV coat protein. (d) LMV coat protein. (e) Mixture of BYMV and PMV coat proteins. To all preparations, cytochrome c (6, with MW = 11,700) was added, and mobilities of all proteins were calculated relative to this marker.

CLASSIFICATION

TABLE 5 AMINO ACID COMPOSITION OF POTYVIRUSES STUDIED -.. . . 1 Kelative molar ratio” Residues per subunit

Amir LO acid

__

;

Ala Arg Asp Glu GUY His Ile Leu LYS Met Phe Pro Selb ThP g5 Val

-’I

359

OF POTYVIRUSES

%YMV PMV

SPMV LM\

21.3 16.7 42.5 33.1 21.3 6.0 14.5 21.9 20.0 9.4 9.0 11.0 15.4 20.3 4.8 11.0 16.5

21.2 17.6 41.6 33.1 21.5 5.1 15.3 20.7 22.0 8.1 8.3 9.8 13.4 16.6 3.7 10.8 15.6

21.9 16.7 39.8 33.3 21.7 3.8 14.5 22.2 19.0 6.6 8.8 11.1 14.7 20.4 3.8 12.9 15.6

Catlculated

m !olecular

25.7 15.9 44.2 31.6 23.4 a.7 12.3 19.8 18.0 11.6 5.9 10.5 12.1 19.3 4.3 13.6 10.5

-

3CMV PWV PVY EiCMV 21.8 16.8 46.6 28.4 18.7 5.7 7.2 19.8 20.0 17.7 7.8 14.5 15.9 15.5 3.2 10.1 18.4

!

26.7 14.5 46.1 31.4 21.2 5.2 7.4 21.2 22.1 12.8 7.9 10.4 14.4 17.6 3.6 10.5 19.3

25.6 16.1 32.5 33.9 18.2 6.4 14.8 18.4 ,18.0 / 7.2 5.6 18.2 17.8 23.8 3.4 10.4 16.2

3YMV

‘MV

;PMI

LMI

ICMV

‘WI

'VY 3CMV -

21 17 42 33 21 6 14 22 20 9 9 11 15 20 5 11 16

22 17 40 33 22 4 15 22 19 7 9 11 15 20 4 13 16

21 18 42 33 22 5 15 21 22 8 8 10 13 20 4 11 16

26 16 44 32 23 9 12 20 18 12 6 11 12 19 4 14 11

22 17 47 28 19 6 7 20 20 18 8 15 16 16 3 10 18

27 14 46 31 21 5 7 21 22 18 8 10 14 18 3 10 19

26 16 33 34 18 6 15 18 18 7 6 18 18 24 3 10 16

26 16 47 25 19 6 11 16 22 9 9 11 22 19 4 11 16

292 33.1

189 3‘2.6

289 32.7

289 32.6

290 32.8

389 2!86 12.2 3 1.7

289 32.3

25.6 15.7 47.3 24.7 18.6 6.0 10.8 16.0 22.0 8.5 8.6 11.8 22.0 19.0 3.5 11.0 15.7

Total amino acid residues weight polypeptide x 10e3

r ’ Means of 24-, 48-, and 72-hr hydrolyses analyzed in duplicate. b Extrapolated to zero hydrolysis time. c Determined spectrophotometrically as described in Beaven and Holiday TABLE CA

BYM’

6

.LCULI~TED PEARSON’S (c:IRRELAT )N COEFFICIEI :s OF POTYVIRU: AMINO ACID COMPOSITUIN ~ 7 ‘MV” ISPMV” SCMV” TEVb PVY’ PVYd TuMV” -1MDMV’ LMV” BCMV’ .994 ?MV”

.992 .987 iPMV”

,945 .937 .944 LMV”

,874 ,835 .849 .829 BCMV”

,889 .978 ,876 ,800 ,837 ,875 ,873 SCMV”

I 1 .899 .870 ,870 ,893 ,923 ,922 ,850 .766 TEVb

L

a Data b Data o Data d Data e Data I Data

(1952).

,925 ,912 ,914 ,867 .850 ,869 ,947 ,824 ,878 PVY’

.875 .849 ,847 ,820 ,854 .840 ,908 .751 .921 ,933 PVYd

.928 ,913 ,923 .910 .826 ,874 ,836 ,759 ,906 ,824 .865 TuMV’

.788 ,790 ,754 ,824 ,710 .763 ,750 .775 .778 .742 ,691 ,706

from Table 5. for tobacco etch virus after Damirdagh and Shepherd (1970). for potato virus Y after State-Smith and Tremaine (1970). for potato virus Y after Miki and Oshima (1972). for turnip mosaic virus after Hill and Shepherd (1972). for maize dwarf mosaic virus after Hill et al. (1973).

bromide fragments, Miki and Oshima (1972) concluded that PVY coat protein contains 187 amino acid residues and has a molecular weight of 21,300. Tryptic peptide maps and amino acid data led Damir-

dagh and Shepherd (1970) to conclude that tobacco etch virus (TEV) protein consists of 194 amino acid residues and a molecular weight of 22,000; Hill and Shepherd (19721, that turnip mosaic virus (TuMV) protein

360

MOGHAL

AND

has 231 amino acids and a molecular weight of 26,000; and Hill et al. (1973), that maize dwarf mosaic virus (MDMV) protein has 264 amino acids and a molecular weight of 28,500. However, protein size determinations by polyacrylamide-gel electrophoresis in SDS on the potyviruses reported so far, have yielded higher values, ranging from 32,000 daltons for TEV (Hiebert and McDonald, 1973) to 36,000 daltons for MDMV (Hill et al., 1973). If it is assumed that potyvirus coat proteins undergo partial degradation in vitro, it would appear that proteins of the viruses investigated here have subunit molecular weights around 33,000. This value is in reasonable agreement with those reported by others; for example, 34,000 for BYMV, LMV, and PVY (Huttinga, 1975; Hiebert and McDonald, 1973), and 35,000 for BYMV and BCMV (Uyemoto et al., 1972). Although the figure of about 33,000 daltons has been used in calculating the number of amino acid residues per protein coat subunit (Table 5), it must be considered only as a tentative value awaiting more comprehensive studies on the viral proteins. Because of the problems associated with the isolation of undegraded potyvirus coat proteins, the reported amino acid data (Table 5) may lack precision. However, it seems significant that similarities in amino acid composition are more marked among viruses which appear to be closely related serologically. On the contrary, viruses which appear to be only distantly related serologically do not seem to show greater similarities in amino acid composition than those which appear to be totally unrelated. A preliminary inspection of published amino acid data for viruses with elongated particles belonging to other major groups indicate that such data may be useful in virus classification generally. The apparent partial degradation of potyvirus proteins in highly purified virus preparations also raises important implications in relation to serological studies in that such degradation is likely to alter the antigenic properties of the viruses. Hence, results of serological reactions may depend on the degree to which the viral proteins used as immunogen and as test antigen

FRANCKI

were altered prior to and during the tests. They may also continue to be degraded during immunization. Immunodiffusion tests with plant viruses offer many advantages over other techniques. However, diffusion of potyvirus particles is too slow for easily recognizable immunoprecipitin lines to be formed. To overcome this, ultrasonically fragmented viruses (Tomlinson and Walkley, 1967) or proteins prepared by chemical dissociation of viruses (Purcifull and Sheperd, 1964; Shepard and Grogan, 1967) have been used. In the latter approach, pyrrolidine-dissociated proteins have been especially popular (Shepard et al ., 1974). Data presented in this paper (Table 4) illustrate two problems in using chemically dissociated potyvirus coat proteins for serological studies. Firstly, the antigenic specificity of dissociated proteins can change in such a way that they can fail to be recognised by antibodies elicited in response to intact virus. Secondly, the dissociated proteins appear to be poor immunogens which has previously been observed for tobacco mosaic virus protein (Loor, 1967) and turnip yellow mosaic virus empty protein shells (Marbrook and Matthews, 1966). Our results with two of the BYMV isolates (BYMV and PMV) illustrate problems associated with the detection of minor antigenic differences among potyviruses and the dangers of drawing conclusions from data obtained using only one antiserum. It would appear from the homologous and heterologous titres of a single rabbit antiserum to each of the viruses, that BYMV and PMV are antigenically distinct (Table 3). This conclusion was substantiated by results of an experiment in which antisera from several mice bled at various times after immunization were tested (Fig. 4). However, no differences were detected in intragel absorption tests (Table 3) or in qualitative immunodiffusion tests (Fig. 2) using the same antisera which detected differences in homologous and heterologous titres. The above considerations indicate that considerable caution is needed in the interpretation of serological data used for the classification of potyviruses. The problems

CLASSIFICATION

mentioned may account, at least in part, for some of the apparently conflicting observations reported in the literature (Edwardson, 1974). ACKNOWLEDGMENTS We thank the colleagues mentioned in Table 1 for providing us with virus cultures and antisera; Mrs. L. Wichman for preparing the illustrations; Mrs. G. Bishop for the statistical analyses; Mr. K. W. Jones for supply and maintenance of plants. The facilities of the Biochemistry Department, University of Adelaide, were kindly made available for the amino acid analyses, and we thank Mr. M. Cichorz for carrying out the determinations. One of us (S.M.M.) was supported by a grant under the Australian Commonwealth Scholarship and Fellowship Plan. REFERENCES AGRAWAL, H. O., and TREMAINE, J. H. (1972). Proteins of cowpea chlorotic mottle, broad bean mottle and brome mosaic viruses. Virology 47,8-20. BEAVEN, G. H., and HOLIDAY, E. R. (1952). Ultraviolet absorption spectra of proteins and amino acids. Aduan. Protein Chem. 7, 319-386. Bos, L. (1970). Bean yellow mosaic virus. C.M.Z.l A .A .B. Description of Plant Viruses, No. 40. Bos, L., KAWAL~KA, Cz., and MAAT, D. 2. (1974). The identification of bean mosaic, pea yellow mosaic and pea necrosis strains of bean yellow mosaic virus. Net/z. J. Plant Puthol 80, 173-191. BRUNT, A. A. (1970). “Annual Report, Glasshouse Crops Research Institute,” Sussex, 1970. DAMIRDAGH, I. S., and SHEPHERD, R. J. (1970). Some of the chemical properties of the tobacco etch virus and its protein and nucleic acid components. Virology 40, 84-89. DELANGE, R. J., FAMBROUGH, D. M., SMITH, E. L., and BONNER, J. (1969). Calf and pea histone IV. II. The complete amino acid sequence of calf thymus histone IV, presence of c-N-acetyllysine. J. Biol. Chem. 244, 319-334. EDWARDSON, J. R. (1974). Some properties of the potato virus Y group. Florida Agr. &a. Monograph, Sr. 4, pp. 398. 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 MCLEAN, G. D. (1968). Purification of potato virus X and preparation of infectious ribonucleic acid by degradation with LiCl. Aust. J. Biol. Sci. 21, 1311-1318. GIBBS, A. (1969). Plant virus classification. Adu. Virus Res. 14, 263-328. HARRISON, B. D., FINCH, J. T., GIBBS, A. J., HOLLINGS, M., SHEPHERD, R. J., VALENTA, V., and WETTER, C. (1971). Sixteen groups of plant viruses. Virology 45, 356-363.

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