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Identity and location of a minor protein component in virions of southern bean mosaic virus
VIROLOGY
77, I.-11
(1977)
Identity and Location of a Minor Protein Component Southern Bean Mosaic Virus 0. B. SEHGAL’ Department
of Plant
Pathology,
AND
University Accepted
C.
of Missouri,
October
in Virions
of
H. HSU Columbia,
Missouri
65201
4, 1976
Polyacrylamide-gel electrophoretic analysis of southern bean mosaic virus (SBMV) protein isolated with different procedures revealed the presence of a minor protein comlponent (MW 66,000) besides the major or capsid protein (MW 29,000). The amount of the minor protein in virions was ca. one-tenth the amount of the major protein. The minor SBMV protein remained stable when heated in the presence of sodium dodecyl sulfate, urea, and reducing agents (P-mercaptoethanol or dithiothreitol) or upon succinylation, reduction-carboxymethylation, or performic acid oxidation. Controlled virion disassembly revealed that all of the minor protein and some major protein subunits were intimately linked to the SBMV-RNA. The two proteins were separated and purified by preparative gel electrophoresis for subsequent characterization. The minor and major SBMV proteins possessed an identical amino acid composition. In gel diffusion tests, the minor and the major proteins developed two strong and confluent precipitin bands when reacted with an antiserum prepared against the purified major protein. Treating purified SBMV major protein with cross-linking reagents (dimethyl adipimidate, formaldehyde, or glutaraldehydel resulted in the formation of a protein component comigrating with the minor protein isolated from the virions. When SBMV virions were iodinated (NaY) using lactoperoxidase or chloramine-T, radioactivity was incorporated into the minor and major proteins. These results suggest that the minor SBMV protein is a covalently linked coat protein dimer involving e-amino groups of lysyl residues and resides in the virion capsid. On the basis of these studies, a tentative structural model for .the SBMV capsid is proposed.
reaction with protein cross-linking reagents, and radioiodination suggests that the minor SBMV protein is a stable dimer of the coat protein and resides in the virion capsid. On the basis of these studies, a structural model for the SBMV capsid is proposed.
INTRODUCTION
Virions of cowpea chlorotic mottle, satellite tobacco necrosis, bromegrass mosaic, and tobacco mosaic strain flavum contain a minor protein component in addition to the coat protein (Rice, 1974; Bancroft and Smith, 1975; Oxelfelt 1976). Some evidence suggests tlhat the minor protein in these viruses is a stable dimer of the coat ‘protein, but its functional significance in virion construction or structure is unknown. Recently, we reported (Hsu et al., 1976) that virions of southern bean mosaic virus (SBMV) contain a minor protein in addition to the coat protein. In this study, the identity and location of the minor protein in the virus particles is reported. Evidence based on serology, chemical composition, ’ Address
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MATERIALS
AND
METHODS
Virions of SBMV (bean strain) were purified from Phaseolus vulgaris L. “bountiful” by a modification of the procedure of Hsu et al. (1976). Following dialysis and low speed centrifugation of the 8% polyethylene glycol-precipitated SBMV, the preparations were adjusted to pH 4.5 (with 0.1 N HCl), maintained at 5” for 60 min, and centrifuged (10,000 g, 20 min). The resulting supernatant fraction was adjusted to pH 7.0 (with 0.1 N NaOH), and virions
Sehgal. 1
Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
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SEHGAL
were sedimented by ultracentrifugation (105,000 g, 2.5 hr), suspended in 0.02 M sodium phosphate buffer, pH 7.0, and clarified by low speed centrifugation (10,000 g, 20 min). The differential centrifugation procedure was repeated two or three times and the final virion preparations stored (5”) in 0.02 A4 phosphate buffer, pH 7.0. In some experiments, virions were purified further by sucrose density gradient or isopycnic CsCl centrifugations (Sehgal and Das, 1975). Virions recovered from the density gradient columns were dialyzed (12 hr, 5”) against 0.02 M phosphate buffer, pH 7.0, sedimented by ultracentrifugation, and suspended in 0.02 M phosphate buffer, pH 7.0. Infectivity assays were performed on P. vulgaris “Pinto” (Sehgal, 1973a). Virions of tobacco mosaic virus (strain Ul) were purified from Nicotiana tabacum L. “Samsun” (Sehgal, 1973b). Concentrations of virions or viral components were estimated spectrophotometrically. SBMV protein was prepared by treating virions with several chemical reagents known to be effective in isolating plant viral proteins. These included warm formic acid (Miki and Knight, 1965), cold acetic acid (Fraenkel-Conrat, 1957a), 1.0 N HCl (Ghabrial et al., 1967), phenol (Anderer, 1959), 0.1 M sodium carbonate-bicarbonate buffer, pH 10.5 (Knight, 1975), guanidine hydrochloride (Reichmann and State-Smith, 1959), and cold calcium chloride (Yamazaki and Kaesberg, 1963). SBMV protein was also isolated by degrading virions with pyridine (Shalla and Shepard, 1970) or pyrrolidine (Shepard et al., 1974), but in these procedures formaldehyde was omitted from the dialyzing buffer. SBMV protein isolated with 1.0 N HCl, or the intact virions were succinylated (Fraenkel-Conrat, 1957b), reducedcarboxymethylated (Crestfield et al., 1963), or oxidized with performic acid (Hirs, 1967). Sucrose density gradient analysis revealed that virions were degraded upon reduction-carboxymethylation or performic acid oxidation but not by succinylation. In routine studies, SBMV virions were dissociated to yield minor and major proteins by heating (loo”, 5 min) in 1% sodium dodecyl sulfate (SDS) contain-
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ing 0.1% P-mercaptoethanol. Virions or the isolated SBMV protein were reacted (60 min, 5”) with formaldehyde or glutaraldehyde at pH 7.0, while dimethyl adipimidate (Pierce Chemical Company, Rockford, Illinois) treatment was performed at pH 8.5 or 9.1, according to Bancroft and Smith (1975). The samples were dialyzed (12 hr, 5”) thereafter against 0.02 M phosphate buffer, pH 7.0. Polyacrylamide-gel electrophoresis (7.5 mA/tube, 3 hr, 25’) was performed routinely in 5% gels (6.0 x 0.6 cm) containing 0.1% SDS according to Maize1 (1969). For molecular weight determinations, polyacrylamide gels of four different concentrations (5, 7.5, 10.0, and 15%) along with several marker proteins were used (Weber et al., 1972). Preparative separation of the minor and major SBMV proteins was achieved by electrophoresis in large (10 x 1.5 cm) polyacrylamide-gel (5%) columns. One milliliter of sample (2-3 mg of viral protein) was layered onto each gel and electrophoresed at 7.5 ma/tube for 1 hr and thereafter at 20 mAltube for 8 hr. After electrophoresis, the gels were removed and maintained at 5” for 12-14 hr, at which time the separated proteins appeared as prominent white bands. Gel sections containing the major and minor SBMV proteins were excised, the segments from several gels were combined, and the proteins were eluted electrophoretically with a slight modification of the procedure described by Sarkar (1973). The protein samples were dialyzed (12 hr, 5”) against deionized water and lyophilized. Subsequent electrophoretic analysis revealed that these two protein preparations were homogeneous. Isolation and purification of the major SBMV protein for use in the preparation of antiserum were done as follows. Virions (3 mg/ml) were treated with 0.1 M phosphate buffer, pH 7.5, containing 15 mM EDTA for 60 min at 5”, then NaCl (0.4 M) was added and the samples were stored at 5” for an additional 60 min. This sample (3.0 ml) was layered onto a 2-ml cushion of 10% sucrose containing 0.4 M NaCl and centrifuged (105,000 g, 4 hr). The top 2.5 ml of solution was removed and dialyzed (12 hr,
CAPSID
STRUCTURE
5”) against 0.02 M phosphate buffer, pH 7.0. Polyacrylamide-gel electrophoresis of this sample (400-500 pg) revealed the presence of only the major SBMV protein. Approximately 1.5-2.0 mg of the purified major protein was injected intravenously into a rabbit, and this injection schedule was repeated three times at weekly intervals. Four clays after the last injection, the rabbit was bled and the y-globulin fraction isolated by ammonium sulfate precipitation. Immunodiffusion tests were performed using the polyacrylamide-gel sections containing the minor and major proteins separated by preparative electrophoresis. These gel sections were embedded in warm 1% algarose containing 0.85% NaCl and 0.025% sodium azide, and the agarose was allowed to solidify. Two wells were punched into the agarose bed between the polyacrylarnide-gel sections and were charged with the sera. The reaction was allowed to proceed at 25” in a humid chamber. SBMV virions were iodinated using Na’““I (Industrial Nuclear Co., St. Louis, Missouri)- with the procedures of Montelaro and Rueckert (1975). When the chloramine-T procedure was followed, the reaction mixture contained 0.5 mCi of Na”“1, 0.5 M sodium phosphate, pH 7.4 (0.2 ml), 2.0 mg of SBMV in 0.02 M phosphate buffer, pH 7.0 (0.2 ml), and 0.5% (w/ v) chlorarnine-T in 0.05 M phosphate buffer, pH 7.4 (0.2 ml). After 40-60 set (25”), the reaction was stopped by adding 1 ml of 0.1% (w/v) sodium metabisulfite dissolved in 0.02 M phosphate buffer, pH 7.5. The iodinated virions were immediately passed through a column (1 x 2 cm) of an anion-exchLange (AGl-X10) resin (Bio-Rad Laboratories, Richmond, California) equilibrated with deionized water (Felber, 19741, and. the virions were sedimented with sucrose density gradient centrifugation. Virions recovered from the gradients were dialyzed (12 hr, 5”) against several changes of 0.02 M phosphate buffer, pH 7.0. Iodinaltion mediated with lactoperoxidase was performed by adding, in order, 0.5 mCi of Na12”I, 2 mg of SBMV (0.2 ml), 0.27% (w/v) lactoperoxidase (0.1 ml), and 0.88 mM hydrogen peroxide (0.01 ml).
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After 60 set, 0.2 ml of 0.067 M phosphate buffer, pH 7.0, was added, and the virions were passed through the anion-exchange resin column and then subjected to sucrose density gradient centrifugation. The amino acid compositions of the major and minor SBMV proteins were determined according to Benson and Patterson (1971) with a Beckman Ml21 amino acid analyzer. Uranyl formate-stained samples were examined with a JEOL 100B electron microscope (Hsu et al., 1976). RESULTS
AND
DISCUSSION
Virions dissociated by heating in SDSp-mercaptoethanol in the absence or presence of urea yielded the minor and major SBMV proteins upon electrophoresis (Figs. 1A and E). Similarly, SBMV protein isolated with formic acid, acetic acid, or 1.0 N HCl showed the presence of both these protein components (Figs. 1 B-D). The phenol-isolated viral protein showed an additional slowly migrating protein component (Fig. 1F). While virion treatment with guanidine hydrochloride or calcium chloride yielded highly aggregated SBMV protein (Figs. 1H and J), it was readily dissociated upon heating in SDS-p-mercaptoethanol and yielded the minor and major proteins (Figs. 11 and K). The pyridine-isolated SBMV protein showed two or three additional high molecular weight polymers which were dissociated by heating, but there was some indication of a limited degradation of the minor and major proteins by pyridine (Figs. 1L and M). For SBMV protein isolated with pyrrolidine, several additional and discrete protein polymers were observed which were dissociated largely by heating (Figs. 1N and 0). SBMV protein prepared by prolonged dialysis (3 days, 5”) of virions against 0.1 M sodium carbonate-bicarbonate buffer, pH 10.5, however, yielded only the major protein (Fig. 1G). Similar results were obtained when SBMV protein was prepared by dialyzing virions against 0.5 N NaOH (4”, 24 hr) followed by additional dialysis against 1.0% ammonium carbonate (Ghabrial et al., 1967). These results indicate that the minor SBMV protein is sensitive to prolonged exposure to
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A
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DE
FOh
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I
J
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M
N
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FIG. 1. Polyacrylamide-gel electrophoretic analysis of SBMV protein. The isolated virion protein was dissolved in 0.02 M phosphate buffer, pH 7.0, containing 0.1% SDS and 0.01% P-mercaptoethanol. (A), Virions dissociated by heating (loo”, 5-30 min) in 1% SDS and 0.1% fi-mercaptoethanol; SBMV protein isolated with (B), warm formic acid; CC), cold acetic acid; CD), 1 N HCI; iE), by exposure (45”, 1 hr or loo”, 5 min) to 0.02 M phosphate buffer, pH 7.0, containing 1% SDS, 0.1% P-mercaptoethanol, and 4 M urea; (FI, with phenol, followed by protein precipitation from the phenol phase with methanol and sodium acetate. (G), with 0.1 M sodium carbonateebicarbonate buffer, pH 10.5; cH), guanidine hydrochloride; (I). guanidine hydrochloride-isolated protein, heated (100”. 5 min); (J). cold calcium chloride; (K). calcium chloride-isolated protein, heated; CL), pyridine; (Ml, pyridine-isolated, heated; (N), pyrrolidine; IO), pyrrolidine-isolated, heated. Approximately 100 /*g of the dissociated virions or 120 pg of isolated protein were electrophoresed (3 hr, 7.5 mA/tube) in 5% polyacrylamide gels (6.0 x 0.6 cm) containing 0.1% SDS in Maizel’s SDS-phosphate buffer system. The gels were stained with Coomassie brilliant blue and then destained with a methanolacetic acid mixture. The gels were maintained in 2.5% acetic acid.
strongly alkaline conditions. The average molecular weights determined by electrophoresis in 515% polyacrylamide gels for the minor and major SBMV proteins were, respectively, 66,000 and 29,000. Quantitative estimation based on staining intensity (Weber et al., 1972) employing 40-120 Fg of SBMV protein revealed that the amount of minor protein in virions was ca. onetenth the amount of the major protein. Only freshly prepared virion preparations (l-2 weeks following purification) were used in these studies. Gel-electrophoretic analysis of somewhat older (4-5 weeks) SBMV samples showed the presence of an additional protein band similar to the phenol-isolated SBMV protein (Fig. 1F). Preliminary studies show that virions of the cowpea SBMV strain contain a similar minor protein component. Factors affecting the dissociation of protein from SBMV virions and the stability of the viral proteins were investigated in detail. In the presence of 0.02 M phosphate buffer, pH 7.0, containing O.l-1.0% SDS and 0.1% ,&mercaptoethanol, the virions (0.5-2 mg/ml) were dissociated completely, yielding the minor and major proteins
when either heated (100”) for 3-30 min or exposed to SDS-p-mercaptoethanol at 45” for 60 min. Similarly, virions were dissociated completely at pH 5.0 or 9.1 by heating in 1% SDS. SBMV virions purified further with sucrose density gradient or isopycnic CsCl centrifugations yielded the minor and major proteins when dissociated by heating in SDS-P-mercaptoethanol. Both these proteins remained stable when heated (loo”, 3-5 min) in 1% SDS containing 1% sodium sulfite, lo-100 mJ4 dithiothreitol, and 5 mM glutathione or in 1% SDS containing 0.1% P-mercaptoethano1 and 8.0 M urea. Additionally, the minor and major SBMV proteins remained completely stable upon succinylation, reduction-carboxymethylation, or performic acid oxidation. When tobacco mosaic virus (TMV) virions (l-2 mgiml) were dissociated by heating in 0.1% SDS and subsequently electrophoresed, a minor protein (MW 35,000) component was observed as well as the capsid protein (MW 17,500). This minor TMV protein disappeared, however, when virions were heated in SDS containing 0.1% ,&mercaptoethanol or 10 mM dithiothreitol, indicating that it was a
CAPSID
STRUCTURE
disulfide-linked dimer of the TMV coat protein. Since the minor SBMV protein resisted dissociation upon heating in SDS containing reducing agents and urea, it seems unlikely that it is a coat protein dimer involving disulfide linkages. Following exposure to 0.1 M phosphate buffer, pH 7.5, containing EDTA and bentonite, SBMV virions degrade into a disCrete 47-50 S entity comprised of intact SBMV-RNA and ca. one-third of the total viral protein (Sehgal and Sinha, 1974). Similar results can be obtained by substituting bentonite with 0.4 M NaCl or KC1 (see below). To determine whether the mi-
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nor SBMV protein was linked to SBMVRNA, virions were degraded and the intermediate macromolecular components analyzed. Figure 2 is a composite illustration showing sedimentation behavior, morphology, and polyacrylamide-gel electrophoresis of the variously treated SBMV. Untreated virions sedimented sharply, possessed discrete spheroidal (diameter, 30 nm) morphology, excluded stain from penetrating the core, and yielded the major and minor proteins upon electrophoresis. The EDTA-treated virions exhibited a slight reduction (ca. 12%) in their sedimentation rate; the particles appeared
P
SVE +
n Fraction11
RELATIVE
DEPTH
FIG. 2. A composite illustration showing sedimentation behavior, morphology, and SDS-polyacrylamide-gel electrophoretic analysis of the untreated or variously treated SBMV. Approximately 500 pg of untreated SBMV (0.3 ml) or treated virions, as indicated, were layered onto 530% linear sucrose density gradients and centrifuged at 105,000 g for 2 hr at 5”. The peak fractions from several runs were pooled and dialyzed against 0.02 M phosphate buffer, pH 7.0. Samples of these preparations were stained with uranyl formate for electron microscopy. Aliquots of the same samples were heated (loo”, 5 min) in SDS-Dmercaptoethanol and then analyzed in 5% polyacrylamide gels. Note absence of the minor protein in the electropherograms of Fraction I of the bottom sedimentation profile.
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spheroidal but were rendered permeable to the negative stain and yielded the minor and major proteins upon electrophoresis (Hsu et al., 1976). Exposure of EDTAtreated SBMV to 0.4 A4 NaCl degraded it into the 47-50 S ribonucleoproteinaceous complex (Fraction II) and viral protein (Fraction I) which remained at the top of the centrifuged sucrose density-gradient columns. Particles of the 47-50 S SBMVRNA-protein complex (Fraction II) appeared “loose” with an irregular outline (diameter, 15-25 nm) as reported previously (Sehgal and Sinha, 1974) and yielded the minor and major proteins, although the amount of the major protein associated with it was somewhat less compared to the untreated SBMV. The SBMV protein (Fraction I) was aggregated largely as spheroidal shells (diameter, 25-30 nm), although many collapsed particles were also observed, but gel electrophoresis indicated the presence of only the major SBMV (29,000 daltons) protein in this fraction. These preparations (2-3 mg/ml) were noninfectious and exhibited an ultraviolet light absorption spectrum typical for proteins with a maximum extinction at 275280 nm and a minimum extinction at 250 nm (Sehgal, 1973a). Two conclusions are apparent from these results. First, the spheroidal SBMV protein particles contain only the major SBMV protein and are devoid of SBMV-RNA. Second, all the minor SBMV protein and some major protein subunits are linked to the viral RNA. The identity of the minor SBMV protein was determined as follows. When the electrophoretically separated minor and major proteins were reacted with an antiserum prepared against the purified SBMV major protein, two strong and confluent precipitin bands developed (Fig. 3). Possibly, conformational heterogeneity of the SBMV major protein used for antisera production led to the production of distinctive antibodies which upon reaction with these protein antigens produced two precipitin bands. These results demonstrate that the minor and major SBMV proteins are serologically related. Additionally, the amino acid compositions of the separated minor and major proteins
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are similar (Table l), suggesting that these two proteins are also chemically identical. Our results on the amino acid analysis of SBMV agree with those reported by Tremaine (1966). Another approach concerning identification of the minor SBMV protein involved treating virions or the purified major pro-
FIG. 3. Immunodiffusion patterns employing polyacrylamide-gel sections containing the minor and major SBMV proteins upon reaction with the antiserum prepared against the major protein (AS) or normal serum (NS). The sera were fractionated with ammonium sulfate to isolate the y-globulin fraction. TABLE AMINO Amino
ACID COMPOSITION SOUTHERN BEAN acid Unfractionated
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hi&dine Lysine Arginine
1 OF THE PROTEINS MOSAIC VIRUS
OF
Electrophoretically separated”
Virion protein”
Minor prokin
Major protein
7.9’ 11.4 8.4 8.1 4.8 4.5 6.3 1.5 7.1 3.2 4.0 11.4 6.0 1.9 1.0 3.2 9.3
8.0 11.4 8.6 8.4 4.7 4.6 6.6 1.4 6.6 3.1 3.5 11.0 5.8 2.0 1.0 3.4 9.9
8.4 11.1 9.0 8.0 4.3 5.2 6.2 1.4 7.0 3.2 3.6 11.1 6.0 2.3 1.3 3.4 8.3
” SBMV protein was prepared by treating virions with 1.0 N HCl (Ghabrial et al., 1967). ’ Protein isolated with 1.ON HCl was electrophoretically resolved into the minor and major protein components. c These values (averages of two or three experiments) represent relative molar ratios. Tryptophan was not determined.
CAPSID
STRUCTURE
tein with slelected protein cross-linking reagents and analyzing the engendered products by SDS-polyacrylamide-gel electrophoresis (Fig. 4). Whereas untreated virions showed the presence of the minor and major proteins only (Fig. 4A), those virions treated with dimethyl adipimidate, formaldehyde, or glutaraldehyde yielded several additional high molecular weight protein polymers (Figs. 4B-D). Treating the major SBMV protein (Fig. 4E) with dimethyl a.dipimidate caused formation of a protein entity comigrating with the minor protein (Fig. 4F), while exposure to formaldehyde or glutaraldehyde resulted in the formlation of several additional high molecular weight polymers (Figs. 4 G and H). Subsequently, it was determined that dimethyl adipimidate treatment at pH 9.1, compared to pH 8.5, was more effective in protein cross-linking. Whereas these three cross-linking reagents were effective for protein polymerization for virions, glutaraldehyde was the most effective for the isolated m,ajor SBMV protein. The reason for the increased cross-linking in virions compared to isolated SBMV major protein with dimethyl adipimidate is not known. A similar observation has been made by Bickle et al. where bis(19721, (methyl)su.berimidate cross-linked ribosomal proteins in the intact ribosomes but not free ribosomal proteins. The polymerized produlcts engendered by treating virions or the isolated SBMV protein with dimethyl ,adipimidate, formaldehyde, or glutaraldehyde resisted dissociation upon heating in SDS-p-mercaptoethanol. On the basis of the known specificity of the reaction of dimethyl adipimidate with proteins (Bancroft and Smith, 1975; Means and Feeney, 19711, it appears that the minor SBMV protein is a stable dimer of the SBMV capsid protein involving covalent cross-links between the e-amino groups of the lysyl rfesidues. Such cross-links can be broken by exposing proteins to highly alkaline con’ditions (Bickle et al., 19721, and this may explain the absence of a minor protein component in the SBMV protein samples prepared by prolonged dialysis of virions against 0.1 M carbonate buffer, pH 10.5, or 0.5 N NaOH followed by additional
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E
F
GH
FIG. 4. Polyacrylamide-gel electrophoresis of virions or the major SBMV protein following treatment with protein cross-linking reagents. After the appropriate treatment, the samples were heated (loo”, 5 nun) in SDS-P-mercaptoethanol and then electrophoresed. (A), Untreated virions; (B), dimethyl adipimidate-treated virions; (0, virions treated with 1% formaldehyde; (D), virions treated with 0.2% glutaraldehyde; (El, SBMV major protein, untreated; (Fl, dimethyl adipimidate-treated protein; (G), protein treated with 1.0% formaldehyde; (H), protein treated with 0.2% glutaraldehyde. Conditions for electrophoresis were the same as in Fig. 1.
dialysis against 1.0% ammonium carbonate. The reason for the observed discrepancy in the molecular weight of the minor protein, which is slightly more than the expected value for the SBMV coat protein dimer, is not clear. The presence of chemical cross-links can result in a reduced binding of SDS to proteins and decrease their electrophoretic mobilities in the polyacrylamide gels (Davies and Stark, 1970). Additional studies demonstrated that dimethyl adipimidate treatment caused no change in the serological properties or specific infectivity of SBMV. However, dimethyl adipimidate-treated cowpea chlorotic mottle and bromegrass mosaic viruses possessed low specific infectivities, but the RNAs isolated from the treated virions were highly infectious (Bancroft and Smith, 1975). It was suggested that the reduced specific infectivity of such virions was due to their inability to uncoat on the leaf surface to cause infection. The location of the minor SBMV protein in virions was determined with radioiodination, which has proved to be a useful technique for examining the distribution and organization of proteins in several en-
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veloped and nonenveloped animal viruses (e.g., Montelaro and Rueckert, 1975; Sangar et al., 1976). When iodinated SBMV virions were dissociated and electrophoresed, the radioactivity was recovered in the minor and major viral proteins (Fig. 5). The relative proportion of 12zI incorporation in the minor and major SBMV proteins (1:8) compared favorably with the quantitative estimation based on the staining intensity of these two proteins in virions. Radioiodination mediated with chloramine-T or lactoperoxidase gave similar results. Iodinated SBMV sedimented exactly the same as the untreated virions in the sucrose density gradient columns, indicating that this treatment was not deleterious for virion structure or integrity. These results indicate that the minor SBMV protein resides in the virion capsid. On the basis of the known molecular weight values for SBMV and SBMV-RNA, 6.6 x 10” and 1.4 x 10” respectively, (Shepherd, 1971), the mass of SBMV protein is calculated as 5.2 x 10”. This value accounts for and is consistent with the presence of 180 protein subunits (MW 29,000) in each virus particle. We propose that the entire complement of SBMV protein constitutes the viral capsid consisting of ca. 12 dimers (MW 66,000) and ca. 156 monomers (MW 29,000) on the basis of the relative proportion of these proteins in each virion. According to the quasi-equivalence concept of packing of coat protein subunits in an icosahedron (Caspar and Klug, 1962), the 180 SBMV structural subunits are probably arranged in clusters of pentamers at the 12 vertices and clusters of hexamers at the 20 faces of the capsid as in other isometric viruses with T = 3. Since SBMV-RNA intimately contacts ca. onethird of the total viral protein (ca. 60 subunits) including all dimers, supportive evidence for which is presented, a possibility may be considered that protein subunits interacting with RNA are arranged as pentamers (one dimer and three monomers) on the capsid surface. This is consistent with data on the RNA-protein content of the 47-50 S ribonucleoproteinaceous complex isolated from SBMV virions (Sehgal and Sinha, 1974). The remaining
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66 K 29 K
lL0
5
10
1251 ctdmin
15
;o
(x 104)
FIG. 5. lviI incorporation into the minor and major SBMV proteins. Following radioiodination, the virions were freed of unreacted iodine as described in Materials and Methods, disrupted with heating in SDS-P-mercaptoethanol, and electrophoresed in 5% polyacrylamide gels. The gels were stained with Coomassie brillant blue, destained, and then sliced into 30 equal segments. The radioactivity in each gel segment was determined with a Pickard Model 3001 Tri-Carb scintillation spectrometer.
two-thirds of the major SBMV protein is organized as clusters of hexamers (devoid of pentamers) and shows no interaction with SBMV-RNA. The stability of these spheroidal SBMV protein shells may be derived from strong hydrophobic interactions between the constituent protein subunits, but this needs additional study. The pentamers located at the 12 vertices of adenovirus 2 virions can be selectively removed, and yet the icosahedral form of the virus particles is fully retained (Laver et al., 1969). Since exposure of SBMV to EDTA and NaCl causes it to dissociate into the 47-50 S nucleoproteinaceous complex and spheroidal protein particles, it appears that the interactions between pentamers and hexamers are less stable than those between protein subunits within a pentamer and hexamer, as in adenovirus 2 (Prage et al., 1970). Figure 6 schematically shows the arrangement of SBMV coat protein subunits on the capsid surface and virion sensitivity to EDTA and KCl. Since the SBMV protein shells retain their spheroidal form in the absence of the viral RNA, the virion stability is apparently de-
FIG. 6. A schematic representation of the clustering of SBMV coat protein subunits on the capsid surface and virion dissociation with EDTAKC1 treatment. The dotted subunits on the pentamers represent the coat protein dimers. The black strand in the “loose nucleoproteinaceous complex” (lower right) represents SBMV-RNA attached to the pentamers (only five pentamers shown). This figure represents a modified version of an illustration in the article entitled “Stabilization of Brome Mosaic Virus” by Pfeiffer, Herzog, and Hirth (Phil. Trans. ofthe Roy. Sot. Discuss., in press).
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rived from strong protein-protein interactions, which is consistent with previous reports (Sehgal, 1973a; Sehgal and Das, 1975; Hsu et al., 1976; Boatman and Kaper, 1976). The presence of coat protein dimers involving lysyl residues may provide an added degree of stability to the SBMV capsid. The importance of the lysyl residues of the coat protein subunits in maintaining the stability of tobacco mosaic and cucumber mosaic viruses has been suggested (Caspar, 1963; Kaper, 1976). An interesting aspect concerning the disassembly pathway of SBMV is evident from the present study and from earlier observations. Apparently, in vitro SBMV disassembly involves as a first preparatory step a virion conformational change by a slight “swelling” of the capsid (Sehgal and Sinha, 1974; Hsu et al., 1976) followed by its dissociation into two distinctive subviral entities, namely the 47-50 S proteinRNA complex and spheroidal protein shells. That these two entities are discrete intermediates engendered during virion disassembly is indirectly supported by the observation that dialysis against divalent metal ions (15 n-&I Mg”+ plus 5.0 mM Ca’+ plus 1.0 mM Ni”+) causes these entities to coassociate into stable 115 S virus particles, similar to native virions (unpublished). Consequently, SBMV dissociation in vitro proceeds sequentially and is not an “all or none” disassembly process, a feature which appears novel among the isometric plant viruses examined so far. ACKNOWLEDGMENTS We thank Drs. L. Wall and C. Gehrke for the amino acid analyses and Dr. R. Mitra for help with the radioiodination techniques. Competent technical assistance by Don Lee and Mark Behrens is also appreciated. Missouri Agricultural Experiment Station Project No. 293, Journal Series No. 7627. REFERENCES ANDERER, F. A. (1959). Das Molekulargewicht der Peptideinheit im Protein des Tabakmosaikvirus. Z. Naturforsch. 14b, 24-28. BANCROFT, J. B., and SMITH, D. B. (1975). The effect of dimethyladipimidate on stability of cowpea chlorotic mottle and brome mosaic viruses. J. Gen. Viral. 27. 257-261. BENSON, J. V., and PATTERSON, J. A. (1971). Chromatographic advances in amino acids and peptide
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analysis using spherical resins and their applications in biochemistry and medicine. Iri “New Techniques in Amino Acid, Peptide and Protein Analysis” I A. Niederweiser and G. Pataki, eds. 1, pp. l-73. Ann Arbor Science, Ann Arbor, Mich. BIcKLE,‘~.A..HERsHEY.J.W.B..andTaAU’r,R.R. (1972). Spatial arrangement of ribosomal proteins: Reaction of the Escherichm co/i 30 S subunit with his-Imidoesters. Proc. Nat. Acnd. Sci. USA 69. 1327-1331. BOATMAN, S., and KAPER, J. M. 11976). Molecular organization and stabilizing forces of simple RNA viruses. IV. Selective interference with proteinRNA interactions by use of sodium dodecyl sulfate. Virology TO, 1-16. CASPAR. D. I,. D. (1963). Assembly and stability of the tobacco mosaic particle. Aduan. Protein Chern. 18, 37-121. CASPAR, D. I,. D., and KLUG. A. (19621. Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Qua&. Blol. 27. l-24. CRESTFIELD, A. M., MOORE, S., and STEIN, W. H. (1963). The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. Biol. Chem. 238, 622-627. DAVIES, G. E., and STARK, G. R. (1970). Use of dimethyl suberimidate. a cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc. Nat. Acad. Sci. USA 66, 651-656. FELBER. J. P. (1974). Radioimmunoassay of polypeptide hormones and enzymes. In “Methods of Biochemical Analysis” (D. Glick, ed.) pp. I-94. John Wiley, New York. FRAENKEL-CONRAT, H. (1957a). Degradation of tobacco mosaic virus with acetic acid. Virology 4, l4. FRAENKEL-CONRAT, H. (1957b3. Methods for investigating the essential groups for enzyme activity. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 4, PP. 247-269. Academic Press, New York. GHABRIAL, S. A., SHEPHERD, R. J., and GROGAN, R. G. (1967). Chemical properties of three strains of southern bean mosaic virus. Virology 33, 17-25. HIRS, C. H. W. (1967). Cleavage of disultide bonds. Performic acid oxidation. In “Methods in Enzymology” (C. H. W. Hirs, ed.), Vol. 11, pp. 1977199. Academic Press, New York. HSU, C. H., SEHGAL, 0. P., and PICKETT, E. E. (1976). Stabilizing effect of diavalent metal ions on virions of southern bean mosaic virus. Viro10g~v 69, 587-595. KAPER, J. M. (1976). Molecular organization and stabilizing forces of simple RNA viruses. V. The role of lysyl residues in the stabilization of cucumber mosaic virus strain S. Virology 71, 185-198. KNIGHT, C. A. (1975). “Chemistry of Viruses,” 2nd Ed. Springer-Verlag. New York.
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STRUCTURE
LAVER,W.G.,WRIGLEY,N.G.,~~~PEREIRA,H.G. (1969). Removal of pentons from particles of adenovirus type 2. Virology 39, 599-605. MAIZEL, J. V. I 19691. Acrylamide gel electrophoresis of proteins and nucleic acids. In “Fundamental Techniques m Virology” (K. Habel and N. P. Salzman, eds.), pp. 334-362. Academic Press, New York. MEANS, G. A.. and FEENEY, R. E. (19711. “Chemical Modification of Proteins.” Holden-Day, San Francisco MIKI, T., and KNIGHT, C. A. (1965). Preparation of broad bean mottle virus protein. Virology 25, 478481. MONTELARO, R. C., and RUECKERT, R. R. (1975). On the use of chloramine-T to iodinate specifically the surface proteins of intact enveloped viruses. J. Gen. Viral. 29, 127-131. OXELFELT, P. (1976). Stable dimers of the coat protein of a strain of tobacco mosaic virus. Virology 69, 346-349. PRAGE, L., PETTERSSON, U., HOGLUND, S., LONBERGHOLM, K., and PHILIPSON, L. (1970). Structural proteins of adenoviruses. IV. Sequential degradation of the ,adenovirus type 2 virion. Virology 42, 341-358. REICHMANN, M. E., and STACE-SMITH, R. (1959). Preparation of infectious ribonucleic acid from potato virus X by means of guanidine denaturation. Virology 9, 710-712. RICE, R. H. (1974). Minor protein components in cowpea chlorotic mottle virus and satellite of tobacco necrosis virus. Virology 61, 249-255. SANGAR, D. V., ROWLANDS, D. J., CAVANAGH, D., and BROWN, F. (19761. Characterization of the minor polypeptides in the foot-and-mouth disease particle. J. Gen. Viral. 31, 35-46. SARKAR, S. (1973). Recovery of the nucleic acids of tobacco mosaic and potato X viruses from polyacrylamide gel and evidence for a single infectious
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component in each of the two viruses. Z. Naturforsch. 28, 618-625. SEHGAL, 0. P. (1973al. Inactivation of southern bean mosaic virus and its ribonucleic acid by nitrous acid and ultraviolet light. J. Gen. Viral. 18, l-10. SEHGAL, 0. P. (1973b3. Biological and physico-chemical properties of an atypical mutant of tobacco mosaic virus. Mol. Gen. Genet. 121, 15-34. SEHGAL, 0. P., and DAS, P. D. (19751. Effect of freezing on conformation and stability of the virions of southern bean mosaic virus. Virology 61, 180-186. SEHGAL, 0. P., and SINHA, R. C. (1974). Characteristics of a nucleoproteinaceous subviral entity resulting from partial degradation of southern bean mosaic virus. Virology 59, 499-508. SHALLA, T. A., and SHEPARD, J. F. (19701. An antigenie analysis of potato virus X and of its degraded protein. II. Evidence for a conformational change associated with the depolymerization of structural protein. Virology 42, 835-847. SHEPARn,J.F.,SEcoR,G.A., andPURCIFULI.,D. E. (1974). Immunochemical cross-reactivity between the dissociated capsid proteins of PVY group plant viruses. Virology 58, 464-475. SHEPHERD, R. J. (1971). Southern bean mosaic virus. CMIiAAB Descriptions of Plant Viruses, No. 57. TREMAINE, J. H. (1966). The amino acid and nucleotide composition of the bean and cowpea strains of southern bean mosaic virus. VirologLy 30, 348-354. WEBER, J., PRINGLE, J. R., and OSBORN, M. (1972). Measurement of molecular weights by electrophoresis on SDS-acrylamide gels. In “Methods in Enzymology” (C. H. W. Hirs and S. N. Timasheff, eds.), Vol. 26, pp. 3-27. Academic Press, New York. YAMAZAKI, H., and KAESBERG, P. (19631. Degradation of bromegrass mosaic virus with calcium chloride and the isolation of its protein and nucleic acid. J. Mol. Bid. 7, 760-762.
77, I.-11
(1977)
Identity and Location of a Minor Protein Component Southern Bean Mosaic Virus 0. B. SEHGAL’ Department
of Plant
Pathology,
AND
University Accepted
C.
of Missouri,
October
in Virions
of
H. HSU Columbia,
Missouri
65201
4, 1976
Polyacrylamide-gel electrophoretic analysis of southern bean mosaic virus (SBMV) protein isolated with different procedures revealed the presence of a minor protein comlponent (MW 66,000) besides the major or capsid protein (MW 29,000). The amount of the minor protein in virions was ca. one-tenth the amount of the major protein. The minor SBMV protein remained stable when heated in the presence of sodium dodecyl sulfate, urea, and reducing agents (P-mercaptoethanol or dithiothreitol) or upon succinylation, reduction-carboxymethylation, or performic acid oxidation. Controlled virion disassembly revealed that all of the minor protein and some major protein subunits were intimately linked to the SBMV-RNA. The two proteins were separated and purified by preparative gel electrophoresis for subsequent characterization. The minor and major SBMV proteins possessed an identical amino acid composition. In gel diffusion tests, the minor and the major proteins developed two strong and confluent precipitin bands when reacted with an antiserum prepared against the purified major protein. Treating purified SBMV major protein with cross-linking reagents (dimethyl adipimidate, formaldehyde, or glutaraldehydel resulted in the formation of a protein component comigrating with the minor protein isolated from the virions. When SBMV virions were iodinated (NaY) using lactoperoxidase or chloramine-T, radioactivity was incorporated into the minor and major proteins. These results suggest that the minor SBMV protein is a covalently linked coat protein dimer involving e-amino groups of lysyl residues and resides in the virion capsid. On the basis of these studies, a tentative structural model for .the SBMV capsid is proposed.
reaction with protein cross-linking reagents, and radioiodination suggests that the minor SBMV protein is a stable dimer of the coat protein and resides in the virion capsid. On the basis of these studies, a structural model for the SBMV capsid is proposed.
INTRODUCTION
Virions of cowpea chlorotic mottle, satellite tobacco necrosis, bromegrass mosaic, and tobacco mosaic strain flavum contain a minor protein component in addition to the coat protein (Rice, 1974; Bancroft and Smith, 1975; Oxelfelt 1976). Some evidence suggests tlhat the minor protein in these viruses is a stable dimer of the coat ‘protein, but its functional significance in virion construction or structure is unknown. Recently, we reported (Hsu et al., 1976) that virions of southern bean mosaic virus (SBMV) contain a minor protein in addition to the coat protein. In this study, the identity and location of the minor protein in the virus particles is reported. Evidence based on serology, chemical composition, ’ Address
reprint
requests
to Dr.
MATERIALS
AND
METHODS
Virions of SBMV (bean strain) were purified from Phaseolus vulgaris L. “bountiful” by a modification of the procedure of Hsu et al. (1976). Following dialysis and low speed centrifugation of the 8% polyethylene glycol-precipitated SBMV, the preparations were adjusted to pH 4.5 (with 0.1 N HCl), maintained at 5” for 60 min, and centrifuged (10,000 g, 20 min). The resulting supernatant fraction was adjusted to pH 7.0 (with 0.1 N NaOH), and virions
Sehgal. 1
Copyright All rights
0 1977 by Academic Press, of reproduction in any form
Inc. reserved.
ISSN
0042-6822
2
SEHGAL
were sedimented by ultracentrifugation (105,000 g, 2.5 hr), suspended in 0.02 M sodium phosphate buffer, pH 7.0, and clarified by low speed centrifugation (10,000 g, 20 min). The differential centrifugation procedure was repeated two or three times and the final virion preparations stored (5”) in 0.02 A4 phosphate buffer, pH 7.0. In some experiments, virions were purified further by sucrose density gradient or isopycnic CsCl centrifugations (Sehgal and Das, 1975). Virions recovered from the density gradient columns were dialyzed (12 hr, 5”) against 0.02 M phosphate buffer, pH 7.0, sedimented by ultracentrifugation, and suspended in 0.02 M phosphate buffer, pH 7.0. Infectivity assays were performed on P. vulgaris “Pinto” (Sehgal, 1973a). Virions of tobacco mosaic virus (strain Ul) were purified from Nicotiana tabacum L. “Samsun” (Sehgal, 1973b). Concentrations of virions or viral components were estimated spectrophotometrically. SBMV protein was prepared by treating virions with several chemical reagents known to be effective in isolating plant viral proteins. These included warm formic acid (Miki and Knight, 1965), cold acetic acid (Fraenkel-Conrat, 1957a), 1.0 N HCl (Ghabrial et al., 1967), phenol (Anderer, 1959), 0.1 M sodium carbonate-bicarbonate buffer, pH 10.5 (Knight, 1975), guanidine hydrochloride (Reichmann and State-Smith, 1959), and cold calcium chloride (Yamazaki and Kaesberg, 1963). SBMV protein was also isolated by degrading virions with pyridine (Shalla and Shepard, 1970) or pyrrolidine (Shepard et al., 1974), but in these procedures formaldehyde was omitted from the dialyzing buffer. SBMV protein isolated with 1.0 N HCl, or the intact virions were succinylated (Fraenkel-Conrat, 1957b), reducedcarboxymethylated (Crestfield et al., 1963), or oxidized with performic acid (Hirs, 1967). Sucrose density gradient analysis revealed that virions were degraded upon reduction-carboxymethylation or performic acid oxidation but not by succinylation. In routine studies, SBMV virions were dissociated to yield minor and major proteins by heating (loo”, 5 min) in 1% sodium dodecyl sulfate (SDS) contain-
AND
HSU
ing 0.1% P-mercaptoethanol. Virions or the isolated SBMV protein were reacted (60 min, 5”) with formaldehyde or glutaraldehyde at pH 7.0, while dimethyl adipimidate (Pierce Chemical Company, Rockford, Illinois) treatment was performed at pH 8.5 or 9.1, according to Bancroft and Smith (1975). The samples were dialyzed (12 hr, 5”) thereafter against 0.02 M phosphate buffer, pH 7.0. Polyacrylamide-gel electrophoresis (7.5 mA/tube, 3 hr, 25’) was performed routinely in 5% gels (6.0 x 0.6 cm) containing 0.1% SDS according to Maize1 (1969). For molecular weight determinations, polyacrylamide gels of four different concentrations (5, 7.5, 10.0, and 15%) along with several marker proteins were used (Weber et al., 1972). Preparative separation of the minor and major SBMV proteins was achieved by electrophoresis in large (10 x 1.5 cm) polyacrylamide-gel (5%) columns. One milliliter of sample (2-3 mg of viral protein) was layered onto each gel and electrophoresed at 7.5 ma/tube for 1 hr and thereafter at 20 mAltube for 8 hr. After electrophoresis, the gels were removed and maintained at 5” for 12-14 hr, at which time the separated proteins appeared as prominent white bands. Gel sections containing the major and minor SBMV proteins were excised, the segments from several gels were combined, and the proteins were eluted electrophoretically with a slight modification of the procedure described by Sarkar (1973). The protein samples were dialyzed (12 hr, 5”) against deionized water and lyophilized. Subsequent electrophoretic analysis revealed that these two protein preparations were homogeneous. Isolation and purification of the major SBMV protein for use in the preparation of antiserum were done as follows. Virions (3 mg/ml) were treated with 0.1 M phosphate buffer, pH 7.5, containing 15 mM EDTA for 60 min at 5”, then NaCl (0.4 M) was added and the samples were stored at 5” for an additional 60 min. This sample (3.0 ml) was layered onto a 2-ml cushion of 10% sucrose containing 0.4 M NaCl and centrifuged (105,000 g, 4 hr). The top 2.5 ml of solution was removed and dialyzed (12 hr,
CAPSID
STRUCTURE
5”) against 0.02 M phosphate buffer, pH 7.0. Polyacrylamide-gel electrophoresis of this sample (400-500 pg) revealed the presence of only the major SBMV protein. Approximately 1.5-2.0 mg of the purified major protein was injected intravenously into a rabbit, and this injection schedule was repeated three times at weekly intervals. Four clays after the last injection, the rabbit was bled and the y-globulin fraction isolated by ammonium sulfate precipitation. Immunodiffusion tests were performed using the polyacrylamide-gel sections containing the minor and major proteins separated by preparative electrophoresis. These gel sections were embedded in warm 1% algarose containing 0.85% NaCl and 0.025% sodium azide, and the agarose was allowed to solidify. Two wells were punched into the agarose bed between the polyacrylarnide-gel sections and were charged with the sera. The reaction was allowed to proceed at 25” in a humid chamber. SBMV virions were iodinated using Na’““I (Industrial Nuclear Co., St. Louis, Missouri)- with the procedures of Montelaro and Rueckert (1975). When the chloramine-T procedure was followed, the reaction mixture contained 0.5 mCi of Na”“1, 0.5 M sodium phosphate, pH 7.4 (0.2 ml), 2.0 mg of SBMV in 0.02 M phosphate buffer, pH 7.0 (0.2 ml), and 0.5% (w/ v) chlorarnine-T in 0.05 M phosphate buffer, pH 7.4 (0.2 ml). After 40-60 set (25”), the reaction was stopped by adding 1 ml of 0.1% (w/v) sodium metabisulfite dissolved in 0.02 M phosphate buffer, pH 7.5. The iodinated virions were immediately passed through a column (1 x 2 cm) of an anion-exchLange (AGl-X10) resin (Bio-Rad Laboratories, Richmond, California) equilibrated with deionized water (Felber, 19741, and. the virions were sedimented with sucrose density gradient centrifugation. Virions recovered from the gradients were dialyzed (12 hr, 5”) against several changes of 0.02 M phosphate buffer, pH 7.0. Iodinaltion mediated with lactoperoxidase was performed by adding, in order, 0.5 mCi of Na12”I, 2 mg of SBMV (0.2 ml), 0.27% (w/v) lactoperoxidase (0.1 ml), and 0.88 mM hydrogen peroxide (0.01 ml).
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3
SBMV
After 60 set, 0.2 ml of 0.067 M phosphate buffer, pH 7.0, was added, and the virions were passed through the anion-exchange resin column and then subjected to sucrose density gradient centrifugation. The amino acid compositions of the major and minor SBMV proteins were determined according to Benson and Patterson (1971) with a Beckman Ml21 amino acid analyzer. Uranyl formate-stained samples were examined with a JEOL 100B electron microscope (Hsu et al., 1976). RESULTS
AND
DISCUSSION
Virions dissociated by heating in SDSp-mercaptoethanol in the absence or presence of urea yielded the minor and major SBMV proteins upon electrophoresis (Figs. 1A and E). Similarly, SBMV protein isolated with formic acid, acetic acid, or 1.0 N HCl showed the presence of both these protein components (Figs. 1 B-D). The phenol-isolated viral protein showed an additional slowly migrating protein component (Fig. 1F). While virion treatment with guanidine hydrochloride or calcium chloride yielded highly aggregated SBMV protein (Figs. 1H and J), it was readily dissociated upon heating in SDS-p-mercaptoethanol and yielded the minor and major proteins (Figs. 11 and K). The pyridine-isolated SBMV protein showed two or three additional high molecular weight polymers which were dissociated by heating, but there was some indication of a limited degradation of the minor and major proteins by pyridine (Figs. 1L and M). For SBMV protein isolated with pyrrolidine, several additional and discrete protein polymers were observed which were dissociated largely by heating (Figs. 1N and 0). SBMV protein prepared by prolonged dialysis (3 days, 5”) of virions against 0.1 M sodium carbonate-bicarbonate buffer, pH 10.5, however, yielded only the major protein (Fig. 1G). Similar results were obtained when SBMV protein was prepared by dialyzing virions against 0.5 N NaOH (4”, 24 hr) followed by additional dialysis against 1.0% ammonium carbonate (Ghabrial et al., 1967). These results indicate that the minor SBMV protein is sensitive to prolonged exposure to
4
SEHGAL
A
BC
DE
FOh
AND
HSU
I
J
K
1
M
N
0
FIG. 1. Polyacrylamide-gel electrophoretic analysis of SBMV protein. The isolated virion protein was dissolved in 0.02 M phosphate buffer, pH 7.0, containing 0.1% SDS and 0.01% P-mercaptoethanol. (A), Virions dissociated by heating (loo”, 5-30 min) in 1% SDS and 0.1% fi-mercaptoethanol; SBMV protein isolated with (B), warm formic acid; CC), cold acetic acid; CD), 1 N HCI; iE), by exposure (45”, 1 hr or loo”, 5 min) to 0.02 M phosphate buffer, pH 7.0, containing 1% SDS, 0.1% P-mercaptoethanol, and 4 M urea; (FI, with phenol, followed by protein precipitation from the phenol phase with methanol and sodium acetate. (G), with 0.1 M sodium carbonateebicarbonate buffer, pH 10.5; cH), guanidine hydrochloride; (I). guanidine hydrochloride-isolated protein, heated (100”. 5 min); (J). cold calcium chloride; (K). calcium chloride-isolated protein, heated; CL), pyridine; (Ml, pyridine-isolated, heated; (N), pyrrolidine; IO), pyrrolidine-isolated, heated. Approximately 100 /*g of the dissociated virions or 120 pg of isolated protein were electrophoresed (3 hr, 7.5 mA/tube) in 5% polyacrylamide gels (6.0 x 0.6 cm) containing 0.1% SDS in Maizel’s SDS-phosphate buffer system. The gels were stained with Coomassie brilliant blue and then destained with a methanolacetic acid mixture. The gels were maintained in 2.5% acetic acid.
strongly alkaline conditions. The average molecular weights determined by electrophoresis in 515% polyacrylamide gels for the minor and major SBMV proteins were, respectively, 66,000 and 29,000. Quantitative estimation based on staining intensity (Weber et al., 1972) employing 40-120 Fg of SBMV protein revealed that the amount of minor protein in virions was ca. onetenth the amount of the major protein. Only freshly prepared virion preparations (l-2 weeks following purification) were used in these studies. Gel-electrophoretic analysis of somewhat older (4-5 weeks) SBMV samples showed the presence of an additional protein band similar to the phenol-isolated SBMV protein (Fig. 1F). Preliminary studies show that virions of the cowpea SBMV strain contain a similar minor protein component. Factors affecting the dissociation of protein from SBMV virions and the stability of the viral proteins were investigated in detail. In the presence of 0.02 M phosphate buffer, pH 7.0, containing O.l-1.0% SDS and 0.1% ,&mercaptoethanol, the virions (0.5-2 mg/ml) were dissociated completely, yielding the minor and major proteins
when either heated (100”) for 3-30 min or exposed to SDS-p-mercaptoethanol at 45” for 60 min. Similarly, virions were dissociated completely at pH 5.0 or 9.1 by heating in 1% SDS. SBMV virions purified further with sucrose density gradient or isopycnic CsCl centrifugations yielded the minor and major proteins when dissociated by heating in SDS-P-mercaptoethanol. Both these proteins remained stable when heated (loo”, 3-5 min) in 1% SDS containing 1% sodium sulfite, lo-100 mJ4 dithiothreitol, and 5 mM glutathione or in 1% SDS containing 0.1% P-mercaptoethano1 and 8.0 M urea. Additionally, the minor and major SBMV proteins remained completely stable upon succinylation, reduction-carboxymethylation, or performic acid oxidation. When tobacco mosaic virus (TMV) virions (l-2 mgiml) were dissociated by heating in 0.1% SDS and subsequently electrophoresed, a minor protein (MW 35,000) component was observed as well as the capsid protein (MW 17,500). This minor TMV protein disappeared, however, when virions were heated in SDS containing 0.1% ,&mercaptoethanol or 10 mM dithiothreitol, indicating that it was a
CAPSID
STRUCTURE
disulfide-linked dimer of the TMV coat protein. Since the minor SBMV protein resisted dissociation upon heating in SDS containing reducing agents and urea, it seems unlikely that it is a coat protein dimer involving disulfide linkages. Following exposure to 0.1 M phosphate buffer, pH 7.5, containing EDTA and bentonite, SBMV virions degrade into a disCrete 47-50 S entity comprised of intact SBMV-RNA and ca. one-third of the total viral protein (Sehgal and Sinha, 1974). Similar results can be obtained by substituting bentonite with 0.4 M NaCl or KC1 (see below). To determine whether the mi-
OF
SBMV
5
nor SBMV protein was linked to SBMVRNA, virions were degraded and the intermediate macromolecular components analyzed. Figure 2 is a composite illustration showing sedimentation behavior, morphology, and polyacrylamide-gel electrophoresis of the variously treated SBMV. Untreated virions sedimented sharply, possessed discrete spheroidal (diameter, 30 nm) morphology, excluded stain from penetrating the core, and yielded the major and minor proteins upon electrophoresis. The EDTA-treated virions exhibited a slight reduction (ca. 12%) in their sedimentation rate; the particles appeared
P
SVE +
n Fraction11
RELATIVE
DEPTH
FIG. 2. A composite illustration showing sedimentation behavior, morphology, and SDS-polyacrylamide-gel electrophoretic analysis of the untreated or variously treated SBMV. Approximately 500 pg of untreated SBMV (0.3 ml) or treated virions, as indicated, were layered onto 530% linear sucrose density gradients and centrifuged at 105,000 g for 2 hr at 5”. The peak fractions from several runs were pooled and dialyzed against 0.02 M phosphate buffer, pH 7.0. Samples of these preparations were stained with uranyl formate for electron microscopy. Aliquots of the same samples were heated (loo”, 5 min) in SDS-Dmercaptoethanol and then analyzed in 5% polyacrylamide gels. Note absence of the minor protein in the electropherograms of Fraction I of the bottom sedimentation profile.
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SEHGAL
spheroidal but were rendered permeable to the negative stain and yielded the minor and major proteins upon electrophoresis (Hsu et al., 1976). Exposure of EDTAtreated SBMV to 0.4 A4 NaCl degraded it into the 47-50 S ribonucleoproteinaceous complex (Fraction II) and viral protein (Fraction I) which remained at the top of the centrifuged sucrose density-gradient columns. Particles of the 47-50 S SBMVRNA-protein complex (Fraction II) appeared “loose” with an irregular outline (diameter, 15-25 nm) as reported previously (Sehgal and Sinha, 1974) and yielded the minor and major proteins, although the amount of the major protein associated with it was somewhat less compared to the untreated SBMV. The SBMV protein (Fraction I) was aggregated largely as spheroidal shells (diameter, 25-30 nm), although many collapsed particles were also observed, but gel electrophoresis indicated the presence of only the major SBMV (29,000 daltons) protein in this fraction. These preparations (2-3 mg/ml) were noninfectious and exhibited an ultraviolet light absorption spectrum typical for proteins with a maximum extinction at 275280 nm and a minimum extinction at 250 nm (Sehgal, 1973a). Two conclusions are apparent from these results. First, the spheroidal SBMV protein particles contain only the major SBMV protein and are devoid of SBMV-RNA. Second, all the minor SBMV protein and some major protein subunits are linked to the viral RNA. The identity of the minor SBMV protein was determined as follows. When the electrophoretically separated minor and major proteins were reacted with an antiserum prepared against the purified SBMV major protein, two strong and confluent precipitin bands developed (Fig. 3). Possibly, conformational heterogeneity of the SBMV major protein used for antisera production led to the production of distinctive antibodies which upon reaction with these protein antigens produced two precipitin bands. These results demonstrate that the minor and major SBMV proteins are serologically related. Additionally, the amino acid compositions of the separated minor and major proteins
AND
HSC
are similar (Table l), suggesting that these two proteins are also chemically identical. Our results on the amino acid analysis of SBMV agree with those reported by Tremaine (1966). Another approach concerning identification of the minor SBMV protein involved treating virions or the purified major pro-
FIG. 3. Immunodiffusion patterns employing polyacrylamide-gel sections containing the minor and major SBMV proteins upon reaction with the antiserum prepared against the major protein (AS) or normal serum (NS). The sera were fractionated with ammonium sulfate to isolate the y-globulin fraction. TABLE AMINO Amino
ACID COMPOSITION SOUTHERN BEAN acid Unfractionated
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hi&dine Lysine Arginine
1 OF THE PROTEINS MOSAIC VIRUS
OF
Electrophoretically separated”
Virion protein”
Minor prokin
Major protein
7.9’ 11.4 8.4 8.1 4.8 4.5 6.3 1.5 7.1 3.2 4.0 11.4 6.0 1.9 1.0 3.2 9.3
8.0 11.4 8.6 8.4 4.7 4.6 6.6 1.4 6.6 3.1 3.5 11.0 5.8 2.0 1.0 3.4 9.9
8.4 11.1 9.0 8.0 4.3 5.2 6.2 1.4 7.0 3.2 3.6 11.1 6.0 2.3 1.3 3.4 8.3
” SBMV protein was prepared by treating virions with 1.0 N HCl (Ghabrial et al., 1967). ’ Protein isolated with 1.ON HCl was electrophoretically resolved into the minor and major protein components. c These values (averages of two or three experiments) represent relative molar ratios. Tryptophan was not determined.
CAPSID
STRUCTURE
tein with slelected protein cross-linking reagents and analyzing the engendered products by SDS-polyacrylamide-gel electrophoresis (Fig. 4). Whereas untreated virions showed the presence of the minor and major proteins only (Fig. 4A), those virions treated with dimethyl adipimidate, formaldehyde, or glutaraldehyde yielded several additional high molecular weight protein polymers (Figs. 4B-D). Treating the major SBMV protein (Fig. 4E) with dimethyl a.dipimidate caused formation of a protein entity comigrating with the minor protein (Fig. 4F), while exposure to formaldehyde or glutaraldehyde resulted in the formlation of several additional high molecular weight polymers (Figs. 4 G and H). Subsequently, it was determined that dimethyl adipimidate treatment at pH 9.1, compared to pH 8.5, was more effective in protein cross-linking. Whereas these three cross-linking reagents were effective for protein polymerization for virions, glutaraldehyde was the most effective for the isolated m,ajor SBMV protein. The reason for the increased cross-linking in virions compared to isolated SBMV major protein with dimethyl adipimidate is not known. A similar observation has been made by Bickle et al. where bis(19721, (methyl)su.berimidate cross-linked ribosomal proteins in the intact ribosomes but not free ribosomal proteins. The polymerized produlcts engendered by treating virions or the isolated SBMV protein with dimethyl ,adipimidate, formaldehyde, or glutaraldehyde resisted dissociation upon heating in SDS-p-mercaptoethanol. On the basis of the known specificity of the reaction of dimethyl adipimidate with proteins (Bancroft and Smith, 1975; Means and Feeney, 19711, it appears that the minor SBMV protein is a stable dimer of the SBMV capsid protein involving covalent cross-links between the e-amino groups of the lysyl rfesidues. Such cross-links can be broken by exposing proteins to highly alkaline con’ditions (Bickle et al., 19721, and this may explain the absence of a minor protein component in the SBMV protein samples prepared by prolonged dialysis of virions against 0.1 M carbonate buffer, pH 10.5, or 0.5 N NaOH followed by additional
OF
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SBMV
E
F
GH
FIG. 4. Polyacrylamide-gel electrophoresis of virions or the major SBMV protein following treatment with protein cross-linking reagents. After the appropriate treatment, the samples were heated (loo”, 5 nun) in SDS-P-mercaptoethanol and then electrophoresed. (A), Untreated virions; (B), dimethyl adipimidate-treated virions; (0, virions treated with 1% formaldehyde; (D), virions treated with 0.2% glutaraldehyde; (El, SBMV major protein, untreated; (Fl, dimethyl adipimidate-treated protein; (G), protein treated with 1.0% formaldehyde; (H), protein treated with 0.2% glutaraldehyde. Conditions for electrophoresis were the same as in Fig. 1.
dialysis against 1.0% ammonium carbonate. The reason for the observed discrepancy in the molecular weight of the minor protein, which is slightly more than the expected value for the SBMV coat protein dimer, is not clear. The presence of chemical cross-links can result in a reduced binding of SDS to proteins and decrease their electrophoretic mobilities in the polyacrylamide gels (Davies and Stark, 1970). Additional studies demonstrated that dimethyl adipimidate treatment caused no change in the serological properties or specific infectivity of SBMV. However, dimethyl adipimidate-treated cowpea chlorotic mottle and bromegrass mosaic viruses possessed low specific infectivities, but the RNAs isolated from the treated virions were highly infectious (Bancroft and Smith, 1975). It was suggested that the reduced specific infectivity of such virions was due to their inability to uncoat on the leaf surface to cause infection. The location of the minor SBMV protein in virions was determined with radioiodination, which has proved to be a useful technique for examining the distribution and organization of proteins in several en-
8
SEHGAL
veloped and nonenveloped animal viruses (e.g., Montelaro and Rueckert, 1975; Sangar et al., 1976). When iodinated SBMV virions were dissociated and electrophoresed, the radioactivity was recovered in the minor and major viral proteins (Fig. 5). The relative proportion of 12zI incorporation in the minor and major SBMV proteins (1:8) compared favorably with the quantitative estimation based on the staining intensity of these two proteins in virions. Radioiodination mediated with chloramine-T or lactoperoxidase gave similar results. Iodinated SBMV sedimented exactly the same as the untreated virions in the sucrose density gradient columns, indicating that this treatment was not deleterious for virion structure or integrity. These results indicate that the minor SBMV protein resides in the virion capsid. On the basis of the known molecular weight values for SBMV and SBMV-RNA, 6.6 x 10” and 1.4 x 10” respectively, (Shepherd, 1971), the mass of SBMV protein is calculated as 5.2 x 10”. This value accounts for and is consistent with the presence of 180 protein subunits (MW 29,000) in each virus particle. We propose that the entire complement of SBMV protein constitutes the viral capsid consisting of ca. 12 dimers (MW 66,000) and ca. 156 monomers (MW 29,000) on the basis of the relative proportion of these proteins in each virion. According to the quasi-equivalence concept of packing of coat protein subunits in an icosahedron (Caspar and Klug, 1962), the 180 SBMV structural subunits are probably arranged in clusters of pentamers at the 12 vertices and clusters of hexamers at the 20 faces of the capsid as in other isometric viruses with T = 3. Since SBMV-RNA intimately contacts ca. onethird of the total viral protein (ca. 60 subunits) including all dimers, supportive evidence for which is presented, a possibility may be considered that protein subunits interacting with RNA are arranged as pentamers (one dimer and three monomers) on the capsid surface. This is consistent with data on the RNA-protein content of the 47-50 S ribonucleoproteinaceous complex isolated from SBMV virions (Sehgal and Sinha, 1974). The remaining
AND
HSU
66 K 29 K
lL0
5
10
1251 ctdmin
15
;o
(x 104)
FIG. 5. lviI incorporation into the minor and major SBMV proteins. Following radioiodination, the virions were freed of unreacted iodine as described in Materials and Methods, disrupted with heating in SDS-P-mercaptoethanol, and electrophoresed in 5% polyacrylamide gels. The gels were stained with Coomassie brillant blue, destained, and then sliced into 30 equal segments. The radioactivity in each gel segment was determined with a Pickard Model 3001 Tri-Carb scintillation spectrometer.
two-thirds of the major SBMV protein is organized as clusters of hexamers (devoid of pentamers) and shows no interaction with SBMV-RNA. The stability of these spheroidal SBMV protein shells may be derived from strong hydrophobic interactions between the constituent protein subunits, but this needs additional study. The pentamers located at the 12 vertices of adenovirus 2 virions can be selectively removed, and yet the icosahedral form of the virus particles is fully retained (Laver et al., 1969). Since exposure of SBMV to EDTA and NaCl causes it to dissociate into the 47-50 S nucleoproteinaceous complex and spheroidal protein particles, it appears that the interactions between pentamers and hexamers are less stable than those between protein subunits within a pentamer and hexamer, as in adenovirus 2 (Prage et al., 1970). Figure 6 schematically shows the arrangement of SBMV coat protein subunits on the capsid surface and virion sensitivity to EDTA and KCl. Since the SBMV protein shells retain their spheroidal form in the absence of the viral RNA, the virion stability is apparently de-
FIG. 6. A schematic representation of the clustering of SBMV coat protein subunits on the capsid surface and virion dissociation with EDTAKC1 treatment. The dotted subunits on the pentamers represent the coat protein dimers. The black strand in the “loose nucleoproteinaceous complex” (lower right) represents SBMV-RNA attached to the pentamers (only five pentamers shown). This figure represents a modified version of an illustration in the article entitled “Stabilization of Brome Mosaic Virus” by Pfeiffer, Herzog, and Hirth (Phil. Trans. ofthe Roy. Sot. Discuss., in press).
10
SEHGAL
rived from strong protein-protein interactions, which is consistent with previous reports (Sehgal, 1973a; Sehgal and Das, 1975; Hsu et al., 1976; Boatman and Kaper, 1976). The presence of coat protein dimers involving lysyl residues may provide an added degree of stability to the SBMV capsid. The importance of the lysyl residues of the coat protein subunits in maintaining the stability of tobacco mosaic and cucumber mosaic viruses has been suggested (Caspar, 1963; Kaper, 1976). An interesting aspect concerning the disassembly pathway of SBMV is evident from the present study and from earlier observations. Apparently, in vitro SBMV disassembly involves as a first preparatory step a virion conformational change by a slight “swelling” of the capsid (Sehgal and Sinha, 1974; Hsu et al., 1976) followed by its dissociation into two distinctive subviral entities, namely the 47-50 S proteinRNA complex and spheroidal protein shells. That these two entities are discrete intermediates engendered during virion disassembly is indirectly supported by the observation that dialysis against divalent metal ions (15 n-&I Mg”+ plus 5.0 mM Ca’+ plus 1.0 mM Ni”+) causes these entities to coassociate into stable 115 S virus particles, similar to native virions (unpublished). Consequently, SBMV dissociation in vitro proceeds sequentially and is not an “all or none” disassembly process, a feature which appears novel among the isometric plant viruses examined so far. ACKNOWLEDGMENTS We thank Drs. L. Wall and C. Gehrke for the amino acid analyses and Dr. R. Mitra for help with the radioiodination techniques. Competent technical assistance by Don Lee and Mark Behrens is also appreciated. Missouri Agricultural Experiment Station Project No. 293, Journal Series No. 7627. REFERENCES ANDERER, F. A. (1959). Das Molekulargewicht der Peptideinheit im Protein des Tabakmosaikvirus. Z. Naturforsch. 14b, 24-28. BANCROFT, J. B., and SMITH, D. B. (1975). The effect of dimethyladipimidate on stability of cowpea chlorotic mottle and brome mosaic viruses. J. Gen. Viral. 27. 257-261. BENSON, J. V., and PATTERSON, J. A. (1971). Chromatographic advances in amino acids and peptide
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STRUCTURE
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