VIROLOGY
70, 217-220 (1976)
The Coat Proteins R. HULL’ Department
of Plant Pathology,
of Cauliflower AND
Mosaic
Virus
R. J. SHEPHERD
University
of California,
Davis,
California,
95616
Accepted October 21, 1975
Four polypeptides with molecular weights and molar ratios (in parentheses) of 96,000 and 88,000(both 0.08),64,000 (0.15) and 37,000 (0.77) were found in purified preparations of cauliflower mosaic virus (CaMVl. Two further polypeptides were found in some preparations and one was shown to he a degradation product of the major polypeptide. The four proteins found consistently had uniform charge/size ratio and gave positive but faint reaction to periodate-Schiff stain for glycoprotein. It is proposed that CaMV particles are comnosed of a T = 7 shell of the major protein surrounding a T = 1 core of the 64,000 molecular weight protein.
Cauliflower mosaic virus (CaMV) has isometric particles about 50 nm in diameter which contain double-stranded DNA (I -3). Recently, there have been three conflicting reports on the proteins of this virus. Tezuka and Taniguichi (4) considered that the viral protein consisted of a major polypeptide of 33,000 molecular weight; they were uncertain whether a minor component of 68,000 molecular weight was a stable dimer of this or was a different polypeptide. Kelley et al. (5) reported that the protein comprised up to six polypeptides with molecular weights of 27,32,40,67,92, and 100 x 103,the two largest being glycopeptides. They suggested that the 27,000 molecular weight polypeptide might be a degradation product of the 67,000 molecular weight polypeptide. Brunt et al. (6) observed two major and possibly one minor polypeptide with molecular weights of 68,000, 42,000, and 55,000, respectively; they also found up to seven other minor bands which they considered to be either stable aggregates or degradation products. The last two reports were published while the work described in this paper was in progress. We found four polypeptides consistently and frequently two others, 1 Present address: John Innes Institute, Lane, Norwich NR4 7UH, England.
Colney
one of which was a degradation product of the major polypeptide. Six isolates of CaMV were used: Cabbage B, (isolated by Walker et al., 71, CM4184 (derived from Cabbage B through single lesion culture), New York 8153, Campbell, Nome, and Phatak. The virus was purified from turnip (Brassica rapa L.) as described by Hull et al. (8). Virus and marker proteins were prepared for electrophoresis by boiling for 2 min in 5% (w/v) sodium dodecyl sulfate (SDS), 5 M urea, and 5% v/v 2-mercaptoethanol. Gels were loaded with about 20 pg CaMV protein with or without various combinations of 10 pg each of the following marker proteins (molecular weights in parentheses): bovine serum albumin (67,000), ovalbumin (43,000), alcohol dehydrogenase (37,000), carbonic anhydrase (29,000), chymotrypsinogen A (257001, and lysozyme (14,300). The gels, consisting of 10% (w/v) acrylamide (unless otherwise specified) with 0.2% cross-linked (w/v) NN’methylenebisacrylamide, polymerized using 0.016% ammonium persulfate and 0.04% N,iV,iV’fl’-tetramethylethylenediamine and containing 8 M urea, 0.1% SDS and 0.178 M Tris, 0.178 M boric acid, and 2 mM EDTA, pH 8.3 (9) were cast in 10 x 0.6-mm glass tubes; the Tris-boric acid 217
Copyright 8 1976 by Academic Press, Inc. All rights of reproductionin any form reserved.
218
SHORT
COMMUNICATIONS
buffer containing 0.1% SDS was used in the reservoirs. Electrophoresis was at 15 V/cm (about 3 mA/gel) for about 90 min, by which time the bromophenol blue marker reached the end of the gel. Protein bands were located by staining the gels for 1 hr in 0.2% Coumassie brilliant blue in methanol-acetic acid-water (4:1:5 v/v/v) and destaining overnight in methanol-acetic acid-water (2.6:0.75:6.75 v/v/v). The protein of CaMV, dissociated using consistently urea/SDS/mercaptoethanol, gave four bands in polyacrylamide gels (Bands 1, 2,4, and 5; Fig. 1A); on occasion, two other bands were found (bands 3 and 6). No differences were found in number, mobility, or proportion (estimated by scanning stained gels at 550 nm in a Gilford recording spectrophotometer with linear transport attachment) of the polypeptides between the various isolates of CaMV examined. The protein pattern of CaMV which had been isopycnically banded in CsCl was the same as that of untreated virus suggesting that none of the bands represented contamination of the prepara-
FIG. 1. Densitometer traces of CaMV proteins electrophoresed in 10% polyacrylamide gels and the bands stained using Coumassie brilliant blue (A, B) Proteins from virus preparations stored at -20”. (C, D) Proteins from the same virus preparations stored at room temperature for 2 weeks. The numbers in (A) identify the protein bands.
tion with host material. Attempts to dissociate the material further into the slower moving bands by extraction with 67% acetic acid or with 0.1 M ethanolamine, pH 10.5, prior to boiling with urea/SDSlmercaptoethanol or by reduction and carboxymethylation (using the technique of 10) of either whole or dissociated virus were unsuccessful. If any of the constituents of the dissociation medium was omitted or reduced in concentration (i.e., 4 M urea or 1% SDS) or if the boiling was omitted, the virus did not dissociate fully and stained material was left at the top of the gel. Thus, it appears that the pattern shown in Fig. 1 represents the maximum dissociation of CaMV proteins. The possibility that some of the bands were formed by differential binding of SDS or by differences in configuration, as has been suggested for some proteins by Maizel (11) and Tung and Knight (12) was examined by the technique described by Hendrick and Smith (13). CaMV and marker proteins were electrophoresed on 10, 8, 5, and 3% polyacrylamide gels (all with 2% cross-linker) and their mobilities were measured relative to that of lysozyme (Fig. 2). The fact that the slopes for the various polypeptides of CaMV and those of the marker proteins (data not shown) pass through a common origin indicates that all the polypeptides have similar charge/size ratios. The molecular weights of CaMV polypeptides were estimated from their slopes and those of the marker proteins (13; Table 1). The phenomenon of variation of apparent molecular weight with gel concentration reported by Brunt et aZ. (6) was not observed. Both Kelley et al. (5) and Brunt et al. (6) reported that some of the bands found on electrophoresis of CaMV protein probably arose from degradation of viral polypeptides. Figures la, b, c, and d show traces from gels in which protein had been electrophoresed from preparations of CaMV which had been stored either at -20” or at room temperature for 2 weeks. It appeared that band 6 was derived from band 5 and that band 3 perhaps came from band 2. This differs from the interpretation of Kelly et al. (5) who suggested that band 6
219
SHORT COMMUNICATIONS
(their CMVG) was derived from band 4 (their CMVS). Gels, in which relatively large amounts (40 pg) of CaMV protein had been electrophoresed, were stained by the periodateShiff procedure. SDS was removed from the gels by soaking them in a large volume of methanol-acetic acid-water (50.754.25 v/v/v) overnight, washing 5 times for 15 min in distilled water and then following
2 4 6 8 GEL cONCF.NTRATlON
IO %
FIG. 2. The mobilities of CaMV proteins relative to that of lysozyme measured in different concentrations of polyacrylamide gel. 0, Protein 1; 0, Protein 2; 0, Protein 4; W, Protein 5; A, Protein 6.
steps 3-8 of the method described by Zacharius et al. (14). Using this procedure, bands 1, 2, 4, and 5 (61 stained faintly, with the stain in bands 1 and 2 being relatively stronger than that in the other bands. In controls on the staining procedure, human glycoprotein, ovalbumin, and barley stripe mosaic virus coat protein stained whereas the other marker proteins listed earlier did not. The ratio of the densities of staining by the periodate-Shiff procedure to that by Coumassie blue (both measured at 550 nm) for CaMV polypeptides was half or less than that for ovalbumin and barley stripe mosaic virus protein, both of which have about 3% carbohydrate (I 5, IS). Stainability with the periodate-Shiff procedure, especially at the low levels observed, is not direct proof of glycopeptides. Confirmation of the glycosylation of CaMV polypeptides awaits tests by other procedures. To test for the possibility of phosphoproteins, the protein from CaMV which had been grown in the presence of 32Pand contained approximately 106 cpmlmg virus, was electrophoresed on 10% polyacrylamide gels. From counts on slices from these gels there was no evidence of phosphoproteins or of nucleic acid in the regions of the polypeptide bands. From the evidence presented above, it appears that the protein of CaMV can be
TABLE
1
MOLECULAR WEIGHTS AND MOLAR RATIOS OF CaMV POLYPEPTIDES This paper Designation
i
Molecular weight (x 10-J)
2
5
37
6
33 >
Kelley Molar ratio
0.15
Designation
3
et al. (5) Molecular weight (X 10-Y
67
Brunt Molar ratio
0.18
Designation
3A
Molecular weight (x 10-Y
Molar ratio
70
0.2
3B
0.77
4
40
5 6
-
0.07
i 6
ii
0.12
32
0.69
0.62
0.06
7A 7B
42
27
is
0.06
9”
28 15
10
” Value not given.
et al. (6)
1 -
220
SHORT
COMMUNICATIONS
separated into four polypeptides (bands 1, 2,4, and 5); these correspond to bands 1, 2, 3, and 5 of Kelley et al. (5j and bands 1, 2, 3a, and 7a of Brunt et al. (6; Table 1). The differences between our molecular weight estimates and those reported by the above authors are mostly within the 10% error associated with this technique. Polypeptides 4 and 5 are almost certainly structural and comprise over 90% of the viral protein. Molecular weight determinations on CaMV (8) indicate that CaMV contains about 18.9 x 10” of protein. From the molecular weights and molar ratios (Table 1) it can be calculated that each particle has approximately 418 copies of polypeptide 5 and 55 copies of polypeptide 4. The number of copies of polypeptide 5 would fit the number expected for a particle with icosahedral symmetry with triangulation number T = 7 (17); the next permissible number of structural subunits, 540 for T = 9, would account for much more than totals for polypeptides 4 and 5. The particles of papilloma and poIyoma viruses, which are of similar diameter to those of CaMV, have their structural subunits arranged in T = 7 icosahedral surface lattices (18, 19). Since, according to the theory of icosahedral structure (17), polypeptide 4 does not appear to fit into the surface shell of the particle it is possible that it forms a T = 1 (60 subunit) core. From studies of the degradation of CaMV with pronase, Tezuka and Taniguchi (20) suggested that CaMV might have a core. Electron microscope studies (Hull and Plaskitt, unpublished observation) also indicate the possibility of a core of about 20-25 nm diameter, which would be consistent with an icosahedron of T = 1 structure. CaMV particles contain about 12 copies of polypeptides 1 + 2 and, as pointed out by Kelley et al. (5) and Brunt et al. (6) their combined molecular weight would account for more than 90% of the coding capacity of the viral nucleic acid. It is uncertain whether these are host coded or if they are virus coded, whether they are precursor proteins similar to those found in picornaviruses (21-231 or stable aggregates of major polypeptides as suggested by Brunt et al. (6).
ACKNOWLEDGMENTS The authors are indebted to Mr. R. J. Wakeman for technical assistance. This work was supported by National Science Foundation Grant GB39959. REFERENCES 1. PIRONE, T. P., POUND, G. S., and SHEPHERD, R. J., Phytopathology 51, 541-547 (1961). 2. SHEPHERD, R. J., BRUENING, G. E., and WAKEMAN, R., Virology 41, 339-347 (1979). 3. RUSSELL, G. J., FOLLETT, E. A. C., SUBAKSHARPE, J. H., and HARRISON, B. D., J. Gen. Viral. 11, 129-138 (1971). 4. TEZUKA, N., and TANIGUCHI, T., Virology 48, 297-299. 5. KELLEY, D. C., COOPER, V., and WALKEY, D. G. A., Microbios. 10, 239-245 (1974). 6. BRUNT, A. A., BARTON, R. J., TREMAINE, J. H., and STACE-SMITH, R., J. Gen. Viral. 27, 101106 (1975). 7. WALKER, J. C., LEBEAU, F. J., PROPOUND, G. S., J. Agr. Res. 70, 379-404 (1945). 8. HULL, R., SHEPHERD, R. J., and HARVEY, J. D., J. Gen. Virol., in press. 9. PEACOCK, A. C., and DINGMAN, C. W., Biochemistry 6, 1818-1827 (1967). 10. CRESTFIELD, A. M., MOORE, S., and STEIN, W. H., J. Biol. Chem. 238, 622-627 (1963). Il. MAIZEL, J. V., in “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol5, pp. 179-246. Academic Press, New York, 1971. 12. TUNG, J-S., and KNIGHT, C. A., Anal. Biochem. 48, 153-163 (1972). 13. HENDRICK, J. L. and SMITH, A. J., Arch. Biothem. Biophys. 126, 155-164 (1968). 14. ZACHARIUS, R. M., ZELL, T. A., MORRISON, J. H., and WOODLOCK, J. J., Anal. Biochem. 30,148152 (1969). 15. MARSHALL, R. D., and NEUBERGER, A., in “Glycoproteins” (A. Gottschalk, ed.), p. 741. Elsevier, Amsterdam, 1972. 16. PARTRIDGE, J. E., SHANNON, L. M., GUMPF, D. J., and COLBOUGH, P., Nature /London) 247, 391-392 (1974). 17. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, l-24 (1962). 18. KLUG, A., and FINCH, J. T., J. Mol. Biol. 11, 403-423 (1965). 19. FINCH, J. T., J. Gen. Virol., 24,359-364 (1974). 20. TEZUKA, N., and TANIGUCHI, T., Virology 47, 142-146 (1972). 21. HOLLAND, J. J., and KIEHN, E. D., Proc. Nat. Acad. Sci. USA 60, 1015- 1022 (1968). 22. JACOBSON, M. F., and BALTIMORE, D., Proc. Nat. Acad. Sci. USA 61, 77-84 (1968). 23. SUMMERS, D. F., and MAIZEL, J. V., Proc. Nat. Acad. Sci. USA 59, 966-971 (1968).
70, 217-220 (1976)
The Coat Proteins R. HULL’ Department
of Plant Pathology,
of Cauliflower AND
Mosaic
Virus
R. J. SHEPHERD
University
of California,
Davis,
California,
95616
Accepted October 21, 1975
Four polypeptides with molecular weights and molar ratios (in parentheses) of 96,000 and 88,000(both 0.08),64,000 (0.15) and 37,000 (0.77) were found in purified preparations of cauliflower mosaic virus (CaMVl. Two further polypeptides were found in some preparations and one was shown to he a degradation product of the major polypeptide. The four proteins found consistently had uniform charge/size ratio and gave positive but faint reaction to periodate-Schiff stain for glycoprotein. It is proposed that CaMV particles are comnosed of a T = 7 shell of the major protein surrounding a T = 1 core of the 64,000 molecular weight protein.
Cauliflower mosaic virus (CaMV) has isometric particles about 50 nm in diameter which contain double-stranded DNA (I -3). Recently, there have been three conflicting reports on the proteins of this virus. Tezuka and Taniguichi (4) considered that the viral protein consisted of a major polypeptide of 33,000 molecular weight; they were uncertain whether a minor component of 68,000 molecular weight was a stable dimer of this or was a different polypeptide. Kelley et al. (5) reported that the protein comprised up to six polypeptides with molecular weights of 27,32,40,67,92, and 100 x 103,the two largest being glycopeptides. They suggested that the 27,000 molecular weight polypeptide might be a degradation product of the 67,000 molecular weight polypeptide. Brunt et al. (6) observed two major and possibly one minor polypeptide with molecular weights of 68,000, 42,000, and 55,000, respectively; they also found up to seven other minor bands which they considered to be either stable aggregates or degradation products. The last two reports were published while the work described in this paper was in progress. We found four polypeptides consistently and frequently two others, 1 Present address: John Innes Institute, Lane, Norwich NR4 7UH, England.
Colney
one of which was a degradation product of the major polypeptide. Six isolates of CaMV were used: Cabbage B, (isolated by Walker et al., 71, CM4184 (derived from Cabbage B through single lesion culture), New York 8153, Campbell, Nome, and Phatak. The virus was purified from turnip (Brassica rapa L.) as described by Hull et al. (8). Virus and marker proteins were prepared for electrophoresis by boiling for 2 min in 5% (w/v) sodium dodecyl sulfate (SDS), 5 M urea, and 5% v/v 2-mercaptoethanol. Gels were loaded with about 20 pg CaMV protein with or without various combinations of 10 pg each of the following marker proteins (molecular weights in parentheses): bovine serum albumin (67,000), ovalbumin (43,000), alcohol dehydrogenase (37,000), carbonic anhydrase (29,000), chymotrypsinogen A (257001, and lysozyme (14,300). The gels, consisting of 10% (w/v) acrylamide (unless otherwise specified) with 0.2% cross-linked (w/v) NN’methylenebisacrylamide, polymerized using 0.016% ammonium persulfate and 0.04% N,iV,iV’fl’-tetramethylethylenediamine and containing 8 M urea, 0.1% SDS and 0.178 M Tris, 0.178 M boric acid, and 2 mM EDTA, pH 8.3 (9) were cast in 10 x 0.6-mm glass tubes; the Tris-boric acid 217
Copyright 8 1976 by Academic Press, Inc. All rights of reproductionin any form reserved.
218
SHORT
COMMUNICATIONS
buffer containing 0.1% SDS was used in the reservoirs. Electrophoresis was at 15 V/cm (about 3 mA/gel) for about 90 min, by which time the bromophenol blue marker reached the end of the gel. Protein bands were located by staining the gels for 1 hr in 0.2% Coumassie brilliant blue in methanol-acetic acid-water (4:1:5 v/v/v) and destaining overnight in methanol-acetic acid-water (2.6:0.75:6.75 v/v/v). The protein of CaMV, dissociated using consistently urea/SDS/mercaptoethanol, gave four bands in polyacrylamide gels (Bands 1, 2,4, and 5; Fig. 1A); on occasion, two other bands were found (bands 3 and 6). No differences were found in number, mobility, or proportion (estimated by scanning stained gels at 550 nm in a Gilford recording spectrophotometer with linear transport attachment) of the polypeptides between the various isolates of CaMV examined. The protein pattern of CaMV which had been isopycnically banded in CsCl was the same as that of untreated virus suggesting that none of the bands represented contamination of the prepara-
FIG. 1. Densitometer traces of CaMV proteins electrophoresed in 10% polyacrylamide gels and the bands stained using Coumassie brilliant blue (A, B) Proteins from virus preparations stored at -20”. (C, D) Proteins from the same virus preparations stored at room temperature for 2 weeks. The numbers in (A) identify the protein bands.
tion with host material. Attempts to dissociate the material further into the slower moving bands by extraction with 67% acetic acid or with 0.1 M ethanolamine, pH 10.5, prior to boiling with urea/SDSlmercaptoethanol or by reduction and carboxymethylation (using the technique of 10) of either whole or dissociated virus were unsuccessful. If any of the constituents of the dissociation medium was omitted or reduced in concentration (i.e., 4 M urea or 1% SDS) or if the boiling was omitted, the virus did not dissociate fully and stained material was left at the top of the gel. Thus, it appears that the pattern shown in Fig. 1 represents the maximum dissociation of CaMV proteins. The possibility that some of the bands were formed by differential binding of SDS or by differences in configuration, as has been suggested for some proteins by Maizel (11) and Tung and Knight (12) was examined by the technique described by Hendrick and Smith (13). CaMV and marker proteins were electrophoresed on 10, 8, 5, and 3% polyacrylamide gels (all with 2% cross-linker) and their mobilities were measured relative to that of lysozyme (Fig. 2). The fact that the slopes for the various polypeptides of CaMV and those of the marker proteins (data not shown) pass through a common origin indicates that all the polypeptides have similar charge/size ratios. The molecular weights of CaMV polypeptides were estimated from their slopes and those of the marker proteins (13; Table 1). The phenomenon of variation of apparent molecular weight with gel concentration reported by Brunt et aZ. (6) was not observed. Both Kelley et al. (5) and Brunt et al. (6) reported that some of the bands found on electrophoresis of CaMV protein probably arose from degradation of viral polypeptides. Figures la, b, c, and d show traces from gels in which protein had been electrophoresed from preparations of CaMV which had been stored either at -20” or at room temperature for 2 weeks. It appeared that band 6 was derived from band 5 and that band 3 perhaps came from band 2. This differs from the interpretation of Kelly et al. (5) who suggested that band 6
219
SHORT COMMUNICATIONS
(their CMVG) was derived from band 4 (their CMVS). Gels, in which relatively large amounts (40 pg) of CaMV protein had been electrophoresed, were stained by the periodateShiff procedure. SDS was removed from the gels by soaking them in a large volume of methanol-acetic acid-water (50.754.25 v/v/v) overnight, washing 5 times for 15 min in distilled water and then following
2 4 6 8 GEL cONCF.NTRATlON
IO %
FIG. 2. The mobilities of CaMV proteins relative to that of lysozyme measured in different concentrations of polyacrylamide gel. 0, Protein 1; 0, Protein 2; 0, Protein 4; W, Protein 5; A, Protein 6.
steps 3-8 of the method described by Zacharius et al. (14). Using this procedure, bands 1, 2, 4, and 5 (61 stained faintly, with the stain in bands 1 and 2 being relatively stronger than that in the other bands. In controls on the staining procedure, human glycoprotein, ovalbumin, and barley stripe mosaic virus coat protein stained whereas the other marker proteins listed earlier did not. The ratio of the densities of staining by the periodate-Shiff procedure to that by Coumassie blue (both measured at 550 nm) for CaMV polypeptides was half or less than that for ovalbumin and barley stripe mosaic virus protein, both of which have about 3% carbohydrate (I 5, IS). Stainability with the periodate-Shiff procedure, especially at the low levels observed, is not direct proof of glycopeptides. Confirmation of the glycosylation of CaMV polypeptides awaits tests by other procedures. To test for the possibility of phosphoproteins, the protein from CaMV which had been grown in the presence of 32Pand contained approximately 106 cpmlmg virus, was electrophoresed on 10% polyacrylamide gels. From counts on slices from these gels there was no evidence of phosphoproteins or of nucleic acid in the regions of the polypeptide bands. From the evidence presented above, it appears that the protein of CaMV can be
TABLE
1
MOLECULAR WEIGHTS AND MOLAR RATIOS OF CaMV POLYPEPTIDES This paper Designation
i
Molecular weight (x 10-J)
2
5
37
6
33 >
Kelley Molar ratio
0.15
Designation
3
et al. (5) Molecular weight (X 10-Y
67
Brunt Molar ratio
0.18
Designation
3A
Molecular weight (x 10-Y
Molar ratio
70
0.2
3B
0.77
4
40
5 6
-
0.07
i 6
ii
0.12
32
0.69
0.62
0.06
7A 7B
42
27
is
0.06
9”
28 15
10
” Value not given.
et al. (6)
1 -
220
SHORT
COMMUNICATIONS
separated into four polypeptides (bands 1, 2,4, and 5); these correspond to bands 1, 2, 3, and 5 of Kelley et al. (5j and bands 1, 2, 3a, and 7a of Brunt et al. (6; Table 1). The differences between our molecular weight estimates and those reported by the above authors are mostly within the 10% error associated with this technique. Polypeptides 4 and 5 are almost certainly structural and comprise over 90% of the viral protein. Molecular weight determinations on CaMV (8) indicate that CaMV contains about 18.9 x 10” of protein. From the molecular weights and molar ratios (Table 1) it can be calculated that each particle has approximately 418 copies of polypeptide 5 and 55 copies of polypeptide 4. The number of copies of polypeptide 5 would fit the number expected for a particle with icosahedral symmetry with triangulation number T = 7 (17); the next permissible number of structural subunits, 540 for T = 9, would account for much more than totals for polypeptides 4 and 5. The particles of papilloma and poIyoma viruses, which are of similar diameter to those of CaMV, have their structural subunits arranged in T = 7 icosahedral surface lattices (18, 19). Since, according to the theory of icosahedral structure (17), polypeptide 4 does not appear to fit into the surface shell of the particle it is possible that it forms a T = 1 (60 subunit) core. From studies of the degradation of CaMV with pronase, Tezuka and Taniguchi (20) suggested that CaMV might have a core. Electron microscope studies (Hull and Plaskitt, unpublished observation) also indicate the possibility of a core of about 20-25 nm diameter, which would be consistent with an icosahedron of T = 1 structure. CaMV particles contain about 12 copies of polypeptides 1 + 2 and, as pointed out by Kelley et al. (5) and Brunt et al. (6) their combined molecular weight would account for more than 90% of the coding capacity of the viral nucleic acid. It is uncertain whether these are host coded or if they are virus coded, whether they are precursor proteins similar to those found in picornaviruses (21-231 or stable aggregates of major polypeptides as suggested by Brunt et al. (6).
ACKNOWLEDGMENTS The authors are indebted to Mr. R. J. Wakeman for technical assistance. This work was supported by National Science Foundation Grant GB39959. REFERENCES 1. PIRONE, T. P., POUND, G. S., and SHEPHERD, R. J., Phytopathology 51, 541-547 (1961). 2. SHEPHERD, R. J., BRUENING, G. E., and WAKEMAN, R., Virology 41, 339-347 (1979). 3. RUSSELL, G. J., FOLLETT, E. A. C., SUBAKSHARPE, J. H., and HARRISON, B. D., J. Gen. Viral. 11, 129-138 (1971). 4. TEZUKA, N., and TANIGUCHI, T., Virology 48, 297-299. 5. KELLEY, D. C., COOPER, V., and WALKEY, D. G. A., Microbios. 10, 239-245 (1974). 6. BRUNT, A. A., BARTON, R. J., TREMAINE, J. H., and STACE-SMITH, R., J. Gen. Viral. 27, 101106 (1975). 7. WALKER, J. C., LEBEAU, F. J., PROPOUND, G. S., J. Agr. Res. 70, 379-404 (1945). 8. HULL, R., SHEPHERD, R. J., and HARVEY, J. D., J. Gen. Virol., in press. 9. PEACOCK, A. C., and DINGMAN, C. W., Biochemistry 6, 1818-1827 (1967). 10. CRESTFIELD, A. M., MOORE, S., and STEIN, W. H., J. Biol. Chem. 238, 622-627 (1963). Il. MAIZEL, J. V., in “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol5, pp. 179-246. Academic Press, New York, 1971. 12. TUNG, J-S., and KNIGHT, C. A., Anal. Biochem. 48, 153-163 (1972). 13. HENDRICK, J. L. and SMITH, A. J., Arch. Biothem. Biophys. 126, 155-164 (1968). 14. ZACHARIUS, R. M., ZELL, T. A., MORRISON, J. H., and WOODLOCK, J. J., Anal. Biochem. 30,148152 (1969). 15. MARSHALL, R. D., and NEUBERGER, A., in “Glycoproteins” (A. Gottschalk, ed.), p. 741. Elsevier, Amsterdam, 1972. 16. PARTRIDGE, J. E., SHANNON, L. M., GUMPF, D. J., and COLBOUGH, P., Nature /London) 247, 391-392 (1974). 17. CASPAR, D. L. D., and KLUG, A., Cold Spring Harbor Symp. Quant. Biol. 27, l-24 (1962). 18. KLUG, A., and FINCH, J. T., J. Mol. Biol. 11, 403-423 (1965). 19. FINCH, J. T., J. Gen. Virol., 24,359-364 (1974). 20. TEZUKA, N., and TANIGUCHI, T., Virology 47, 142-146 (1972). 21. HOLLAND, J. J., and KIEHN, E. D., Proc. Nat. Acad. Sci. USA 60, 1015- 1022 (1968). 22. JACOBSON, M. F., and BALTIMORE, D., Proc. Nat. Acad. Sci. USA 61, 77-84 (1968). 23. SUMMERS, D. F., and MAIZEL, J. V., Proc. Nat. Acad. Sci. USA 59, 966-971 (1968).
