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The coat protein subunits of cucumber viruses 3 and 4 and a comparison of methods for determining their molecular weights
VIROLOGY
48, 574-581 (1972)
The Coat
Protein
Subunits
Comparison
of Cucumber
of Methods
for
Molecular JWU-SHENG Department
oj” lMolecular Biology
Viruses
3 and
Determining
Their
a
Weights’
TUNG
AND
C. A. KNIGHT
and Virus Laboratory, University
Berkeley,
4 and
California
of California,
9JZ?O
Accepted Fehruaj y 17, l9Y2 Chemical analyses (amino acid composition coupled with peptide mapping), gel chromatography and SDS-polyacrylamide gel electrophoresis were used to determine the molecular weights of the coat protein subunitsof several strains of cucumber viruses 3 and 4 (CV3 and CV4). The results obtained by means of amino acid analyses and peptide mapping were 17,100 for two strains of CV3 and 16,100 for two strains of CV4. These values are considered accurate to within &l$!& and if they are considered to be the actual values, the results obtained on these same strains of CV3 and CV4 by C-terminal analyses, SDS-polyacrylamide gel electrophoresis, and gel chromatography show deviations ranging from -0.6% to +47%. Gel chromatography in guanidine hydrochloride appeared to be next in reliability to amino acid analyses and peptide mapping.
INTRODUCTION
Cucumber viruses 3 and 4 (CV3 and CV4) resemble tobacco mosaic virus (TMV) in size and shape of particles and in content of RNA (Knight, 1955). There seems also to be a positive serological relationship between these cucumber viruses and strains of TMV (Bawden and Pirie, 1937; van Regenmortel, 1967). However, there are striking chemical differences between them, and they show profound differences in their host range specificities (Knight, 1955; Kado and Knight, 1970). The present investigation was undertaken partly to obtain basic information which might later illuminate some aspects of the host range specificity of plant viruses, em1 This investigation was supported in part by the U.S. Public Health Service Research Grant AI 00634 from the National Institute of Allergy and Infectious Diseases and Training Grant CA 05028 from the National Cancer Institute; taken in part from a thesis submitted by J.-S. Tung in partial fulfillment of the requirements for the Ph.D. degree in Molecular Biology, University of California, Berkeley, California, 1971.
ploying the cucumber viruses as models, and partly to compare the protein coats of the classical CV3 and CV4 (Ainsworth, 1935; Bawden and Pirie, 1937; Knight and Stanley, 1941) with two other more recently described members of this group of viruses (BrEak et al., 1962; Inouye et al., 1967). In addition, the availability of the diverse coat proteins of these viruses prompted us to test the reliability of four methods currently employed for determining molecular weights of viral proteins. MATERIALS
l7iru.s source. CV3, Berkeley strain (Berk CV3, and CV4, Berkeley strain (Berk CV4) were both obtained from England in the late 1930’s by workers at the Rockefeller Institute for Medical Research, Princeton branch. Cultures were maintained at Princeton until 1948 when they were brought to Berkeley, where they have been maintained by occasional passage in cucumber plants ever since. CV3, Japan strain (Japan CV3) was originally isolated by Inouye et al. in 1967 (cucumber green mottle mosaic virus,
574 Copyright
@ 1972 by Academic
Press,
Ine.
AND METHODS
COAT
PROTEIPiS
CGMIMV), and was a gift from M. H. V. egenmortel. CV4, Czechoslovakia Czech CV4) was obtained from J. &E&k In Czechoslovakia (Br&Lk et al., 1962). Ml viruses were cultured in Cucumis Sativua, L. cv. White spme and the infected plants were harvested about 3 wk after infection. The virus was purified by two to three cycles eentrifugation in 0.1 M phos7 (Knight, 1963), after which pigments were removed by homogenizing the virus in 0.1 iv phosphate buffer at pW 7 wit,h an equal volume of nbutanol: chloroform (I: 1, v/v) for 2-3 min room temperature in a Waring Blendor. e aqueous layer, containing the virus, was separated from the organic phase by eentrifugation at 900 g, and the virus was then subjected to one or two more cycles of differential centrifugation taking up the pellets in water (Kado and Knight, 1970). Ektein preparation. Goat proteins of both CV3 and CV4 were prepared by the acetic method of Fraenkel-Conrat (1957). Amino acid analyses. The amino acid analyses were ma by ion-exchange chromatography using a eckman 12OC ammo acid analyzer (Moore and Stein, 1963). Cysteine was estimated as cysteie acid by the method of Hirs (4967), and tryptophan was estimated by the method of Spies and Chambers (1949). Mappiny oj” tryptic peptides. The protein was digested with Worthington TPCKtrypsin, and tryptic peptides were mapped as previously reported (Woody and Knight, 1959). CaTbox&terminal analysis with carboxypeptidase -4. The carboxypeptidase A was obtained as crystalline aqueous suspensions from Worthington Biochemical Co. Just before use, a portion of the enzyme suspension was brought into solution by diluting with PO volumes of 10 % LiCl and stirring for I hr at 0”. Virus was suspended in 0.1 M Tris buffer at pII 7.6 at a concentration of 20-48 mg/ml. The enzyme was then added to the virus solution with the enzyme : virus ratio about I: 25 in the final reaction mixture. The reaction mixture was then incubated at 37”. Aliquots (0.2 ml) of the reaction mixture were withdrawn at suitable
OF CV3 AND
‘37.3
CV4
intervals and diluted with 1.0 ml of 0.2 I!$ sodium citrate buffer at 2.2. After I^<‘moval of the virus by een gation, supernatant solutions were analyzed for t,heir amino acid contents with a Beckman 12OC ammo acid analyzer. SDS-polyacryiamide gel elec6rophoresi.s. The electrophoresis was carried out in 10 o/d gels according to Weber and &born (E&XI). The isolated proteins were used or sometimes the whole virus was dissociated into protein subunits and nucleic acid by boiling for 5 min in I % sodium dodecyl sulfate (SDS), and the mixture was applied directly to the gel. The nucleic acid apparently did not mte,trfere with the subsequent e~~ctro~b~~es~~ since essentially t,he same results were obtamed wit,h protein and whole virus. Gel chromatography in 6 M g~~~~~~~n,~ hyddrochlode and in 0.1% SDS. Gel chrornaOography on 6% agarose @is-Rad A5M) was performed according to Fish et al. (1969) using 0.01 M sodium phosphate buffer at pH 7.0 in the elating mixture. RESULTS Amino Acid Composition The results of amino acid analyses are presented in Table 1. The highest ammo acid value of hydrolyzates was taken as the correct value. In the case of serine and threanine, the values were obtained by extrapolation t,o time zero. Minimal molecular weights rounded off to the nearest 10 culated for Berk CV3, Japan CV3, CV4, and Czech CV4 proteins were I for the CV3 strains and 16,100 for the strams (Table 3) s Peptide Mapping 1) showed 14 spots Peptide maps ( erk CV3, Berk CV4? for tryptic digests ns and 10 spots and Czech CV4 p Japan GV3 protein. While these values not coincide exactly with the number expected from contents of the basic ammo acids in the polypeptides of minimal molecular weight (from ammo acid analyses), t,hey are easily close enough to indicate a factor of one. Thus the minimal molecular w&g calculat,ed for the protein subunits by sum-
576
TUNG
AND TABLE
KNIGHT 1
AMINO ACID COMPOSITIONS OF CV3 AND CV4 PROTEINS Relative Amino acid
Lysine Histidine Arginine Aspartic acid Threoninee Serinee Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Total
amino
Residues per subuni@
Berk CV3 Japan CV3 Berk CV4 Czech CV4 Berk CV3 proteinb protei@ protein5 proteid protein 3.59 0 10.16 17.53 12.10 22.60 10.13 8.55 5.15 20.00 13.60 0 7.36 12.23 4.26 10.69 1.00 0
Tryptophanf Cysteineg
molar ratioa
4.02 1.00 8.29 20.67 10.20 23.68
10.00 5.78 9.05 21.19 6.86 0 6.83 18.40 4.09 9.01 2.03 0
3.14 0 9.30 16.93 11.68 22.20 9.54 8.41 4.68 18.40 13.77 0 5.30 12.70 4.02 10.36 1.00 0
acid
residues Molecular weight
3.26 0 9.19 17.10 11.72 22.34 9.87 8.21 4.96 18.12 13.98 0 5.32 13.20 4.21 10.24 1.00 0
4 0 10 18 12 23 10 9 5 20 14 0 7 12 4 11 1 0 160 17,106
Japan CV3 protein
Berk and Czech CV4 proteins
4 1 8 21 (26)h 10 24 10 6 9 21 7 0 7 18 4 9 2 0 161 17,055
3 0 9 17 12 22 10 8 5 18 14 0 5 13 4 10 1 0 151 16,102
calculated
a Average of the maximum values obtained by analyses on three different virus preparations. bBased on tryptophan as one. c Based on histidine as one. d Relative molar ratios multiplied by one and rounded off to nearest whole number. eValues obtained by extrapolation to time zero from the data of 24- and 72-hr hydrolyzates. f Determined independently by calorimetric procedure. g Determined as cysteic acid. h Twentv - aspartic acid residues were reported by the Funatsu and Funatsu (1968) from analysis of the tryptic peptides of Japan CV3 protein. mation of amino acid residues are also the actual molecular weights. C-Terminal
Amino Acid Analysis
Kinetic studies of the sort shown for Czech CV4 in Fig. 2 were made for each virus, and it was found that alanine, serine, and threonine were the main products resulting from the action of carboxypeptidase A on whole virus. The amino acid released in greatest amount from the protein of whole virus, and which is taken to be Cterminal, was alanine in the case of Berk CV4, Czech CV4, and Japan CV3, but threonine was found to be the C-terminal
amino acid of Berk CV3 protein. The maximum quantities of C-terminal ammo acid released (micromoles per milligram of virus) and the calculated molecular weights of the subunits based on these figures were 0.0990 and 10,100 for Berk CV3,0.0403 and 25,000 for Japan CV3, 0.0750 and 13,300 for Berk CV4, 0.0601 and 16,700 for Czech CV4 (Table 3). SDS-Polyacrylamide
Gel Electrophoresis
The protein markers used showed a linear relationship between the logarithm of their molecular weights and their mobilities on the gel (Fig. 3). Berk CV3, Berk CV4, and
COAT
PROTEINS
Origin
____
OF
571
CV3 AND 04
Table 3). The latter result was not due to experimental err5r since a mixture of and Japan 6863 proteins separated distinctly in SDS-gel.
The distribution coefficients, && ) for proteins tested are presented in Table 2. A linear relationship of the logarithm of molecular weights against K,, values was obtained for the marker proteins in the range between 66,000 and 13,700 daltons. The K,, values for all four of the CV3 and CV4 proteins were the same and Ied to a calculated molecular weight of 16,000 (Table 3). Gel ~~~roy~aa~ogra~~l~ in 0.1 % SDS The I&, value obtained for Japan CV3 protein was 0.6198 and for Berk CV4! Czech CV4, and Berk CV3 proteins, 63.6381 (Table 2). The molecular weights corresponding with these two values are _hS,OoO and 14,000, respect’ively (Table 3). A feature that is outstanding in the results of amino acid analyses made on kobaccsl mosaic virus (T&V) is the apparent occur.
0,*-. ..:
i -4
D -
-
Electrophoresis
-
+
FIG. 1. Tryptic peptide maps of: (A) Berk CV3 protein, (B) Japan CV3 protein, (C) Berk CV4 and Czech CV4 proteins, and (D) a mixture of Berk CV4 and Japan CV3 proteins; areas enclosed by dashed lines were identified as tryptic peptides of Japa,n 073 protein.
Czech @V4 proteins were shown to have identical electrophoret’ic mobilities in SDSgel, but these three proteins were clearly separable from Japan CK3 protein. Wnen mixed with markers, the molecular weights were estimated to be 14,200 for Berk CV3, Berk CV4, and Czech CV4 proteins but 16,000 for Japan CV’A protein (Fig. 3 and
0
I Enzyme
2
3 reaction
4 (hri
FIG. 2. Kefease of amino acids from Czech CW4 by the action of carboxypeptidase A: , threonine; ) serina.
TUNG
578
AND KNIGHT TABLE
2
DISTRIBUTION COEFFICIENTS (IL.) OF PROTEINS ELUTED FROM A 6% AGAROSE GEL FILTRATION COLUMN
r guani-
12 , ...
D
3
4
6 911
2 3
4
6 IO II
7 6-
ruvate
4
kinase
-A -.a---..s--
(I)
bonic
anhydrose
hymotrypsinogen
II
0
Jopan
CV3
protein
(8)
CV4
protein
(IO)
’ 3
’ 4
’ I
’ 2 Distance
moved
OFT SDS”
0.0862
0.3913
66,000
0.1709 0.2534 0.3001
0.4636 0.5340
46,000 34,500 25,700
8 c
0.4036 0.4100
0.6120 0.6380
Japan CV3 protein
0.4149
0.6198
Berk CV4 protein
0.4125
0.6381
Czech CV4 protein
0.4125
0.6385
Lysozyme Ribonuclease Cytochrome e
0.4872
0.6376 0.6404 -
A
__
.Q___. ,,
5-
Czech
dine HCl Bovine serum albumin Ovalbumin Carboxypeptidase Chymotrypsinogen A TMV protein Berk CV3
,-:‘..
Molecular weighta
In 6 M
Protein
(4) (5)
,
I 5 toward
1 6 anode
I 7
I 8
(cm)
FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoregrams of CV3 and CV4 proteins. The lower half is a semilogarithmic plot of molecular weights of protein markers versus the distances moved toward the anode.
17,500 16,000b; 14,000” 16,000b; 16,000° 16,000b; 14,000” 16,000b; 14,oooc 14,300 13) 700 11,700
a Molecular weights taken from Weber and Osborn (1969) and Dunker and Rueckert (1969). b Calculated from semilogarithmic plot of marker proteins for K,, values in 6 M guanidine HCl. c Calculated from K,, values in 0.1% SDS. TABLE
3
MOLECULARWEIGHTSOFCOATPROTEINSUBUNITS OF CUCUMBER VIRUSES DETERMINED BY DIFFERENT METHODS of determination
rence of the same total number of amino acid residues in the coat protein subunits of different strains, namely, 158 residues (Tsugita, 1962; Wittmann and WittmannLiebold, 1966). Only one unquestioned strain of TMV, the HR strain, and Japan CV3 (CGMMV) have until now been reported to deviate from this norm (Funatsu and Funatsu, 1968). HR was found to have 156 residues, and Japan CV3, 160. These results indicate, respectively, a small deletion from and a small addition to the coat
Berk CV3 Japan cv3 Berk CV4 Czech CV4
17,100 lO,lO( 14,200 16,000 14,000 17,100 25,00( 16,000 16,000 16,000 16,100 13,30(1 14,200 16,000 14,000 16,100 16,70(1 14,200 16,000 14,000
-
-
COAT
PROTEINS
protein gene of common TMV. In a similar comparison, our results indicate small additions to the coat protein genes of both Japan CV3 and Berk CV3 and rather great deletions from the coat protein genes of Be& CV4 and Czech CV4. If Berk CV3 is considered to be the prototype of these viruses, then the coat protein gene of Japan CV3 is about the same size as that of Berk CV3 while the coat protein genes of both CV4 si;rains are substantially smaller. We have no explanation for the discrepancies between the present analyses of Berk CV4 and those reported by van Regenmortel (1967) unless the strain analyzed by van egenmortel had been mislabeled and was actually what is called here Berk CV3 rather than CV4. The residual differences might then be accounted for by variations in technical aspects of the analyses. uahtativeiy, the results summarized in that the coat proteins of the ns have identical compositions whi.ch differ somewhat from Berk CV3 prot,ein and very much from Japan CV3 protein. Japan CV3 protein is also distinguished from the others by possession of a histidine residue, an amino acid absent from t,he other proteins. Nozu et al. (1971) have recently reported the amino acid composition of t,he coat protein of a watermelon strain of Japan CV3. The composition of this protein appears to be different from the others shown in Table 1 but closer t’o Berk CV4 than to the cucumber strain of Japan CV3. 0ur olecular weight values for the protein subunits of four cucumber viruses obtained different methods are list,ed in Table 3, consider the values based on ammo acid analysis coupled with mapping to be the most accurate. The amino acid data have an accuracy of &3 70, and from these data one derives a minimal molecular weight. The factor by which. the minimal molecular weight is multiplied to give the actual molecular weight is obtained by comparing the number of spots on the tryptic peptide map with the expected number based on summation of the lysine and arginine residues (plus one for the C-terminal pep tide) of the minimal molecular weight unit. Exactness is unimportant here. If 15
OF CV3 AND
CV4
.579
peptide spots are expected and 14 or 16 are observed, the factor is obviously ~IK: and not two. Accuracy of the arnrno acid analysispeptide mapping procedure’ is affected by such factors as homogeneity of the protein, completeness of the tryptic digestion, overlapping of spots on the peptide maps (the risk of overlapping spots increases greatly above 25 to 20 spots) and the compositors of the protein (for example, when there arc 17 to 33 amino acid residues of one kind the 3 % accuracy of the amino acid analysis comes equivalent to ICI ammo acid residue. Also slight errors are introduced by not distinguishing between the dizarboxylic ammo acids and t’heir amides). Kane of those possibilities is considered to be a serious source of error in determining the molecular weights of the cucumber virus coat proteins for the following reasons. Homogeneity of viral coat proteins can be conveniently assessed by polyacrylamidc gel electrophoresis. In the current study this was done, and single components were observed for all samples even when 5G100 pg of prot’ein was applied to the gd. Ii seems probable that tryptie digestion was essentially complete and that httle overlap of spots occurred on the peptide maps since the numbers of peptide spots predicted on the basis of complete dig&ion and no overlaps and the numbers of spots observed are close. There might have been some overlap of spots in the case of the CV3 strains and perhaps one incompletely sphit bond in the case of the CV4 strains; but the closeness of the observed to the predicted numbers of spots based on arginine and Ivsine contents makes t’hese postulatjcd Y anomalies negligible. Some uncertainty is attached to the analyses for aspartic acid: alanine, and srrine on the basis of the large numbers of residues but even if each vahre given for these three amino acids in Table I. is off one residue, the error in the molecular weight is not very great. Completely ac6urate analyses for molecular weight, except, for the amide groups, can be obtained pro vided that the tryptic peptides C~LI be separated. The composition of the pcptidcs can usually be determined without question 1 and summation gives the molecular weighty
580
TUNG AND KNIGHT
of the protein subunit. Such analyses were made by Funatsu and Funatsu (1968) on Japan CV3; our results on analyses of the whole protein (Table 1) agree completely with theirs except for aspartic acid, which differs by only 1 residue. In order to account for amide groups, it is necessary to analyze enzymatic rather than acid hydrolyzates of protein or tryptic peptides. However, failure to account for amide groups has an almost negligible effect on molecular weight values. In summary, the molecular weight values in the first column of Table 3 probably differ from the actual molecular weights by no more than the weight of one or two amino acid residues. Clearly, the coat proteins of the two CV3 strains have a substantially higher molecular weight than the coat proteins of the two strains of CV4. This is not evident, however, from the results of any of the other methods of analysis (Table 3), even though some of them, especially the gel chromatography in 6 M guanidine HCl, give a good approximation of the actual molecular weights. The C-terminal analyses were most erratic, ranging from a good coincidence with the results of amino acid analyses for the Czech CV4 to very poor agreement with the CV3 strains; we have no reasonable explanation at present for this variability. The molecular weights obtained by SDSpolyacrylamide gel electrophoresis deviate from the actual values by a little more than the =t 10 % accuracy usually ascribed to this method in 3 of t.he 4 cases, and fall within the &lo % range only for the Japan CV3 protein. With respect to reliability of different methods for determining molecular weights, the most significant deviation observed in the SDS-polyacrylamide gel results was the apparent difference in molecular weights of Berk CV3 and Japan CV3 proteins indicated by this procedure. The results of amino acid analyses (cohrmn 2, Table 3) show that there is virtually no difference in molecular weight of the two coat proteins whereas the results of analyses made by SDS-polyacrylamide gel electrophoresis indicate an almost 13 % difference. Evidently the electrophoretic migrations of Berk CV3 protein and Berk and Czech
CV4 proteins are significantly influenced by factors other than molecular weight. As discussed elsewhere (Tung and Knight, 1971)) the elec trophoretic migrations of proteins of similar size in SDS-polyacrylamide gels is a closely related function of their molecular weights only when they hate the same hydrodynamic shape and charge to mass ratio. Most proteins exist in concentrated guanidine HCl as linear random coils after disulfide linkages have been disrupted. Hence, polypeptide chain length and therefore molecular weight is the exclusive factor governing the elution of a protein from a gel chromatography column (Tanford, 1968; Fish et al., 1969). In the linear portion of the calibration curve, molecular weight range 7000 to 40,000, the accuracy has been reported to be &7%. We found this to hold in the present investigation with standard proteins ranging in molecular weight between 11,700 and 66,000. All four of the cucumber virus proteins appeared to have the same molecular weight (Table 3) upon gel chromatography in 6 M guanidine HCl, and the common molecular weight observed, 16,000, is within 7 % or less of the actual molecular weights determined by amino acid analysis. On the other hand, gel chromatography in 0.1% SDS led to spuriously low values for molecular weights of three of the four proteins (Table 3). Presumably, the st.andard proteins and Japan CV3 protein were transformed into rodlike protein-SDS complexes whose lengths are regularly related to molecular weight (Reynolds and Tanford, 1970) whereas the other cucumber virus proteins retained some tertiary structure which caused them to migrate on the gel columns at anomalous rates with respect to the standard proteins. It seems to us in conclusion that the most straightforward and accurate procedure for determining the molecular weight of homogeneous viral proteins such as those examined here is the combination of amino acid analysis with peptide mapping. Almost as good, but less accurate, are gel chromatography in 6 M guanidine HCl and electrophoresis in SDS-polyacrylamide gels. Both of the last two methods have anomalies, those associated with gel chromatography
COAT
PROTEINS
being more predictable and controllable than the ones associated with SDS-polyaerylamide gel electrophoresis. Molecular weights based on C-terminal analyses seem generally least dependable. Confidence in the results of any method is of course enhanced if the application of several methods yields essentially the same result. In this connection, gel chromatography and SDSpolyacrylamide gel electrophoresis might for peptide mapping in well substitute conjunction with amino acid analysis. In addition, the chromatography and electrophoresis methods can help in evaluating the homogeneity of viral protein preparaiions. IJlt,imately Lhe choice of method or methods will probably depend on convenience and degree of accuracy sought. ACKSOWLEDGMENT The authors are indebted to Larry Seiple for his excellent technical assistance in the amino acid analyses. REFERENCES
AINSWORTIX,6. C. (1935). Mosaic
diseases of the cucumber. Ann. Appl. Biol. 22, 55-67. BAWDEN, F. C., and PIEIE, N. W. (1937). The relationship between liquid crystalline preparations of cucumber viruses 3 and 4 and st’rains of tobacco mosaic virus. Brit. J. Exp. Pathol. 28, 275-291. BeE& J., ULRYCHOV~, M., and (?ECH, M. (1962). Infection of tobacco and some Chenopodium species by the CV4 (and 3) and by its nucleic acid. viroloyv 16, 105-114. DTJSKER, A. K., and RUECKERT, R. R. (1969). Observations on molecular weight determinations on polyacrylamide gel. J. Biol. Chem. 244 ) 5074-5080. FISH, W. W., MANN-, K. G., and TARTFORD, C. (1969). The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol.Chem. 244, 49894994. FIXENHEL-CoNRST, H. (1957). Degradation of tobacco mosaic virus with acetic acid. XTirology 4,14. FUN.~TSU, G., and FcNaTSu, M. (1968). Chemical studies on proteins from two tobacco mosaic virus strains. Proceedings of the 1967 InterIlational Symposium on Biochemical Regulation in Piseased Plants or Injury, Tokyo, Japan, pp* l-9.
OF CV3 AND
CV4
381
HIRS, C. H. W. (1967). Determination of cysti~le as cysteic acid. Methods Enzymol. 3il, 59-62. IXOZYE, T., INOUYE, N., AMTANI, M., and, MITSUHATA, K. (1967). Studies on cucumber green mottle mosaic virus in Japan. Xogni-u Kenkyu 53, 175-i86. KADO, C. I., and KNIGIIT, C. A. (197h)). Hcsr, specificity of plant viruses. I. Cucumber virus 4. virozogy 40, 997-1007. KNIGHT, C. A. (1955). Are cucumber viruses 3 and 4 strains of tobacco mosaic virus? Vi~olr~n?/ I;, 261-267. KNIGHT, 6. A. (1963). Chemistry of viruses. Protoplasmntologic: KNIGHT, C. A.,
KV.
2,
I-177.
and STAX~EY, W. M. (1941). Preparation and properties of cucumber virus 4. J. Biol. Chem. 141, 2938. MOORE, S., and STEIN, W. H. (1963). Chromategraphic determination of amino acids by the use of automatic recording equipment. ‘Meihods Enzymol. 6, 819-831. Nozu, Y., TOCHIH~UU, H., KOMURO, I”.; and OK~DA, Y. (1971). Chemical and immunological CharacTerization of cucumber green mettle mosaic virus (watermelon strain) protein. Virology 45, 577-585. REVNOLDS, 3. A., and T~XFOR~, C. (1970). The gross conformation of protein sodium dodecyi sulfate complexes. J. Biol. Chenz. 245, X61-5165. SPIES, j. R., and CH~~MBERS, D. C. (1949). Chemi-. cal determination of tryptophan in proteins. Anal. Chem. 21, i249-1266. TANFORD, C. (1968). Prot’ein deaaturation. A&an. Protein Chem. 23, 121-282. TSUGITA, 9. (1962). The proteins of murants of TMV: Classification of spontaneous and chemically evoked strains. J. UoZ. Biol. 5, 293-300. TUNG, J.-S., and KNIGET, 6. A. (1971). Effect of charge on the determination of molecular weight of proteins by gel electrophoresis in 9DS. Biochem. Biophys. Res. Cbmmun. 42, 1117-1121. VAN REGENMORTEL, M. H. V. (1967). Sero1ogica.I studies on naturally occurring strains and chemically induced mutants of TMV. virology 31,467480. WEBER, K., and OSBORX, M. (1969). The reliability of molecular weight determination by Sl%polyacrylamide gel electrophoresis. J. Riol. Chenz. 244, 44064412. WITTMANX, N. G., and
WITTMANX-LIEBGD,
B.
(1966). Protein chemical studies oi two RN.4 viruses and their mutants. iTold Spriny Igarbor Symp. Qua~zt. Biol. 31, 163-172. WOODY, B. R., and K;NIGNT, C. A. (1959). Peptide maps obtained with tryplic digests ol the proteins of some strains of tobacco mosaic virus. Virology 9, 359374.
48, 574-581 (1972)
The Coat
Protein
Subunits
Comparison
of Cucumber
of Methods
for
Molecular JWU-SHENG Department
oj” lMolecular Biology
Viruses
3 and
Determining
Their
a
Weights’
TUNG
AND
C. A. KNIGHT
and Virus Laboratory, University
Berkeley,
4 and
California
of California,
9JZ?O
Accepted Fehruaj y 17, l9Y2 Chemical analyses (amino acid composition coupled with peptide mapping), gel chromatography and SDS-polyacrylamide gel electrophoresis were used to determine the molecular weights of the coat protein subunitsof several strains of cucumber viruses 3 and 4 (CV3 and CV4). The results obtained by means of amino acid analyses and peptide mapping were 17,100 for two strains of CV3 and 16,100 for two strains of CV4. These values are considered accurate to within &l$!& and if they are considered to be the actual values, the results obtained on these same strains of CV3 and CV4 by C-terminal analyses, SDS-polyacrylamide gel electrophoresis, and gel chromatography show deviations ranging from -0.6% to +47%. Gel chromatography in guanidine hydrochloride appeared to be next in reliability to amino acid analyses and peptide mapping.
INTRODUCTION
Cucumber viruses 3 and 4 (CV3 and CV4) resemble tobacco mosaic virus (TMV) in size and shape of particles and in content of RNA (Knight, 1955). There seems also to be a positive serological relationship between these cucumber viruses and strains of TMV (Bawden and Pirie, 1937; van Regenmortel, 1967). However, there are striking chemical differences between them, and they show profound differences in their host range specificities (Knight, 1955; Kado and Knight, 1970). The present investigation was undertaken partly to obtain basic information which might later illuminate some aspects of the host range specificity of plant viruses, em1 This investigation was supported in part by the U.S. Public Health Service Research Grant AI 00634 from the National Institute of Allergy and Infectious Diseases and Training Grant CA 05028 from the National Cancer Institute; taken in part from a thesis submitted by J.-S. Tung in partial fulfillment of the requirements for the Ph.D. degree in Molecular Biology, University of California, Berkeley, California, 1971.
ploying the cucumber viruses as models, and partly to compare the protein coats of the classical CV3 and CV4 (Ainsworth, 1935; Bawden and Pirie, 1937; Knight and Stanley, 1941) with two other more recently described members of this group of viruses (BrEak et al., 1962; Inouye et al., 1967). In addition, the availability of the diverse coat proteins of these viruses prompted us to test the reliability of four methods currently employed for determining molecular weights of viral proteins. MATERIALS
l7iru.s source. CV3, Berkeley strain (Berk CV3, and CV4, Berkeley strain (Berk CV4) were both obtained from England in the late 1930’s by workers at the Rockefeller Institute for Medical Research, Princeton branch. Cultures were maintained at Princeton until 1948 when they were brought to Berkeley, where they have been maintained by occasional passage in cucumber plants ever since. CV3, Japan strain (Japan CV3) was originally isolated by Inouye et al. in 1967 (cucumber green mottle mosaic virus,
574 Copyright
@ 1972 by Academic
Press,
Ine.
AND METHODS
COAT
PROTEIPiS
CGMIMV), and was a gift from M. H. V. egenmortel. CV4, Czechoslovakia Czech CV4) was obtained from J. &E&k In Czechoslovakia (Br&Lk et al., 1962). Ml viruses were cultured in Cucumis Sativua, L. cv. White spme and the infected plants were harvested about 3 wk after infection. The virus was purified by two to three cycles eentrifugation in 0.1 M phos7 (Knight, 1963), after which pigments were removed by homogenizing the virus in 0.1 iv phosphate buffer at pW 7 wit,h an equal volume of nbutanol: chloroform (I: 1, v/v) for 2-3 min room temperature in a Waring Blendor. e aqueous layer, containing the virus, was separated from the organic phase by eentrifugation at 900 g, and the virus was then subjected to one or two more cycles of differential centrifugation taking up the pellets in water (Kado and Knight, 1970). Ektein preparation. Goat proteins of both CV3 and CV4 were prepared by the acetic method of Fraenkel-Conrat (1957). Amino acid analyses. The amino acid analyses were ma by ion-exchange chromatography using a eckman 12OC ammo acid analyzer (Moore and Stein, 1963). Cysteine was estimated as cysteie acid by the method of Hirs (4967), and tryptophan was estimated by the method of Spies and Chambers (1949). Mappiny oj” tryptic peptides. The protein was digested with Worthington TPCKtrypsin, and tryptic peptides were mapped as previously reported (Woody and Knight, 1959). CaTbox&terminal analysis with carboxypeptidase -4. The carboxypeptidase A was obtained as crystalline aqueous suspensions from Worthington Biochemical Co. Just before use, a portion of the enzyme suspension was brought into solution by diluting with PO volumes of 10 % LiCl and stirring for I hr at 0”. Virus was suspended in 0.1 M Tris buffer at pII 7.6 at a concentration of 20-48 mg/ml. The enzyme was then added to the virus solution with the enzyme : virus ratio about I: 25 in the final reaction mixture. The reaction mixture was then incubated at 37”. Aliquots (0.2 ml) of the reaction mixture were withdrawn at suitable
OF CV3 AND
‘37.3
CV4
intervals and diluted with 1.0 ml of 0.2 I!$ sodium citrate buffer at 2.2. After I^<‘moval of the virus by een gation, supernatant solutions were analyzed for t,heir amino acid contents with a Beckman 12OC ammo acid analyzer. SDS-polyacryiamide gel elec6rophoresi.s. The electrophoresis was carried out in 10 o/d gels according to Weber and &born (E&XI). The isolated proteins were used or sometimes the whole virus was dissociated into protein subunits and nucleic acid by boiling for 5 min in I % sodium dodecyl sulfate (SDS), and the mixture was applied directly to the gel. The nucleic acid apparently did not mte,trfere with the subsequent e~~ctro~b~~es~~ since essentially t,he same results were obtamed wit,h protein and whole virus. Gel chromatography in 6 M g~~~~~~~n,~ hyddrochlode and in 0.1% SDS. Gel chrornaOography on 6% agarose @is-Rad A5M) was performed according to Fish et al. (1969) using 0.01 M sodium phosphate buffer at pH 7.0 in the elating mixture. RESULTS Amino Acid Composition The results of amino acid analyses are presented in Table 1. The highest ammo acid value of hydrolyzates was taken as the correct value. In the case of serine and threanine, the values were obtained by extrapolation t,o time zero. Minimal molecular weights rounded off to the nearest 10 culated for Berk CV3, Japan CV3, CV4, and Czech CV4 proteins were I for the CV3 strains and 16,100 for the strams (Table 3) s Peptide Mapping 1) showed 14 spots Peptide maps ( erk CV3, Berk CV4? for tryptic digests ns and 10 spots and Czech CV4 p Japan GV3 protein. While these values not coincide exactly with the number expected from contents of the basic ammo acids in the polypeptides of minimal molecular weight (from ammo acid analyses), t,hey are easily close enough to indicate a factor of one. Thus the minimal molecular w&g calculat,ed for the protein subunits by sum-
576
TUNG
AND TABLE
KNIGHT 1
AMINO ACID COMPOSITIONS OF CV3 AND CV4 PROTEINS Relative Amino acid
Lysine Histidine Arginine Aspartic acid Threoninee Serinee Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
Total
amino
Residues per subuni@
Berk CV3 Japan CV3 Berk CV4 Czech CV4 Berk CV3 proteinb protei@ protein5 proteid protein 3.59 0 10.16 17.53 12.10 22.60 10.13 8.55 5.15 20.00 13.60 0 7.36 12.23 4.26 10.69 1.00 0
Tryptophanf Cysteineg
molar ratioa
4.02 1.00 8.29 20.67 10.20 23.68
10.00 5.78 9.05 21.19 6.86 0 6.83 18.40 4.09 9.01 2.03 0
3.14 0 9.30 16.93 11.68 22.20 9.54 8.41 4.68 18.40 13.77 0 5.30 12.70 4.02 10.36 1.00 0
acid
residues Molecular weight
3.26 0 9.19 17.10 11.72 22.34 9.87 8.21 4.96 18.12 13.98 0 5.32 13.20 4.21 10.24 1.00 0
4 0 10 18 12 23 10 9 5 20 14 0 7 12 4 11 1 0 160 17,106
Japan CV3 protein
Berk and Czech CV4 proteins
4 1 8 21 (26)h 10 24 10 6 9 21 7 0 7 18 4 9 2 0 161 17,055
3 0 9 17 12 22 10 8 5 18 14 0 5 13 4 10 1 0 151 16,102
calculated
a Average of the maximum values obtained by analyses on three different virus preparations. bBased on tryptophan as one. c Based on histidine as one. d Relative molar ratios multiplied by one and rounded off to nearest whole number. eValues obtained by extrapolation to time zero from the data of 24- and 72-hr hydrolyzates. f Determined independently by calorimetric procedure. g Determined as cysteic acid. h Twentv - aspartic acid residues were reported by the Funatsu and Funatsu (1968) from analysis of the tryptic peptides of Japan CV3 protein. mation of amino acid residues are also the actual molecular weights. C-Terminal
Amino Acid Analysis
Kinetic studies of the sort shown for Czech CV4 in Fig. 2 were made for each virus, and it was found that alanine, serine, and threonine were the main products resulting from the action of carboxypeptidase A on whole virus. The amino acid released in greatest amount from the protein of whole virus, and which is taken to be Cterminal, was alanine in the case of Berk CV4, Czech CV4, and Japan CV3, but threonine was found to be the C-terminal
amino acid of Berk CV3 protein. The maximum quantities of C-terminal ammo acid released (micromoles per milligram of virus) and the calculated molecular weights of the subunits based on these figures were 0.0990 and 10,100 for Berk CV3,0.0403 and 25,000 for Japan CV3, 0.0750 and 13,300 for Berk CV4, 0.0601 and 16,700 for Czech CV4 (Table 3). SDS-Polyacrylamide
Gel Electrophoresis
The protein markers used showed a linear relationship between the logarithm of their molecular weights and their mobilities on the gel (Fig. 3). Berk CV3, Berk CV4, and
COAT
PROTEINS
Origin
____
OF
571
CV3 AND 04
Table 3). The latter result was not due to experimental err5r since a mixture of and Japan 6863 proteins separated distinctly in SDS-gel.
The distribution coefficients, && ) for proteins tested are presented in Table 2. A linear relationship of the logarithm of molecular weights against K,, values was obtained for the marker proteins in the range between 66,000 and 13,700 daltons. The K,, values for all four of the CV3 and CV4 proteins were the same and Ied to a calculated molecular weight of 16,000 (Table 3). Gel ~~~roy~aa~ogra~~l~ in 0.1 % SDS The I&, value obtained for Japan CV3 protein was 0.6198 and for Berk CV4! Czech CV4, and Berk CV3 proteins, 63.6381 (Table 2). The molecular weights corresponding with these two values are _hS,OoO and 14,000, respect’ively (Table 3). A feature that is outstanding in the results of amino acid analyses made on kobaccsl mosaic virus (T&V) is the apparent occur.
0,*-. ..:
i -4
D -
-
Electrophoresis
-
+
FIG. 1. Tryptic peptide maps of: (A) Berk CV3 protein, (B) Japan CV3 protein, (C) Berk CV4 and Czech CV4 proteins, and (D) a mixture of Berk CV4 and Japan CV3 proteins; areas enclosed by dashed lines were identified as tryptic peptides of Japa,n 073 protein.
Czech @V4 proteins were shown to have identical electrophoret’ic mobilities in SDSgel, but these three proteins were clearly separable from Japan CK3 protein. Wnen mixed with markers, the molecular weights were estimated to be 14,200 for Berk CV3, Berk CV4, and Czech CV4 proteins but 16,000 for Japan CV’A protein (Fig. 3 and
0
I Enzyme
2
3 reaction
4 (hri
FIG. 2. Kefease of amino acids from Czech CW4 by the action of carboxypeptidase A: , threonine; ) serina.
TUNG
578
AND KNIGHT TABLE
2
DISTRIBUTION COEFFICIENTS (IL.) OF PROTEINS ELUTED FROM A 6% AGAROSE GEL FILTRATION COLUMN
r guani-
12 , ...
D
3
4
6 911
2 3
4
6 IO II
7 6-
ruvate
4
kinase
-A -.a---..s--
(I)
bonic
anhydrose
hymotrypsinogen
II
0
Jopan
CV3
protein
(8)
CV4
protein
(IO)
’ 3
’ 4
’ I
’ 2 Distance
moved
OFT SDS”
0.0862
0.3913
66,000
0.1709 0.2534 0.3001
0.4636 0.5340
46,000 34,500 25,700
8 c
0.4036 0.4100
0.6120 0.6380
Japan CV3 protein
0.4149
0.6198
Berk CV4 protein
0.4125
0.6381
Czech CV4 protein
0.4125
0.6385
Lysozyme Ribonuclease Cytochrome e
0.4872
0.6376 0.6404 -
A
__
.Q___. ,,
5-
Czech
dine HCl Bovine serum albumin Ovalbumin Carboxypeptidase Chymotrypsinogen A TMV protein Berk CV3
,-:‘..
Molecular weighta
In 6 M
Protein
(4) (5)
,
I 5 toward
1 6 anode
I 7
I 8
(cm)
FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoregrams of CV3 and CV4 proteins. The lower half is a semilogarithmic plot of molecular weights of protein markers versus the distances moved toward the anode.
17,500 16,000b; 14,000” 16,000b; 16,000° 16,000b; 14,000” 16,000b; 14,oooc 14,300 13) 700 11,700
a Molecular weights taken from Weber and Osborn (1969) and Dunker and Rueckert (1969). b Calculated from semilogarithmic plot of marker proteins for K,, values in 6 M guanidine HCl. c Calculated from K,, values in 0.1% SDS. TABLE
3
MOLECULARWEIGHTSOFCOATPROTEINSUBUNITS OF CUCUMBER VIRUSES DETERMINED BY DIFFERENT METHODS of determination
rence of the same total number of amino acid residues in the coat protein subunits of different strains, namely, 158 residues (Tsugita, 1962; Wittmann and WittmannLiebold, 1966). Only one unquestioned strain of TMV, the HR strain, and Japan CV3 (CGMMV) have until now been reported to deviate from this norm (Funatsu and Funatsu, 1968). HR was found to have 156 residues, and Japan CV3, 160. These results indicate, respectively, a small deletion from and a small addition to the coat
Berk CV3 Japan cv3 Berk CV4 Czech CV4
17,100 lO,lO( 14,200 16,000 14,000 17,100 25,00( 16,000 16,000 16,000 16,100 13,30(1 14,200 16,000 14,000 16,100 16,70(1 14,200 16,000 14,000
-
-
COAT
PROTEINS
protein gene of common TMV. In a similar comparison, our results indicate small additions to the coat protein genes of both Japan CV3 and Berk CV3 and rather great deletions from the coat protein genes of Be& CV4 and Czech CV4. If Berk CV3 is considered to be the prototype of these viruses, then the coat protein gene of Japan CV3 is about the same size as that of Berk CV3 while the coat protein genes of both CV4 si;rains are substantially smaller. We have no explanation for the discrepancies between the present analyses of Berk CV4 and those reported by van Regenmortel (1967) unless the strain analyzed by van egenmortel had been mislabeled and was actually what is called here Berk CV3 rather than CV4. The residual differences might then be accounted for by variations in technical aspects of the analyses. uahtativeiy, the results summarized in that the coat proteins of the ns have identical compositions whi.ch differ somewhat from Berk CV3 prot,ein and very much from Japan CV3 protein. Japan CV3 protein is also distinguished from the others by possession of a histidine residue, an amino acid absent from t,he other proteins. Nozu et al. (1971) have recently reported the amino acid composition of t,he coat protein of a watermelon strain of Japan CV3. The composition of this protein appears to be different from the others shown in Table 1 but closer t’o Berk CV4 than to the cucumber strain of Japan CV3. 0ur olecular weight values for the protein subunits of four cucumber viruses obtained different methods are list,ed in Table 3, consider the values based on ammo acid analysis coupled with mapping to be the most accurate. The amino acid data have an accuracy of &3 70, and from these data one derives a minimal molecular weight. The factor by which. the minimal molecular weight is multiplied to give the actual molecular weight is obtained by comparing the number of spots on the tryptic peptide map with the expected number based on summation of the lysine and arginine residues (plus one for the C-terminal pep tide) of the minimal molecular weight unit. Exactness is unimportant here. If 15
OF CV3 AND
CV4
.579
peptide spots are expected and 14 or 16 are observed, the factor is obviously ~IK: and not two. Accuracy of the arnrno acid analysispeptide mapping procedure’ is affected by such factors as homogeneity of the protein, completeness of the tryptic digestion, overlapping of spots on the peptide maps (the risk of overlapping spots increases greatly above 25 to 20 spots) and the compositors of the protein (for example, when there arc 17 to 33 amino acid residues of one kind the 3 % accuracy of the amino acid analysis comes equivalent to ICI ammo acid residue. Also slight errors are introduced by not distinguishing between the dizarboxylic ammo acids and t’heir amides). Kane of those possibilities is considered to be a serious source of error in determining the molecular weights of the cucumber virus coat proteins for the following reasons. Homogeneity of viral coat proteins can be conveniently assessed by polyacrylamidc gel electrophoresis. In the current study this was done, and single components were observed for all samples even when 5G100 pg of prot’ein was applied to the gd. Ii seems probable that tryptie digestion was essentially complete and that httle overlap of spots occurred on the peptide maps since the numbers of peptide spots predicted on the basis of complete dig&ion and no overlaps and the numbers of spots observed are close. There might have been some overlap of spots in the case of the CV3 strains and perhaps one incompletely sphit bond in the case of the CV4 strains; but the closeness of the observed to the predicted numbers of spots based on arginine and Ivsine contents makes t’hese postulatjcd Y anomalies negligible. Some uncertainty is attached to the analyses for aspartic acid: alanine, and srrine on the basis of the large numbers of residues but even if each vahre given for these three amino acids in Table I. is off one residue, the error in the molecular weight is not very great. Completely ac6urate analyses for molecular weight, except, for the amide groups, can be obtained pro vided that the tryptic peptides C~LI be separated. The composition of the pcptidcs can usually be determined without question 1 and summation gives the molecular weighty
580
TUNG AND KNIGHT
of the protein subunit. Such analyses were made by Funatsu and Funatsu (1968) on Japan CV3; our results on analyses of the whole protein (Table 1) agree completely with theirs except for aspartic acid, which differs by only 1 residue. In order to account for amide groups, it is necessary to analyze enzymatic rather than acid hydrolyzates of protein or tryptic peptides. However, failure to account for amide groups has an almost negligible effect on molecular weight values. In summary, the molecular weight values in the first column of Table 3 probably differ from the actual molecular weights by no more than the weight of one or two amino acid residues. Clearly, the coat proteins of the two CV3 strains have a substantially higher molecular weight than the coat proteins of the two strains of CV4. This is not evident, however, from the results of any of the other methods of analysis (Table 3), even though some of them, especially the gel chromatography in 6 M guanidine HCl, give a good approximation of the actual molecular weights. The C-terminal analyses were most erratic, ranging from a good coincidence with the results of amino acid analyses for the Czech CV4 to very poor agreement with the CV3 strains; we have no reasonable explanation at present for this variability. The molecular weights obtained by SDSpolyacrylamide gel electrophoresis deviate from the actual values by a little more than the =t 10 % accuracy usually ascribed to this method in 3 of t.he 4 cases, and fall within the &lo % range only for the Japan CV3 protein. With respect to reliability of different methods for determining molecular weights, the most significant deviation observed in the SDS-polyacrylamide gel results was the apparent difference in molecular weights of Berk CV3 and Japan CV3 proteins indicated by this procedure. The results of amino acid analyses (cohrmn 2, Table 3) show that there is virtually no difference in molecular weight of the two coat proteins whereas the results of analyses made by SDS-polyacrylamide gel electrophoresis indicate an almost 13 % difference. Evidently the electrophoretic migrations of Berk CV3 protein and Berk and Czech
CV4 proteins are significantly influenced by factors other than molecular weight. As discussed elsewhere (Tung and Knight, 1971)) the elec trophoretic migrations of proteins of similar size in SDS-polyacrylamide gels is a closely related function of their molecular weights only when they hate the same hydrodynamic shape and charge to mass ratio. Most proteins exist in concentrated guanidine HCl as linear random coils after disulfide linkages have been disrupted. Hence, polypeptide chain length and therefore molecular weight is the exclusive factor governing the elution of a protein from a gel chromatography column (Tanford, 1968; Fish et al., 1969). In the linear portion of the calibration curve, molecular weight range 7000 to 40,000, the accuracy has been reported to be &7%. We found this to hold in the present investigation with standard proteins ranging in molecular weight between 11,700 and 66,000. All four of the cucumber virus proteins appeared to have the same molecular weight (Table 3) upon gel chromatography in 6 M guanidine HCl, and the common molecular weight observed, 16,000, is within 7 % or less of the actual molecular weights determined by amino acid analysis. On the other hand, gel chromatography in 0.1% SDS led to spuriously low values for molecular weights of three of the four proteins (Table 3). Presumably, the st.andard proteins and Japan CV3 protein were transformed into rodlike protein-SDS complexes whose lengths are regularly related to molecular weight (Reynolds and Tanford, 1970) whereas the other cucumber virus proteins retained some tertiary structure which caused them to migrate on the gel columns at anomalous rates with respect to the standard proteins. It seems to us in conclusion that the most straightforward and accurate procedure for determining the molecular weight of homogeneous viral proteins such as those examined here is the combination of amino acid analysis with peptide mapping. Almost as good, but less accurate, are gel chromatography in 6 M guanidine HCl and electrophoresis in SDS-polyacrylamide gels. Both of the last two methods have anomalies, those associated with gel chromatography
COAT
PROTEINS
being more predictable and controllable than the ones associated with SDS-polyaerylamide gel electrophoresis. Molecular weights based on C-terminal analyses seem generally least dependable. Confidence in the results of any method is of course enhanced if the application of several methods yields essentially the same result. In this connection, gel chromatography and SDSpolyacrylamide gel electrophoresis might for peptide mapping in well substitute conjunction with amino acid analysis. In addition, the chromatography and electrophoresis methods can help in evaluating the homogeneity of viral protein preparaiions. IJlt,imately Lhe choice of method or methods will probably depend on convenience and degree of accuracy sought. ACKSOWLEDGMENT The authors are indebted to Larry Seiple for his excellent technical assistance in the amino acid analyses. REFERENCES
AINSWORTIX,6. C. (1935). Mosaic
diseases of the cucumber. Ann. Appl. Biol. 22, 55-67. BAWDEN, F. C., and PIEIE, N. W. (1937). The relationship between liquid crystalline preparations of cucumber viruses 3 and 4 and st’rains of tobacco mosaic virus. Brit. J. Exp. Pathol. 28, 275-291. BeE& J., ULRYCHOV~, M., and (?ECH, M. (1962). Infection of tobacco and some Chenopodium species by the CV4 (and 3) and by its nucleic acid. viroloyv 16, 105-114. DTJSKER, A. K., and RUECKERT, R. R. (1969). Observations on molecular weight determinations on polyacrylamide gel. J. Biol. Chem. 244 ) 5074-5080. FISH, W. W., MANN-, K. G., and TARTFORD, C. (1969). The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol.Chem. 244, 49894994. FIXENHEL-CoNRST, H. (1957). Degradation of tobacco mosaic virus with acetic acid. XTirology 4,14. FUN.~TSU, G., and FcNaTSu, M. (1968). Chemical studies on proteins from two tobacco mosaic virus strains. Proceedings of the 1967 InterIlational Symposium on Biochemical Regulation in Piseased Plants or Injury, Tokyo, Japan, pp* l-9.
OF CV3 AND
CV4
381
HIRS, C. H. W. (1967). Determination of cysti~le as cysteic acid. Methods Enzymol. 3il, 59-62. IXOZYE, T., INOUYE, N., AMTANI, M., and, MITSUHATA, K. (1967). Studies on cucumber green mottle mosaic virus in Japan. Xogni-u Kenkyu 53, 175-i86. KADO, C. I., and KNIGIIT, C. A. (197h)). Hcsr, specificity of plant viruses. I. Cucumber virus 4. virozogy 40, 997-1007. KNIGHT, C. A. (1955). Are cucumber viruses 3 and 4 strains of tobacco mosaic virus? Vi~olr~n?/ I;, 261-267. KNIGHT, 6. A. (1963). Chemistry of viruses. Protoplasmntologic: KNIGHT, C. A.,
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and STAX~EY, W. M. (1941). Preparation and properties of cucumber virus 4. J. Biol. Chem. 141, 2938. MOORE, S., and STEIN, W. H. (1963). Chromategraphic determination of amino acids by the use of automatic recording equipment. ‘Meihods Enzymol. 6, 819-831. Nozu, Y., TOCHIH~UU, H., KOMURO, I”.; and OK~DA, Y. (1971). Chemical and immunological CharacTerization of cucumber green mettle mosaic virus (watermelon strain) protein. Virology 45, 577-585. REVNOLDS, 3. A., and T~XFOR~, C. (1970). The gross conformation of protein sodium dodecyi sulfate complexes. J. Biol. Chenz. 245, X61-5165. SPIES, j. R., and CH~~MBERS, D. C. (1949). Chemi-. cal determination of tryptophan in proteins. Anal. Chem. 21, i249-1266. TANFORD, C. (1968). Prot’ein deaaturation. A&an. Protein Chem. 23, 121-282. TSUGITA, 9. (1962). The proteins of murants of TMV: Classification of spontaneous and chemically evoked strains. J. UoZ. Biol. 5, 293-300. TUNG, J.-S., and KNIGET, 6. A. (1971). Effect of charge on the determination of molecular weight of proteins by gel electrophoresis in 9DS. Biochem. Biophys. Res. Cbmmun. 42, 1117-1121. VAN REGENMORTEL, M. H. V. (1967). Sero1ogica.I studies on naturally occurring strains and chemically induced mutants of TMV. virology 31,467480. WEBER, K., and OSBORX, M. (1969). The reliability of molecular weight determination by Sl%polyacrylamide gel electrophoresis. J. Riol. Chenz. 244, 44064412. WITTMANX, N. G., and
WITTMANX-LIEBGD,
B.
(1966). Protein chemical studies oi two RN.4 viruses and their mutants. iTold Spriny Igarbor Symp. Qua~zt. Biol. 31, 163-172. WOODY, B. R., and K;NIGNT, C. A. (1959). Peptide maps obtained with tryplic digests ol the proteins of some strains of tobacco mosaic virus. Virology 9, 359374.