VIROLOGY 66,336-340
Amino
(1975)
Acid Analysis
of Alfalfa
Mosaic
An Aid for Viral Strain
Virus Coat Proteins:
Identification
B. KRAAL’ Department
of Biochemistry,
State University, Accepted
Wassenaarseweg
March
64, Leiden,
The Netherlands
18, 1975
Amino acid analysis of the coat proteins from different strains of alfalfa mosaic virus (AMV) can be used as a tool for strain characterization. This method together with carboxy-terminal amino acid analysis of 11 different AMV strains allowed three groups of closely related AMV coat proteins to be distinguished. A rough estimation of the evolutionary relationships among these coat proteins is presented. New chemical evidence is provided in support of previous genetic experiments that have localized the coat protein cistron on the Tb-component RNA.
Quite a number of different strains of alfalfa mosaic virus (AMV) have been described on the basis of various properties such as symptomatology, host range, serology and component ratio of virus preparations (1, 2). This variability among AMV strains may be partially explained (3,4) by the multipartite genome (5) of this multicomponent virus, which facilitates exchange of genetic material. As the coat protein is the only viral gene product which is easily obtained, strains with different coat proteins have been very useful for genetic work (5, 6). Besides serological tests, more knowledge about the primary structure of coat protein molecules (7, 8) undoubtedly facilitates characterization of the various AMV strains and hybrids, as will be demonstrated by this communication. The following strains and hybrids were analyzed: AMV isolate 425 of Hagedorn and Hanson (9), yellow spot mosaic virus (YSMV) isolated by Zaumeyer (IO), isolates 15/64 and VRU from Hull (I, II), isolate Strasbourg (S) from Hirth (I2), and isolate AA-l from Iizuka and Iida (13). The hybrid strain B,M,Tb,,, was obtained by ’ Present address: Department of Biochemistry, Imperial College of Science and Technology, University of London, London, SW7 2AZ, U.K. 336 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
combining bottom component (B) and middle component (M) of YSMV with top component b (Tb) of 425. In the same way, hybrid B,,,M,,,Tb, was constructed from B and M components of AMV 425 and Tb component of YSMV (5). Tb-tsl was isolated as a thermosensitive mutant of 425, the mutation being localized in the Tbcomponent RNA (Van Vloten-Doting et al., to be published). Amino acid data on AMV strains A and P were obtained from Tremaine and State-Smith (14). All the virus strains used were isolated from tobacco and further purified by sucrose density gradient centrifugation as described before (15). Coat protein solutions for amino acid analysis were obtained by precipitating the RNA with 0.5 M MgCl, (16). All the protein preparations were checked for homogeneity by sodium dodecyl sulfate - polyacrylamide-gel electrophoresis (17), and they all displayed the same electrophoretic mobility, which corresponded to a molecular weight of about 25,000 (16). Amino acid analyses of 24-hr hydrolysates of the proteins were performed as described before (7). Carboxy-terminal amino acid determinations were carried out as follows. For each analysis, 2 mg of AMV (corresponding to about 65 nmoles of coat protein) were
SHORT
COMMUNICATIONS
used in 0.375 ml of 10 mM NaH,PO,, 1 mM disodium ethylenediaminetetraacetate and 1 mM NaN,, pH 7.0, at 0”. After the addition of 0.040 ml of 5% (w/v) sodium dodecyl sulfate and 0.025 ml of 20 mM ZnSO,, a non-opalescent, clear solution was obtained. To this solution was added 0.050 ml of a buffer solution of 1 M NaCl and 1 M NaHCO, at pH 8.5 , in which 2.5 units of carboxypeptidase A had been dissolved. Subsequently, 0.010 ml (2.5 U) of carboxypeptidase B solution in 0.1 A4 NaCl was also added. Both carboxypeptidase preparations (DFP-treated) were purchased from Sigma Chemical Co. (St. Louis, MO). The complete reaction mixture was transferred from 0 to 37” and, after 1-hr incubation at 37”, the reaction was stopped by adding 0.500 ml of 14% (w/v) trichloroacetic acid, 0.2 A4 sodium acetate. The macromolecular precipitate was removed by centrifugation and the supernatant fluid was directly applied to an amino acid analyzer for the determination of released amino acids. Blank experiments with either virus or carboxypeptidases alone led to only minor corrections. The results of the amino acid analyses and end-group determinations of the coat proteins from the various AMV strains are presented in Table 1. All the amino acid compositions are similar. Nevertheless, some subgroups having more closely related compositions can be discerned. This subdivision is accentuated by the results of the carboxypeptidase A and B degradations. As was found for 425 protein (7), coat proteins from B,M,Tb,,, and Tb-tsl also have a carboxy-terminal - Arg - His sequence. The amino acid compositions of 425 and B,M,Tb,,,coat proteins are identical. The only difference between 425 and Tb-tsl proteins is the replacement of one Ala and one His by one Thr and one Tyr, which may be caused by single pointmutations in the respective RNA triplets. As was found for 15/64 (18), VRU coat protein also has a carboxy-terminal - Ser Arg sequence. In the case of YSMV, B,25M,25Tby, AA-l and S coat protein, no released carboxy-terminal amino acids could be detected. No reports of end-group
337
determinations were available for P and A proteins. The amino acid compositions of YSMV and B,,,M,,,Tb, proteins are identical. This finding and the above-mentioned identity of B,M,Tb,,, and 425 coat proteins provide new evidence in support of previous genetic experiments that have localized the coat protein cistron on the Tb-component RNA (5). A similar experimental approach was used by others (19) for the comparable allocation of the coat protein cistron of tobacco rattle virus to its short-particle RNA. Table 2 shows the numbers of amino acid differences in the overall compositions of coat proteins from different AMV strains. As a matter of course, these overall differences do not exactly reflect all the genetic differences. Even considering amino acid sequences, rather profound differences may be caused by just a slight alteration of the genetic information such as a frameshift or a nonsense mutation (21). Nevertheless, one could make a rough estimation of the degree of relationship between the coat proteins by merely comparing the overall amino acid compositions (20). Using the data from Table 2 a phylogenetic tree may be tentatively designed (Fig. l), as has been thoroughly calculated for coat proteins of different strains of tobacco mosaic virus (21). Three subgroups of viral coat proteins with closely related compositions and similar endgroups can be discerned, namely YSMV, P, A, S, AA-l; 425, Tb-tsl; and VRU, 15/64. Serological characterization partially supports this subdivision. A double diffusion test with antiserum against 425 revealed distinct spur formation with YSMV and S proteins (5, 22). However, in similar tests 425 and 15164or VRU proteins were serologically indistinguishable (1 I). Further support for the classification presented in Table 2 is given by the strainspecific (or coat-specific) aggregation behaviour of AMV particles in vivo as seen with the electron microscope by Hull et al. (2, 4). In this way, 15/64, VRU and a group with YSMV were each classified in different categories. Compared to all the AMV proteins stud-
(23) (12) (18) (19) (16) (16) (23) .(3) (12) (3) (5) (23) (4) (17) (2) (15) (6) (11)
Arg 1.0 Ser 0.8
227.7 (228)
22.8 12.4 17.6 19.4 16.2 15.7 23.0 2.9 12.0 3.0 5.1 22.7 3.7 17.2 1.7 15.2 6.2 10.9
15164
-
(20) (13) (15) (19) (16) (17) (21) (3) (12) (3) (5) (22) (5) (16) (2) (14) (6) (11)
-
20.0 13.3 15.3 19.0 15.8 17.1 21.2 2.9 12.1 3.0 5.0 22.2 4.9 15.8 1.6 13.8 5.8 10.7
Arg 1.0 Ser 0.9
(20) (13) (15) (19) (16) (17) (21) (3) (12) (3) (5) (22) (5) (16) (2) (14) (6) (11) 219.5 (220)
20.1 13.1 15.1 18.9 16.3 16.8 21.3 2.7 11.8 3.0 5.0 22.2 4.9 15.8 1.8 14.0 6.1 10.8 219.7 (220)
(22) (12) (18) (19) (16) (16) (24) (3) (11) (3) (5) (23) (3) (17) (2) (14j (7) (12)
YSMV (20) (13) (15) (20) (17) (18) (20) (3) (14) (3) (5) (22) (5) (16) (2) (14) (6) (10)
-
222.9 (223)
20.0 13.0 15.0 20.1 16.6 17.7 20.1 3.2 13.6 3.0 5.0 22.1 5.0 16.2 2.0 14.0 6.0 10.3
AA-1 (20) (13) (15) (20) (17) (17) (21) (3) (13) (3) (5) (23) (5) (16) (2) (14) (6) (10)
-
223.1 (223)
19.8 13.1 15.1 20.2 16.9 17.4 21.1 3.1 12.7 3.1 5.1 22.6 5.1 16.1 2.0 13.9 5.8 10.0
S
GROUP@ OF COAT PROTEINS
226.8 (227)
22.1 12.2 18.0 19.0 16.3 16.0 24.1 2.9 10.6 3.0 5.1 23.1 2.8 16.8 1.8 13.9 7.1 12.0
VRU
AMV strain
TABLE 1 ACID COMPOSITIONS~ AND CARBOXY-TERMINAL FROM 11 DIFFERENT AMV STRAINS
n.d.’
220.1 (221)
(20) (13) (16) (20) (18) (17) (21) (3) (12) (3) (5) (21) (5) (15) (2) (14) (6) (10)
n.d.’
220.3 (221)
20.0 12.7 15.8 20.2 17.9 17.3 21.3 3.0 12.3 2.6 5.0 20.8 5.0 15.3 1.3 13.8‘ 6.1 9.9
19.9 13.1 15.3 20.1 17.9 17.2 21.3 2.4 11.8 2.7 5.0 21.3 5.0 15.6 1.4 13.9 6.1 10.1
(20) (13) (15) (20) (18) (17) (21) (3) (12) (3) (5) (21) (5) (16) (2) (14) (6) (10)
A
P
n The amino acid composition of each type of coat protein, given in residues per molecule, is the average result of four-analyses. The composition of coat protein 425 was published earlier (7) as the result of about 30 analyses. The pattern of amino acid peaks from this protein could therefore conveniently be taken as the calibration standard instead of the usual equimolar aminoacid calibration mixture for calculating the amino acid peaks of the related coat protein hydrolysates. Cysteine and tryptophan contents were each determined in separate analyses (7). Values are calculated as residues per molecule (with the nearest integers in parentheses) according to a molecular weight of about 25,000. The amino acid data for strains A and P were taken from the literature (14). ’ Carboxy-terminal degradation was obtained by incubation with carboxypeptidases A and B. Values below 0.1 amino acid residue per molecule were omitted. For further experimental details see text. L Cysteine was determined as cystejc acid after acid hydrolysis in the presence of 0.21 M dimethylsulphoxide. d Tryptophan was protected during acid hydrolysis by the presence of 4% (v/v) thioglycoiic acid. (’ n.d., not determined.
His 1.0 Arg 0.7
His 1.0 Arg 0.8
His 1.0 Arg 0.8
(21) (14) (15) (20) (17) (17) (19) (3) (13) (3) (5) (21) (5) (18) (2) (14) (6) (11)
223.7 (224)
20.9 14.0 15.3 20.0 16.6 16.7 19.1 2.8 13.0 3.1 5.3 21.2 5.0 18.0 1.7 14.0 6.0 11.0
224.2 (224)
20.6 (21) 12.9 (13) 14.9 (15) 20.3 (20) 17.0 (17) 17.2 (17) 20.3 (20) 3.1 (3) 13.0 (13) 3.3 (3) 5.2 (5) 21.2 (21) 3.8 (4) 17.9 (18) 1.8 (2) 14.0 (14) 6.9 (7) 10.8 (11)
Tb-tsl
223.9 (224)
(21) (13) (15) (20) (17) (17) (20) (3) (13) (3) (5) (21) (4) (18) (2) (14) (7) (11)
B,M,Tb.,,
Total number of residues Carboxy-terminal degradation*
425
21.0 13.0 14.9 20.1 17.1 17.0 20.2 3.1 13.1 2.9 4.9 21.0 3.9 17.9 1.8 14.0 7.0 11.0
r
BETWEEN THE AMINO
Aspartic Acid Threonine Swine Glutamic acid Pmline Glycine Alanine Cysteine’ Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophand Lysine Histidine Arginine
Amino acid
COMPARISON
339
SHORT COMMUNICATIONS TABLE
2
NUMBERS OF AMINO ACID DIFFERENCESIN THE OVERALL COMPOSITIONS~OF COAT PROTEINS FROM DIFFERENT AMV STRAINS
AMV strain (chain length, residues/molecule)
I {
YSMV’ (220) P (221) A (221) S (223) AA-l (223) 425b (224) Tb-tsl (224) VRU (227) 15/64 (228)
YSMV” (2201
P
A
S
(221)
(221)
(223)
AA-l
425”
(223)
(224)
Tb-tsl (224)
VRU
15/64
(227)
(228)
5 7 5 7 10 10 17 14
2 4 6 9 9 22 19
6 8 11 11 22 19
4 9 9 20 17
9 9 24 21
4 19 18
23 20
7
-
aThe amino acid compositions used are listed in Table 1. bThe compositions of B,,,M,,,Tb, and B,M,Tb,,, proteins are identical with those of YSMV and 425, respectively, and consequently they are omitted from this table. Subgroups with closely related compositions and similar end-groups are bracketed.
tion as listed in Table 2 may be localized in the carboxy-terminal region of the protein chain and are partially due to differences in chain length. This probable carboxy-terminal variability is indicated by the results of carboxypeptidase A and B degradation and by preliminary studies on the coat protein of YSMV (23). Comparing the amino acid compositions of the corresponding CNBr cleavage products of YSMV and 425 proteins, eight amino acid changes FIG. 1. Hypothetical evolutionary relationships be- were revealed in the carboxy-terminal tween the coat proteins of different AMV strains. CNBr fragment consisting of about 65 The measured distances along the lines drawn from residues. In contrast, the two internal one strain to another are roughly proportional to the CNBr fragments containing the preceding numbers of amino acid changes in the protein chains, 60 residues were essentially the same in as listed in Table 2. Subgroups with closely related both cases. Finally, in the amino-terminal amino acid compositions and similar end-groups are CNBr fragment of about 100 residues there indicated by dashed contours. were only four amino acid changes. As a common feature, the blocked amino-termiied, 15164 and VRU coat proteins both nal region (about 25 residues) of this fraghave the most deviating amino acid com- ment contains a lot of basic amino acids positions. Especially the protein subunits clustered together. As was similarly found of VRU and, to some extent, those of 15/64 for 425, YSMV, S, 15/64, and VRU, this have a tendency to form very long tubular region could be specifically removed by virus particles in addition to the common mild tryptic treatment of intact virus parmixture of shorter components observed in ticles (I 7). preparations of 425 or YSMV. Therefore, it In conclusion, amino acid analysis and would be of interest to compare the pri- especially carboxy-terminal amino acid mary structure of VRU coat protein with analysis should be preferred to serological the well-known structure of 425 protein (8) tests as tools for the characterization of to obtain more data concerning their differ- different AMV strains. As the differences ent functional behaviour in architecture. between total amino acid compositions are The differences in amino acid composi- rather small, reliable results can only be
340
SHORT COMMUNICATIONS
L., Eur. J. Biochem. 28, 20-29 (1972). obtained from a series of analyses in order 8. VAN BEYNUM,G. M. A., KRAAL, B., DE GRAAF,J. to eliminate random instrumental errors. M., and BOSCH,L., Eur. J. Biochem., 52, 231In contrast, carboxy-terminal analysis can 238 (1975). present a much quicker and simpler way of 9. HAGEDORN, D. J., and HANSON,E. W., Phytopadetecting differences between subgroups of thology 53, 188-192 (1963). AMV strains, as the all-or-none response is 10. ZAUMEYER,W. J. Phytopathology 53, 444-449 also satisfactory in a qualitative assay. (1963). Depending on the sensitivity of detection of 11. HULL, R., Virology 42, 283-292 (1970). G., FRAENKEL-CONRAT, H., WURTZ,M., 12. LEBEURIER, released amino acids, even submilligram and HIRTH, L., Virology 43, 51-61 (1971). amounts of viral material may be easily 13. IIZUKA, N., and IIDA, W., Bull. Tohoku Nat. Agr. analyzed within one working day. ACKNOWLEDGMENTS I wish to thank Dr. L. van Vloten-Doting and Dr. A. M. Dingjan-Versteegh for the kind gifts of purified virus preparations, Dr. E. M. J. Jaspars for valuable discussions and Dr. L. Bosch for his continuous interest.
Exp. Sta. 37, 43-122 (1969). 14. TREMAINE,J. H., and STACE-SMITH,R., Phytopathology 59, 521-522 (1969). E. M. J., 15. VAN VLOTEN-DOTING,L., and JASPAF~S, Virology 48, 699-708 (1972).
16. KRUSEMAN,J., KRAAL, B., JASPARS,E. M. J., BOL, J. F., BREDERODE,F. TH., and VELDSTRA,H., Biochemistry 10, 447-455 (1971). F. TH., 17. B~L, J. F., KRAAL, B., and BREDERODE, REFERENCES Virology 58, 101-110 (1974). 18, HULL, R., REES, M. W., and SHORT, M. N., 1. HULL, R., Aduun. Virus Res. 15, 365-433 (1969). 2. HULL, R., HILLS, G. J., and PLASKITT,A., Virology Virology 37, 404-415 (1969). 42,153-712 (1970). 19. GHABRIAL,S. A., and LISTER,R. M., Virology 52, 3. VAN VLOTEN-DOTING,L., Ph.D. thesis, University l-12 (1973). of Leiden, 1968. J. J.. and WELTMAN.J. K.. Camp. 20. MARCHALONIS. 4. HULL, R., and PLASKI’IT, A., Virology 42, 773-776 Biochem. khysioi. 38B, 609-625’(1971j. (1970). M. 0. (Ed.), “Atlas of Protein Sequence 21. DAYHOFF, 5. DINGJAN-VERSTEECH, A. M., VAN VLOTEN-DOTING, and Structure,” Vol. 5, D-283. National BioL., and JASPARS,E. M. J., Virology 49.716-722 medical Research Foundation, Silver Spring, (1972). MD., 1972. 6. DINGJAN-VERSTEEGH, A. M., VAN VLOTEN-DOTING, 22. BOL, J. F., and VAN VLOTEN-DOTING,L., Virology L., and JASPARS,E. M. J., Virology 59,328-330 51, 102-108 (1973). (1974). 23. KRAAL, B., Ph.D. thesis, University of Leiden, 7. KRAAL, B., DE GRAAF,J. M., BAKKER,T. A., VAN 1972. BEYNUM,G. M. A., GOEDHART,M., and BOSCH,
Amino
(1975)
Acid Analysis
of Alfalfa
Mosaic
An Aid for Viral Strain
Virus Coat Proteins:
Identification
B. KRAAL’ Department
of Biochemistry,
State University, Accepted
Wassenaarseweg
March
64, Leiden,
The Netherlands
18, 1975
Amino acid analysis of the coat proteins from different strains of alfalfa mosaic virus (AMV) can be used as a tool for strain characterization. This method together with carboxy-terminal amino acid analysis of 11 different AMV strains allowed three groups of closely related AMV coat proteins to be distinguished. A rough estimation of the evolutionary relationships among these coat proteins is presented. New chemical evidence is provided in support of previous genetic experiments that have localized the coat protein cistron on the Tb-component RNA.
Quite a number of different strains of alfalfa mosaic virus (AMV) have been described on the basis of various properties such as symptomatology, host range, serology and component ratio of virus preparations (1, 2). This variability among AMV strains may be partially explained (3,4) by the multipartite genome (5) of this multicomponent virus, which facilitates exchange of genetic material. As the coat protein is the only viral gene product which is easily obtained, strains with different coat proteins have been very useful for genetic work (5, 6). Besides serological tests, more knowledge about the primary structure of coat protein molecules (7, 8) undoubtedly facilitates characterization of the various AMV strains and hybrids, as will be demonstrated by this communication. The following strains and hybrids were analyzed: AMV isolate 425 of Hagedorn and Hanson (9), yellow spot mosaic virus (YSMV) isolated by Zaumeyer (IO), isolates 15/64 and VRU from Hull (I, II), isolate Strasbourg (S) from Hirth (I2), and isolate AA-l from Iizuka and Iida (13). The hybrid strain B,M,Tb,,, was obtained by ’ Present address: Department of Biochemistry, Imperial College of Science and Technology, University of London, London, SW7 2AZ, U.K. 336 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
combining bottom component (B) and middle component (M) of YSMV with top component b (Tb) of 425. In the same way, hybrid B,,,M,,,Tb, was constructed from B and M components of AMV 425 and Tb component of YSMV (5). Tb-tsl was isolated as a thermosensitive mutant of 425, the mutation being localized in the Tbcomponent RNA (Van Vloten-Doting et al., to be published). Amino acid data on AMV strains A and P were obtained from Tremaine and State-Smith (14). All the virus strains used were isolated from tobacco and further purified by sucrose density gradient centrifugation as described before (15). Coat protein solutions for amino acid analysis were obtained by precipitating the RNA with 0.5 M MgCl, (16). All the protein preparations were checked for homogeneity by sodium dodecyl sulfate - polyacrylamide-gel electrophoresis (17), and they all displayed the same electrophoretic mobility, which corresponded to a molecular weight of about 25,000 (16). Amino acid analyses of 24-hr hydrolysates of the proteins were performed as described before (7). Carboxy-terminal amino acid determinations were carried out as follows. For each analysis, 2 mg of AMV (corresponding to about 65 nmoles of coat protein) were
SHORT
COMMUNICATIONS
used in 0.375 ml of 10 mM NaH,PO,, 1 mM disodium ethylenediaminetetraacetate and 1 mM NaN,, pH 7.0, at 0”. After the addition of 0.040 ml of 5% (w/v) sodium dodecyl sulfate and 0.025 ml of 20 mM ZnSO,, a non-opalescent, clear solution was obtained. To this solution was added 0.050 ml of a buffer solution of 1 M NaCl and 1 M NaHCO, at pH 8.5 , in which 2.5 units of carboxypeptidase A had been dissolved. Subsequently, 0.010 ml (2.5 U) of carboxypeptidase B solution in 0.1 A4 NaCl was also added. Both carboxypeptidase preparations (DFP-treated) were purchased from Sigma Chemical Co. (St. Louis, MO). The complete reaction mixture was transferred from 0 to 37” and, after 1-hr incubation at 37”, the reaction was stopped by adding 0.500 ml of 14% (w/v) trichloroacetic acid, 0.2 A4 sodium acetate. The macromolecular precipitate was removed by centrifugation and the supernatant fluid was directly applied to an amino acid analyzer for the determination of released amino acids. Blank experiments with either virus or carboxypeptidases alone led to only minor corrections. The results of the amino acid analyses and end-group determinations of the coat proteins from the various AMV strains are presented in Table 1. All the amino acid compositions are similar. Nevertheless, some subgroups having more closely related compositions can be discerned. This subdivision is accentuated by the results of the carboxypeptidase A and B degradations. As was found for 425 protein (7), coat proteins from B,M,Tb,,, and Tb-tsl also have a carboxy-terminal - Arg - His sequence. The amino acid compositions of 425 and B,M,Tb,,,coat proteins are identical. The only difference between 425 and Tb-tsl proteins is the replacement of one Ala and one His by one Thr and one Tyr, which may be caused by single pointmutations in the respective RNA triplets. As was found for 15/64 (18), VRU coat protein also has a carboxy-terminal - Ser Arg sequence. In the case of YSMV, B,25M,25Tby, AA-l and S coat protein, no released carboxy-terminal amino acids could be detected. No reports of end-group
337
determinations were available for P and A proteins. The amino acid compositions of YSMV and B,,,M,,,Tb, proteins are identical. This finding and the above-mentioned identity of B,M,Tb,,, and 425 coat proteins provide new evidence in support of previous genetic experiments that have localized the coat protein cistron on the Tb-component RNA (5). A similar experimental approach was used by others (19) for the comparable allocation of the coat protein cistron of tobacco rattle virus to its short-particle RNA. Table 2 shows the numbers of amino acid differences in the overall compositions of coat proteins from different AMV strains. As a matter of course, these overall differences do not exactly reflect all the genetic differences. Even considering amino acid sequences, rather profound differences may be caused by just a slight alteration of the genetic information such as a frameshift or a nonsense mutation (21). Nevertheless, one could make a rough estimation of the degree of relationship between the coat proteins by merely comparing the overall amino acid compositions (20). Using the data from Table 2 a phylogenetic tree may be tentatively designed (Fig. l), as has been thoroughly calculated for coat proteins of different strains of tobacco mosaic virus (21). Three subgroups of viral coat proteins with closely related compositions and similar endgroups can be discerned, namely YSMV, P, A, S, AA-l; 425, Tb-tsl; and VRU, 15/64. Serological characterization partially supports this subdivision. A double diffusion test with antiserum against 425 revealed distinct spur formation with YSMV and S proteins (5, 22). However, in similar tests 425 and 15164or VRU proteins were serologically indistinguishable (1 I). Further support for the classification presented in Table 2 is given by the strainspecific (or coat-specific) aggregation behaviour of AMV particles in vivo as seen with the electron microscope by Hull et al. (2, 4). In this way, 15/64, VRU and a group with YSMV were each classified in different categories. Compared to all the AMV proteins stud-
(23) (12) (18) (19) (16) (16) (23) .(3) (12) (3) (5) (23) (4) (17) (2) (15) (6) (11)
Arg 1.0 Ser 0.8
227.7 (228)
22.8 12.4 17.6 19.4 16.2 15.7 23.0 2.9 12.0 3.0 5.1 22.7 3.7 17.2 1.7 15.2 6.2 10.9
15164
-
(20) (13) (15) (19) (16) (17) (21) (3) (12) (3) (5) (22) (5) (16) (2) (14) (6) (11)
-
20.0 13.3 15.3 19.0 15.8 17.1 21.2 2.9 12.1 3.0 5.0 22.2 4.9 15.8 1.6 13.8 5.8 10.7
Arg 1.0 Ser 0.9
(20) (13) (15) (19) (16) (17) (21) (3) (12) (3) (5) (22) (5) (16) (2) (14) (6) (11) 219.5 (220)
20.1 13.1 15.1 18.9 16.3 16.8 21.3 2.7 11.8 3.0 5.0 22.2 4.9 15.8 1.8 14.0 6.1 10.8 219.7 (220)
(22) (12) (18) (19) (16) (16) (24) (3) (11) (3) (5) (23) (3) (17) (2) (14j (7) (12)
YSMV (20) (13) (15) (20) (17) (18) (20) (3) (14) (3) (5) (22) (5) (16) (2) (14) (6) (10)
-
222.9 (223)
20.0 13.0 15.0 20.1 16.6 17.7 20.1 3.2 13.6 3.0 5.0 22.1 5.0 16.2 2.0 14.0 6.0 10.3
AA-1 (20) (13) (15) (20) (17) (17) (21) (3) (13) (3) (5) (23) (5) (16) (2) (14) (6) (10)
-
223.1 (223)
19.8 13.1 15.1 20.2 16.9 17.4 21.1 3.1 12.7 3.1 5.1 22.6 5.1 16.1 2.0 13.9 5.8 10.0
S
GROUP@ OF COAT PROTEINS
226.8 (227)
22.1 12.2 18.0 19.0 16.3 16.0 24.1 2.9 10.6 3.0 5.1 23.1 2.8 16.8 1.8 13.9 7.1 12.0
VRU
AMV strain
TABLE 1 ACID COMPOSITIONS~ AND CARBOXY-TERMINAL FROM 11 DIFFERENT AMV STRAINS
n.d.’
220.1 (221)
(20) (13) (16) (20) (18) (17) (21) (3) (12) (3) (5) (21) (5) (15) (2) (14) (6) (10)
n.d.’
220.3 (221)
20.0 12.7 15.8 20.2 17.9 17.3 21.3 3.0 12.3 2.6 5.0 20.8 5.0 15.3 1.3 13.8‘ 6.1 9.9
19.9 13.1 15.3 20.1 17.9 17.2 21.3 2.4 11.8 2.7 5.0 21.3 5.0 15.6 1.4 13.9 6.1 10.1
(20) (13) (15) (20) (18) (17) (21) (3) (12) (3) (5) (21) (5) (16) (2) (14) (6) (10)
A
P
n The amino acid composition of each type of coat protein, given in residues per molecule, is the average result of four-analyses. The composition of coat protein 425 was published earlier (7) as the result of about 30 analyses. The pattern of amino acid peaks from this protein could therefore conveniently be taken as the calibration standard instead of the usual equimolar aminoacid calibration mixture for calculating the amino acid peaks of the related coat protein hydrolysates. Cysteine and tryptophan contents were each determined in separate analyses (7). Values are calculated as residues per molecule (with the nearest integers in parentheses) according to a molecular weight of about 25,000. The amino acid data for strains A and P were taken from the literature (14). ’ Carboxy-terminal degradation was obtained by incubation with carboxypeptidases A and B. Values below 0.1 amino acid residue per molecule were omitted. For further experimental details see text. L Cysteine was determined as cystejc acid after acid hydrolysis in the presence of 0.21 M dimethylsulphoxide. d Tryptophan was protected during acid hydrolysis by the presence of 4% (v/v) thioglycoiic acid. (’ n.d., not determined.
His 1.0 Arg 0.7
His 1.0 Arg 0.8
His 1.0 Arg 0.8
(21) (14) (15) (20) (17) (17) (19) (3) (13) (3) (5) (21) (5) (18) (2) (14) (6) (11)
223.7 (224)
20.9 14.0 15.3 20.0 16.6 16.7 19.1 2.8 13.0 3.1 5.3 21.2 5.0 18.0 1.7 14.0 6.0 11.0
224.2 (224)
20.6 (21) 12.9 (13) 14.9 (15) 20.3 (20) 17.0 (17) 17.2 (17) 20.3 (20) 3.1 (3) 13.0 (13) 3.3 (3) 5.2 (5) 21.2 (21) 3.8 (4) 17.9 (18) 1.8 (2) 14.0 (14) 6.9 (7) 10.8 (11)
Tb-tsl
223.9 (224)
(21) (13) (15) (20) (17) (17) (20) (3) (13) (3) (5) (21) (4) (18) (2) (14) (7) (11)
B,M,Tb.,,
Total number of residues Carboxy-terminal degradation*
425
21.0 13.0 14.9 20.1 17.1 17.0 20.2 3.1 13.1 2.9 4.9 21.0 3.9 17.9 1.8 14.0 7.0 11.0
r
BETWEEN THE AMINO
Aspartic Acid Threonine Swine Glutamic acid Pmline Glycine Alanine Cysteine’ Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophand Lysine Histidine Arginine
Amino acid
COMPARISON
339
SHORT COMMUNICATIONS TABLE
2
NUMBERS OF AMINO ACID DIFFERENCESIN THE OVERALL COMPOSITIONS~OF COAT PROTEINS FROM DIFFERENT AMV STRAINS
AMV strain (chain length, residues/molecule)
I {
YSMV’ (220) P (221) A (221) S (223) AA-l (223) 425b (224) Tb-tsl (224) VRU (227) 15/64 (228)
YSMV” (2201
P
A
S
(221)
(221)
(223)
AA-l
425”
(223)
(224)
Tb-tsl (224)
VRU
15/64
(227)
(228)
5 7 5 7 10 10 17 14
2 4 6 9 9 22 19
6 8 11 11 22 19
4 9 9 20 17
9 9 24 21
4 19 18
23 20
7
-
aThe amino acid compositions used are listed in Table 1. bThe compositions of B,,,M,,,Tb, and B,M,Tb,,, proteins are identical with those of YSMV and 425, respectively, and consequently they are omitted from this table. Subgroups with closely related compositions and similar end-groups are bracketed.
tion as listed in Table 2 may be localized in the carboxy-terminal region of the protein chain and are partially due to differences in chain length. This probable carboxy-terminal variability is indicated by the results of carboxypeptidase A and B degradation and by preliminary studies on the coat protein of YSMV (23). Comparing the amino acid compositions of the corresponding CNBr cleavage products of YSMV and 425 proteins, eight amino acid changes FIG. 1. Hypothetical evolutionary relationships be- were revealed in the carboxy-terminal tween the coat proteins of different AMV strains. CNBr fragment consisting of about 65 The measured distances along the lines drawn from residues. In contrast, the two internal one strain to another are roughly proportional to the CNBr fragments containing the preceding numbers of amino acid changes in the protein chains, 60 residues were essentially the same in as listed in Table 2. Subgroups with closely related both cases. Finally, in the amino-terminal amino acid compositions and similar end-groups are CNBr fragment of about 100 residues there indicated by dashed contours. were only four amino acid changes. As a common feature, the blocked amino-termiied, 15164 and VRU coat proteins both nal region (about 25 residues) of this fraghave the most deviating amino acid com- ment contains a lot of basic amino acids positions. Especially the protein subunits clustered together. As was similarly found of VRU and, to some extent, those of 15/64 for 425, YSMV, S, 15/64, and VRU, this have a tendency to form very long tubular region could be specifically removed by virus particles in addition to the common mild tryptic treatment of intact virus parmixture of shorter components observed in ticles (I 7). preparations of 425 or YSMV. Therefore, it In conclusion, amino acid analysis and would be of interest to compare the pri- especially carboxy-terminal amino acid mary structure of VRU coat protein with analysis should be preferred to serological the well-known structure of 425 protein (8) tests as tools for the characterization of to obtain more data concerning their differ- different AMV strains. As the differences ent functional behaviour in architecture. between total amino acid compositions are The differences in amino acid composi- rather small, reliable results can only be
340
SHORT COMMUNICATIONS
L., Eur. J. Biochem. 28, 20-29 (1972). obtained from a series of analyses in order 8. VAN BEYNUM,G. M. A., KRAAL, B., DE GRAAF,J. to eliminate random instrumental errors. M., and BOSCH,L., Eur. J. Biochem., 52, 231In contrast, carboxy-terminal analysis can 238 (1975). present a much quicker and simpler way of 9. HAGEDORN, D. J., and HANSON,E. W., Phytopadetecting differences between subgroups of thology 53, 188-192 (1963). AMV strains, as the all-or-none response is 10. ZAUMEYER,W. J. Phytopathology 53, 444-449 also satisfactory in a qualitative assay. (1963). Depending on the sensitivity of detection of 11. HULL, R., Virology 42, 283-292 (1970). G., FRAENKEL-CONRAT, H., WURTZ,M., 12. LEBEURIER, released amino acids, even submilligram and HIRTH, L., Virology 43, 51-61 (1971). amounts of viral material may be easily 13. IIZUKA, N., and IIDA, W., Bull. Tohoku Nat. Agr. analyzed within one working day. ACKNOWLEDGMENTS I wish to thank Dr. L. van Vloten-Doting and Dr. A. M. Dingjan-Versteegh for the kind gifts of purified virus preparations, Dr. E. M. J. Jaspars for valuable discussions and Dr. L. Bosch for his continuous interest.
Exp. Sta. 37, 43-122 (1969). 14. TREMAINE,J. H., and STACE-SMITH,R., Phytopathology 59, 521-522 (1969). E. M. J., 15. VAN VLOTEN-DOTING,L., and JASPAF~S, Virology 48, 699-708 (1972).
16. KRUSEMAN,J., KRAAL, B., JASPARS,E. M. J., BOL, J. F., BREDERODE,F. TH., and VELDSTRA,H., Biochemistry 10, 447-455 (1971). F. TH., 17. B~L, J. F., KRAAL, B., and BREDERODE, REFERENCES Virology 58, 101-110 (1974). 18, HULL, R., REES, M. W., and SHORT, M. N., 1. HULL, R., Aduun. Virus Res. 15, 365-433 (1969). 2. HULL, R., HILLS, G. J., and PLASKITT,A., Virology Virology 37, 404-415 (1969). 42,153-712 (1970). 19. GHABRIAL,S. A., and LISTER,R. M., Virology 52, 3. VAN VLOTEN-DOTING,L., Ph.D. thesis, University l-12 (1973). of Leiden, 1968. J. J.. and WELTMAN.J. K.. Camp. 20. MARCHALONIS. 4. HULL, R., and PLASKI’IT, A., Virology 42, 773-776 Biochem. khysioi. 38B, 609-625’(1971j. (1970). M. 0. (Ed.), “Atlas of Protein Sequence 21. DAYHOFF, 5. DINGJAN-VERSTEECH, A. M., VAN VLOTEN-DOTING, and Structure,” Vol. 5, D-283. National BioL., and JASPARS,E. M. J., Virology 49.716-722 medical Research Foundation, Silver Spring, (1972). MD., 1972. 6. DINGJAN-VERSTEEGH, A. M., VAN VLOTEN-DOTING, 22. BOL, J. F., and VAN VLOTEN-DOTING,L., Virology L., and JASPARS,E. M. J., Virology 59,328-330 51, 102-108 (1973). (1974). 23. KRAAL, B., Ph.D. thesis, University of Leiden, 7. KRAAL, B., DE GRAAF,J. M., BAKKER,T. A., VAN 1972. BEYNUM,G. M. A., GOEDHART,M., and BOSCH,
