VIROLOGY
69, 346-349
WX)
Stable Dimers of the Coat Protein of a Strain of Tobacco
Mosaic Virus
PER OXELFELT Department
ofPlant
Pathology
and Entomology, Accepted
Agricultural August
College
ofsweden,
S-750 07 Uppsala
7, Sweden
19,1975
A large proportion of the coat protein of the tobacco mosaic virus, strainflaoum, which accumulates in an insoluble form in infected tissue is present as a dimer. The dimer resists disaggregation by heating in the presence of sodium dodecyl sulfate, mercaptoethanol, and urea, and it cannot be completely dissociated by performic acid oxidation.
Characteristic changes in the synthesis of host proteins brought about by various strains and mutants of TMV (tobacco mosaic virus) have been reported (1, 2). In conjunction with the earlier study (1) that described the changes in soluble proteins, other subcellular fractions were also investigated. The present report describes the anomalous behavior of the coat protein of the flavum strain extracted from infected tissue. Three TMV strains were used in the study, uulgare, flauum and DT2. The latter strain, kindly supplied by Dr. S. Sarkar, Tiibingen, is a defective strain, which is derived from PM2 (3) and which differs in producing an insoluble defective coat protein. Flavum is known to produce excess coat protein that accumulates in an insoluble form (4) whereas excess uulgare coat protein accumulates in a soluble form (1). Samsun tobacco plants (Nicotiana tabacum L.) were grown and inoculated by standard procedures; in the case of DT2 the inoculum consisted of infected leaf tissue homogenized in cold pH 8.6 Tris-phosphate buffer with bentonite (5). The inoculated leaves were used 7-10 days after inoculation. Infected and comparable healthy leaf tissue was ground in the pH 8 extraction medium used earlier (1) with the modification that it also contained 0.01 M MgCl,. The homogenate was filtered through cheesecloth and centrifuged at 2500 g for 10 min. The pellet was resuspended in extraction medium to which 2% Triton X-100 was added, incubated in the
cold for 1 hr and then centrifuged at 2500g for 10 min. The pellet thereby obtained, here called the insoluble fraction, was prepared for analysis by the following alternative procedures: (a) It was resuspended in 0.01 M Tris, 0.0004 M EDTA, 0.1 M diethanoldisulfide, 0.01 M 2-mercaptoethanol, and 1% sodium dodecyl sulfate (SDS), pH 8, and incubated at 50” for 1 hr or in a boiling-water bath for 10 min; in most cases the resuspending medium also contained 8 M urea; (b) the pellet was resuspended in 0.01 M Tris, 0.0004 M EDTA, and 1% SDS, pH 8, and heated at 50” for 1 hr; after clarification, the solubilized proteins were oxidized with performic acid according to Hirs (6) and then dialyzed against the resuspending medium; (c) the resuspending medium consisted of the same Tris-EDTA medium as above containing 4 M guanidinium chloride, 0.1 M diethanoldisulfide, 0.01 M 2-mercaptoethanol, and 1% cetyltrimethylammonium bromide (CTAB); the mixture was incubated at 50” for 1 hr. After clarification the preparations obtained by procedures (a) and (b) were electrophoresed in 10% polyacrylamide-SDS gels as described previously (I); preparation (cl was electrophoresed in 10% polyacrylamide gels in the presence of 0.1% CTAB (7). Staining was carried out as described earlier (1) except that 0.025% Coomassie brilliant blue was used. The pattern of the proteins in the insoluble fraction is shown in Figs. la-h. There is no difference between the healthy control and the vulgare-infected sample. In 346
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
I ,,“. I-
--
d
k
_- _- _- --
--
_- _- _- --
--- --
_- _- =-
“.- --
--
--
--
--
--
--
--
--
--
-,- --
_“. -..- --
-
FIG. 1. Electrophoretic patterns in stained 10% polyacrylamide gels. The samples in the gels indicated by letters are as follows: (a)-(d), Insoluble fraction from (a) healthy leaves, (b) uulgare-infected leaves, (c) DTB-infected leaves, (d) fla uum-infected leaves; (e), x band isolated by gel electrophoresis and reelectrophoresed after heating in a boiling-water bath for 10 min; (0, insoluble fraction from flauum-infected leaves electrophoresed in the presence of CTAB; (g), flauum coat protein electrophoresed in the presence of CTAB; (h), insoluble fraction from flooaminfected leaves after oxidation with performic acid and heating in a boiling-water bath; (0 fluuum coat protein prepared by phenol extraction and heating at 50” for 3 hr; (i), same as (i) but heated in a boiling-water bath for 10 min; (k), lyophilized coat protein after heating in a boiling-water bath for 10 min. Except where otherwise stated the samples were incubated at 50”for 1 hr, as described in the text, and electrophoresed in the presence of SDS.
b
w lb 4
346
SHORT
COMMUNICATIONS
the sample from DTZinfected tissue there is a very prominent band in the position of TMV coat protein. This band is also present in the preparation from flavum-infected tissue, but in addition a strong band (marked x) is found in a position corresponding to a molecular weight of -35,000 where hardly any protein was detectable by staining in the other preparations. The intensity of this band was not affected by the presence of 8 M urea during the pretreatment of the sample or by heating in a boiling-water bath instead of at 50”. To isolate this protein, 1 x 10 X 25cm blocks of 10% polyacrylamide were cast. Preparations of the insoluble fraction from flavuminfected leaves were separated under the same conditions as in the small cylindrical gels. Strips were cut from the block for staining while the rest of the block was kept frozen. The portion of the gel block that contained the x band was cut into small pieces, and protein was eluted with 67% acetic acid, the liquid was dialyzed against distilled water, and the protein was recovered by lyophilization (8). It was resuspended and treated according to procedure (a) and analysed by gel electrophoresis. A single band was produced in the position of the x band (Fig. le). Incubation of the insoluble fraction with mercaptoethanol in the presence of guanidinium chloride followed by polyacrylamide-CTAB-gel electrophoresis gave essentially the same pattern as the corresponding SDS gels (Fig. 10; flavum coat protein prepared and analyzed by the same procedure is shown in Fig. lg. The amount of protein in the x band was reduced considerably by performic acid oxidation but it was never completely abolished (Fig. lh). When flavum-infected leaves were allowed to take up [35Slsulfate or [3H]leucine, both the coat protein band and the x band incorporated large amounts of radioactivity. When radioactive histidine or methionine (these two amino acids are absent from the coat protein (9)) was used for labeling, none of the two bands incorporated radioactivity detectably. Coat protein and x band protein were isolated by block electrophoresis, as described
above, from a preparation labeled with [35Slsulfate. The concentration was determined spectrophotometrically and the radioactivity was measured by using a scintillation liquid containing Triton X-100. The preparation from the x band contained 8248 -e 125 cpm/lOO pg of protein, and the coat protein preparation contained 9783 2 181 cpm/lOO pg of protein. It should be noted (Figs. la-d) that two host proteins coelectrophorese with coat protein under these conditions, which probably accounts for the slightly higher radioactivity of the coat protein preparation. These results lead to the conclusion that the x: band consists of a dimer of flavum coat protein. However, the failure to dissociate it by heat treatment in the presence of SDS, mercaptoethanol and urea or guanidinium chloride shows it is unusually stable. When intact virus of vulgare or flavum was disrupted under the conditions used for solubilizing the insoluble fraction and analyzed according to method (a) or cc), usually no dimer was found. If a very large amount (>200 pg) of virus disrupted at 50 was applied on a gel there was sometimes a faint dimer band; this could be abolished by pretreatment in a boiling-water bath. Preparations of coat proteins obtained by phenol extraction of vulgar-e or flavum and pretreated at 50” for several hours contained varying amounts of dimer (Fig. li), however, this dimer band could be almost completely eliminated by pretreatment at 100” for 10 min (Fig. lj). Coat protein preparations of both strains that had been lyophilized contained dimers which were sometimes difficult to eliminate completely even by pretreatment in a boiling-water bath (Fig. lk). In conclusion, a large proportion of the coat protein found in the insoluble fraction of flavum-infected tissue is present in the form of dimers. These dimers are extremely stable and resist disaggregation by conventional procedures for breaking disulfide bonds. The possible occurrence of dimers held together by nondisulfide covalent bonds has been reported by Rice (71, who presented evidence that the coat proteins of cowpea chlorotic mottle virus and satellite tobacco necrosis virus may be
349
SHORT COMMUNICATIONS
partly present as dimers in the intact particle whereas this was not the case with TMV, in agreement with results presented here. The fact that the amount of flaoun coat protein dimer in the insoluble fraction can be reduced considerably by performic acid oxidation indicates that it is a disulfide-bonded dimer which however resists disaggregation by treatment with thiol and diethanoldisulfide in the presence of SDS and urea, a method which is commonly used and which, in most cases, appears to be effective (10). It is not possible to determine if the small proportion remaining after performic acid treatment is held together by a different kind of bond. Performic acid oxidation was apparently not employed in the investigation by Rice (7).
The fact that more or less stable dimers can be produced by certain methods of preparing coat protein calls for a great deal of caution in interpreting gel-electrophoretic patterns. In particular, the effect of lyophilization deserves attention; lyophilization of myoglobin has been shown to yield nondissociable dimers (II ). Nevertheless, several facts indicate that the dimer of flauum coat protein found in the insoluble fraction is not a preparation artifact but is probably present before extraction. The amount of dimer was not affected by the presence of 8 M urea and/or treatment of the preparation in a boiling-water bath instead of at 50”. In contrast, the same variations in sample treatment prior to electrophoresis eliminated, or considerably reduced the amount of, dimers found in coat protein preparations that had been extracted with phenol or lyophilized. Vulgare produces excess coat protein that accumulates in a soluble form but there is no indication of dimer formation (1). The insoluble coat
protein of the defective strain DT2 does not form detectable amounts of dimer, showing that accumulation in an insoluble form does not in itself lead to dimer formation. The flavum coat protein differs from that of uulgure only in having an alanine instead of an aspartic acid in position 19 (9). Although the exact nature of the dimer remains obscure it appears that its formation is in some way favoured by this amino acid replacement. ACKNOWLEDGMENTS I thank Miss Lena Widnersson for excellent technical assistance. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research. REFERENCES 1. OXELFELT, (1972). 2. SINGER, B.,
P., Phytopathol. and CONDIT,
2.
C., Virology
74,
131-140 57, 42-48
(1974). 3. SIEGEL, Proc.
A., ZAITLIN, Nat. Acad.
M., and SEHGAL, 0. P., USA 40, 1845-1851
Sci.
(1962). 4. JOCKIJSCH, H., and JOCKIJSCH, B., Mol. Gen. Genet. 102, 204-209 (1968). 5. SARKAR, S., Virology 20, 185-193 (1963). 6. HIRS, C. H. W. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 11, pp. 197-199. Academic Press, New York, 1967. 7. RICE, R. H., Virology 61, 249-255 (1974). 8. HARII-IARASUBRAMANIAN, V., SMITH, R. C., and ZAITLIN, M., Virology 55, 202-210 (1973). 9. WITTMANN, H. G., WITTMANN-LIEBOLD, B., and JAUREGUI-ADELL, J., 2. Naturforsch. 20B, 1224-1234 (1965). IO. MAIZEL, J. V., In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 180-246. Academic Press, New York, 1971. Il. VAN DEN OORD, A. H. A., WESDORP, J. J., VAN DAM, A. F., and VERHEIJ, J. A., Eur. J. Biothem. 10, 140-145 (1969).
69, 346-349
WX)
Stable Dimers of the Coat Protein of a Strain of Tobacco
Mosaic Virus
PER OXELFELT Department
ofPlant
Pathology
and Entomology, Accepted
Agricultural August
College
ofsweden,
S-750 07 Uppsala
7, Sweden
19,1975
A large proportion of the coat protein of the tobacco mosaic virus, strainflaoum, which accumulates in an insoluble form in infected tissue is present as a dimer. The dimer resists disaggregation by heating in the presence of sodium dodecyl sulfate, mercaptoethanol, and urea, and it cannot be completely dissociated by performic acid oxidation.
Characteristic changes in the synthesis of host proteins brought about by various strains and mutants of TMV (tobacco mosaic virus) have been reported (1, 2). In conjunction with the earlier study (1) that described the changes in soluble proteins, other subcellular fractions were also investigated. The present report describes the anomalous behavior of the coat protein of the flavum strain extracted from infected tissue. Three TMV strains were used in the study, uulgare, flauum and DT2. The latter strain, kindly supplied by Dr. S. Sarkar, Tiibingen, is a defective strain, which is derived from PM2 (3) and which differs in producing an insoluble defective coat protein. Flavum is known to produce excess coat protein that accumulates in an insoluble form (4) whereas excess uulgare coat protein accumulates in a soluble form (1). Samsun tobacco plants (Nicotiana tabacum L.) were grown and inoculated by standard procedures; in the case of DT2 the inoculum consisted of infected leaf tissue homogenized in cold pH 8.6 Tris-phosphate buffer with bentonite (5). The inoculated leaves were used 7-10 days after inoculation. Infected and comparable healthy leaf tissue was ground in the pH 8 extraction medium used earlier (1) with the modification that it also contained 0.01 M MgCl,. The homogenate was filtered through cheesecloth and centrifuged at 2500 g for 10 min. The pellet was resuspended in extraction medium to which 2% Triton X-100 was added, incubated in the
cold for 1 hr and then centrifuged at 2500g for 10 min. The pellet thereby obtained, here called the insoluble fraction, was prepared for analysis by the following alternative procedures: (a) It was resuspended in 0.01 M Tris, 0.0004 M EDTA, 0.1 M diethanoldisulfide, 0.01 M 2-mercaptoethanol, and 1% sodium dodecyl sulfate (SDS), pH 8, and incubated at 50” for 1 hr or in a boiling-water bath for 10 min; in most cases the resuspending medium also contained 8 M urea; (b) the pellet was resuspended in 0.01 M Tris, 0.0004 M EDTA, and 1% SDS, pH 8, and heated at 50” for 1 hr; after clarification, the solubilized proteins were oxidized with performic acid according to Hirs (6) and then dialyzed against the resuspending medium; (c) the resuspending medium consisted of the same Tris-EDTA medium as above containing 4 M guanidinium chloride, 0.1 M diethanoldisulfide, 0.01 M 2-mercaptoethanol, and 1% cetyltrimethylammonium bromide (CTAB); the mixture was incubated at 50” for 1 hr. After clarification the preparations obtained by procedures (a) and (b) were electrophoresed in 10% polyacrylamide-SDS gels as described previously (I); preparation (cl was electrophoresed in 10% polyacrylamide gels in the presence of 0.1% CTAB (7). Staining was carried out as described earlier (1) except that 0.025% Coomassie brilliant blue was used. The pattern of the proteins in the insoluble fraction is shown in Figs. la-h. There is no difference between the healthy control and the vulgare-infected sample. In 346
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
I ,,“. I-
--
d
k
_- _- _- --
--
_- _- _- --
--- --
_- _- =-
“.- --
--
--
--
--
--
--
--
--
--
-,- --
_“. -..- --
-
FIG. 1. Electrophoretic patterns in stained 10% polyacrylamide gels. The samples in the gels indicated by letters are as follows: (a)-(d), Insoluble fraction from (a) healthy leaves, (b) uulgare-infected leaves, (c) DTB-infected leaves, (d) fla uum-infected leaves; (e), x band isolated by gel electrophoresis and reelectrophoresed after heating in a boiling-water bath for 10 min; (0, insoluble fraction from flauum-infected leaves electrophoresed in the presence of CTAB; (g), flauum coat protein electrophoresed in the presence of CTAB; (h), insoluble fraction from flooaminfected leaves after oxidation with performic acid and heating in a boiling-water bath; (0 fluuum coat protein prepared by phenol extraction and heating at 50” for 3 hr; (i), same as (i) but heated in a boiling-water bath for 10 min; (k), lyophilized coat protein after heating in a boiling-water bath for 10 min. Except where otherwise stated the samples were incubated at 50”for 1 hr, as described in the text, and electrophoresed in the presence of SDS.
b
w lb 4
346
SHORT
COMMUNICATIONS
the sample from DTZinfected tissue there is a very prominent band in the position of TMV coat protein. This band is also present in the preparation from flavum-infected tissue, but in addition a strong band (marked x) is found in a position corresponding to a molecular weight of -35,000 where hardly any protein was detectable by staining in the other preparations. The intensity of this band was not affected by the presence of 8 M urea during the pretreatment of the sample or by heating in a boiling-water bath instead of at 50”. To isolate this protein, 1 x 10 X 25cm blocks of 10% polyacrylamide were cast. Preparations of the insoluble fraction from flavuminfected leaves were separated under the same conditions as in the small cylindrical gels. Strips were cut from the block for staining while the rest of the block was kept frozen. The portion of the gel block that contained the x band was cut into small pieces, and protein was eluted with 67% acetic acid, the liquid was dialyzed against distilled water, and the protein was recovered by lyophilization (8). It was resuspended and treated according to procedure (a) and analysed by gel electrophoresis. A single band was produced in the position of the x band (Fig. le). Incubation of the insoluble fraction with mercaptoethanol in the presence of guanidinium chloride followed by polyacrylamide-CTAB-gel electrophoresis gave essentially the same pattern as the corresponding SDS gels (Fig. 10; flavum coat protein prepared and analyzed by the same procedure is shown in Fig. lg. The amount of protein in the x band was reduced considerably by performic acid oxidation but it was never completely abolished (Fig. lh). When flavum-infected leaves were allowed to take up [35Slsulfate or [3H]leucine, both the coat protein band and the x band incorporated large amounts of radioactivity. When radioactive histidine or methionine (these two amino acids are absent from the coat protein (9)) was used for labeling, none of the two bands incorporated radioactivity detectably. Coat protein and x band protein were isolated by block electrophoresis, as described
above, from a preparation labeled with [35Slsulfate. The concentration was determined spectrophotometrically and the radioactivity was measured by using a scintillation liquid containing Triton X-100. The preparation from the x band contained 8248 -e 125 cpm/lOO pg of protein, and the coat protein preparation contained 9783 2 181 cpm/lOO pg of protein. It should be noted (Figs. la-d) that two host proteins coelectrophorese with coat protein under these conditions, which probably accounts for the slightly higher radioactivity of the coat protein preparation. These results lead to the conclusion that the x: band consists of a dimer of flavum coat protein. However, the failure to dissociate it by heat treatment in the presence of SDS, mercaptoethanol and urea or guanidinium chloride shows it is unusually stable. When intact virus of vulgare or flavum was disrupted under the conditions used for solubilizing the insoluble fraction and analyzed according to method (a) or cc), usually no dimer was found. If a very large amount (>200 pg) of virus disrupted at 50 was applied on a gel there was sometimes a faint dimer band; this could be abolished by pretreatment in a boiling-water bath. Preparations of coat proteins obtained by phenol extraction of vulgar-e or flavum and pretreated at 50” for several hours contained varying amounts of dimer (Fig. li), however, this dimer band could be almost completely eliminated by pretreatment at 100” for 10 min (Fig. lj). Coat protein preparations of both strains that had been lyophilized contained dimers which were sometimes difficult to eliminate completely even by pretreatment in a boiling-water bath (Fig. lk). In conclusion, a large proportion of the coat protein found in the insoluble fraction of flavum-infected tissue is present in the form of dimers. These dimers are extremely stable and resist disaggregation by conventional procedures for breaking disulfide bonds. The possible occurrence of dimers held together by nondisulfide covalent bonds has been reported by Rice (71, who presented evidence that the coat proteins of cowpea chlorotic mottle virus and satellite tobacco necrosis virus may be
349
SHORT COMMUNICATIONS
partly present as dimers in the intact particle whereas this was not the case with TMV, in agreement with results presented here. The fact that the amount of flaoun coat protein dimer in the insoluble fraction can be reduced considerably by performic acid oxidation indicates that it is a disulfide-bonded dimer which however resists disaggregation by treatment with thiol and diethanoldisulfide in the presence of SDS and urea, a method which is commonly used and which, in most cases, appears to be effective (10). It is not possible to determine if the small proportion remaining after performic acid treatment is held together by a different kind of bond. Performic acid oxidation was apparently not employed in the investigation by Rice (7).
The fact that more or less stable dimers can be produced by certain methods of preparing coat protein calls for a great deal of caution in interpreting gel-electrophoretic patterns. In particular, the effect of lyophilization deserves attention; lyophilization of myoglobin has been shown to yield nondissociable dimers (II ). Nevertheless, several facts indicate that the dimer of flauum coat protein found in the insoluble fraction is not a preparation artifact but is probably present before extraction. The amount of dimer was not affected by the presence of 8 M urea and/or treatment of the preparation in a boiling-water bath instead of at 50”. In contrast, the same variations in sample treatment prior to electrophoresis eliminated, or considerably reduced the amount of, dimers found in coat protein preparations that had been extracted with phenol or lyophilized. Vulgare produces excess coat protein that accumulates in a soluble form but there is no indication of dimer formation (1). The insoluble coat
protein of the defective strain DT2 does not form detectable amounts of dimer, showing that accumulation in an insoluble form does not in itself lead to dimer formation. The flavum coat protein differs from that of uulgure only in having an alanine instead of an aspartic acid in position 19 (9). Although the exact nature of the dimer remains obscure it appears that its formation is in some way favoured by this amino acid replacement. ACKNOWLEDGMENTS I thank Miss Lena Widnersson for excellent technical assistance. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research. REFERENCES 1. OXELFELT, (1972). 2. SINGER, B.,
P., Phytopathol. and CONDIT,
2.
C., Virology
74,
131-140 57, 42-48
(1974). 3. SIEGEL, Proc.
A., ZAITLIN, Nat. Acad.
M., and SEHGAL, 0. P., USA 40, 1845-1851
Sci.
(1962). 4. JOCKIJSCH, H., and JOCKIJSCH, B., Mol. Gen. Genet. 102, 204-209 (1968). 5. SARKAR, S., Virology 20, 185-193 (1963). 6. HIRS, C. H. W. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 11, pp. 197-199. Academic Press, New York, 1967. 7. RICE, R. H., Virology 61, 249-255 (1974). 8. HARII-IARASUBRAMANIAN, V., SMITH, R. C., and ZAITLIN, M., Virology 55, 202-210 (1973). 9. WITTMANN, H. G., WITTMANN-LIEBOLD, B., and JAUREGUI-ADELL, J., 2. Naturforsch. 20B, 1224-1234 (1965). IO. MAIZEL, J. V., In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 180-246. Academic Press, New York, 1971. Il. VAN DEN OORD, A. H. A., WESDORP, J. J., VAN DAM, A. F., and VERHEIJ, J. A., Eur. J. Biothem. 10, 140-145 (1969).
