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RNA-binding properties of nonstructural polypeptide G of encephalomyocarditis virus
VIROLOGY
88,
183-185
(1978)
RNA-Binding
Properties of Nonstructural Polypeptide Encephalomyocarditis Virus’
A. E. GORBALENYA, Institute of Poliomyelitis
K. M. CHUMAKOV,
AND
G of
V. I. AGOL’
and Viral Encephalitides of the USSR Academy of Medical State University, Moscow, USSR
Sciences and Moscow
Accepted March 9, 1978 Extracts of encephalomyocarditis (EMC) virus-infected Krebs II cells were subjected to chromatography on either EMC virus RNA or different homopolyribonucleotides immobilized on cellulose. Among several EMC virus-specific proteins, the nonstructural polypeptide G was shown to bind selectively to RNA. This binding appears to be relatively unspecific with respect to nucleotide sequence.
In the course of replication of picornaviruses, different virus-specific polypeptides are expected to interact with the viral RNA. We have attempted to identify RNA-binding proteins in extracts of Krebs II cells infected with encephalomyocarditis (EMC) virus. The extracts were subjected to chromatography on RNA-cellulose columns, a procedure shown to be very efficient in a number of systems ( 1,2). Since EMC virus RNA contains poly(A) and poly(C) sequences at the 3’ and 5’ ends of the molecule, respectively (3), we used as affinity sorbents not only viral RNA but also different homopolyribonucleotides immobilized on cellulose. It was found that nonstructural polypeptide G, or a complex containing this polypeptide, binds selectively to RNA. This binding appears to be relatively unspecific with respect to nucleotide sequence. Krebs II cells infected with EMC virus at a multiplicity of 10 to 100 PFU/cell were incubated with a mixture of ‘*C-labeled valine, leucine, lysine, and phenylalanine (2-5 @i/ml of each; specific activity of 175 to 315 mCi/mmol; UPVVVR, Czechoslovakia) between 3 and 4 hr after infection. The suspension was chilled and all subsequent I Reported in part at the USSR-France Symposium, “Interactions of Nucleic Acids and Proteins,” Tashkent, September 6-8, 1977. ’ To whom requests for reprints should be sent.
operations were carried out at 0 to 4”. Cells were pelleted by low-speed centrifugation, and 5 x lo8 cells were suspended in 7 to 8 ml of a hypotonic buffer (0.01 M NaCl, 0.0015 M Mg-acetate, 0.01 M Tris-HCl, pH 7.4; SMT). After swelling, the cells were disrupted in a Dounce homogenizer, and nuclei were sedimented by centrifugation at 800 g for 5 min. The pellet was resuspended in 3 ml of SMT and again sedimented under the same conditions. A solution of 6 M LiCl was added to the combined supernatants to a final concentration of 2 M (this treatment was aimed at dissociation of some protein and nucleoprotein complexes as well as at precipitation of single-stranded RNAs) . After standing overnight, the suspension was centrifuged at 10,000 g for 10 min, and the supernatant obtained was dialyzed against 0.05 M Tris-HCl, 0.005 M 2-mercaptoethanol, pH 7.8 (TM buffer). The dialyzed extract was centrifuged at 4000 g for 5 min, and the supernatant was stored at -70” until used. RNA-cellulose (CF-11) sorbents were prepared by a uv irradiation method (4). A portion of the extract containing about 2 X lo6 cpm (equivalent to 5 X 10’ cells; up to 5 mg of protein) was loaded onto a column containing 250 to 300 mg of the sorbent (l-5 mg of RNA), and the column was washed overnight with 50 to 100 ml of TM buffer. The elution was performed by a linear NaCl concentration gradient from 0 183 0042~6822/78/X381-0183$02.00/O Copyright 0 1978 by Academic Press, Inc. AII rights of reproduction in any form reserved.
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to 1 M, the solutions being prepared in TM buffer. After chromatography the column was regenerated by a solution containing 8 M urea and 2 M NaCl in TM buffer. Labeled extracts from the mock-infected cells were treated similarly. In addition, extracts from both infected and uninfected cells were chromatographed on unmodified CF11 cellulose. Proteins contained in different fractions were precipitated by 5% trichloroacetic acid, washed with acetone, and dried. The precipitates were suspended in a solution containing 8 M urea, 3% SDS, 5% 2-mercaptoethanol in 0.05 M Tris-HCl, pH 6.8, and were heated at 100” for 5 min. Electrophoresis in 10 or 12.5% polyacrylamide slab gels was performed by a slight modification of Laemmli’s (5) method. The chromatographic profiles of extracts from uninfected or EMC virus-infected cells on either unmodified or EMC virus RNA-cellulose are presented in Fig. 1. It is seen that a peak eluting at 0.3-0.4 M NaCl appeared when the extract of the infected cells was chromatographed on EMC virus .I0
-2
5 !
A !
CPM 4 3
2 I----
0
. NaCl.
Lr&1.
s
RNA-cellulose; this peak is absent from the other control profiles. The polypeptide composition of different fractions was studied by electrophoresis in polyacrylamide gel slabs. A detailed description of the results will be presented elsewhere. Here, we would like to report the results obtained with the extracts from virus-infected cells chromatographed on viral RNA-cellulose. The radioactivity in the first, unspecific peak eluting at a low NaCl concentration is represented largely by viral structural polypeptides (Fig. 2A, lanes b and c). The second peak eluting at 0.3-0.4 M NaCl is considerably enriched with the nonstructural virus-specific polypeptide G of 16,500 MW. This polypeptide, indeed, is the only major labeled (that is virus specific) component of the peak (Fig. 2A, lane e), although a number of nonlabeled (cellular) proteins could be revealed by the Coomassie staining (Fig. 2B, lane e). Essentially the same results
a bcdef
abcdef
B AE50K-
D
G-
i!!!!?? OJ02 030105060.7a8
moles
FIG. 1. Chromatography of extracts from mock-in. fected (A and C) and EMC virus-infected (B and D) Krebs II cells on columns of unmodified (A and B) and EMC viral (C and D) RNA-cellulose. For preparation of extracts and columns, see text. Extracts were applied onto columns in TM buffer, and elution was carried out by a linear NaCl concentration gradient.
A
B
FIG. 2. Electrophoretic analysis in slabs of 10% polyacrylamide gel of fractions from RNA-cellulose chromatography of extracts from EMC virus-infected Erebs II cells. (A) Autoradiography; (B) staining with Coomasaie brilliant blue. (a) The unfractionated extract; fractions b-f are defined in Fig. 1D.
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were obtained when poly(A)-, poly(C)-, or poly(U)-celluloses were used instead of EMC virus RNA-cellulose: In all these cases a peak enriched with polypeptide G was eluted in a similar (although for different sorbents slightly different) region of the gradient, the set of contaminating celIular proteins being somewhat dependent on the hind of RNA-cellulose used (not shown). Thus, polypeptide G, or a complex containing this polypeptide, possesses RNAbinding properties, and this binding appears to be largely base sequence independent. A preparation enriched with polypeptide G, obtained by RNA-cellulose chromatography, has been tested for the capacity to bind to either cation or anion exchangers. It was found that, at 0.1 M NaCl and pH 7.8 (TM buffer), polypeptide G was adsorbed on CM-Sephadex but not on DEAESephadex; the elution of this polypeptide could be achieved by using a higher NaCl concentration (0.4 &f’). Thus, polypeptide G, or a G-containing complex, behaves as a basic protein, which may explain its RNAbinding capacity. The physiological function of polypeptide G is unknown although its binding
to ribosomes in the infected cell has been reported (6). By using purified preparations of this polypeptide it is possible to study its function in different cell-free systems. The results reported here should not necessarily be interpreted to mean that polypeptide G is the only RNA-binding EMC virus-specific protein. First, other RNAbinding proteins may be present in our extracts in an unsuitable form, i.e., insoluble or complexed with other macromolecules. Second, the binding conditions used (specifkahy, the absence of Mg”) may be not adequate to reveal the binding property of other proteins. REFERENCES 1. CARMICHAEL, G., J. Biol. Chem. 250, 6160-6167 (1975). 2. HUISMANS, H., and JOKLIK, W. K., Virology 70, 411-424 (1976). 3. CHUMAKOV, K. M., and AGOL, V. I., B&hem. Biophys. Res. Commun. 71,551-557 (1976). 4. SHELDON, R., JURALE, C., and KATES, J., Proc. Nut. Acad. Sci. USA 69,417-421 (1972). 5. LAEMMLI, U. K., Nature (London) 227, 680-665 (1970). 6. MEDVEDKINA, 0. A., SCARLAT, I. V., KALININA, N. O., and AGOL, V. I., FEBS Lett. 39, 4-9 (1974).
88,
183-185
(1978)
RNA-Binding
Properties of Nonstructural Polypeptide Encephalomyocarditis Virus’
A. E. GORBALENYA, Institute of Poliomyelitis
K. M. CHUMAKOV,
AND
G of
V. I. AGOL’
and Viral Encephalitides of the USSR Academy of Medical State University, Moscow, USSR
Sciences and Moscow
Accepted March 9, 1978 Extracts of encephalomyocarditis (EMC) virus-infected Krebs II cells were subjected to chromatography on either EMC virus RNA or different homopolyribonucleotides immobilized on cellulose. Among several EMC virus-specific proteins, the nonstructural polypeptide G was shown to bind selectively to RNA. This binding appears to be relatively unspecific with respect to nucleotide sequence.
In the course of replication of picornaviruses, different virus-specific polypeptides are expected to interact with the viral RNA. We have attempted to identify RNA-binding proteins in extracts of Krebs II cells infected with encephalomyocarditis (EMC) virus. The extracts were subjected to chromatography on RNA-cellulose columns, a procedure shown to be very efficient in a number of systems ( 1,2). Since EMC virus RNA contains poly(A) and poly(C) sequences at the 3’ and 5’ ends of the molecule, respectively (3), we used as affinity sorbents not only viral RNA but also different homopolyribonucleotides immobilized on cellulose. It was found that nonstructural polypeptide G, or a complex containing this polypeptide, binds selectively to RNA. This binding appears to be relatively unspecific with respect to nucleotide sequence. Krebs II cells infected with EMC virus at a multiplicity of 10 to 100 PFU/cell were incubated with a mixture of ‘*C-labeled valine, leucine, lysine, and phenylalanine (2-5 @i/ml of each; specific activity of 175 to 315 mCi/mmol; UPVVVR, Czechoslovakia) between 3 and 4 hr after infection. The suspension was chilled and all subsequent I Reported in part at the USSR-France Symposium, “Interactions of Nucleic Acids and Proteins,” Tashkent, September 6-8, 1977. ’ To whom requests for reprints should be sent.
operations were carried out at 0 to 4”. Cells were pelleted by low-speed centrifugation, and 5 x lo8 cells were suspended in 7 to 8 ml of a hypotonic buffer (0.01 M NaCl, 0.0015 M Mg-acetate, 0.01 M Tris-HCl, pH 7.4; SMT). After swelling, the cells were disrupted in a Dounce homogenizer, and nuclei were sedimented by centrifugation at 800 g for 5 min. The pellet was resuspended in 3 ml of SMT and again sedimented under the same conditions. A solution of 6 M LiCl was added to the combined supernatants to a final concentration of 2 M (this treatment was aimed at dissociation of some protein and nucleoprotein complexes as well as at precipitation of single-stranded RNAs) . After standing overnight, the suspension was centrifuged at 10,000 g for 10 min, and the supernatant obtained was dialyzed against 0.05 M Tris-HCl, 0.005 M 2-mercaptoethanol, pH 7.8 (TM buffer). The dialyzed extract was centrifuged at 4000 g for 5 min, and the supernatant was stored at -70” until used. RNA-cellulose (CF-11) sorbents were prepared by a uv irradiation method (4). A portion of the extract containing about 2 X lo6 cpm (equivalent to 5 X 10’ cells; up to 5 mg of protein) was loaded onto a column containing 250 to 300 mg of the sorbent (l-5 mg of RNA), and the column was washed overnight with 50 to 100 ml of TM buffer. The elution was performed by a linear NaCl concentration gradient from 0 183 0042~6822/78/X381-0183$02.00/O Copyright 0 1978 by Academic Press, Inc. AII rights of reproduction in any form reserved.
SHORT
184
COMMUNICATIONS
to 1 M, the solutions being prepared in TM buffer. After chromatography the column was regenerated by a solution containing 8 M urea and 2 M NaCl in TM buffer. Labeled extracts from the mock-infected cells were treated similarly. In addition, extracts from both infected and uninfected cells were chromatographed on unmodified CF11 cellulose. Proteins contained in different fractions were precipitated by 5% trichloroacetic acid, washed with acetone, and dried. The precipitates were suspended in a solution containing 8 M urea, 3% SDS, 5% 2-mercaptoethanol in 0.05 M Tris-HCl, pH 6.8, and were heated at 100” for 5 min. Electrophoresis in 10 or 12.5% polyacrylamide slab gels was performed by a slight modification of Laemmli’s (5) method. The chromatographic profiles of extracts from uninfected or EMC virus-infected cells on either unmodified or EMC virus RNA-cellulose are presented in Fig. 1. It is seen that a peak eluting at 0.3-0.4 M NaCl appeared when the extract of the infected cells was chromatographed on EMC virus .I0
-2
5 !
A !
CPM 4 3
2 I----
0
. NaCl.
Lr&1.
s
RNA-cellulose; this peak is absent from the other control profiles. The polypeptide composition of different fractions was studied by electrophoresis in polyacrylamide gel slabs. A detailed description of the results will be presented elsewhere. Here, we would like to report the results obtained with the extracts from virus-infected cells chromatographed on viral RNA-cellulose. The radioactivity in the first, unspecific peak eluting at a low NaCl concentration is represented largely by viral structural polypeptides (Fig. 2A, lanes b and c). The second peak eluting at 0.3-0.4 M NaCl is considerably enriched with the nonstructural virus-specific polypeptide G of 16,500 MW. This polypeptide, indeed, is the only major labeled (that is virus specific) component of the peak (Fig. 2A, lane e), although a number of nonlabeled (cellular) proteins could be revealed by the Coomassie staining (Fig. 2B, lane e). Essentially the same results
a bcdef
abcdef
B AE50K-
D
G-
i!!!!?? OJ02 030105060.7a8
moles
FIG. 1. Chromatography of extracts from mock-in. fected (A and C) and EMC virus-infected (B and D) Krebs II cells on columns of unmodified (A and B) and EMC viral (C and D) RNA-cellulose. For preparation of extracts and columns, see text. Extracts were applied onto columns in TM buffer, and elution was carried out by a linear NaCl concentration gradient.
A
B
FIG. 2. Electrophoretic analysis in slabs of 10% polyacrylamide gel of fractions from RNA-cellulose chromatography of extracts from EMC virus-infected Erebs II cells. (A) Autoradiography; (B) staining with Coomasaie brilliant blue. (a) The unfractionated extract; fractions b-f are defined in Fig. 1D.
SHORT
185
COMMUNICATIONS
were obtained when poly(A)-, poly(C)-, or poly(U)-celluloses were used instead of EMC virus RNA-cellulose: In all these cases a peak enriched with polypeptide G was eluted in a similar (although for different sorbents slightly different) region of the gradient, the set of contaminating celIular proteins being somewhat dependent on the hind of RNA-cellulose used (not shown). Thus, polypeptide G, or a complex containing this polypeptide, possesses RNAbinding properties, and this binding appears to be largely base sequence independent. A preparation enriched with polypeptide G, obtained by RNA-cellulose chromatography, has been tested for the capacity to bind to either cation or anion exchangers. It was found that, at 0.1 M NaCl and pH 7.8 (TM buffer), polypeptide G was adsorbed on CM-Sephadex but not on DEAESephadex; the elution of this polypeptide could be achieved by using a higher NaCl concentration (0.4 &f’). Thus, polypeptide G, or a G-containing complex, behaves as a basic protein, which may explain its RNAbinding capacity. The physiological function of polypeptide G is unknown although its binding
to ribosomes in the infected cell has been reported (6). By using purified preparations of this polypeptide it is possible to study its function in different cell-free systems. The results reported here should not necessarily be interpreted to mean that polypeptide G is the only RNA-binding EMC virus-specific protein. First, other RNAbinding proteins may be present in our extracts in an unsuitable form, i.e., insoluble or complexed with other macromolecules. Second, the binding conditions used (specifkahy, the absence of Mg”) may be not adequate to reveal the binding property of other proteins. REFERENCES 1. CARMICHAEL, G., J. Biol. Chem. 250, 6160-6167 (1975). 2. HUISMANS, H., and JOKLIK, W. K., Virology 70, 411-424 (1976). 3. CHUMAKOV, K. M., and AGOL, V. I., B&hem. Biophys. Res. Commun. 71,551-557 (1976). 4. SHELDON, R., JURALE, C., and KATES, J., Proc. Nut. Acad. Sci. USA 69,417-421 (1972). 5. LAEMMLI, U. K., Nature (London) 227, 680-665 (1970). 6. MEDVEDKINA, 0. A., SCARLAT, I. V., KALININA, N. O., and AGOL, V. I., FEBS Lett. 39, 4-9 (1974).