VIROLOGY
78, 359-362
Isoelectric
(1977)
Focusing
A. HAMANN, Heinrich-Pette-Institut
and 2D-Analysis K. J. WIEGERS,
fiir experimentelle Martinistrasse
52,200O
Virologie Hamburg
Accepted
und 20,
January
of Poliovirus AND
Proteins
R. DRZENIEK’
Immunologie an der Universitit Republic of Germany
Hamburg,
Federal
29,1977
The polypeptides of poliovirus were isoelectrically focused in urea containing polyacrylamide gels after dissociation of virus particles in urea. The bands obtained were correlated to the known four structural polypeptides of poliovirus by the two-dimensional technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The measured isoelectric points are: VPl, 8.1; VP2, 6.4; VP3, 6.0; VP4, 7.3. Additional bands causedby charge modification of the polypeptides during storage of the virus can be found.
The analysis of poliovirus proteins by polyacrylamide gel electrophoresis (PAGE) revealed four nonidentical structural polypeptides in poliovirus particles (1,2). Beginning with the latter publication, PAGE in the presence of sodium dodecyl sulfate (SDS) was found to be a powerful tool for the analysis of viral proteins (3). However, the usefulness of PAGE is limited because of its principle of separation relying on the size (molecular weight) of the substance under investigation. Isoelectric focusing (4) reveals a high resolution of proteins similar to PAGE (5), but the separation is based on differences in the isoelectric points. Little effort has been made to apply isoelectric focusing to the analysis of proteins of virus particles. A prerequisite for this application is the use of an appropriate dissociating system. In contrast to charged agents like SDS or guanidine HCl, urea can be used for isoelectric focusing under dissociating conditions. Bacterial proteins, membrane proteins, and ribosomes were analyzed by focusing in the presence of urea, combined with electrophoresis to a two-dimensional analysis (6-8). This paper describes the isoelectric focusing of poliovirus proteins obtained by dissociation of the virus by urea (9, 10). The bands obtained by isoelectric focusing are correlated to the four structural poly1 To whom
reprint
requests
should
be addressed.
peptides VP1 to VP4 by combination with PAGE to a two-dimensional (2D) analysis. Poliovirus, type I, strain Mahoney (unlabeled and 14C-labeled virus) was grown and purified as described earlier (11,121. Isoelectric focusing was carried out in flat bed gels (13,141 containing 5% acrylamide with a portion of 2.6% bisacrylamide, 2% ampholyte (SERVA, Heidelberg, FRG), and 9 M urea. The ampholyte was composed of equal parts of solutions of the pH ranges 5-7, 7-9, and 2-11. After dissociation of poliovirus for 1 hr at 25” in 9 M urea in the presence of 0.1 mglml of bovine pancreatic ribonuclease A (Ribonuclease 1:EC 3.1.4.22), samples (20 ~1, 5-15 pg of virus) were layered directly on prefocused gels near the anode. Focusing was carried out at 25” with carbon electrodes lying directly on the gel as described elsewhere (15). The pH gradient was determined on gel pieces eluted with 9 M urea solution containing 10 mM KC1 (16). Two-dimensional analysis was carried out similar to procedures described recently (6, 7): isoelectric focusing was performed in slab gels in the first dimension and electrophoresis (SDS-PAGE) according to (3) in the second dimension. The focused sample was cut out from the gel as a 5- to lo-mm broad strip. The strips were equilibrated in SDS sample buffer for lo-20 min, heated for 2 min at loo”, and polymerized onto the stacking gel of a slab gel.
359 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
360
SHORT
COMMUNICATIONS
Gels were stained with Coomassie Blue BL in a mixture given recently (16), but with 48% methanol. For autoradiography gels containing 5000-10000 cpm of l*C were dried (3) and left on X-ray film (Kodak Royal X-O mat) for several days. As can be seen in Fig. 1, isoelectric focusing in the presence of urea can be used successfully for the separation of poliovirus proteins obtained by dissociation of virus particles by urea (9, 10). Autoradiography or staining of the gels reveals three (Fig. la) and four (Fig. lb) strong bands and a few weak bands. The differences between the patterns are discussed below. With the two-dimensional technique bands obtained by isoelectric focusing can be correlated to the four structural poly-
VP 2
peptides of poliovirus (2,3) by SDS-PAGE (Fig. 2). All bands can clearly be assigned to the viral polypeptides VP1 to VP4, which are expressed in the nomination of the bands of Fig. 1. It was found that VP1 with an isoelectric point (~1) of 8.1 in urea is the most basic polypeptide of the poliovirus particle. Of interest in this respect is the recent finding that VP1 is bound exclusively to the negatively charged RNA (I 7). A small part of VP1 occurs as secondary band in a region of lower pH. This weak band is not distinguishable in all cases, since VP1 shows a tendency to smear due to precipitation on the surface of the gel. VP2 is slightly acid, having a pI of 6.4. As can be seen in Fig. lb and Fig. 2, a second strong band with a lower p1 of 6.2 (VP2,) can be found, however, in some
6.4
-
VP2 VP 2L
VP3
6.0
VP3 %“_”
a) FIG. 1. Isoelectric focusing of poliovirus polypeptides W-labeled virus, fresh preparation (autoradiography);
in 9 M urea. Two different (b) stored virus preparation
virus preparations: (stained gel).
(a)
361
SHORT COMMUNICATIONS
VP1
-
F32
=
VP4
,*I*
I, r,*
._ FIG. 2. Two-dimensional separation of poliovirus polypeptides. Horizontal: isoelectric focusing in 9 M urea; top: reference strip; vertical: SDS-disc electrophoresis in 14% polyacrylamide; left side: reference of SDS-dissociated poliovirus.
experiments. The occurrence of this secondary band is dependent on the virus preparation used and is not due to artifacts of the focusing procedure, as seen by analysis of different preparations parallel in one gel. VP2, is detected when poliovirus preparations were stored for longer periods (weeks or months) in isotonic phosphatebuffered saline at -20”. VP2, is absent in fresh poliovirus preparations and in samples stored in CsCl at -20”. VP2, is therefore considered a modification of VP2, having the same molecular weight but a lowered isoelectric point (Fig. 2). The modification of VP2 to VP2, is at the moment obscure; deamidation of glutamine or asparagine to the appropriate amino acid might be an explanation for the shift in the isoelectric point of VP2 to VP2,. Although the polypeptide VP2 seems to be the most easily modified, the polypeptides VP1 and VP3 also reveal similar shifts in their isoelectric points, but in a smaller percentage than VP2. The detection of secondary bands demonstrates that subtle changes in poliovirus polypeptides are found by isoelectric focusing which are not detectable by gel electrophoresis.
VP3 is the most acid polypeptide of poliovirus with a p1 of 6.0. Only occasionally a faint secondary band of VP3 is found. A p1 of 7.3 was measured for VP4. Due to its small size it does not focus as sharply as the other polypeptides and is also easily lost from the large-pore gels during the staining procedure. Therefore, one has to use autoradiography or the 2D-technique for detection. It should be mentioned that the isoelectric points were measured in urea. In spite of possible charge differences between the native and denatured state of a protein, the isoelectric points measured in 9 M urea have to be lowered by about half a pH unit if compared with measurements in ureafree solutions (18,19). ACKNOWLEDGMENTS The skillful technical assistance of Miss Marianne Hilbrig is gratefully acknowledged. This work was supported by the Bundesministerium fur Forschung und Technologie (BCT/SO). REFERENCES 1. MAIZEL, mm.
J. V., Biochem. 13, 483-489 (1963).
Biophys.
Res.
Com-
362 2.
3.
4. 5.
6. 7. 8. 9. 10. 11.
SHORT
COMMUNICATIONS
D. F., MAIZEL, J. V., JR., and DARNELL, J. E., JR., Proc. Nat. Acad. Sci. USA 54, 505-513 (1965). MAIZEL, J. V. In “Methods in Virology” (K. Maramorosch and K. Koprowski, eds.), Vol. 5, pp. 179-244. Academic Press, New York and London, 1971. SVENS~ON, H., Acta Chem. &and. 15, 325 (1961). RILBE, H., Ann. N.Y. Acad. Sci. 209, 11-22 (1973). O’FARRELL, P. H., J. Biol. Chem. 250,4007-4021 (1975). ADIES, G. F. -L., and NIKAIDO, K., Biochemistry 15, 616-623 (1976). CZEMPIEL, W., KLOSE, J., and BASS, R., FEBS Letters 62, 259-262 (1976). DRZENIEK, R., and BILELLO, P., Nature New Biol. 240, 118-122 (1972). DRZENIEK, R., 2. Naturforsch. 3Oc, 523-531 (1975). YAMAGUCHI-KOLL, U., WIEGERS, K. J., and DRZENIEK, R., J. Gen. Viral. 26, 307-319 (1975). SUMMERS,
12. 13.
14. 15.
16. 17.
18. 19.
DRZENIEK, R., and BILELLO, P., J. Gen. Virol. 25, 125-132 (1974). AWDEH, 2. L., WILLIAMSON, A. R., and AsKONAS, B. A., Nature (London) 219, 66-67 (1968). VESTERBERG, O., Biochim. Biophys. Acta 257, 11-19 (1972). HAMANN, A., In “Proceedings of the Third International Symposium on Electrofocusing and Isotachophoresis” (D. G. Graesslin and B. J. Radola, eds.). De Gruyter, Berlin and New York (in press). RIGHETTI, P. G., and DRYSDALE, J. W., J. Chromatogr. 98, 271-321 (1974). WIEGERS, K. J., YAMAGUCHI-KOLL, U., and DRZENIEK, R., Biochem. Biophys. Res. Comman. 71, 1308-1312 (1976). UI, N., Biochim. Biophys. Acta 229, 567-581 (1971). RIGHETTI, P. G., In “Proceedings of the Third International Symposium on Electrofocusing and Isotachophoresis” (D. G. Graesslin and B. J. Radola, eds.). De Gruyter, Berlin and New York (in press).
Announcement The National Foundation-March of Dimes will entertain a limited number of research proposals for the support of basic and clinical research in birth defects. The National Foundation defines a birth defect as an abnormality of structure, function, or metabolism, whether genetically determined or a result of environmental interference during embryonic or fetal life. Requests dealing with the structure and function of chromosomes, their subunits, genes, supporting structures, repressor substances, and the like could be included. For further information contact: Vice President for Research, The National Foundation-March of Dimes, 1275 Mamaroneck Avenue, White Plains, New York 10605.
78, 359-362
Isoelectric
(1977)
Focusing
A. HAMANN, Heinrich-Pette-Institut
and 2D-Analysis K. J. WIEGERS,
fiir experimentelle Martinistrasse
52,200O
Virologie Hamburg
Accepted
und 20,
January
of Poliovirus AND
Proteins
R. DRZENIEK’
Immunologie an der Universitit Republic of Germany
Hamburg,
Federal
29,1977
The polypeptides of poliovirus were isoelectrically focused in urea containing polyacrylamide gels after dissociation of virus particles in urea. The bands obtained were correlated to the known four structural polypeptides of poliovirus by the two-dimensional technique with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The measured isoelectric points are: VPl, 8.1; VP2, 6.4; VP3, 6.0; VP4, 7.3. Additional bands causedby charge modification of the polypeptides during storage of the virus can be found.
The analysis of poliovirus proteins by polyacrylamide gel electrophoresis (PAGE) revealed four nonidentical structural polypeptides in poliovirus particles (1,2). Beginning with the latter publication, PAGE in the presence of sodium dodecyl sulfate (SDS) was found to be a powerful tool for the analysis of viral proteins (3). However, the usefulness of PAGE is limited because of its principle of separation relying on the size (molecular weight) of the substance under investigation. Isoelectric focusing (4) reveals a high resolution of proteins similar to PAGE (5), but the separation is based on differences in the isoelectric points. Little effort has been made to apply isoelectric focusing to the analysis of proteins of virus particles. A prerequisite for this application is the use of an appropriate dissociating system. In contrast to charged agents like SDS or guanidine HCl, urea can be used for isoelectric focusing under dissociating conditions. Bacterial proteins, membrane proteins, and ribosomes were analyzed by focusing in the presence of urea, combined with electrophoresis to a two-dimensional analysis (6-8). This paper describes the isoelectric focusing of poliovirus proteins obtained by dissociation of the virus by urea (9, 10). The bands obtained by isoelectric focusing are correlated to the four structural poly1 To whom
reprint
requests
should
be addressed.
peptides VP1 to VP4 by combination with PAGE to a two-dimensional (2D) analysis. Poliovirus, type I, strain Mahoney (unlabeled and 14C-labeled virus) was grown and purified as described earlier (11,121. Isoelectric focusing was carried out in flat bed gels (13,141 containing 5% acrylamide with a portion of 2.6% bisacrylamide, 2% ampholyte (SERVA, Heidelberg, FRG), and 9 M urea. The ampholyte was composed of equal parts of solutions of the pH ranges 5-7, 7-9, and 2-11. After dissociation of poliovirus for 1 hr at 25” in 9 M urea in the presence of 0.1 mglml of bovine pancreatic ribonuclease A (Ribonuclease 1:EC 3.1.4.22), samples (20 ~1, 5-15 pg of virus) were layered directly on prefocused gels near the anode. Focusing was carried out at 25” with carbon electrodes lying directly on the gel as described elsewhere (15). The pH gradient was determined on gel pieces eluted with 9 M urea solution containing 10 mM KC1 (16). Two-dimensional analysis was carried out similar to procedures described recently (6, 7): isoelectric focusing was performed in slab gels in the first dimension and electrophoresis (SDS-PAGE) according to (3) in the second dimension. The focused sample was cut out from the gel as a 5- to lo-mm broad strip. The strips were equilibrated in SDS sample buffer for lo-20 min, heated for 2 min at loo”, and polymerized onto the stacking gel of a slab gel.
359 Copyright All rights
Q 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0042-6822
360
SHORT
COMMUNICATIONS
Gels were stained with Coomassie Blue BL in a mixture given recently (16), but with 48% methanol. For autoradiography gels containing 5000-10000 cpm of l*C were dried (3) and left on X-ray film (Kodak Royal X-O mat) for several days. As can be seen in Fig. 1, isoelectric focusing in the presence of urea can be used successfully for the separation of poliovirus proteins obtained by dissociation of virus particles by urea (9, 10). Autoradiography or staining of the gels reveals three (Fig. la) and four (Fig. lb) strong bands and a few weak bands. The differences between the patterns are discussed below. With the two-dimensional technique bands obtained by isoelectric focusing can be correlated to the four structural poly-
VP 2
peptides of poliovirus (2,3) by SDS-PAGE (Fig. 2). All bands can clearly be assigned to the viral polypeptides VP1 to VP4, which are expressed in the nomination of the bands of Fig. 1. It was found that VP1 with an isoelectric point (~1) of 8.1 in urea is the most basic polypeptide of the poliovirus particle. Of interest in this respect is the recent finding that VP1 is bound exclusively to the negatively charged RNA (I 7). A small part of VP1 occurs as secondary band in a region of lower pH. This weak band is not distinguishable in all cases, since VP1 shows a tendency to smear due to precipitation on the surface of the gel. VP2 is slightly acid, having a pI of 6.4. As can be seen in Fig. lb and Fig. 2, a second strong band with a lower p1 of 6.2 (VP2,) can be found, however, in some
6.4
-
VP2 VP 2L
VP3
6.0
VP3 %“_”
a) FIG. 1. Isoelectric focusing of poliovirus polypeptides W-labeled virus, fresh preparation (autoradiography);
in 9 M urea. Two different (b) stored virus preparation
virus preparations: (stained gel).
(a)
361
SHORT COMMUNICATIONS
VP1
-
F32
=
VP4
,*I*
I, r,*
._ FIG. 2. Two-dimensional separation of poliovirus polypeptides. Horizontal: isoelectric focusing in 9 M urea; top: reference strip; vertical: SDS-disc electrophoresis in 14% polyacrylamide; left side: reference of SDS-dissociated poliovirus.
experiments. The occurrence of this secondary band is dependent on the virus preparation used and is not due to artifacts of the focusing procedure, as seen by analysis of different preparations parallel in one gel. VP2, is detected when poliovirus preparations were stored for longer periods (weeks or months) in isotonic phosphatebuffered saline at -20”. VP2, is absent in fresh poliovirus preparations and in samples stored in CsCl at -20”. VP2, is therefore considered a modification of VP2, having the same molecular weight but a lowered isoelectric point (Fig. 2). The modification of VP2 to VP2, is at the moment obscure; deamidation of glutamine or asparagine to the appropriate amino acid might be an explanation for the shift in the isoelectric point of VP2 to VP2,. Although the polypeptide VP2 seems to be the most easily modified, the polypeptides VP1 and VP3 also reveal similar shifts in their isoelectric points, but in a smaller percentage than VP2. The detection of secondary bands demonstrates that subtle changes in poliovirus polypeptides are found by isoelectric focusing which are not detectable by gel electrophoresis.
VP3 is the most acid polypeptide of poliovirus with a p1 of 6.0. Only occasionally a faint secondary band of VP3 is found. A p1 of 7.3 was measured for VP4. Due to its small size it does not focus as sharply as the other polypeptides and is also easily lost from the large-pore gels during the staining procedure. Therefore, one has to use autoradiography or the 2D-technique for detection. It should be mentioned that the isoelectric points were measured in urea. In spite of possible charge differences between the native and denatured state of a protein, the isoelectric points measured in 9 M urea have to be lowered by about half a pH unit if compared with measurements in ureafree solutions (18,19). ACKNOWLEDGMENTS The skillful technical assistance of Miss Marianne Hilbrig is gratefully acknowledged. This work was supported by the Bundesministerium fur Forschung und Technologie (BCT/SO). REFERENCES 1. MAIZEL, mm.
J. V., Biochem. 13, 483-489 (1963).
Biophys.
Res.
Com-
362 2.
3.
4. 5.
6. 7. 8. 9. 10. 11.
SHORT
COMMUNICATIONS
D. F., MAIZEL, J. V., JR., and DARNELL, J. E., JR., Proc. Nat. Acad. Sci. USA 54, 505-513 (1965). MAIZEL, J. V. In “Methods in Virology” (K. Maramorosch and K. Koprowski, eds.), Vol. 5, pp. 179-244. Academic Press, New York and London, 1971. SVENS~ON, H., Acta Chem. &and. 15, 325 (1961). RILBE, H., Ann. N.Y. Acad. Sci. 209, 11-22 (1973). O’FARRELL, P. H., J. Biol. Chem. 250,4007-4021 (1975). ADIES, G. F. -L., and NIKAIDO, K., Biochemistry 15, 616-623 (1976). CZEMPIEL, W., KLOSE, J., and BASS, R., FEBS Letters 62, 259-262 (1976). DRZENIEK, R., and BILELLO, P., Nature New Biol. 240, 118-122 (1972). DRZENIEK, R., 2. Naturforsch. 3Oc, 523-531 (1975). YAMAGUCHI-KOLL, U., WIEGERS, K. J., and DRZENIEK, R., J. Gen. Viral. 26, 307-319 (1975). SUMMERS,
12. 13.
14. 15.
16. 17.
18. 19.
DRZENIEK, R., and BILELLO, P., J. Gen. Virol. 25, 125-132 (1974). AWDEH, 2. L., WILLIAMSON, A. R., and AsKONAS, B. A., Nature (London) 219, 66-67 (1968). VESTERBERG, O., Biochim. Biophys. Acta 257, 11-19 (1972). HAMANN, A., In “Proceedings of the Third International Symposium on Electrofocusing and Isotachophoresis” (D. G. Graesslin and B. J. Radola, eds.). De Gruyter, Berlin and New York (in press). RIGHETTI, P. G., and DRYSDALE, J. W., J. Chromatogr. 98, 271-321 (1974). WIEGERS, K. J., YAMAGUCHI-KOLL, U., and DRZENIEK, R., Biochem. Biophys. Res. Comman. 71, 1308-1312 (1976). UI, N., Biochim. Biophys. Acta 229, 567-581 (1971). RIGHETTI, P. G., In “Proceedings of the Third International Symposium on Electrofocusing and Isotachophoresis” (D. G. Graesslin and B. J. Radola, eds.). De Gruyter, Berlin and New York (in press).
Announcement The National Foundation-March of Dimes will entertain a limited number of research proposals for the support of basic and clinical research in birth defects. The National Foundation defines a birth defect as an abnormality of structure, function, or metabolism, whether genetically determined or a result of environmental interference during embryonic or fetal life. Requests dealing with the structure and function of chromosomes, their subunits, genes, supporting structures, repressor substances, and the like could be included. For further information contact: Vice President for Research, The National Foundation-March of Dimes, 1275 Mamaroneck Avenue, White Plains, New York 10605.
