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Isolation and characterization of low-molecular-weight DNA-binding proteins from retroviruses
VIROLOGY
104, 491-496 (1980)
isolation
and Characterization DNA-Binding Proteins
of Low-Molecular-Weight from Retroviruses’
C. W. LONG,2 L. E. HENDERSON, Biological
Carcinogenesis Program,
Frederick
AND
S. OROSZLAN
Cancer Research Center,
Frederick,
Maryland
21701
Accepted March 18, 1980
The DNA-binding proteins from Rauscher leukemia virus, baboon leukemia virus, endogenous cat virus, woolly monkey virus, bovine leukemia virus, equine infectious anemia virus, Rous sarcoma virus, Prague strain, mouse mammary tumor virus, and Mason-Pfizer monkey virus were purified to homogeneity by molecular sieving chromatography and DNA affinity chromatography. The amino acid composition shows each to be a basic protein.
compared electrophoretically and according to amino acid composition. Purification of DNA-binding proteins was carried out as reported previously with some modifications (6,12). Sucrose banded, pelleted virus was solubiiized overnight in buffer consisting of 20 mM HEPES-NaOH, pH 8.0, 0.2% P-mercaptoethanol, 0.1 mM EDTA, 0.2% Triton X-100, and 6 M guanidine hydrochloride. Samples were then made 10%in sucrose and applied to a Bio-Gel 1.5m column (2.5 x 80 cm) equilibrated with the same buffer. Fractions of 1.5 ml were collected at a flow rate of 10 ml/hr and assayed for binding to single-stranded calf thymus DNA (6 >. Fractions containing the low-molecular-weight DNA-binding peak were pooled and dialyzed overnight at 4” against low-salt buffer (LSB) consisting of 20 mM HEPES-NaOH, pH 7.8,20 mM KCl, 0.05% P-mercaptoethanol, 10%glycerol, and 0.1% Triton X-100. All further operations were carried out at 4”. Following dialysis, the DNA-binding pool was loaded onto a 16-ml bed volume column of DNA Sepharose equilibrated with LSB containing 0.05 M KCl, and elution was carried out with LSB containing 0.1 KCl, and 1.5 M KCl. The binding activity in the high-salt eluant was pooled, dialyzed against 0.1 M ammonium bicarbonate, and stored at -70 I The U.S. Government’s right to retain a nonexclufor further analysis. sive royalty-free license in and to the copyright Electrophoretic separation of viral procovering this paper, for governmental purposes, is teins was performed according to a proacknowledged. cedure previously described (12 ). Protein * To whom reprint requests should be addressed.
Murine type C RNA viruses contain a structural component of about 8000 to 10,000 molecular weight (~10) (1, 2). The gag region of the viral genome codes for this component which initially is included in a precursor polyprotein containing the structural proteins ~30, ~15, and p12 (3). Immunochemical and tryptic peptide mapping studies have shown that ~10s isolated from various murine viruses are highly related (4-S). p10 was shown to be the most basic virion protein and to be closely associated with viral RNA (7-9). Subsequent in vitro studies demonstrated the interaction of Rauscher leukemia virus (RLV) p10 with various nucleic acids and indicated a preference for single-stranded DNA and RNA (9, 10). It was also reported that the gag gene precursor polyprotein (Pr70) bound to single-stranded nucleic acid possibly through the p10 component (11). More recently, DNA-binding proteins have been isolated from woolly monkey (SSV-1) and mouse mammary tumor viruses (MMTV), and immunological relatedness has been examined (6,12). In the present study, several DNAbinding proteins have been purified to homogeneity from type C, B, and D retroviruses by affinity chromatography, and
491 004%6S22/80/100491-06$02.00/O
492
SHORT COMMUNICATIONS
was determined according to the method of Lowry et al. (13) using bovine serum albumin as a standard. Standards for electrophoresis included: heavy-chain y-globulin, light-chain y-globulin, ribonuclease, cytochrome c, and trasylol. Two-dimensional analysis of viral proteins was performed according to O’Farrell et aZ. (14). Amino acid analysis was carried out with a Beekman Spinco automatic amino acid analyzer Model lZl(Z5) adapted for high sensitivity (16 ), or on a Durram Model D560 analyzer. The low-molecular-weight DNA-binding proteins purified by affinity chromatography from several retroviruses are represented in Fig. 1. The binding proteins isolated from the type C viruses, RLV, baboon leukemia virus (BaLV), SSV-1, and endogenous cat virus (RD114), each migrated as the fastest virion component (~10) when compared to disrupted whole virus (Fig. 1A). In each of these eases the binding protein migrated in SDS-gels at a position intermediate to trasylol (6000~ and cytochrome c (11,500). The binding protein isolated from the type B retrovirus, MMTV, corresponded to the second fastest migrating virion protein designated as p14 when compared to whole disrupted virus. This protein migrated in SDS-gels at a position between cytochrome c and ribonuclease. Mason-Pfizer monkey virus (MPMV), a type D primate virus, contained a binding protein with a molecular weight similar to MMTV ~14, which corresponded to the second fastest migrating virion protein. The binding protein from Rous sarcoma virus (Prague strain) (Pr-RSV) migrated faster than MMTV p14 with a rate approaching that of cytochrome c, and corresponded to the fastest migrating virion protein. Equine infectious anemia virus (EIAV) and bovine leukemia virus (BLV) contained binding proteins which migrated at positions between trasylol and cytochrome c comparable in size to RLV ~10. The EIAV protein was the fastest migrating virion protein whereas the BLV protein corresponded to the second fastest virion protein. Squirrel monkey retrovirus (SMRV), a type D primate virus, contained two major DNA-binding proteins with molecular weights of approximately 15,000 and 20,000.
The gels of purified binding proteins from BaLV, SSV-1, and RSV also revealed a second larger-molecular-weight component. These bands were cut out from the gels and an amino acid analysis was performed on each. In each case the composition was identical to the one for low-molecular-weight component, showing these to be dimers. The amino acid composition data for each retrovirus nucleic acid-binding protein given in Table 1 was generated by computerized analysis of the raw amino acid analyzer data. The computer was pro~ammed to choose the total number of residues for each protein which would give the least departure from integral values for each residue (16, 17’). The calculation does not require any knowledge of the protein molecular weight but does require good analytical data on a pure protein. There is good correspondence between the amino acid composition of RLV p10 as determined by computer-assisted amino acid analysis and by complete amino acid sequence analysis (Henderson et al., manuscript in preparation). The amino acid compositional data presented in Table 1 strongly suggest that the retrovirus nucleic acid-binding proteins have isoelectric points above pH 7. In all cases except SSV-1, the sum of the basic residues (lysine, histidine, and arginine) is equal to or greater than the sum of the acidic residues (aspartic acid and glutamic acid). The total number of acidic residues is a maximum number which would be reduced by the number of amides in the protein. The amide content was not determined, but in most cases it is probably greater than zero. In the case of RLV ~10, the complete amino acid sequence of the protein reveals five amides (Henderson et al., manuscript in preparation). The cysteine content of the proteins was not determined directly, but the quantities detected in the amino acid analysis of each protein suggested that all the proteins listed in Table 1 contain more than one cysteine. RLV p10 is known to contain three cysteine and Pr-RSV ~12 is known to contain six cysteine residues (18 ). The amino acid composition of the Pr-RSV protein agrees with previous analysis of avian ~12 (19 >except that here the computer
SHORT COMMUNICATIONS
493
-
RSV
Mbnv
Ribonuclease
EIAV
_ Ribonuclease - Cytochrome C
FIG. 1. (A) Numbering lanes from left to right, 1,4,7, and 10 contain the standards heavy- and lightchain y-globulin, ribonucleaae, cytochrome c, and trasylol, respectively. Lane 2 contains RLV whole virus, lane 5, BaLV whole virus, and lanes 3, 6, 8 and 9 contain purified DNA-binding proteins from RLV, BaLV, RD114, and SSV-1, respectively. (B) From left to right lanes 1,4,7, and 10 contain standards, while lanes 25, and 8 contain whole viruses from RSV, MMTV, and EIAV, respectively, and lanes 3, 6, and 9 contain DNA-binding proteins from RSV, MMTV, and EIAV, respectively. (C) Lanes 1,4,7, and 10 contain standards, lanes 2,5, and 8 contain whole virus from MPMV, BoLV, and SMRV, respectively, and Ianes 3,6, and 9 contain DNA-bin~~ proteins from MPMV, BoLV, and SMRV, respectively.
494
SHORT COMMUNICATIONS TABLE 1 AMINO ACID COMPOSITIONS OF RETROVIRUS DNA-BINDING
Amino acid
EIAV
MPMV
Pr-RSV
PROTEINS~
MMTV
BLV
RD114
BaLV
ssv-1
RLV
5 1 6 7 3 4 6 5 5 5 3 0 1 4 1 1
5 1 8 4 1 2 s 5 5 4 2 0 0 3 1 0
57
49
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine VaIine Methionine Isoleucine Leucine Tyrosine Phenylalanine
10 2 3 2 3 4 9 7 11 5 2 0 0 4 1 4
13 5 3 11 2 2 10 8 10 7 2 0 1 2 1 3
7 2 8 7 3 6 10 6 11 6 4 1 2 5 2 1
18 2 5 9 6 IO 12 9 23 6 2 1 I 5 3 4
6 2 4 4 4 3 3 16 7 3 2 1 1 2 1 0
10 1 7 5 5 ;5 4 3 3 0 2 2 1 1
10 1 8 5 3 3 6 6 4 5 4 0 1 2 1 0
Total residues”
67
80
81
116
59
61
59
0 Data expressed as moles of residue per mole of protein. Tryptophan and cysteine were not determined and are omitted from total. The data for each protein are the averaged results of several analysis performed on 24- and 48-hr hydrolysates. The amino acid composition was determined from the raw data by a computer programmed to give the nearest integer minimum molecular weight.
analysis of the primary data indicates a molecular weight of about 10,000 while the previously published compositions were calculated assuming a molecular weight of 12,000. Based upon the DNA affinity isolation procedure employed here and reported sequence homology (201, the avian ~12 protein is shown to be the ~nctional homolog of the murine ~10. The overall composition of the proteins from BLV and EIAV appear quite different from RLV, BaLV, and RD114. A comparison of the percentage of polar amino acids relative to total amino acid content (15) showed that the type C p10 proteins, RD114 (48.8%), RLV (48.2%), and BaLV (4’7.6%), had the highest content of polar amino acids, followed by MPMV (43.7%), SSV-1(37.8%), and MMTV (37.3%). In the case of RLV ~10, the complete amino acid sequence of the protein has been determined (Henderson et al., manuscript in preparation). The amino acid composition as deter~n~d by sequence analysis is in complete agreement with the data given in Table 1. The molecular weight
of RLV p10 as determined by sequence analysis is 6060. The molecular weight of RLV ~10, as determined by its relative mobility in SDS-gel electrophoresis, is 6000 to 7000. Similarly, for all the proteins listed in Table 1, the molecular weights as estimated by SDS-gel electrophoresis are in good agreement with the molecular weights indicated by amino acid composition. The data presented here suggest the following approximate molecular weights for the viral nucleic acid-binding proteins: BaLV (7~0), SSV-1 (7500), RD114 (7~0}, BLV (7000), EIAV (SOOO),MPMV (lO$OO), RSV (10,500), and MMTV (14,000). Since the isolated proteins were mostly of a basic nature, a two-dimensional gel analysis was made on each whole disrupted virus to confirm the presence of a lowmolecular-weight protein with a high isoelectric point relative to the other virion components. As shown in Figs. 2 and 3, each virus contained a basic low-molecularweight protein corresponding closely in molecular weight to that isolated by DNA
SHORT
FIGS. 2 AND 3. Two-dimensional
COMMUNICATIONS
electrophoresis
of retrovirnses.
FIG. 2. (A) BaLV; (B) RLV; (C) MMTV; (D) MPMV; (E) EIAV; (F) BoLV. Acidic part of gel is to the left and basic on the right. The standards to the right of each gel are heavy and light chains of y-globulin, ribonuclease, cytochrome c, and trasylol. FIG. 3. (A) A-MuLV; (B) AKR; (C) FeLV; (D) RSV; (E) REV; (F) SMRV. Acidic part of gel is to the left and basic on the right. The standards on the right of each gel are heavy and light chains of y-globulin, ribonuclease, cytochrome c, and trasylol.
affinity chromatography. Two-dimensional analysis of purified DNA-binding proteins from RLV, BaLV, MMTV, MPMV, and RSV showed each to consist of the most basic low-molecular-weight virion protein. This establishes the presence in all retroviruses of a basic low-molecular-weight protein with an affinity for nucleic acid. The exact function of these proteins in viral maturation remains to be determined.
4. 5.
6. 7. 8.
ACKNOWLEDGMENTS We wish to thank M. Ramsburg, R. Snead, and G. Smythers for excellent technical assistance. This work was supported by Contract NOl-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20205. REFERENCES 1. AUGUST, J. T., E., GILDEN, Virology 60, 2. NOWINSKI, R. N. H., and (1972). 8. BARBACID, M.,
BOLOGNESI, D. P., FLEISSNER, R. V., and NOWINSKI, R. C., 1595 (1974). C., FLEISSNER, E., SARKAR, AOKI, T., J. Viral. 9, 359-366
9. IO. 11. 12. 13.
14. 15.
STEPHENSON, J. R. and AARON-
SON, S. A., Nature (London) 262, 554-559 (1976). BUCHHAGEN, D. L., STUTMAN, O., and FLEISSNER, E., J. Viral. 15, 1148-1157 (1975). BARBACID, M., STEPHENSON, J. R., and AARONSON, S. A., J. Biol. Chem. 251, 4859-4866 (1976). LONG, C. W., BERZINSKI, R., and GILDEN, R. V., Znt. J. Cancer 19, 843-850 (1977). BOLOGNESI, D. P., LUFTIG, R., and SHAPER, J. H., Virology 56, 549-564 (1973). FLEISSNER, E., and TRESS, E., J. Viral. 12, 1612, 1615 (1973). SCHULEIN, M., BURNETTE, W. N., and AUGUST, J. T., J. Viral. 26, 54 (1978). DAVIS, J., SCHERER, M., TSAI, W. P., and LONG, C., J. Viral. 18, 709-718 (1976). OROSZLAN, S., LONG, C. W., and GILDEN, R. V., Virology 72, 523-526 (1976). ARTHUR, L. O., LONG, C. W., SMITH, G. H., and FINE, D. L., Znt. J. Cancer 22,433-440 (1978). LOWRY, 0. H., ROSENBROUGH, W. J., FARR, A. L., and RANDALL, R. J., J. Biol. Chem. 193, 265-275 (1951). O’FARRELL, P. Z., GOODMAN, H. M., and O’FARRELL, P. H., Cell 12, 1133-1142 (1977). OROSZLAN, S., HENDERSON, L. E., STEPHENSON, J. R., COPELAND, T. D., LONG, C. W., IHLE,
496
SHORT COMMUNICATIONS
J. N., and GILDEN, R. V., Proe. Nat. ACULL Sci. USA 75, 1404-1408 (1978). 16. HENDERSON, L. E., COPELAND, T. D., SMIIYTHERS, G. W., MARQUARDI, H., and OROSZLAN, S., Virology 85, 319-322 (1978). 17. BOYER, S. H., NOYES, A. N., BOYER, M. L., and MARR, K., J. Biol. Chem. 248, 992-1003 (1973). 18. MISONO, K. S., SHARIEF, F. S., and LEIS, J., in
“Fed. Proc.” Vol. 39, No. 6, Abs. No. 60, pp. 1611 (1980). 19. HERMAN, A. C., GREEN, R. W., BOLOGNEX, D. P., and BANAMAN, T. C., Virology 64, 339-348 (1975). 20. OROSZLAN, S., and GILDEN, R. V., In “Molecular Biology of RNA Tumor Viruses” (J. R. Stephenson, ed.), pp. 299-344. Academic Press, New York/London (1980).
104, 491-496 (1980)
isolation
and Characterization DNA-Binding Proteins
of Low-Molecular-Weight from Retroviruses’
C. W. LONG,2 L. E. HENDERSON, Biological
Carcinogenesis Program,
Frederick
AND
S. OROSZLAN
Cancer Research Center,
Frederick,
Maryland
21701
Accepted March 18, 1980
The DNA-binding proteins from Rauscher leukemia virus, baboon leukemia virus, endogenous cat virus, woolly monkey virus, bovine leukemia virus, equine infectious anemia virus, Rous sarcoma virus, Prague strain, mouse mammary tumor virus, and Mason-Pfizer monkey virus were purified to homogeneity by molecular sieving chromatography and DNA affinity chromatography. The amino acid composition shows each to be a basic protein.
compared electrophoretically and according to amino acid composition. Purification of DNA-binding proteins was carried out as reported previously with some modifications (6,12). Sucrose banded, pelleted virus was solubiiized overnight in buffer consisting of 20 mM HEPES-NaOH, pH 8.0, 0.2% P-mercaptoethanol, 0.1 mM EDTA, 0.2% Triton X-100, and 6 M guanidine hydrochloride. Samples were then made 10%in sucrose and applied to a Bio-Gel 1.5m column (2.5 x 80 cm) equilibrated with the same buffer. Fractions of 1.5 ml were collected at a flow rate of 10 ml/hr and assayed for binding to single-stranded calf thymus DNA (6 >. Fractions containing the low-molecular-weight DNA-binding peak were pooled and dialyzed overnight at 4” against low-salt buffer (LSB) consisting of 20 mM HEPES-NaOH, pH 7.8,20 mM KCl, 0.05% P-mercaptoethanol, 10%glycerol, and 0.1% Triton X-100. All further operations were carried out at 4”. Following dialysis, the DNA-binding pool was loaded onto a 16-ml bed volume column of DNA Sepharose equilibrated with LSB containing 0.05 M KCl, and elution was carried out with LSB containing 0.1 KCl, and 1.5 M KCl. The binding activity in the high-salt eluant was pooled, dialyzed against 0.1 M ammonium bicarbonate, and stored at -70 I The U.S. Government’s right to retain a nonexclufor further analysis. sive royalty-free license in and to the copyright Electrophoretic separation of viral procovering this paper, for governmental purposes, is teins was performed according to a proacknowledged. cedure previously described (12 ). Protein * To whom reprint requests should be addressed.
Murine type C RNA viruses contain a structural component of about 8000 to 10,000 molecular weight (~10) (1, 2). The gag region of the viral genome codes for this component which initially is included in a precursor polyprotein containing the structural proteins ~30, ~15, and p12 (3). Immunochemical and tryptic peptide mapping studies have shown that ~10s isolated from various murine viruses are highly related (4-S). p10 was shown to be the most basic virion protein and to be closely associated with viral RNA (7-9). Subsequent in vitro studies demonstrated the interaction of Rauscher leukemia virus (RLV) p10 with various nucleic acids and indicated a preference for single-stranded DNA and RNA (9, 10). It was also reported that the gag gene precursor polyprotein (Pr70) bound to single-stranded nucleic acid possibly through the p10 component (11). More recently, DNA-binding proteins have been isolated from woolly monkey (SSV-1) and mouse mammary tumor viruses (MMTV), and immunological relatedness has been examined (6,12). In the present study, several DNAbinding proteins have been purified to homogeneity from type C, B, and D retroviruses by affinity chromatography, and
491 004%6S22/80/100491-06$02.00/O
492
SHORT COMMUNICATIONS
was determined according to the method of Lowry et al. (13) using bovine serum albumin as a standard. Standards for electrophoresis included: heavy-chain y-globulin, light-chain y-globulin, ribonuclease, cytochrome c, and trasylol. Two-dimensional analysis of viral proteins was performed according to O’Farrell et aZ. (14). Amino acid analysis was carried out with a Beekman Spinco automatic amino acid analyzer Model lZl(Z5) adapted for high sensitivity (16 ), or on a Durram Model D560 analyzer. The low-molecular-weight DNA-binding proteins purified by affinity chromatography from several retroviruses are represented in Fig. 1. The binding proteins isolated from the type C viruses, RLV, baboon leukemia virus (BaLV), SSV-1, and endogenous cat virus (RD114), each migrated as the fastest virion component (~10) when compared to disrupted whole virus (Fig. 1A). In each of these eases the binding protein migrated in SDS-gels at a position intermediate to trasylol (6000~ and cytochrome c (11,500). The binding protein isolated from the type B retrovirus, MMTV, corresponded to the second fastest migrating virion protein designated as p14 when compared to whole disrupted virus. This protein migrated in SDS-gels at a position between cytochrome c and ribonuclease. Mason-Pfizer monkey virus (MPMV), a type D primate virus, contained a binding protein with a molecular weight similar to MMTV ~14, which corresponded to the second fastest migrating virion protein. The binding protein from Rous sarcoma virus (Prague strain) (Pr-RSV) migrated faster than MMTV p14 with a rate approaching that of cytochrome c, and corresponded to the fastest migrating virion protein. Equine infectious anemia virus (EIAV) and bovine leukemia virus (BLV) contained binding proteins which migrated at positions between trasylol and cytochrome c comparable in size to RLV ~10. The EIAV protein was the fastest migrating virion protein whereas the BLV protein corresponded to the second fastest virion protein. Squirrel monkey retrovirus (SMRV), a type D primate virus, contained two major DNA-binding proteins with molecular weights of approximately 15,000 and 20,000.
The gels of purified binding proteins from BaLV, SSV-1, and RSV also revealed a second larger-molecular-weight component. These bands were cut out from the gels and an amino acid analysis was performed on each. In each case the composition was identical to the one for low-molecular-weight component, showing these to be dimers. The amino acid composition data for each retrovirus nucleic acid-binding protein given in Table 1 was generated by computerized analysis of the raw amino acid analyzer data. The computer was pro~ammed to choose the total number of residues for each protein which would give the least departure from integral values for each residue (16, 17’). The calculation does not require any knowledge of the protein molecular weight but does require good analytical data on a pure protein. There is good correspondence between the amino acid composition of RLV p10 as determined by computer-assisted amino acid analysis and by complete amino acid sequence analysis (Henderson et al., manuscript in preparation). The amino acid compositional data presented in Table 1 strongly suggest that the retrovirus nucleic acid-binding proteins have isoelectric points above pH 7. In all cases except SSV-1, the sum of the basic residues (lysine, histidine, and arginine) is equal to or greater than the sum of the acidic residues (aspartic acid and glutamic acid). The total number of acidic residues is a maximum number which would be reduced by the number of amides in the protein. The amide content was not determined, but in most cases it is probably greater than zero. In the case of RLV ~10, the complete amino acid sequence of the protein reveals five amides (Henderson et al., manuscript in preparation). The cysteine content of the proteins was not determined directly, but the quantities detected in the amino acid analysis of each protein suggested that all the proteins listed in Table 1 contain more than one cysteine. RLV p10 is known to contain three cysteine and Pr-RSV ~12 is known to contain six cysteine residues (18 ). The amino acid composition of the Pr-RSV protein agrees with previous analysis of avian ~12 (19 >except that here the computer
SHORT COMMUNICATIONS
493
-
RSV
Mbnv
Ribonuclease
EIAV
_ Ribonuclease - Cytochrome C
FIG. 1. (A) Numbering lanes from left to right, 1,4,7, and 10 contain the standards heavy- and lightchain y-globulin, ribonucleaae, cytochrome c, and trasylol, respectively. Lane 2 contains RLV whole virus, lane 5, BaLV whole virus, and lanes 3, 6, 8 and 9 contain purified DNA-binding proteins from RLV, BaLV, RD114, and SSV-1, respectively. (B) From left to right lanes 1,4,7, and 10 contain standards, while lanes 25, and 8 contain whole viruses from RSV, MMTV, and EIAV, respectively, and lanes 3, 6, and 9 contain DNA-binding proteins from RSV, MMTV, and EIAV, respectively. (C) Lanes 1,4,7, and 10 contain standards, lanes 2,5, and 8 contain whole virus from MPMV, BoLV, and SMRV, respectively, and Ianes 3,6, and 9 contain DNA-bin~~ proteins from MPMV, BoLV, and SMRV, respectively.
494
SHORT COMMUNICATIONS TABLE 1 AMINO ACID COMPOSITIONS OF RETROVIRUS DNA-BINDING
Amino acid
EIAV
MPMV
Pr-RSV
PROTEINS~
MMTV
BLV
RD114
BaLV
ssv-1
RLV
5 1 6 7 3 4 6 5 5 5 3 0 1 4 1 1
5 1 8 4 1 2 s 5 5 4 2 0 0 3 1 0
57
49
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine VaIine Methionine Isoleucine Leucine Tyrosine Phenylalanine
10 2 3 2 3 4 9 7 11 5 2 0 0 4 1 4
13 5 3 11 2 2 10 8 10 7 2 0 1 2 1 3
7 2 8 7 3 6 10 6 11 6 4 1 2 5 2 1
18 2 5 9 6 IO 12 9 23 6 2 1 I 5 3 4
6 2 4 4 4 3 3 16 7 3 2 1 1 2 1 0
10 1 7 5 5 ;5 4 3 3 0 2 2 1 1
10 1 8 5 3 3 6 6 4 5 4 0 1 2 1 0
Total residues”
67
80
81
116
59
61
59
0 Data expressed as moles of residue per mole of protein. Tryptophan and cysteine were not determined and are omitted from total. The data for each protein are the averaged results of several analysis performed on 24- and 48-hr hydrolysates. The amino acid composition was determined from the raw data by a computer programmed to give the nearest integer minimum molecular weight.
analysis of the primary data indicates a molecular weight of about 10,000 while the previously published compositions were calculated assuming a molecular weight of 12,000. Based upon the DNA affinity isolation procedure employed here and reported sequence homology (201, the avian ~12 protein is shown to be the ~nctional homolog of the murine ~10. The overall composition of the proteins from BLV and EIAV appear quite different from RLV, BaLV, and RD114. A comparison of the percentage of polar amino acids relative to total amino acid content (15) showed that the type C p10 proteins, RD114 (48.8%), RLV (48.2%), and BaLV (4’7.6%), had the highest content of polar amino acids, followed by MPMV (43.7%), SSV-1(37.8%), and MMTV (37.3%). In the case of RLV ~10, the complete amino acid sequence of the protein has been determined (Henderson et al., manuscript in preparation). The amino acid composition as deter~n~d by sequence analysis is in complete agreement with the data given in Table 1. The molecular weight
of RLV p10 as determined by sequence analysis is 6060. The molecular weight of RLV ~10, as determined by its relative mobility in SDS-gel electrophoresis, is 6000 to 7000. Similarly, for all the proteins listed in Table 1, the molecular weights as estimated by SDS-gel electrophoresis are in good agreement with the molecular weights indicated by amino acid composition. The data presented here suggest the following approximate molecular weights for the viral nucleic acid-binding proteins: BaLV (7~0), SSV-1 (7500), RD114 (7~0}, BLV (7000), EIAV (SOOO),MPMV (lO$OO), RSV (10,500), and MMTV (14,000). Since the isolated proteins were mostly of a basic nature, a two-dimensional gel analysis was made on each whole disrupted virus to confirm the presence of a lowmolecular-weight protein with a high isoelectric point relative to the other virion components. As shown in Figs. 2 and 3, each virus contained a basic low-molecularweight protein corresponding closely in molecular weight to that isolated by DNA
SHORT
FIGS. 2 AND 3. Two-dimensional
COMMUNICATIONS
electrophoresis
of retrovirnses.
FIG. 2. (A) BaLV; (B) RLV; (C) MMTV; (D) MPMV; (E) EIAV; (F) BoLV. Acidic part of gel is to the left and basic on the right. The standards to the right of each gel are heavy and light chains of y-globulin, ribonuclease, cytochrome c, and trasylol. FIG. 3. (A) A-MuLV; (B) AKR; (C) FeLV; (D) RSV; (E) REV; (F) SMRV. Acidic part of gel is to the left and basic on the right. The standards on the right of each gel are heavy and light chains of y-globulin, ribonuclease, cytochrome c, and trasylol.
affinity chromatography. Two-dimensional analysis of purified DNA-binding proteins from RLV, BaLV, MMTV, MPMV, and RSV showed each to consist of the most basic low-molecular-weight virion protein. This establishes the presence in all retroviruses of a basic low-molecular-weight protein with an affinity for nucleic acid. The exact function of these proteins in viral maturation remains to be determined.
4. 5.
6. 7. 8.
ACKNOWLEDGMENTS We wish to thank M. Ramsburg, R. Snead, and G. Smythers for excellent technical assistance. This work was supported by Contract NOl-CO-75380 with the National Cancer Institute, NIH, Bethesda, Maryland 20205. REFERENCES 1. AUGUST, J. T., E., GILDEN, Virology 60, 2. NOWINSKI, R. N. H., and (1972). 8. BARBACID, M.,
BOLOGNESI, D. P., FLEISSNER, R. V., and NOWINSKI, R. C., 1595 (1974). C., FLEISSNER, E., SARKAR, AOKI, T., J. Viral. 9, 359-366
9. IO. 11. 12. 13.
14. 15.
STEPHENSON, J. R. and AARON-
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