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Equine infectious anemia virus, a putative lentivirus, contains polypeptides analogous to prototype-C oncornaviruses
VIROLOGY
lo:,
Equine
BHARAT
520-52<5
(1980)
Infectious
Anemia Virus, a Putative Analogous to Prototype-C
PAREKH,* Departments
CHARLES
of *Rioch,emistry Baton
J. ISSEL,t
Lentivirus, Contains Oncornaviruses AND
RONALD
and tVeterin,ary Science, Louisiana Rouge, Louisiana 7’0803
Accepted
August
Polypeptides
C. MONTELARO*,’ State
University,
19, 1980
The polypeptide composition of purified radioactive leucine or glucosamine-labeled equine infectious anemia virus (EIAV) was investigated using guanidine hydrochloride gel filtration (GHCI-GF) and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to Friend murine leukemia virus (FLV), a prototype-C oncornavirus. The apparent molecular weights and relative abundance of each EIAV polypeptide were calculated. The results of these studies indicate that EIAV contains four major nonglycosylated proteins (~26, ~15, pll, and p9) and two glycoproteins (gp90 and gp45), which together account for greater than 95% of the total virion protein. Four minor polypeptides of unknown significance were also detected reproducibly. EIAV gp90 appears to be the more heavily glycosylated polypeptide. while the gp45 component aggregates in 6 M GHCl, evidently reflecting a hydrophobic character. No disulfide linkages were detected between the EIAV glycoproteins. These observations demonstrate for the first time that EIAV contains polypeptides analogous to prototype-C oncornaviruses, such as FLV. However, the demonstrated serological unrelatedness between EIAV and FLV was reflected in biochemical differences in protein apparent molecular weights and by the resistance of EIAV nonglycosylated proteins to dissociation in GHCl, a property shared by visna virus.
Equine infectious anemia virus (EIAV) has recently been proposed as a member of the Lentivirus subfamily of Retroviridae which also includes visna, progressive pneumonia and maedi viruses (1-z). Like prototype retroviruses, EIAV matures by budding from cytoplasmic membranes (4, .i), displays a complex morphology characteristic of type-C viruses (6), contains a reverse transcriptase enzyme (7), and a high-molecular-weight (60-70 S) RNA genome composed of subunits (8). Some ultrastructural studies, however, suggest that EIAV more closely resembles visna virus than any of the leukemia-sarcoma viruses (6). In addition, serologic comparisons have failed to detect any relatedness between EIAV and a number of other retroviruses (1, 2). These observations have led to the suggestion that the polypeptide composition of EIAV may be distinct from type-C retroviruses (1, 9). Preliminary descriptions of EIAV polypeptide composition have been contradictory and ’ Author
to whom
reprint
requests
0042-6822/80/160526-07$02.00/O Copyright All rights
F 1980 by Academlr Press. Inc. of rqn-nduetion in any form rrserved.
should
be sent. 520
thus have been unable to resolve the exact relationship of EIAV to other retroviruses. Previous experience with retroviruses has consistently emphasized the need to utilize several analytical techniques in identifying virion structural polypeptides. Two procedures, guanidine hydrochloride gel filtration (GHCl-GF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) have been particularly effective in elucidating retrovirus polypeptide composition and have therefore been chosen as a basis for deriving a nomenclature for virion polypeptides (10). Yet the analysis of EIAV polypeptides by both procedures remains heretofore unreported. Thus we have analyzed the polypeptides of purified radioactive leucineand glucosamine-labeled EIAV by SDSPAGE and GHCl-GF to obtain the apparent molecular weights and relative abundance of the virion structural components. For these studies, the cell-adapted Wyoming strain of EIAV (11) was propagated in equine dermal cells CCL-57 or in equine kidney cells grown in sterile
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FIG. 1. Analysis of purified EIAV polypeptides. (A) GHCI-GF of [‘4C]leucine EIAV (--) with [RH]leucine FLV (- - -) as a marker. All procedures for sample preparation, chromatography, and radioactivity analysis have been described in detail (13). The FLV marker proteins (gp71, ~30, ~16. ~12, and ~10) are designated in italics; the EIAV proteins (~26, ~15, pll, and p9) are labeled with block letters. V, represents the void volume fractions, while DNP-alanine serves as the dye marker for the total column volume. (B) SDS-PAGE of [“Clleucine EIAV (--) and L:‘H]leucine FLV (- - -) as described previously (18). As in (A), FLV proteins are designated in italics (gp71, ~30, p15E. p15C, ~12, p12E, and ~10): EIAV proteins are labeled in block numbers (gp90, ~70, ~61, gp45, ~30, ~23, ~15, pll, and ~9). (C) GHCl-GF of [“Hlglucosamine EIAV under conditions identical to those employed above in (A). (D) SDS-PAGE of [“Hlglucosamine EIAV under conditions identical to those described in (B)
Eagle’s minimal essential medium with the addition of 25 mM Hepes buffer, antibiotics and 10% virus-screened bovine fetal serum as described previously (12). Radioactively labeled EIAV was propagated in leucinefree medium containing either approximately 25 $Zi/ml of [“Hlleucine (New England Nuclear, NET-135H) or 2.5 pCi/ml of [“Clleucine (New England Nuclear, NEC-279E) or in medium reduced to ‘/l&h the normal glucose concentration and supplemented with 25 $Xml [3H]glucosamine (New England Nuclear, NET-190). After 24-hr intervals of growth in the radioactive medium, the virus-containing media was centrifuged to remove cellular debris and the virus purified from the supernatant fluid essentially as described for Friend murine leukemia virus (FLV) (13).
Purified radioactive virus was then analyzed by GHCl-GF and by SDS-PAGE as described previously (13, 14). The chromatographic radioactivity profile of [ “C]leucine-labeled EIAV is presented in Fig. lA, while the electrophoretic radioactivity profile of the same virus preparation is presented in Fig. 1R. In both procedures, [“Hlleucine-labeled FLV was analyzed simultaneously for comparative purposes and as a molecular weight calibration standard. The apparent molecular weights and relative abundance of each EIAV polypeptide resolved by the two procedures is summarized in Table 1. The chromatographic analysis of the radioactive EIAV reveals six distinct peaks of radioactivity (Fig. 1A). As observed with other oncornaviruses, the first protein(s) to
522
SHORT
Polypeptide” Major gP90 gp45
~26 P15 pll P9 Minor p70
~‘31 p30 p23
Percentage total radioactivity”
6.0 3.0 45.0 28.<5 5.5 7.5
1.0 1.0 1.5 1.0
COMMUNICATIONS
Apparent GHCl-GF
74,000 ~100,000 26,000 15,000 11,000 9,000
-
molecular
xeight’
SDS-
PAGE
Estimated polypeptide chains per virion”
90.000 45,000 26,000 15,000 12,000 10.000
300 300 6900 7600 2000 3400
70,000 60,000 30,000 23,000
60 70 200 170
” Protein nomenclature based on recommendations of August et rtl. (IO). h Calculated from the data presented in Fig. 1 averaging percentage cpm from GHCI-GF ’ Calculated by GHCl-GF and SDS-PAGE using FLV as a molecular weight standard ” Calculated assuming that virion protein mass is 4 x 10’ daltons as described by Davis
elute from GHCl-GF are contained in the void volume fractions of the column indicating an apparent molecular weight of at least 100,000. The next EIAV protein elutes as a relatively broad peak with an apparent molecular weight of about 74,000. This component is followed by the major EIAV structural polypeptide (~26) which displays a molecular weight of 26,000 and, in succession, by three additional components of 15,000 (p15), 11,000 (~11) and 9000 (p9) molecular weight, respectively. A comparison of the respective polypeptide profiles of FLV and EIAV shown in Fig. 1A indicates that the two viruses contain remarkably similar structural components. However, two differences can be reproducibly noted between the profiles of EIAV and FLV: (i) a larger percentage of the total EIAV radioactivity elutes in the void volume fractions relative to that observed with FLV and (ii) the EIAV and FLV polypeptides display slightly different apparent molecular weights (Fig. lA, Table 1). SDS-PAGE analysis of [14C]leucineEIAV (Fig. 1B) also reveals four major lowmolecular-weight polypeptides (~26, ~15, pll, and p9) and further emphasizes their variance in apparent molecular weight
and SDS-PAGE, as described (1.j). and Rueckert (1:;).
from the corresponding proteins of [“HIleucine-FLV. In addition to the four major EIAV polypeptides, SDS-PAGE reproducibly resolves several high-molecularweight components. These include two polypeptides of 90,000 (gp90) and 45,000 (gp45) molecular weight which are glycosylated (see below) and four minor nonglycosylated proteins designated as ~70, ~61, ~30, and p23 (Table 1). It is well established that certain oncornavirus proteins (e.g., RSV pl5 and ~12) display different apparent molecular weights when analyzed by SDS- PAGE and by GHCl-GF (IO). Thus to establish a definitive correlation between the EIAV proteins resolved by these two analytical systems, each radioactive component resolved by GHCl-GF of [“Hlleucine EIAV (cf. Fig. 1A) was isolated and subjected to high-resolution SDS-PAGE. The results of this experiment are presented in Fig. 2 and can be compared to the electrophoretic profile of purified EIAV shown in Fig. 1B. The data in Fig. 2 demonstrate that the GHCl-GF components designated as EIAV ~26, ~15, pll, and p9 (Fig. 1A) contain a single homogeneous peak of radioactivity upon analysis by SDS-PAGE (Figs. 2C-F, respectively). In contrast, the void volume fractions from
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FIG. 2. SDS-PAGE of the individual structural components isolated by GHCl-GF of [“Hlleucine EIAV as described in Fig. 1A. Samples of each radioactive peak fractionated by GHCl-GF (cf. Fig. 1A) were isolated and prepared for SDS-PAGE analysis ( 23). (A)(F) correspond to the six radioactive components in the order of their elution from the gel filtration column.
GHCl-GF reveal a complex profile in SDSPAGE (Fig. 2A) including the major proteins designated gp90, gp45, ~26, and ~15, as well as certain high-molecular-weight
523
minor polypeptides. The presence of large quantities of p26 and p15 in the void volume fractions is surprising and suggests that even treatment of the virus with 8 M GHCl at 100” fails to completely disrupt the intermolecular interactions between some of the EIAV polypeptides. The presence of gp90 in the void volume may be explained by the fact that gp90 elutes immediately after the void volume fractions of the column (Fig. ZB), along with several of the minor highmolecular-weight virion components. On the other hand, gp45 elutes exclusively in the void volume fractions, apparently in an aggregated state analogous to murine p15E/ p12E and avian gp35 (13), as described below. To identify which of the above polypeptides were glycosylated, [3H]glucosamineEIAV was prepared in equine kidney cells, purified, and subjected to GHCl-GF (Fig. 1C) and SDS-PAGE (Fig. 1D) as described above for radioactive leucine-labeled EIAV. Once again the proteins resolved by both procedures were correlated by isolating each GHCl-GF component and analyzing the isolated material by high-resolution SDS-PAGE (Fig. 3). Both the chromatographic profile of [3H]glucosamine-EIAV (Fig. 1C) and the electrophoretic profile of the virus (Fig. 1D) reveal two distinct glycoproteins, designated on the basis of their apparent molecular weight as gp90 and gp45. The void volume fractions from the GHCl-GF analysis (Fig. lC> reveals some of the virion gp90 and all of the virion gp45 upon analysis by SDS-PAGE (Fig. 3A). In contrast, the second glycosylated chromatographic peak to elute from the GHCl column contains only radioactive gp90 as demonstrated by SDS-PAGE (Fig. 3B). Quantitation of the data in Fig. 1D reveals that gp90 contains 80% of the carbohydrate radiolabel, while gp45 contains only 20% of the label. Since gp90 and gp45 are evidently present in the virus in similar amounts (Table 1, 300 copies per virion), gp90 would appear to be the more heavily glycosylated protein analogous to murine gp71 and avian gp85, while EIAV gp45 would appear to be analogous to the hydrophobic murine p15EiplZE and avian gp35. Nonreducing SDS-PAGE analysis of [3H]glucosamineEIAV failed to detect any disulfide link-
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ages between the virion glycoproteins (data not shown). Some descriptions of EIAV polypeptide composition have been reported previously, but have been inconsistent. The major nonglycosylated protein of EIAV, which is employed in standard immunodiagnostic tests, is evidently a group specific antigen with a molecular weight of 25,000-29,000 (8, 15, IS), although one report claims a molecular weight of only 7600 (I?). Cheevers et al. (18) reported further that 80% of EIAV structural protein is accounted for by five polypeptides, including two nonglycosylated components (p29 and ~13) which comprise one-half of the virion protein and three glycoproteins (gp77179, gp64, and gp40). They also describe eight or nine minor polypeptides of unknown significance. Ishizaki et ol. (16) detected three major nonglycosylated proteins (~25, ~14, and pll), but only two glycoproteins (gp80 and gp40). While both of the above investigators employed SDS-PAGE as the sole analytical procedure, Charman ct 01. (19) used isoelectric focusing to detect four major nonglycosylated, components [p25, ~12, p10 (acidic), and ~10 (basic)], but made no statements concerning virion glycoproteins. The experiments presented here, which employ the complementary high resolution SDS-PAGE and GHCl-GF analytical techniques, demonstrate clearly that purified EIAV contains four major nonglycosylated components (~26, ~15, pll, and p9) and two glycoproteins (gp90 and gp45). Moreover the quantitation summarized in Table 1 reveals that the relative amount of each EIAV component is similar to that determined previously for prototype-C avian and murine oncornaviruses (14). The remarkable similarity between EIAV and FLV polypeptide profiles further strengthens the relationship between the Oncornavirus and Lentivirus subfamilies of Retroviridae. Now that the polypeptides of EIAV have been identified, experiments are in progress to localize these components in the intact virion and to compare this localization to the proposed model for type-C virion structure (14). Although we have thus far emphasized the similarities between EIAV and FLV
A
50
100 150 DISTANCE MIGRATED (mm)
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FIG. 3. SDS-PAGE; analysis of the glycoprotein components isolated by GHCI-GF of purified [“HIglucosamine labeled EIAV (cf. Fig. IC). (A) and (B) correspond to the two radioactivity peaks resolved by gel filtration in the order of their elution from the column.
polypeptides, several important differences should also be noted. In contrast to FLV where disulfide linkages between its major surface components, gp71 and pl5E, have been detected (13), disulfide linkages were not detectable between EIAV glycoproteins. In this regard, EIAV resembles other Lentiviruses, such as visna (20) and type-B oncornaviruses such as murine mammary tumor virus (21, %), which evidently lack disulfide linkages between structural glycoproteins. Moreover, the apparent molecular weights of EIAVpolypeptides differ from those of FLV, perhaps indicating biochemical differences (e.g., amino acid content) which are responsible for the serological unrelatedness of these viruses. One final intriguing difference between EIAV and FLV is that the major non-glycosylated proteins of the EIAV are incompletely dissociated by rigorous treatments with GHCI, whereas FLV is readily dissociated by the same treatment. In fact, we have observed that as much as 50% of the total virus protein will elute in the void volume fractions obtained from GHCI-GF if the EIAV is not treated with 8 M GHCI at 100” for 3-4 min prior to incubation at
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45”. This apparent resistance to dissociation by GHCI treatment has been previously reported for visna virus (23) and may be a characteristic of the Lentiviruses. Detailed biochemical characterizations such as amino acid composition and sequence of EIAV polypeptides may help to elucidate the basis for these unique solubility properties. Some biochemical characterization of other Lentiviruses have been reported recently (20, 23, 24). Lin (25) employed GHCI-GF and SDS-PAGE procedures to analyze the polypeptide composition of purified, radioactive visna virus. These studies indicate that visna contains about 25 proteins ranging in molecular weight from about 7000 to 110,000, including two glycoproteins (gp175 and gp115). A eomparison of the visna polypeptide profiles and polypeptide stoichiometry reported by Lin reveals little resemblance to the EIAV polypeptides described in this report. Other investigators have suggested less complex polypeptide compositions for visna virus. For example, Bruns and Frenzel (20) employed isoelectric focusing and gel filtration to isolate three homogeneous proteins designated as ~15, ~24, and gp70, while Scott et al. (24) discuss only one glycoprotein (gp135) and three nonglycosylated polypeptides (~30, ~16, and ~14). Although it is tempting to compare EIAV polypeptides with these latter characterizations of visna virus, one cannot accomplish the comparison since neither of these papers include a complete cataloging of all virion proteins detected by their analytical procedures. Analysis of other Lentiviruses such as visna by the techniques employed here with EIAV would serve to clarify further the relationship of the various subfamilies of Retroviruses and ident.ify the correct members of each grouping. ACKNOWLEDGMENTS The authors acknowledge the excellent technical assistance of Helen Dvorin and Nancy Lohrey and the advice of Dr. Grace Amborski for optimal cell culture propagation of EIAV. This study was supported in part by the Louisiana Agricultural Experiment Station, by USDA Cooperative Agreement No. 1214-100-9068(45), and by National Institutes of Health Biomedical Research Development Grants l-508RR09087-01 and 5-507-RR07039-08.
525 REFERENCES
1. CHARMAN, H. P., BLADEN, S., GILDEN, R. V., and COGGINS, L., b. b’irol. 19, 1073-10’79 (1976). 2 STOWRING, L., HAASE, A. T., and CHARMAN, H. P., J. Viral. 29, 523-528 (1979). 3. &EL, C. J., and COGGINS, L., JAVMA 174, 72’7-733 (1979). 4. MCCONNELL, M. B., KATADA, M., and McCONNELL, S., Amer. d. Vet. Res. 38, 2067-2069 (1977). 5. WEILAND, F., MATHEKA, H. D., COGGINS, L., and HARTNER, D., Arch. Viral. 55,335-340 (1977). 6. GONDA, M. A., CHARMAN, H. P., WALKER, J. L., and COCGINS, L., Am. b. Vet. Res. 39,431-740 (1978). 7. ARCHER, B. G., CRAU’FORD, T. B., MCGUIRE, T. C., and FRAZIER, M. E., J. Vi&. 22, 16-22 (1077). 8. CHEEVERS, W. P., ARCHER, B. G., and CRAWFORD, T. B., J. Viral. 24, 289-297 (1977). 9. RICE, N. R., SIMEK, S., RYDER, 0. A., and COGGINS, L. J. vird. 26, 577-583 (1978). 10. AUGUST, J. T., BOLOGNESI, D. P., FLEISSNER, E., GILDEN, R. V., and NOWINSKI, R. C., Virology 60, 595-601 (1974). Il. MALMQUIST, W. A., BARNETT, D., and BECVAR, C. S., Arch. Gesamte Virusforsch 42, 361-370 (1973). 12. AMBORSKI, G. F., JEFFERS, G., AMBORSKI, R. L., and ISSEL, C. J., Amer. J. Vet. Res. 40, 302-304 (1979). 1Y. MONTELARO, R. C., SULLIVAN, and BOLOGNESI. D. P., Virology 84, 19-31 (1978). R. C., and BOLOGNESI, D. P., Adv. 14. MO~XTELARO, Cmcer Res. 28, 63-89 (1978). 15. BARTA, V. and ISSEL, C. J., Amer. J. Vet. Res. 39, 1856- 1857 (1978). 16. ISHIZAKI, R., GREEN, R. W., and BOLOGNESI, D. P., Intervirology 9, 286-294 (1978). 17. HART, L. T., BRAYMER, H. D., and LARSON, A. D., Prep. Riochew. 6, 193-211 (1976). 18. CHEEVERS, W. P., ACKLEY, C. M., and CRAWFORD, T. B., J. Viral. 28, 997-1001 (1978). 19. CHARMAN, H., LONG, C.. and COGGINS, L., Irlfect. Immun. 23, 472-478 (1979). 20. BRIJNS, M. and FRENZEL. B., Virology 97, 207211 (1979). il. YAGI, M. J., and COMPANS, R. W., tirology 76, 751-766 (1977). 22. MARCUS, S. L., SMITH, S. W., RACEVSKIS, J., and SARKAR, N. H., Virology 86, 398-412 (1978). 23. LIN, F. H., /. Viral. 25, 204-214 (1978). 24. SCOTT, J. V., STOWRING, L., HAASE, A. T., NARAYAN, 0.. and VIGNE. R., Cell 18. 321x27 (1979). 2.5. DAVIS, N. S., and RUECKERT, R. R., J. Viro/. 10, 1010-1020 (1972).
lo:,
Equine
BHARAT
520-52<5
(1980)
Infectious
Anemia Virus, a Putative Analogous to Prototype-C
PAREKH,* Departments
CHARLES
of *Rioch,emistry Baton
J. ISSEL,t
Lentivirus, Contains Oncornaviruses AND
RONALD
and tVeterin,ary Science, Louisiana Rouge, Louisiana 7’0803
Accepted
August
Polypeptides
C. MONTELARO*,’ State
University,
19, 1980
The polypeptide composition of purified radioactive leucine or glucosamine-labeled equine infectious anemia virus (EIAV) was investigated using guanidine hydrochloride gel filtration (GHCI-GF) and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and compared to Friend murine leukemia virus (FLV), a prototype-C oncornavirus. The apparent molecular weights and relative abundance of each EIAV polypeptide were calculated. The results of these studies indicate that EIAV contains four major nonglycosylated proteins (~26, ~15, pll, and p9) and two glycoproteins (gp90 and gp45), which together account for greater than 95% of the total virion protein. Four minor polypeptides of unknown significance were also detected reproducibly. EIAV gp90 appears to be the more heavily glycosylated polypeptide. while the gp45 component aggregates in 6 M GHCl, evidently reflecting a hydrophobic character. No disulfide linkages were detected between the EIAV glycoproteins. These observations demonstrate for the first time that EIAV contains polypeptides analogous to prototype-C oncornaviruses, such as FLV. However, the demonstrated serological unrelatedness between EIAV and FLV was reflected in biochemical differences in protein apparent molecular weights and by the resistance of EIAV nonglycosylated proteins to dissociation in GHCl, a property shared by visna virus.
Equine infectious anemia virus (EIAV) has recently been proposed as a member of the Lentivirus subfamily of Retroviridae which also includes visna, progressive pneumonia and maedi viruses (1-z). Like prototype retroviruses, EIAV matures by budding from cytoplasmic membranes (4, .i), displays a complex morphology characteristic of type-C viruses (6), contains a reverse transcriptase enzyme (7), and a high-molecular-weight (60-70 S) RNA genome composed of subunits (8). Some ultrastructural studies, however, suggest that EIAV more closely resembles visna virus than any of the leukemia-sarcoma viruses (6). In addition, serologic comparisons have failed to detect any relatedness between EIAV and a number of other retroviruses (1, 2). These observations have led to the suggestion that the polypeptide composition of EIAV may be distinct from type-C retroviruses (1, 9). Preliminary descriptions of EIAV polypeptide composition have been contradictory and ’ Author
to whom
reprint
requests
0042-6822/80/160526-07$02.00/O Copyright All rights
F 1980 by Academlr Press. Inc. of rqn-nduetion in any form rrserved.
should
be sent. 520
thus have been unable to resolve the exact relationship of EIAV to other retroviruses. Previous experience with retroviruses has consistently emphasized the need to utilize several analytical techniques in identifying virion structural polypeptides. Two procedures, guanidine hydrochloride gel filtration (GHCl-GF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) have been particularly effective in elucidating retrovirus polypeptide composition and have therefore been chosen as a basis for deriving a nomenclature for virion polypeptides (10). Yet the analysis of EIAV polypeptides by both procedures remains heretofore unreported. Thus we have analyzed the polypeptides of purified radioactive leucineand glucosamine-labeled EIAV by SDSPAGE and GHCl-GF to obtain the apparent molecular weights and relative abundance of the virion structural components. For these studies, the cell-adapted Wyoming strain of EIAV (11) was propagated in equine dermal cells CCL-57 or in equine kidney cells grown in sterile
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FIG. 1. Analysis of purified EIAV polypeptides. (A) GHCI-GF of [‘4C]leucine EIAV (--) with [RH]leucine FLV (- - -) as a marker. All procedures for sample preparation, chromatography, and radioactivity analysis have been described in detail (13). The FLV marker proteins (gp71, ~30, ~16. ~12, and ~10) are designated in italics; the EIAV proteins (~26, ~15, pll, and p9) are labeled with block letters. V, represents the void volume fractions, while DNP-alanine serves as the dye marker for the total column volume. (B) SDS-PAGE of [“Clleucine EIAV (--) and L:‘H]leucine FLV (- - -) as described previously (18). As in (A), FLV proteins are designated in italics (gp71, ~30, p15E. p15C, ~12, p12E, and ~10): EIAV proteins are labeled in block numbers (gp90, ~70, ~61, gp45, ~30, ~23, ~15, pll, and ~9). (C) GHCl-GF of [“Hlglucosamine EIAV under conditions identical to those employed above in (A). (D) SDS-PAGE of [“Hlglucosamine EIAV under conditions identical to those described in (B)
Eagle’s minimal essential medium with the addition of 25 mM Hepes buffer, antibiotics and 10% virus-screened bovine fetal serum as described previously (12). Radioactively labeled EIAV was propagated in leucinefree medium containing either approximately 25 $Zi/ml of [“Hlleucine (New England Nuclear, NET-135H) or 2.5 pCi/ml of [“Clleucine (New England Nuclear, NEC-279E) or in medium reduced to ‘/l&h the normal glucose concentration and supplemented with 25 $Xml [3H]glucosamine (New England Nuclear, NET-190). After 24-hr intervals of growth in the radioactive medium, the virus-containing media was centrifuged to remove cellular debris and the virus purified from the supernatant fluid essentially as described for Friend murine leukemia virus (FLV) (13).
Purified radioactive virus was then analyzed by GHCl-GF and by SDS-PAGE as described previously (13, 14). The chromatographic radioactivity profile of [ “C]leucine-labeled EIAV is presented in Fig. lA, while the electrophoretic radioactivity profile of the same virus preparation is presented in Fig. 1R. In both procedures, [“Hlleucine-labeled FLV was analyzed simultaneously for comparative purposes and as a molecular weight calibration standard. The apparent molecular weights and relative abundance of each EIAV polypeptide resolved by the two procedures is summarized in Table 1. The chromatographic analysis of the radioactive EIAV reveals six distinct peaks of radioactivity (Fig. 1A). As observed with other oncornaviruses, the first protein(s) to
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Polypeptide” Major gP90 gp45
~26 P15 pll P9 Minor p70
~‘31 p30 p23
Percentage total radioactivity”
6.0 3.0 45.0 28.<5 5.5 7.5
1.0 1.0 1.5 1.0
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Apparent GHCl-GF
74,000 ~100,000 26,000 15,000 11,000 9,000
-
molecular
xeight’
SDS-
PAGE
Estimated polypeptide chains per virion”
90.000 45,000 26,000 15,000 12,000 10.000
300 300 6900 7600 2000 3400
70,000 60,000 30,000 23,000
60 70 200 170
” Protein nomenclature based on recommendations of August et rtl. (IO). h Calculated from the data presented in Fig. 1 averaging percentage cpm from GHCI-GF ’ Calculated by GHCl-GF and SDS-PAGE using FLV as a molecular weight standard ” Calculated assuming that virion protein mass is 4 x 10’ daltons as described by Davis
elute from GHCl-GF are contained in the void volume fractions of the column indicating an apparent molecular weight of at least 100,000. The next EIAV protein elutes as a relatively broad peak with an apparent molecular weight of about 74,000. This component is followed by the major EIAV structural polypeptide (~26) which displays a molecular weight of 26,000 and, in succession, by three additional components of 15,000 (p15), 11,000 (~11) and 9000 (p9) molecular weight, respectively. A comparison of the respective polypeptide profiles of FLV and EIAV shown in Fig. 1A indicates that the two viruses contain remarkably similar structural components. However, two differences can be reproducibly noted between the profiles of EIAV and FLV: (i) a larger percentage of the total EIAV radioactivity elutes in the void volume fractions relative to that observed with FLV and (ii) the EIAV and FLV polypeptides display slightly different apparent molecular weights (Fig. lA, Table 1). SDS-PAGE analysis of [14C]leucineEIAV (Fig. 1B) also reveals four major lowmolecular-weight polypeptides (~26, ~15, pll, and p9) and further emphasizes their variance in apparent molecular weight
and SDS-PAGE, as described (1.j). and Rueckert (1:;).
from the corresponding proteins of [“HIleucine-FLV. In addition to the four major EIAV polypeptides, SDS-PAGE reproducibly resolves several high-molecularweight components. These include two polypeptides of 90,000 (gp90) and 45,000 (gp45) molecular weight which are glycosylated (see below) and four minor nonglycosylated proteins designated as ~70, ~61, ~30, and p23 (Table 1). It is well established that certain oncornavirus proteins (e.g., RSV pl5 and ~12) display different apparent molecular weights when analyzed by SDS- PAGE and by GHCl-GF (IO). Thus to establish a definitive correlation between the EIAV proteins resolved by these two analytical systems, each radioactive component resolved by GHCl-GF of [“Hlleucine EIAV (cf. Fig. 1A) was isolated and subjected to high-resolution SDS-PAGE. The results of this experiment are presented in Fig. 2 and can be compared to the electrophoretic profile of purified EIAV shown in Fig. 1B. The data in Fig. 2 demonstrate that the GHCl-GF components designated as EIAV ~26, ~15, pll, and p9 (Fig. 1A) contain a single homogeneous peak of radioactivity upon analysis by SDS-PAGE (Figs. 2C-F, respectively). In contrast, the void volume fractions from
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FIG. 2. SDS-PAGE of the individual structural components isolated by GHCl-GF of [“Hlleucine EIAV as described in Fig. 1A. Samples of each radioactive peak fractionated by GHCl-GF (cf. Fig. 1A) were isolated and prepared for SDS-PAGE analysis ( 23). (A)(F) correspond to the six radioactive components in the order of their elution from the gel filtration column.
GHCl-GF reveal a complex profile in SDSPAGE (Fig. 2A) including the major proteins designated gp90, gp45, ~26, and ~15, as well as certain high-molecular-weight
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minor polypeptides. The presence of large quantities of p26 and p15 in the void volume fractions is surprising and suggests that even treatment of the virus with 8 M GHCl at 100” fails to completely disrupt the intermolecular interactions between some of the EIAV polypeptides. The presence of gp90 in the void volume may be explained by the fact that gp90 elutes immediately after the void volume fractions of the column (Fig. ZB), along with several of the minor highmolecular-weight virion components. On the other hand, gp45 elutes exclusively in the void volume fractions, apparently in an aggregated state analogous to murine p15E/ p12E and avian gp35 (13), as described below. To identify which of the above polypeptides were glycosylated, [3H]glucosamineEIAV was prepared in equine kidney cells, purified, and subjected to GHCl-GF (Fig. 1C) and SDS-PAGE (Fig. 1D) as described above for radioactive leucine-labeled EIAV. Once again the proteins resolved by both procedures were correlated by isolating each GHCl-GF component and analyzing the isolated material by high-resolution SDS-PAGE (Fig. 3). Both the chromatographic profile of [3H]glucosamine-EIAV (Fig. 1C) and the electrophoretic profile of the virus (Fig. 1D) reveal two distinct glycoproteins, designated on the basis of their apparent molecular weight as gp90 and gp45. The void volume fractions from the GHCl-GF analysis (Fig. lC> reveals some of the virion gp90 and all of the virion gp45 upon analysis by SDS-PAGE (Fig. 3A). In contrast, the second glycosylated chromatographic peak to elute from the GHCl column contains only radioactive gp90 as demonstrated by SDS-PAGE (Fig. 3B). Quantitation of the data in Fig. 1D reveals that gp90 contains 80% of the carbohydrate radiolabel, while gp45 contains only 20% of the label. Since gp90 and gp45 are evidently present in the virus in similar amounts (Table 1, 300 copies per virion), gp90 would appear to be the more heavily glycosylated protein analogous to murine gp71 and avian gp85, while EIAV gp45 would appear to be analogous to the hydrophobic murine p15EiplZE and avian gp35. Nonreducing SDS-PAGE analysis of [3H]glucosamineEIAV failed to detect any disulfide link-
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ages between the virion glycoproteins (data not shown). Some descriptions of EIAV polypeptide composition have been reported previously, but have been inconsistent. The major nonglycosylated protein of EIAV, which is employed in standard immunodiagnostic tests, is evidently a group specific antigen with a molecular weight of 25,000-29,000 (8, 15, IS), although one report claims a molecular weight of only 7600 (I?). Cheevers et al. (18) reported further that 80% of EIAV structural protein is accounted for by five polypeptides, including two nonglycosylated components (p29 and ~13) which comprise one-half of the virion protein and three glycoproteins (gp77179, gp64, and gp40). They also describe eight or nine minor polypeptides of unknown significance. Ishizaki et ol. (16) detected three major nonglycosylated proteins (~25, ~14, and pll), but only two glycoproteins (gp80 and gp40). While both of the above investigators employed SDS-PAGE as the sole analytical procedure, Charman ct 01. (19) used isoelectric focusing to detect four major nonglycosylated, components [p25, ~12, p10 (acidic), and ~10 (basic)], but made no statements concerning virion glycoproteins. The experiments presented here, which employ the complementary high resolution SDS-PAGE and GHCl-GF analytical techniques, demonstrate clearly that purified EIAV contains four major nonglycosylated components (~26, ~15, pll, and p9) and two glycoproteins (gp90 and gp45). Moreover the quantitation summarized in Table 1 reveals that the relative amount of each EIAV component is similar to that determined previously for prototype-C avian and murine oncornaviruses (14). The remarkable similarity between EIAV and FLV polypeptide profiles further strengthens the relationship between the Oncornavirus and Lentivirus subfamilies of Retroviridae. Now that the polypeptides of EIAV have been identified, experiments are in progress to localize these components in the intact virion and to compare this localization to the proposed model for type-C virion structure (14). Although we have thus far emphasized the similarities between EIAV and FLV
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FIG. 3. SDS-PAGE; analysis of the glycoprotein components isolated by GHCI-GF of purified [“HIglucosamine labeled EIAV (cf. Fig. IC). (A) and (B) correspond to the two radioactivity peaks resolved by gel filtration in the order of their elution from the column.
polypeptides, several important differences should also be noted. In contrast to FLV where disulfide linkages between its major surface components, gp71 and pl5E, have been detected (13), disulfide linkages were not detectable between EIAV glycoproteins. In this regard, EIAV resembles other Lentiviruses, such as visna (20) and type-B oncornaviruses such as murine mammary tumor virus (21, %), which evidently lack disulfide linkages between structural glycoproteins. Moreover, the apparent molecular weights of EIAVpolypeptides differ from those of FLV, perhaps indicating biochemical differences (e.g., amino acid content) which are responsible for the serological unrelatedness of these viruses. One final intriguing difference between EIAV and FLV is that the major non-glycosylated proteins of the EIAV are incompletely dissociated by rigorous treatments with GHCI, whereas FLV is readily dissociated by the same treatment. In fact, we have observed that as much as 50% of the total virus protein will elute in the void volume fractions obtained from GHCI-GF if the EIAV is not treated with 8 M GHCI at 100” for 3-4 min prior to incubation at
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45”. This apparent resistance to dissociation by GHCI treatment has been previously reported for visna virus (23) and may be a characteristic of the Lentiviruses. Detailed biochemical characterizations such as amino acid composition and sequence of EIAV polypeptides may help to elucidate the basis for these unique solubility properties. Some biochemical characterization of other Lentiviruses have been reported recently (20, 23, 24). Lin (25) employed GHCI-GF and SDS-PAGE procedures to analyze the polypeptide composition of purified, radioactive visna virus. These studies indicate that visna contains about 25 proteins ranging in molecular weight from about 7000 to 110,000, including two glycoproteins (gp175 and gp115). A eomparison of the visna polypeptide profiles and polypeptide stoichiometry reported by Lin reveals little resemblance to the EIAV polypeptides described in this report. Other investigators have suggested less complex polypeptide compositions for visna virus. For example, Bruns and Frenzel (20) employed isoelectric focusing and gel filtration to isolate three homogeneous proteins designated as ~15, ~24, and gp70, while Scott et al. (24) discuss only one glycoprotein (gp135) and three nonglycosylated polypeptides (~30, ~16, and ~14). Although it is tempting to compare EIAV polypeptides with these latter characterizations of visna virus, one cannot accomplish the comparison since neither of these papers include a complete cataloging of all virion proteins detected by their analytical procedures. Analysis of other Lentiviruses such as visna by the techniques employed here with EIAV would serve to clarify further the relationship of the various subfamilies of Retroviruses and ident.ify the correct members of each grouping. ACKNOWLEDGMENTS The authors acknowledge the excellent technical assistance of Helen Dvorin and Nancy Lohrey and the advice of Dr. Grace Amborski for optimal cell culture propagation of EIAV. This study was supported in part by the Louisiana Agricultural Experiment Station, by USDA Cooperative Agreement No. 1214-100-9068(45), and by National Institutes of Health Biomedical Research Development Grants l-508RR09087-01 and 5-507-RR07039-08.
525 REFERENCES
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