Proc. Natl. Acad. Sci. USA Vol. 75, No. 8, pp. 3644-3648, August 1978 Biochemistry
Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces (tumor virus envelope protein/gp70/endogenous tumor virus proteins/crosslinking/celi surface organization)
L. J. TAKEMOTO*, C. FRED FOX*, FRED C. JENSENt, JOHN H. ELDERt, AND RICHARD A. LERNERt * Department of Microbiology and The Molecular Biology Institute, University of California, Los Angeles, California 90024; and t Department of Cellular and Developmental Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037
Communicated by Paul D. Boyer, May 15,1978
ABSTRACT Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein o mure leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surfaceradioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of'purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One crosslinked product consistently detectedon the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus. Type C RNA tumor viruses have been strongly implicated in the etiology of various neoplasias. This is especially true in the murine system in which general correlation is found between tumorigenicity and the production of endogenous murine leukemia virus (1). The 70,000-dalton glycoprotein (gp 70) is the major constituent of the viral envelope (2, 3), possessing type, group, and interspecies determinants (4). Because of its obvious importance in recognition phenomena involving the intact virion, analysis of its synthesis (5, 6), assembly (7-9), and degradation (10) has been the subject of intense investigation. Further impetus for study has come from the observation that gp7O is expressed in certain classes of normal differentiated murine cells (11-13). These non-virus-producing or "nonproducing" cells can, under certain conditions, produce complete virions, suggesting that in these nonproducing cells gp7O maintains some of the nearest-neighbor interactions that promote virus assembly in producing cells. We report studies in which chemical crosslinking was used in conjunction with immunoadsorption to probe for evidence for nearest-neighbor interactions of gp7O molecules on the surfaces of nonproducing murine cells. Comparative studies with intact virions indicate that gp7O molecules have similar nearest-neighbor interactions in both systems. MATERIALS AND METHODS Cells and Virus. Mouse fibroblasts (LM cells) were grown as described (14). Thymocytes, obtained from thymuses of 6to 8-week-old C57BL/6 (B6) female mice, were gently pushed through a wire screen, treated with 10 ml of 0.80% NH4Cl for
15 min at room temperature, and purified on Ficoll/Hypaque (15). The resultant cells were 97% viable as determined by trypan blue exclusion (16). EL-4 cells, propagated intraperitoneally in B6 mice, were purified in the same fashion. Type C RNA tumor virus was obtained from the culture medium of the chronically infected 60A lymphoblastic cell line, grown in Ham's F-12 medium containing 15% fetal calf serum (Gibco), and purified by the method of Lerner et al. (17). Radioiodination and Detergent Extraction of Cellular Proteins. LM cells were surface-labeled as described (14, 18). The labeled cell monolayer was incubated for 30 min at 00 with 1.0 ml of 0.05 M N-ethylmaleimide/0.5% (vol/vol) Nonidet P40/Dulbecco's phosphate-buffered saline (NEM/NP/Pi/ NaCl). The preparation was centrifuged for 2 X 105 g min. and the supernatant fraction was saved. Cell viability as assessed by trypan blue exclusion (16) did not significantly decrease during this procedure. A similar technique was used to surface-label and extract B6 and EL-4 cells, except that the cells (4.0 X 107) were iodinated in a suspension (2 ml) containing 2.0 mCi of NaI251. Cells were pelleted at 2500 g-min for five to seven wash steps with Dulbecco's phosphate-buffered saline containing equimolar Nal in place of NaCl prior to detergent extraction. Crosslinking of Cells. Dimethyl-3,3'-dithiobispropionimidate dihydrochloride (DTBP; Pierce) was dissolved in Pi/NaCI at 1.5 mg/ml, and the pH was adjusted to 7.8 with 0.1 M NaOH. Each plate of 125I-labeled LM cells was incubated for 25 min at 230 with 1.0 ml of this solution. Reactions were terminated by washing the cells twice with 10 ml of Pi/NaCl containing 5 mM ammonium acetate, and cellular proteins were then extracted with NEM/NP/Pi/NaCl. The B6 and EL-4 cells (4.0 X 107) were treated in identical fashion, except that cells were first suspended in 0.5 ml of Pi/NaCl and then combined with 0.5 ml of DTBP (3.0 mg/ml) in Pi/NaCl. Crosslinking, Radiolabeling, and Extraction of Viral Proteins. Fifty microliters of virus suspension (1 mg/ml as protein in Pi/NaCl) was combined with 200 Al of DTBP (1.8 mg/ml) in Pi/NaCl adjusted to pH 7.8. After a 30-min incubation at room temperature, the reaction was terminated by addition of 25 Al of 1 M ammonium acetate, and 600 Ml of NEM/NP/Pi/NaCl was then added. After a 30-min incubation at 00, the extracted viral protein was radioiodinated essentially by the procedure of Klinman and Taylor (19) by addition of 0.5 mCi of carrier-free Na'25I followed by 25 ,ul of a 1 mg/ml solution of chloramine-T. After a 30-sec incubation, the solution Abbreviations: gp7O, 70,000-dalton glycoprotein; LM cells, mouse fibroblasts; Pi/NaCI, Dulbecco's phosphate-buffered saline; DTBP, dimethyl-3,3'-dithiobispropionimidate dihydrochloride; NaDodSO4, sodium dodecyl sulfate; NEM/NP/Pj/NaCI, 0.05 M N-ethylmaleimide/0.5% (vol/vol) Nonidet P40/Pi/NaCl; NP/Pj/NaCl, 0.5% Nonidet P40/Pj/NaCI; sample buffer, 0.05 M Tris-HCI, pH 6.8/2% NaDodSO4; B6, C57BL/6.
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3644
Biochemistry:
Takemoto et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
was dialyzed against 500 ml of 0.5% Nonidet P40/0.1 M Nal/10 mM sodium phosphate, pH 7.4; 50,1A of the dialyzed solutiont was subjected to immunoadsorption. Immunoadsorption and Electrophoresis. Five microliters of normal goat serum or goat antiserum to purified gp7O of Rauscher leukemia virus was added to 500 ul of a cell or viral extract in NP/Pi/NaCl (these sera were provided by R. Wilsnack, Huntington Research Center). After a S-min incubation at 00, 150 ,l of fixed Staphylococcus aureus (Cowen I strain) suspension (10% in NP/Pi/NaCl) was added for a 10-min incubation at 00, and the bacteria were pelleted at 10,000 g-min at 00 (20). The pellet was washed three times with 5 ml of NP/Pi/NaCl, suspended in 400 Ml of sample buffer [0.05 M
3645
B6 -W
* do
I
-85,000
I
-70,000
I a
Tris-HCl, pH 6.8/2% sodium dodecyl sulfate (NaDodSO4)] containing 0.05 M N-ethylmaleimide, and incubated for 30 min at room temperature. The suspension was then heated at 1000 for 2 min, and the bacteria were sedimented for 3 min in a Beckman Microfuge B. Fifty microliters of the supernatant solution was analyzed by either one- or two-dimensional NaDodSO4/polyacrylamide gel electrophoresis. Prior to electrophoretic analysis, some samples were heated for 1 min at 1000 in the presence of 1% 2-mercaptoethanol. One-dimensional slab-gel electrophoresis was performed by the procedure of Laemmli (21); two-dimensional diagonal electrophoresis and radioautography were as described by Takemoto et al. (14).
a
b
d
c
LM a.
W4
4-
-85,000 70,X00
a
a
I
RESULTS Fig. 1 illustrates the one-dimensional NaDodSO4/polyacrylamide gel electrophoresis profiles of B6 thymocyte-derived iodinated surface components isolated by immunoadsorption. Anti-gp7O specifically bound to a major labeled component of 85,000 Mr (lane b). Treatment of a parallel sample with 2mercaptoethanol resulted in the disappearance of this band, with concomitant appearance of a 70,000 Mr component (lane c). Immunoadsorption with normal serum demonstrated minor nonspecific binding of other components, including one near the top of the resolving geL Similar patterns were obtained with materials from two other cell types: a murine fibroblast line (LM) and a murine thymocyte tumor line, EL-4, derived from
b
a
d
c
E L-4
B6 mice.
These results show that gp7O exists on these cell surfaces linked to another component of Mr 15,000 via native disulfide bonding. (Hereafter this complex will be referred to as gp85). Two-dimensional diagonal electrophoresis demonstrated that gp85 yielded labeled components of Mr 70,000 and approximately 17,000 after treatment with 2-mercaptoethanol (Fig. 2). Identical results were obtained with surface-labeled gp85 of LM and EL-4 cells (data not shown). The nearest-neighbor interactions of gp85 on the cell surface were investigated by using DTBP, a bifunctional and reversible crosslinking reagent of approximately 11-A bridging distance (22, 23). By combining crosslinking with immunoadsorption, we obtained unambiguous off-diagonal patterns of gp85 and its crosslinked components. Fig. 3 top demonstrates that treatment of B6 thymocytes with DTBP (1.5 mg/ml) resulted in the formation of crosslinked oligomers having apparent Mr of 140,000,160,000, and 240,000. The Mr 160,000 and 240,000 oligomers are probably homodimers and homotrimers of gp85. The identity of the Mr 140,000 component is less certain (see Discussion). Crosslinking of transformed mouse fibroblasts (LM) and transformed mouse T-cells (EL-4) with DTBP produced the patterns shown in Fig. 3 middle and bottom. These patterns also demonstrate the formation of Mr 140,000,160,000, and 240,000 oligomers containing gp7O. No oligomeric species of Mr 2 140,000 containing gp7O was observed with any of
2
85,000 -70,000
9 a
a
b
c
d
FIG. 1. NaDodSO4l10% polyacrylamide gel electrophoresis profiles of proteins derived from surface-radioiodinated cells by detergent extraction and immunoadsorption. The Nonidet P40 cell extract (50 Ml) was adjusted to a total volume of 500 Ml with NP/Pi/ NaCl and treated by immunoadsorption before electrophoresis. Kodak No-Screen x-ray film was exposed to the dried gels for approximately 24 hr. Lanes: a, 25 Ad of extract before immunoabsorption, treated with 2-mercaptoethanol; b, extract + anti-gp7O, no reduction; c, extract + anti-gp7O, treated with 2-mercaptoethanol; d, extract + normal serum, treated with 2-mercaptoethanol. (Top) EL-4 cells; (Middle) LM cells; (Bottom) B6 cells.
Biochemistry: Takemoto et al.
3646 1
..
Proc. Natl. Acad. Sci. USA 75 (1978)
1St DIMENSION
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(il
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1st DIMENSION 0
85,000 FIG. 2. Two-dimensional NaDodSO4 gel electrophoresis profile of proteins extracted from B6 cells and immunoadsorbed to anti-gp7O. The 7.5% first-dimension gel was treated with 2-mercaptoethanol and run in a 10% second-dimensional slab gel. After drying of the gel, autoradiography was performed for 3 weeks.
these lines when normal serum was substituted for antiserum to gp7O or when DTBP treatment was omitted (14). Because of the known potential of B6 thymocytes (24), LM fibroblasts (25), and EL-4 lymphocytes (26) to produce type C particles under appropriate conditions, it was of interest to determine if the nearest-neighbor interactions of gp85 in these cell types are also found in mature virions. The pattern in Fig. 4 upper shows that both gp7O and gp85 are present in the type C virus purified from 60A cells. Also present were Mr 160,000 and 240,000 oligomers of apparent disulfide-linked origin. Conspicuously missing in this virus was the Mr 140,000 oligomer detected after crosslinking of all the cell types studies. In addition, we observed two components lying on the diagonal having identical Mr (160,000 and 240,000) in both dimensions
(see arrows). Boiling of the uncrosslinked sample in NaDodSO4 solution for 5 min in the absence of 2-mercaptoethanol followed by identical two-dimensional analysis produced the same pattern as in Fig. 4 upper (data not shown). Similar treatment in the presence of 2-mercaptoethanol followed by one-dimensional NaDodSO4 gel electrophoresis resulted in breakdown of some, but not all, of the Mr 160,000 and 240,000 oligomers into the FIG. 3. Two-dimensional analysis of proteins derived from DTBP-treated cells by extraction with nonionic detergent and immunoadsorption with anti-gp7O or normal serum. Surface-labeled cells were treated with DTBP, and the proteins were electrophoresed on 5% (first dimension) gel and then treated with 2-mercaptoethanol and run on a 5% (second dimension) gel. X-ray film was exposed to the dried gels for 3 weeks. (Top) B6 + anti-gp7O; (Middle) LM + anti-gp7O; (Bottom) EL-4 + anti-gp7O.
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Biochemistry: Takemoto et al. I
Proc. Natl. Acad. Sci. USA 75 (1978)
3647
the result of alterations in secondary structure caused by intramholecular crosslinking or changes in charge that arise from amidination by DTBP and hydrolysis of the distal imidate of DTBP.
DIMENSION st
:!
DISCUSSION Bifunctional crosslinking reagents have proved valuable in the demonstration of nearest-neighbor interactions between biologically important macromolecules (29, 30). The identification and stoichiometry of the components of the crosslinked species in ribosomes (31, 32), erythrocyte membranes (22, 33), viruses (34, 35), and fibroblast cell surface membranes (14) have been facilitated by the development of reversible crosslinking reagents used in conjunction with two-dimensional electropho-
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FIG. 4 Nearest-neighbor interactions of major membrane glycoprotein of Scripps Clinic and Research Foundation 60A murine leukemia virus. Untreated virions and virions treated with DTBP were disrupted with NP/PJINaCl, iodinated by the chloramine-T method, and immunoadsorbed with anti-gp70 sera. The resultant samples were electrophoresed on a 5% (first dimension) gel and on a 5% (second dimension) gel after reduction with 1% 2-mercaptoethanol. X-ray film was exposed to the dried gels for 2 days. (Upper) Virus extract + anti-gp7O (arrows indicate two components having identical Mr in both dimensions, 160,000 and 240,000); (Lower) extract of DTBPtreated virus + anti-gp70.
Mr 70,000 component (data not shown). These results suggest that some oligomers containing gp85 are held together by intermolecular disulfide and possibly other binding, analogous to the hydrophobic interactions reported for the major sialoglycoprotein of the human erythrocyte membrane (27, 28). Fig. 4 lower demonstrates that treatment of virus with DTBP produced increased amounts of the homodimeric and homotrimeric complexes and that these were cleaved entirely in the second dimension by reducing agent. Missing after DTBP treatment are the Mr 160,000 and 240,000 on-diagonal components seen in Fig. 4 upper. Although the reasons for the loss of these components are not known, this phenomenon could be
The identification and resolution of the crosslinked species becomes a limiting factor in the unambiguous identification and resolution of the individual components in complex systems in which many proteins have nearly identical electrophoretic mobilities. Specific immunoadsorption of crosslinked complexes serves two purposes: first, it clearly identifies the primary component; and second, it separates this component from a heterogeneous mixture of proteins, increasing the resolution of the system. When the primary antibody-antigen complex was precipitated in initial experiments with rabbit anti-goat serum, the additional unlabeled protein interfered with the resolving ability of the second-dimension acrylamide gel system (data not shown). Formaldehyde-fixed S. aureus introduces only trivial amounts of solubilized protein, thereby eliminating this problem. Immunoadsorption in Ouchterlony double-diffusion gels provides a satisfactory method for identifying species in complexes crosslinked by irreversible reagents (36). gp7O is both a major constituent of the envelope of RNA type C viruses (3) and an endogenous surface component of certain uninfected, normal murine cells (11-13)-e.g., thymocytes from B6 mice-that produce intact viral particles under appropriate conditions (37). el-4 cells, a T-cell lymphoma (38), possess endogenous gp7O but produce significant amounts of type C particles only when grown in vitro or in immune responsive hosts (26). A similar phenomenon has been observed with the LM cell line. Under the growth conditions used here, LM cells produce only trace amounts of virus (25), indicating that the gp7O labeled and immunoadsorbed in this study is an endogenous plasma membrane component. The surface-labeled gp7O molecules in these nonproducing cells are almost entirely linked to a protein of Mr approximately 15,000-17,000 by disulfide bonding in N-ethylmaleimidetreated or untreated cells. Others have demonstrated that gp7O exists as a single polypeptide chain, sometimes linked by disulfide bonding to a protein of Mr approximately 14,000-15,000 (39). Semiquantitative estimates indicate that equimolar amounts of both proteins exist in purified virions (39) and infected cells (9). Our findings agree with data showing that appreciable quantities of both gp7O and gp85 (gp7O + pi5E) are present in purified type C virus (9, 39). In contrast, the three cell types studied in this report possessed cell surface-displayed gp7O almost entirely in the gp85 form. Crosslinking reveals what appear to be intimate nearestneighbor interactions between gp85 molecules on the cell surface. This association may be an indication of incomplete viral assembly in nonproducing cells. Two-dimensional diagonal electrophoresis of immunoadsorbed gp85 from purified type C virus substantiated this supposition, demonstrating the existence of both native disulfide-linked, as well as DTBP-induced, dimers and trimers of gp85. DTBP treatment of both cells and
3648
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Takemoto et al.
virions produced no crosslinked species of higher multiples than
the apparent homodimers and homotrimers of gp85. Higher concentrations or longer periods of incubation with DTBP failed to produce clearly discernible species representative of homo-oligomers of higher molecular weight (data not shown). Sedimentation analysis of avian tumor virus solubilized with Nonidet P40 has indicated the existence of noncovalent direric and trimeric forms of the major envelope glycoprotein (40), and crosslinking studies with influenza virus (41) and vesicular stomatitus virus (42) have also demonstrated oligomeric forms of the major envelope glycoproteins. All three cell types tested and the virus displayed apparent homotrimeric and homodimeric complexes of Mr 240,000 and 160,000. The cells displayed an additional major oligomeric species of Mr 140,000. Because surface-labeled gp7O in these cell systems existed almost totally linked via native disulfide bonding to a Mr 17,000 species, it is unlikely that the Mr 140,000 species represents a homodimeric form of gp7O or a complex of gp7O and gp85. These results are more consistent with other possibilities. First, gp85 may exist in two different disulfide bond-specified conformations on the cell surface, so that intramolecular crosslinking by DTBP of one of these would result in a homodimeric species with an apparent Mr in NaDodSO4 gel electrophoresis less than the expected 160,000-170,000. Disulfide bonding giving rise to increased relative mobility of proteins in NaDodSO4 gel electrophoresis has been reported (43). Second, the Mr 140,000 species may indicate a nearestneighbor association of gp85 with a hitherto unidentified cellular component with a Mr of approximately 55,000, such as a cell-surface receptor for gp7O, Pr 65gag, the precursor of the major nucleocapsid protein (44), microtubule protein [Mr 50,000 (45)], actin [Mr 43,000 (46)], and the major histocompatibility antigen (H-2) [Mr 43,000-47,000 (47)]. The last of these is of special interest because of recent suggestions that H-2 gene products and gp7O are physically associated in an "altered self" configuration (48, 49). Third, the Mr 140,000 oligomer present in LM, EL-4, and B6 cells may be formed from a surface protein that crossreacts with gp7O antiserum and is not expressed by the host cells that gave rise to the 60A virus used in this study. Finally, the Mr 140,000 oligomer might be formed from crossreacting proteins that are not capable of the associations required for their incorporation into mature viral particles. Peptide mapping of the crosslinked oligomeric complexes is required to distinguish among these possibilities. The authors acknowledge the assistance and advice provided by Dr. T. Miyakawa, currently of The Mitsubishi-Kasei Institute of Life Science, Tokyo, Japan. This work was supported by U.S. Public Health Service Grant GM18233, a contract from the National Cancer Institute, and a grant from-the, Muscular Dystrophy Association, Inc. L.T. is a postdoctoral fellow of that Association. 1. Meier, H., Taylor, B. L, Cherry, M. & Huebner, R. J. (1973) Proc. Natl. Acad. Sci. USA 70,1450-1455. 2. Hunsmann, G., Moennig, V. & Schafer, W. (1975) Virology 66, 327-329. 3. Witte, 0. N., Weissman, I. L. & Kaplan, H. S. (1973) Proc. Natl. Acad. Sci. USA 70,3640. 4. Strand, M. & August, J. T. (1974) J. Virol. 13, 171-180. 5. Vogt, V. M. & Eisenman, R. (1973) Proc. Natl. Acad. Sci. USA 70, 1734-1738. 6. Karshin, W. L., Arcement, L. J., Naso, R. B. & Arlinghaus, R. B. (1977) J. Virol. 23, 787-798. 7. Witte, 0. N. & Weissman, I. L. (1974) Virology 61,575-587. 8. Witte, 0. N. & Weissman, I. L. (1976) Virology 69,464-473. 9. Witte, 0. N., Tsukamoto-Adey, A. & Weissman, I. L. (1977) Virology 76,539-553. 10. Krantz, M. J., Strand, M. & August, J. T. (1977) 804-815.
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11. Del Villano, B. C., Nave, B., Croker, B. P., Lerner, R. A. & Dixon, F. J. (1975) J. Exp. Med. 141, 172-187. 12. Elder, J. H., Jensen, F. C., Bryant, M. L. & Lerner, R. A. (1977) Nature 267,23-28. 13. Cloyd, M. W., Bolognesi, D. P. & Bigner, D. D. (1977) Cancer Res. 37,931-938. 14. Takemoto, L. J., Miyakawa, T. & Fox, C. F. (1977) in Cell Shape and Surface Architecture, eds. Revel, J., Henning, U. & Fox, C. (Alan R. Liss, New York), pp. 605-614. 15. Heininger, D., Touton, M., Chakravarty, A. K. & Clark, W. R.
(1976) J. Immunol. 117,2175-2180. Phillips, H. J. (1973) in Tissue Culture Methods and Applications, eds. Kruse, P. & Patterson, M. (Academic, New York), pp. 406-408. 17. Lerner, R., Jensen, F., Kennel, S. J., Dixon, F. J., Des Roches, G. & Francke, U. (1972) Proc. Natl. Acad. Sci. USA 69, 296516.
2969. 18. Hynes, R. 0. (1973) Proc. Natl. Acad. Sci. USA 70, 31703174. 19. Klinman, N. R. & Taylor, R. B. (1969) Clin. Exp. Immunol. 4, 473-487. 20. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624. 21. Laemmli, V. K. (1970) Nature 227,680-86. 22. Wang, K. & Richards, F. M. (1974) Isr. J. Chem. 12,375-389. 23. Ruoho, A., Bartlett, P. A., Dutton, A. & Singer, S. J. (1975) Biochem. Biophys. Res. Commun. 63,417-423. 24. Kaplan, H. S. (1967) Cancer Res. 27,1325-1340. 25. Kindig, D. A. & Kirsten, W. H. (1968) Proc. Natl. Acad. Sd. USA
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26. Colnaghi, M. I., Pierotti, M. A., Torre, G. D. & Miotti, S. (1977) J. Natl. Cancer Inst. 59,123-130. 27. Tuech, J. K. & Morrison, M. (1974) Biochem. Biophys. Res. Commun. 59,352-360. 28. Furthmayr, H. & Marchesi, V. T. (1976) Biochemistry 15, 1137-1144. 29. Davies, G. E. & Stark, G. R. (1970) Proc. Natl. Acad. Sci. USA
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30. Carpenter, F. H. & Harrington, K. T. (1972) J. Biol. Chem. 247, 5580-5586. 31. Sommer, A. & Traut, R. R. (1974) Proc. Natl. Acad. Sci. USA 71,
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32. Lutter, L. C., Ortanderl, F. & Fasold, H. (1974) FEBS Lett. 48, 288-292. 33. Wang, K. & Richards, F. M. (1974) J. Blol. Chem. 249,80058018. 34. Miyakawa, T., Takemoto, L. J. & Fox, C. F. (1976) in Animal Virology, eds. Baltimore, D., Huang, A. & Fox, C. (Alan R. Liss, New York), pp. 485-497. 35. Dubovi, E. J. & Wagner, R. R. (1977) J. Virol. 22,500-509. 36. Sun, T-T., Traut, R. R. & Kahan, L. (1974) J. Mol. Biol. 87,
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Stockert, E., Boyse, E. A., Obata, Y., Ikeda, H., Sarkar, N. H. & Hoffman, H. A. (1975) J. Exp. Med. 142,512-517. 38. Gorer, P. A. (1950) Br. J. Cancer 4,372-379. 39. Leamnson, R. N., Shander, M. H. M., & Halpern, M. S. (1977) Virology 76,437-439. 40. Leamnson, R. N. & Halpern, M. S. (1976) J. Virol. 18,956-
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Dubovi, E. & Wagner, R. R. (1977) J. Virol. 22,500-9. 43. Phillips, D. R. & Agin, P. P. (1977) J. Biol. Chem. 252,21212126. 44. Witte, 0. N. & Baltimore, D. (1978)J. Virol. 26,750-761. 45. Ostlund, R. & Pastan, I. (1975) Biochemistry 14,4064-4068. 46. Fine, R. E. & Bray, D. (1971) Nature 234, 115-118. 47. Schwarz, B. D., Kato, K., Cullen, S. E. & Nathenson, S. G. (1973) Biochemistry 12, 2157-2164. 48. Zinkernagel, R. M. & Doherty, R. C. (1974) Nature 251, 547548. 49. Schrader, J. W., Cunningham, B. A. & Edelman, G. M. (1975) Proc. Natl. Acad. Sci. USA 72,5066-570.
42.
Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces (tumor virus envelope protein/gp70/endogenous tumor virus proteins/crosslinking/celi surface organization)
L. J. TAKEMOTO*, C. FRED FOX*, FRED C. JENSENt, JOHN H. ELDERt, AND RICHARD A. LERNERt * Department of Microbiology and The Molecular Biology Institute, University of California, Los Angeles, California 90024; and t Department of Cellular and Developmental Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, California 92037
Communicated by Paul D. Boyer, May 15,1978
ABSTRACT Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein o mure leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surfaceradioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of'purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One crosslinked product consistently detectedon the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus. Type C RNA tumor viruses have been strongly implicated in the etiology of various neoplasias. This is especially true in the murine system in which general correlation is found between tumorigenicity and the production of endogenous murine leukemia virus (1). The 70,000-dalton glycoprotein (gp 70) is the major constituent of the viral envelope (2, 3), possessing type, group, and interspecies determinants (4). Because of its obvious importance in recognition phenomena involving the intact virion, analysis of its synthesis (5, 6), assembly (7-9), and degradation (10) has been the subject of intense investigation. Further impetus for study has come from the observation that gp7O is expressed in certain classes of normal differentiated murine cells (11-13). These non-virus-producing or "nonproducing" cells can, under certain conditions, produce complete virions, suggesting that in these nonproducing cells gp7O maintains some of the nearest-neighbor interactions that promote virus assembly in producing cells. We report studies in which chemical crosslinking was used in conjunction with immunoadsorption to probe for evidence for nearest-neighbor interactions of gp7O molecules on the surfaces of nonproducing murine cells. Comparative studies with intact virions indicate that gp7O molecules have similar nearest-neighbor interactions in both systems. MATERIALS AND METHODS Cells and Virus. Mouse fibroblasts (LM cells) were grown as described (14). Thymocytes, obtained from thymuses of 6to 8-week-old C57BL/6 (B6) female mice, were gently pushed through a wire screen, treated with 10 ml of 0.80% NH4Cl for
15 min at room temperature, and purified on Ficoll/Hypaque (15). The resultant cells were 97% viable as determined by trypan blue exclusion (16). EL-4 cells, propagated intraperitoneally in B6 mice, were purified in the same fashion. Type C RNA tumor virus was obtained from the culture medium of the chronically infected 60A lymphoblastic cell line, grown in Ham's F-12 medium containing 15% fetal calf serum (Gibco), and purified by the method of Lerner et al. (17). Radioiodination and Detergent Extraction of Cellular Proteins. LM cells were surface-labeled as described (14, 18). The labeled cell monolayer was incubated for 30 min at 00 with 1.0 ml of 0.05 M N-ethylmaleimide/0.5% (vol/vol) Nonidet P40/Dulbecco's phosphate-buffered saline (NEM/NP/Pi/ NaCl). The preparation was centrifuged for 2 X 105 g min. and the supernatant fraction was saved. Cell viability as assessed by trypan blue exclusion (16) did not significantly decrease during this procedure. A similar technique was used to surface-label and extract B6 and EL-4 cells, except that the cells (4.0 X 107) were iodinated in a suspension (2 ml) containing 2.0 mCi of NaI251. Cells were pelleted at 2500 g-min for five to seven wash steps with Dulbecco's phosphate-buffered saline containing equimolar Nal in place of NaCl prior to detergent extraction. Crosslinking of Cells. Dimethyl-3,3'-dithiobispropionimidate dihydrochloride (DTBP; Pierce) was dissolved in Pi/NaCI at 1.5 mg/ml, and the pH was adjusted to 7.8 with 0.1 M NaOH. Each plate of 125I-labeled LM cells was incubated for 25 min at 230 with 1.0 ml of this solution. Reactions were terminated by washing the cells twice with 10 ml of Pi/NaCl containing 5 mM ammonium acetate, and cellular proteins were then extracted with NEM/NP/Pi/NaCl. The B6 and EL-4 cells (4.0 X 107) were treated in identical fashion, except that cells were first suspended in 0.5 ml of Pi/NaCl and then combined with 0.5 ml of DTBP (3.0 mg/ml) in Pi/NaCl. Crosslinking, Radiolabeling, and Extraction of Viral Proteins. Fifty microliters of virus suspension (1 mg/ml as protein in Pi/NaCl) was combined with 200 Al of DTBP (1.8 mg/ml) in Pi/NaCl adjusted to pH 7.8. After a 30-min incubation at room temperature, the reaction was terminated by addition of 25 Al of 1 M ammonium acetate, and 600 Ml of NEM/NP/Pi/NaCl was then added. After a 30-min incubation at 00, the extracted viral protein was radioiodinated essentially by the procedure of Klinman and Taylor (19) by addition of 0.5 mCi of carrier-free Na'25I followed by 25 ,ul of a 1 mg/ml solution of chloramine-T. After a 30-sec incubation, the solution Abbreviations: gp7O, 70,000-dalton glycoprotein; LM cells, mouse fibroblasts; Pi/NaCI, Dulbecco's phosphate-buffered saline; DTBP, dimethyl-3,3'-dithiobispropionimidate dihydrochloride; NaDodSO4, sodium dodecyl sulfate; NEM/NP/Pj/NaCI, 0.05 M N-ethylmaleimide/0.5% (vol/vol) Nonidet P40/Pi/NaCl; NP/Pj/NaCl, 0.5% Nonidet P40/Pj/NaCI; sample buffer, 0.05 M Tris-HCI, pH 6.8/2% NaDodSO4; B6, C57BL/6.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertusement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
3644
Biochemistry:
Takemoto et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
was dialyzed against 500 ml of 0.5% Nonidet P40/0.1 M Nal/10 mM sodium phosphate, pH 7.4; 50,1A of the dialyzed solutiont was subjected to immunoadsorption. Immunoadsorption and Electrophoresis. Five microliters of normal goat serum or goat antiserum to purified gp7O of Rauscher leukemia virus was added to 500 ul of a cell or viral extract in NP/Pi/NaCl (these sera were provided by R. Wilsnack, Huntington Research Center). After a S-min incubation at 00, 150 ,l of fixed Staphylococcus aureus (Cowen I strain) suspension (10% in NP/Pi/NaCl) was added for a 10-min incubation at 00, and the bacteria were pelleted at 10,000 g-min at 00 (20). The pellet was washed three times with 5 ml of NP/Pi/NaCl, suspended in 400 Ml of sample buffer [0.05 M
3645
B6 -W
* do
I
-85,000
I
-70,000
I a
Tris-HCl, pH 6.8/2% sodium dodecyl sulfate (NaDodSO4)] containing 0.05 M N-ethylmaleimide, and incubated for 30 min at room temperature. The suspension was then heated at 1000 for 2 min, and the bacteria were sedimented for 3 min in a Beckman Microfuge B. Fifty microliters of the supernatant solution was analyzed by either one- or two-dimensional NaDodSO4/polyacrylamide gel electrophoresis. Prior to electrophoretic analysis, some samples were heated for 1 min at 1000 in the presence of 1% 2-mercaptoethanol. One-dimensional slab-gel electrophoresis was performed by the procedure of Laemmli (21); two-dimensional diagonal electrophoresis and radioautography were as described by Takemoto et al. (14).
a
b
d
c
LM a.
W4
4-
-85,000 70,X00
a
a
I
RESULTS Fig. 1 illustrates the one-dimensional NaDodSO4/polyacrylamide gel electrophoresis profiles of B6 thymocyte-derived iodinated surface components isolated by immunoadsorption. Anti-gp7O specifically bound to a major labeled component of 85,000 Mr (lane b). Treatment of a parallel sample with 2mercaptoethanol resulted in the disappearance of this band, with concomitant appearance of a 70,000 Mr component (lane c). Immunoadsorption with normal serum demonstrated minor nonspecific binding of other components, including one near the top of the resolving geL Similar patterns were obtained with materials from two other cell types: a murine fibroblast line (LM) and a murine thymocyte tumor line, EL-4, derived from
b
a
d
c
E L-4
B6 mice.
These results show that gp7O exists on these cell surfaces linked to another component of Mr 15,000 via native disulfide bonding. (Hereafter this complex will be referred to as gp85). Two-dimensional diagonal electrophoresis demonstrated that gp85 yielded labeled components of Mr 70,000 and approximately 17,000 after treatment with 2-mercaptoethanol (Fig. 2). Identical results were obtained with surface-labeled gp85 of LM and EL-4 cells (data not shown). The nearest-neighbor interactions of gp85 on the cell surface were investigated by using DTBP, a bifunctional and reversible crosslinking reagent of approximately 11-A bridging distance (22, 23). By combining crosslinking with immunoadsorption, we obtained unambiguous off-diagonal patterns of gp85 and its crosslinked components. Fig. 3 top demonstrates that treatment of B6 thymocytes with DTBP (1.5 mg/ml) resulted in the formation of crosslinked oligomers having apparent Mr of 140,000,160,000, and 240,000. The Mr 160,000 and 240,000 oligomers are probably homodimers and homotrimers of gp85. The identity of the Mr 140,000 component is less certain (see Discussion). Crosslinking of transformed mouse fibroblasts (LM) and transformed mouse T-cells (EL-4) with DTBP produced the patterns shown in Fig. 3 middle and bottom. These patterns also demonstrate the formation of Mr 140,000,160,000, and 240,000 oligomers containing gp7O. No oligomeric species of Mr 2 140,000 containing gp7O was observed with any of
2
85,000 -70,000
9 a
a
b
c
d
FIG. 1. NaDodSO4l10% polyacrylamide gel electrophoresis profiles of proteins derived from surface-radioiodinated cells by detergent extraction and immunoadsorption. The Nonidet P40 cell extract (50 Ml) was adjusted to a total volume of 500 Ml with NP/Pi/ NaCl and treated by immunoadsorption before electrophoresis. Kodak No-Screen x-ray film was exposed to the dried gels for approximately 24 hr. Lanes: a, 25 Ad of extract before immunoabsorption, treated with 2-mercaptoethanol; b, extract + anti-gp7O, no reduction; c, extract + anti-gp7O, treated with 2-mercaptoethanol; d, extract + normal serum, treated with 2-mercaptoethanol. (Top) EL-4 cells; (Middle) LM cells; (Bottom) B6 cells.
Biochemistry: Takemoto et al.
3646 1
..
Proc. Natl. Acad. Sci. USA 75 (1978)
1St DIMENSION
't DIMENSION N)
:3j a
0
z
A. C
(il
mol
2
70,000--m
lb
24!0001o14o,00J 701000
I.
160,000
_17,000
85,000
1st DIMENSION 0
85,000 FIG. 2. Two-dimensional NaDodSO4 gel electrophoresis profile of proteins extracted from B6 cells and immunoadsorbed to anti-gp7O. The 7.5% first-dimension gel was treated with 2-mercaptoethanol and run in a 10% second-dimensional slab gel. After drying of the gel, autoradiography was performed for 3 weeks.
these lines when normal serum was substituted for antiserum to gp7O or when DTBP treatment was omitted (14). Because of the known potential of B6 thymocytes (24), LM fibroblasts (25), and EL-4 lymphocytes (26) to produce type C particles under appropriate conditions, it was of interest to determine if the nearest-neighbor interactions of gp85 in these cell types are also found in mature virions. The pattern in Fig. 4 upper shows that both gp7O and gp85 are present in the type C virus purified from 60A cells. Also present were Mr 160,000 and 240,000 oligomers of apparent disulfide-linked origin. Conspicuously missing in this virus was the Mr 140,000 oligomer detected after crosslinking of all the cell types studies. In addition, we observed two components lying on the diagonal having identical Mr (160,000 and 240,000) in both dimensions
(see arrows). Boiling of the uncrosslinked sample in NaDodSO4 solution for 5 min in the absence of 2-mercaptoethanol followed by identical two-dimensional analysis produced the same pattern as in Fig. 4 upper (data not shown). Similar treatment in the presence of 2-mercaptoethanol followed by one-dimensional NaDodSO4 gel electrophoresis resulted in breakdown of some, but not all, of the Mr 160,000 and 240,000 oligomers into the FIG. 3. Two-dimensional analysis of proteins derived from DTBP-treated cells by extraction with nonionic detergent and immunoadsorption with anti-gp7O or normal serum. Surface-labeled cells were treated with DTBP, and the proteins were electrophoresed on 5% (first dimension) gel and then treated with 2-mercaptoethanol and run on a 5% (second dimension) gel. X-ray film was exposed to the dried gels for 3 weeks. (Top) B6 + anti-gp7O; (Middle) LM + anti-gp7O; (Bottom) EL-4 + anti-gp7O.
z
C,,
a
70,QQQ-mw.
240!000
1140,000 85,000
160,0
85,000
ist DIMENSiON 0 (jn 2
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t
t
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I9t
85,000
Biochemistry: Takemoto et al. I
Proc. Natl. Acad. Sci. USA 75 (1978)
3647
the result of alterations in secondary structure caused by intramholecular crosslinking or changes in charge that arise from amidination by DTBP and hydrolysis of the distal imidate of DTBP.
DIMENSION st
:!
DISCUSSION Bifunctional crosslinking reagents have proved valuable in the demonstration of nearest-neighbor interactions between biologically important macromolecules (29, 30). The identification and stoichiometry of the components of the crosslinked species in ribosomes (31, 32), erythrocyte membranes (22, 33), viruses (34, 35), and fibroblast cell surface membranes (14) have been facilitated by the development of reversible crosslinking reagents used in conjunction with two-dimensional electropho-
Ir;r z
(1
70,000--'-
resis.
a
+I
A
44
i.
160,000
240,000
." L)mENSION ..
70,000
85,000
. .ow_.
n;
I-
t
A
60,000
240,000
70,000
85,000
FIG. 4 Nearest-neighbor interactions of major membrane glycoprotein of Scripps Clinic and Research Foundation 60A murine leukemia virus. Untreated virions and virions treated with DTBP were disrupted with NP/PJINaCl, iodinated by the chloramine-T method, and immunoadsorbed with anti-gp70 sera. The resultant samples were electrophoresed on a 5% (first dimension) gel and on a 5% (second dimension) gel after reduction with 1% 2-mercaptoethanol. X-ray film was exposed to the dried gels for 2 days. (Upper) Virus extract + anti-gp7O (arrows indicate two components having identical Mr in both dimensions, 160,000 and 240,000); (Lower) extract of DTBPtreated virus + anti-gp70.
Mr 70,000 component (data not shown). These results suggest that some oligomers containing gp85 are held together by intermolecular disulfide and possibly other binding, analogous to the hydrophobic interactions reported for the major sialoglycoprotein of the human erythrocyte membrane (27, 28). Fig. 4 lower demonstrates that treatment of virus with DTBP produced increased amounts of the homodimeric and homotrimeric complexes and that these were cleaved entirely in the second dimension by reducing agent. Missing after DTBP treatment are the Mr 160,000 and 240,000 on-diagonal components seen in Fig. 4 upper. Although the reasons for the loss of these components are not known, this phenomenon could be
The identification and resolution of the crosslinked species becomes a limiting factor in the unambiguous identification and resolution of the individual components in complex systems in which many proteins have nearly identical electrophoretic mobilities. Specific immunoadsorption of crosslinked complexes serves two purposes: first, it clearly identifies the primary component; and second, it separates this component from a heterogeneous mixture of proteins, increasing the resolution of the system. When the primary antibody-antigen complex was precipitated in initial experiments with rabbit anti-goat serum, the additional unlabeled protein interfered with the resolving ability of the second-dimension acrylamide gel system (data not shown). Formaldehyde-fixed S. aureus introduces only trivial amounts of solubilized protein, thereby eliminating this problem. Immunoadsorption in Ouchterlony double-diffusion gels provides a satisfactory method for identifying species in complexes crosslinked by irreversible reagents (36). gp7O is both a major constituent of the envelope of RNA type C viruses (3) and an endogenous surface component of certain uninfected, normal murine cells (11-13)-e.g., thymocytes from B6 mice-that produce intact viral particles under appropriate conditions (37). el-4 cells, a T-cell lymphoma (38), possess endogenous gp7O but produce significant amounts of type C particles only when grown in vitro or in immune responsive hosts (26). A similar phenomenon has been observed with the LM cell line. Under the growth conditions used here, LM cells produce only trace amounts of virus (25), indicating that the gp7O labeled and immunoadsorbed in this study is an endogenous plasma membrane component. The surface-labeled gp7O molecules in these nonproducing cells are almost entirely linked to a protein of Mr approximately 15,000-17,000 by disulfide bonding in N-ethylmaleimidetreated or untreated cells. Others have demonstrated that gp7O exists as a single polypeptide chain, sometimes linked by disulfide bonding to a protein of Mr approximately 14,000-15,000 (39). Semiquantitative estimates indicate that equimolar amounts of both proteins exist in purified virions (39) and infected cells (9). Our findings agree with data showing that appreciable quantities of both gp7O and gp85 (gp7O + pi5E) are present in purified type C virus (9, 39). In contrast, the three cell types studied in this report possessed cell surface-displayed gp7O almost entirely in the gp85 form. Crosslinking reveals what appear to be intimate nearestneighbor interactions between gp85 molecules on the cell surface. This association may be an indication of incomplete viral assembly in nonproducing cells. Two-dimensional diagonal electrophoresis of immunoadsorbed gp85 from purified type C virus substantiated this supposition, demonstrating the existence of both native disulfide-linked, as well as DTBP-induced, dimers and trimers of gp85. DTBP treatment of both cells and
3648
Proc. Natl. Acad. Sci. USA 75 (1978)
Biochemistry: Takemoto et al.
virions produced no crosslinked species of higher multiples than
the apparent homodimers and homotrimers of gp85. Higher concentrations or longer periods of incubation with DTBP failed to produce clearly discernible species representative of homo-oligomers of higher molecular weight (data not shown). Sedimentation analysis of avian tumor virus solubilized with Nonidet P40 has indicated the existence of noncovalent direric and trimeric forms of the major envelope glycoprotein (40), and crosslinking studies with influenza virus (41) and vesicular stomatitus virus (42) have also demonstrated oligomeric forms of the major envelope glycoproteins. All three cell types tested and the virus displayed apparent homotrimeric and homodimeric complexes of Mr 240,000 and 160,000. The cells displayed an additional major oligomeric species of Mr 140,000. Because surface-labeled gp7O in these cell systems existed almost totally linked via native disulfide bonding to a Mr 17,000 species, it is unlikely that the Mr 140,000 species represents a homodimeric form of gp7O or a complex of gp7O and gp85. These results are more consistent with other possibilities. First, gp85 may exist in two different disulfide bond-specified conformations on the cell surface, so that intramolecular crosslinking by DTBP of one of these would result in a homodimeric species with an apparent Mr in NaDodSO4 gel electrophoresis less than the expected 160,000-170,000. Disulfide bonding giving rise to increased relative mobility of proteins in NaDodSO4 gel electrophoresis has been reported (43). Second, the Mr 140,000 species may indicate a nearestneighbor association of gp85 with a hitherto unidentified cellular component with a Mr of approximately 55,000, such as a cell-surface receptor for gp7O, Pr 65gag, the precursor of the major nucleocapsid protein (44), microtubule protein [Mr 50,000 (45)], actin [Mr 43,000 (46)], and the major histocompatibility antigen (H-2) [Mr 43,000-47,000 (47)]. The last of these is of special interest because of recent suggestions that H-2 gene products and gp7O are physically associated in an "altered self" configuration (48, 49). Third, the Mr 140,000 oligomer present in LM, EL-4, and B6 cells may be formed from a surface protein that crossreacts with gp7O antiserum and is not expressed by the host cells that gave rise to the 60A virus used in this study. Finally, the Mr 140,000 oligomer might be formed from crossreacting proteins that are not capable of the associations required for their incorporation into mature viral particles. Peptide mapping of the crosslinked oligomeric complexes is required to distinguish among these possibilities. The authors acknowledge the assistance and advice provided by Dr. T. Miyakawa, currently of The Mitsubishi-Kasei Institute of Life Science, Tokyo, Japan. This work was supported by U.S. Public Health Service Grant GM18233, a contract from the National Cancer Institute, and a grant from-the, Muscular Dystrophy Association, Inc. L.T. is a postdoctoral fellow of that Association. 1. Meier, H., Taylor, B. L, Cherry, M. & Huebner, R. J. (1973) Proc. Natl. Acad. Sci. USA 70,1450-1455. 2. Hunsmann, G., Moennig, V. & Schafer, W. (1975) Virology 66, 327-329. 3. Witte, 0. N., Weissman, I. L. & Kaplan, H. S. (1973) Proc. Natl. Acad. Sci. USA 70,3640. 4. Strand, M. & August, J. T. (1974) J. Virol. 13, 171-180. 5. Vogt, V. M. & Eisenman, R. (1973) Proc. Natl. Acad. Sci. USA 70, 1734-1738. 6. Karshin, W. L., Arcement, L. J., Naso, R. B. & Arlinghaus, R. B. (1977) J. Virol. 23, 787-798. 7. Witte, 0. N. & Weissman, I. L. (1974) Virology 61,575-587. 8. Witte, 0. N. & Weissman, I. L. (1976) Virology 69,464-473. 9. Witte, 0. N., Tsukamoto-Adey, A. & Weissman, I. L. (1977) Virology 76,539-553. 10. Krantz, M. J., Strand, M. & August, J. T. (1977) 804-815.
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42.
