Vol. 16, No. 4 Printed in U.S.A.
JOURNAL OF VIROLOGY, Oct. 1975, p. 1071-1074 Copyright ( 1975 American Society for Microbiology
NOTES Detection of Type A Oncornavirus-Like Structures in Chicken Embryo Cells After Infection with Rous Sarcoma Virus GERD G. MAUL AND LEON J. LEWANDOWSKI* The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
Received for publication 9 June 1975
Electron microscopy examination of Rous sarcoma virus-transformed chicken embryo cells revealed membrane-free nucleoids resembling type A oncornavirus. These particles were not detected in noninfected cells, nor did they accumulate in excess under conditions of glucosamine block in virus-transformed cultures.
The ultrastructural classification of virus particles associated with murine neoplasms into types A, B, C, and D was originally suggested by Bernhard (1). More recently, electron microscopy observations on the maturation of murine RNA tumor virus particles suggested that the B particle-type mouse mammary tumor virus can develop either by incorporation of an intracytoplasmic type A particle into the forming bud, or by the simultaneous formation of the two layers of a viral "nucleoid" just under the plasma membrane of a developing bud (5). The free virions produced by either process are morphologically identical, possessing a centrally located nucleoid, a distinct intermediate layer, and an outer envelope. By comparison, murine C-type virions are believed to develop at the surface of the infected cell only after the appearance of a viral nucleoid in crescentic form under a bulging plasma membrane. Since chicken and murine C-type RNA tumor viruses have many properties in common (8), it was of interest to determine whether avian RNA tumor virus particle maturation was restricted to subsurface formation of the viral nucleoid immediately prior to virus budding. The method that seemed potentially wellsuited for enhancing the detectability of subviral particles was to expose infected cells to glucosamine. In chicken embryo cells transformed by Rous sarcoma virus (RSV), the presence of glucosamine inhibits to nondetectable levels subsequent production of virus (9, 9a). Viral envelope glycoprotein synthesis is aberrant and markedly reduced (9a), whereas the non-glycosylated group-specific (gs) polypeptides that compose the viral core are
synthesized, although also in reduced amounts (9, 9a). It seemed possible that those gs proteins that are synthesized in the presence of glucosamine might accumulate in the form of electron microscopic-detectable intracytoplasmic nucleoids, perhaps analogous to the glycoprotein-free viral "core" structures obtained by detergent treatment in vitro of whole virus
(2).
A clone-purified stock of subgroup C of RSV strain B77 was used to infect C/E chicken embryo cells. Primary cultures were prepared from 11-day-old chicken embryo (SPAFAS, Inc., Storrs, Conn.) as described by Vogt (12). Infection and maintenance of secondary cultures were as described by Duesberg et al. (7). Confluent secondary cultures of both noninfected and RSV-transformed chicken embryo cells, grown in tissue culture chamber/slides (LabTek), were incubated with and without 20 ,umol of glucosamine per ml for 16 h (9a). All cultures were then washed three times with phosphate-buffered saline and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer and 3 mM MgCl2 at pH 7.4 for 20 min at room temperature. -The preparations were again thoroughly washed with phosphate-buffered saline and postfixed for 30 min with 1% OsO, (phosphate buffered; pH 7.4). Before dehydration in ethanol, the cells were stained with 1% uranyl acetate in distilled water for 20 h at 60 C. They were embedded in situ (3) in Epon 812. Thin sections were cut on a LKB Ultratome III and observed (unstained and stained with uranyl acetate and lead citrate) with a Hitachi HU-11D electron microscope operated at 75 kV. Magnifications are noted in
1071
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-
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49'
z
ar
.:
w
$L
$
* k
.-. 'p
r
&
FIG. 1. Survey micrograph of chicken embryo fibroblast transformed with RSV. Note aggregate of densely stained circular particles with a diameter of more than 1 um. The particles have two concentric densely stained rings and translucent core. Some seem to be open and crescent shaped (arrows). Uranyl acetate and lead citrate staining. Magnification x 75,000. FIG. 2. Single corelike particles are mostly found in close association with mitochondria. Magnification
x50,000. FIG. 3-4. Higher magnification of single corelike particles. They seem to be in intimate contact with ribosomes (arrows). Magnification x105,000. 1072
NOTES
VOL. 16, 1975
the figure legends. For immune electron microscopy, a double-labeling technique (11) was used that utilized rabbit antisera (kindly supplied by D. Bolognesi) monospecific for p27 and p19, the two major viral gs proteins, and normal rabbit serum; ferritin conjugated goat-anti-rabbit globulin was the electron-dense marker. RSV-transformed chicken fibroblasts cultured in the absence of glucosamine revealed occasional aggregates of high-electron-dense particles within the cytoplasm (Fig. 1). These aggregates were not glycogen since the uranyl acetate stain would have leached glycogen, and they were not lysosomes since they were not surrounded by a membrane. They were present in -10% of all cells and appeared to be composed of membrane-free nucleoids resembling type A oncornavirus particles, with diameters of -60.0 nm, and electron-lucent centers of -25.0 nm in diameter surrounded by two dense rings that were occasionally open. Nearly all trans-
1073
formed cells contained single particles, some apparently at different stages of completeness, interspersed between mitochondria (Fig. 2). Higher magnification (Fig. 3 and 4) shows ribosome-like material closely associated (arrows). Such ribosome-like particles have been visualized by high-resolution electron microscopy inside RNA tumor viruses associated with the viral core structure (6). To avoid any obscuring effect of surface staining on particle substructure (10), unstained sections were also examined. Figure 5 clearly shows the electronlucent centers of complete particles (left) and what appear to be incomplete particles (right). Figure 6 shows typical type C particles, with a characteristic diameter of 100.0 nm, budding at the surface of these cells. Analysis of transformed cells under conditions of glucosamine block showed little, if any, difference from nontreated transformed cells, i.e., no additional accumulation of these struc1{
V1 !~~~ Ii
~
r
rj
''(i 0 AK, IL
'.S'.-'.2 FIG. 5. Unstained preparation of complete and incomplete corelike structures, emphasizing the electrontranslucent center. Magnification x 75,000. FIG. 6. Several budding C particles. Magnification x100,000.
1074
NOTES
tures. No cells containing nucleoids were found in the noninfected control cultures regardless of glucosamine treatment. Attempts to determine the relatedness of the nucleoids to RSV through immune electron microscopy were unfortunately inconclusive; this was apparently due to an inaccessibility of the antigenic binding sites of the nucleoids which usually appeared covered by an abundant, dense ribosome-like material. It is possible, therefore, that the particles represent an unknown endogenous virus unrelated to the input RSV. Their dimensions and ultrastructural features (dense circular particles with an electron-translucent center and two dense shells) suggest, however, that they are either type A oncornavirus particles or an intracellular nucleoid form of C-type RSV, analogous to the type A particle. If so, it remains to be determined whether they are in the process of being assembled de novo or whether they represent RSV progeny superinfecting the cells. Dales and Hanafusa (4), studying the penetration and intracellular release of the genomes of avian RNA tumor viruses, have observed that infecting virions either individually or in groups are internalized by viropexis and move within vacuoles to the nuclear envelopes; they never found either intact input virions or subviral particles (that might be interpreted to be the equivalent of viral cores) without vacuolar membranes. In contrast, we never found the corelike particles with vacuolar membranes. This fact, combined with the observations that the corelike particles were present in conjunction with mitochondria and usually had ribosome-like structures closely associated with them, suggests that they are either viral cores newly synthesized or infecting virus somehow actively engaged in virus replication. In the future, electron microscope autoradiography applied after the virus synthesized de novo is labeled or after the cells are infected with highly labeled input virus may allow us to distinguish between these possibilities.
J. VIROL. This work was supported by Public Health Service research grants CA-10815, CA-15954, and CA-15464 from the National Cancer Institute, and by grant 1194 from the Damon Runyon Memorial Fund. The technical assistance of Carla Brooks, Patricia Stranen and Joseph Weibel is gratefully acknowledged. LITERATURE CITED 1. Bernhard, W. 1960. The detection and study of tumor viruses with the electron microscope. Cancer Res. 20:712-727. 2. Bolognesi, D. P., R. Luftig, and J. H. Shaper. 1973. Localization of RNA tumor virus polypeptides. I. Isolation of further virus substructures. Virology 56:549-564. 3. Brinkley, B. R., P. Murphy, and L. C. Richardson. 1967. Procedure for embedding in situ selected cells cultured in vitro. J. Cell Biol. 35:279-283. 4. Dales, S., and H. Hanafusa. 1972. Penetration and intracellular release of the genomes of avian RNA tumor viruses. Virology 50:440-458. 5. Dalton, A. J. 1972. Further analysis of the detailed structure of Type B and C particles. J. Natl. Cancer. Inst. 48:1095-1099. 6. De Harven, E., and T. Sato. High resolution electron microscopy: studies of murine leukemia viruses, p. 296-306. In Unifying concepts of leukemia, International Symposium on Comparative Leukemia Research Proceedings V, Padova, Italy. Karger, New York. 7. Duesberg, P. H., G. S. Martin, and P. K. Vogt. 1970. Glycoprotein components of avian and murine RNA tumor viruses. Virology 41:631-646. 8. Green, R. W., D. P. Bolognesi, W. Schafer, L. Pister, G. Hunsmann, and F. De Noronha. 1973. Polypeptides of mammalian oncornaviruses. I. Isolation and serological analysis of polypeptides from murine and feline C-type viruses. Virology 56:565-579. 9. Hunter, E., R. R. Friis, and P. K. Vogt. 1974. Inhibition of avian sarcoma virus replication by glucosamine. Virology 58:449-456. 9a. Lewandowski, L. J., D. P. Bolognesi, R. E. Smith, and M. Halpern. 1975. Viral glycoprotein synthesis under conditions of glucosamine block in cells transformed by avian sarcoma viruses. Virology 66:347355. 10. Locke, M., N. Krishman, and J. T. McMahon. 1971. A routine method for obtaining high contrast without staining sections. J. Cell Biol. 50:540-544. 11. McLean, J. D., and S. Y. Singer. 1970. A general method for the specific staining of intracellular antigens with ferritin-antibody conjugates. Proc. Natl. Acad. Sci. U.S.A. 65:122-128. 12. Vogt, P. K. 1969. Focus assay of Rous sarcoma virus, p. 198-211. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York.
JOURNAL OF VIROLOGY, Oct. 1975, p. 1071-1074 Copyright ( 1975 American Society for Microbiology
NOTES Detection of Type A Oncornavirus-Like Structures in Chicken Embryo Cells After Infection with Rous Sarcoma Virus GERD G. MAUL AND LEON J. LEWANDOWSKI* The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104
Received for publication 9 June 1975
Electron microscopy examination of Rous sarcoma virus-transformed chicken embryo cells revealed membrane-free nucleoids resembling type A oncornavirus. These particles were not detected in noninfected cells, nor did they accumulate in excess under conditions of glucosamine block in virus-transformed cultures.
The ultrastructural classification of virus particles associated with murine neoplasms into types A, B, C, and D was originally suggested by Bernhard (1). More recently, electron microscopy observations on the maturation of murine RNA tumor virus particles suggested that the B particle-type mouse mammary tumor virus can develop either by incorporation of an intracytoplasmic type A particle into the forming bud, or by the simultaneous formation of the two layers of a viral "nucleoid" just under the plasma membrane of a developing bud (5). The free virions produced by either process are morphologically identical, possessing a centrally located nucleoid, a distinct intermediate layer, and an outer envelope. By comparison, murine C-type virions are believed to develop at the surface of the infected cell only after the appearance of a viral nucleoid in crescentic form under a bulging plasma membrane. Since chicken and murine C-type RNA tumor viruses have many properties in common (8), it was of interest to determine whether avian RNA tumor virus particle maturation was restricted to subsurface formation of the viral nucleoid immediately prior to virus budding. The method that seemed potentially wellsuited for enhancing the detectability of subviral particles was to expose infected cells to glucosamine. In chicken embryo cells transformed by Rous sarcoma virus (RSV), the presence of glucosamine inhibits to nondetectable levels subsequent production of virus (9, 9a). Viral envelope glycoprotein synthesis is aberrant and markedly reduced (9a), whereas the non-glycosylated group-specific (gs) polypeptides that compose the viral core are
synthesized, although also in reduced amounts (9, 9a). It seemed possible that those gs proteins that are synthesized in the presence of glucosamine might accumulate in the form of electron microscopic-detectable intracytoplasmic nucleoids, perhaps analogous to the glycoprotein-free viral "core" structures obtained by detergent treatment in vitro of whole virus
(2).
A clone-purified stock of subgroup C of RSV strain B77 was used to infect C/E chicken embryo cells. Primary cultures were prepared from 11-day-old chicken embryo (SPAFAS, Inc., Storrs, Conn.) as described by Vogt (12). Infection and maintenance of secondary cultures were as described by Duesberg et al. (7). Confluent secondary cultures of both noninfected and RSV-transformed chicken embryo cells, grown in tissue culture chamber/slides (LabTek), were incubated with and without 20 ,umol of glucosamine per ml for 16 h (9a). All cultures were then washed three times with phosphate-buffered saline and fixed with 2% glutaraldehyde in 0.1 M phosphate buffer and 3 mM MgCl2 at pH 7.4 for 20 min at room temperature. -The preparations were again thoroughly washed with phosphate-buffered saline and postfixed for 30 min with 1% OsO, (phosphate buffered; pH 7.4). Before dehydration in ethanol, the cells were stained with 1% uranyl acetate in distilled water for 20 h at 60 C. They were embedded in situ (3) in Epon 812. Thin sections were cut on a LKB Ultratome III and observed (unstained and stained with uranyl acetate and lead citrate) with a Hitachi HU-11D electron microscope operated at 75 kV. Magnifications are noted in
1071
S -s'
-
.. ~ ~
49'
z
ar
.:
w
$L
$
* k
.-. 'p
r
&
FIG. 1. Survey micrograph of chicken embryo fibroblast transformed with RSV. Note aggregate of densely stained circular particles with a diameter of more than 1 um. The particles have two concentric densely stained rings and translucent core. Some seem to be open and crescent shaped (arrows). Uranyl acetate and lead citrate staining. Magnification x 75,000. FIG. 2. Single corelike particles are mostly found in close association with mitochondria. Magnification
x50,000. FIG. 3-4. Higher magnification of single corelike particles. They seem to be in intimate contact with ribosomes (arrows). Magnification x105,000. 1072
NOTES
VOL. 16, 1975
the figure legends. For immune electron microscopy, a double-labeling technique (11) was used that utilized rabbit antisera (kindly supplied by D. Bolognesi) monospecific for p27 and p19, the two major viral gs proteins, and normal rabbit serum; ferritin conjugated goat-anti-rabbit globulin was the electron-dense marker. RSV-transformed chicken fibroblasts cultured in the absence of glucosamine revealed occasional aggregates of high-electron-dense particles within the cytoplasm (Fig. 1). These aggregates were not glycogen since the uranyl acetate stain would have leached glycogen, and they were not lysosomes since they were not surrounded by a membrane. They were present in -10% of all cells and appeared to be composed of membrane-free nucleoids resembling type A oncornavirus particles, with diameters of -60.0 nm, and electron-lucent centers of -25.0 nm in diameter surrounded by two dense rings that were occasionally open. Nearly all trans-
1073
formed cells contained single particles, some apparently at different stages of completeness, interspersed between mitochondria (Fig. 2). Higher magnification (Fig. 3 and 4) shows ribosome-like material closely associated (arrows). Such ribosome-like particles have been visualized by high-resolution electron microscopy inside RNA tumor viruses associated with the viral core structure (6). To avoid any obscuring effect of surface staining on particle substructure (10), unstained sections were also examined. Figure 5 clearly shows the electronlucent centers of complete particles (left) and what appear to be incomplete particles (right). Figure 6 shows typical type C particles, with a characteristic diameter of 100.0 nm, budding at the surface of these cells. Analysis of transformed cells under conditions of glucosamine block showed little, if any, difference from nontreated transformed cells, i.e., no additional accumulation of these struc1{
V1 !~~~ Ii
~
r
rj
''(i 0 AK, IL
'.S'.-'.2 FIG. 5. Unstained preparation of complete and incomplete corelike structures, emphasizing the electrontranslucent center. Magnification x 75,000. FIG. 6. Several budding C particles. Magnification x100,000.
1074
NOTES
tures. No cells containing nucleoids were found in the noninfected control cultures regardless of glucosamine treatment. Attempts to determine the relatedness of the nucleoids to RSV through immune electron microscopy were unfortunately inconclusive; this was apparently due to an inaccessibility of the antigenic binding sites of the nucleoids which usually appeared covered by an abundant, dense ribosome-like material. It is possible, therefore, that the particles represent an unknown endogenous virus unrelated to the input RSV. Their dimensions and ultrastructural features (dense circular particles with an electron-translucent center and two dense shells) suggest, however, that they are either type A oncornavirus particles or an intracellular nucleoid form of C-type RSV, analogous to the type A particle. If so, it remains to be determined whether they are in the process of being assembled de novo or whether they represent RSV progeny superinfecting the cells. Dales and Hanafusa (4), studying the penetration and intracellular release of the genomes of avian RNA tumor viruses, have observed that infecting virions either individually or in groups are internalized by viropexis and move within vacuoles to the nuclear envelopes; they never found either intact input virions or subviral particles (that might be interpreted to be the equivalent of viral cores) without vacuolar membranes. In contrast, we never found the corelike particles with vacuolar membranes. This fact, combined with the observations that the corelike particles were present in conjunction with mitochondria and usually had ribosome-like structures closely associated with them, suggests that they are either viral cores newly synthesized or infecting virus somehow actively engaged in virus replication. In the future, electron microscope autoradiography applied after the virus synthesized de novo is labeled or after the cells are infected with highly labeled input virus may allow us to distinguish between these possibilities.
J. VIROL. This work was supported by Public Health Service research grants CA-10815, CA-15954, and CA-15464 from the National Cancer Institute, and by grant 1194 from the Damon Runyon Memorial Fund. The technical assistance of Carla Brooks, Patricia Stranen and Joseph Weibel is gratefully acknowledged. LITERATURE CITED 1. Bernhard, W. 1960. The detection and study of tumor viruses with the electron microscope. Cancer Res. 20:712-727. 2. Bolognesi, D. P., R. Luftig, and J. H. Shaper. 1973. Localization of RNA tumor virus polypeptides. I. Isolation of further virus substructures. Virology 56:549-564. 3. Brinkley, B. R., P. Murphy, and L. C. Richardson. 1967. Procedure for embedding in situ selected cells cultured in vitro. J. Cell Biol. 35:279-283. 4. Dales, S., and H. Hanafusa. 1972. Penetration and intracellular release of the genomes of avian RNA tumor viruses. Virology 50:440-458. 5. Dalton, A. J. 1972. Further analysis of the detailed structure of Type B and C particles. J. Natl. Cancer. Inst. 48:1095-1099. 6. De Harven, E., and T. Sato. High resolution electron microscopy: studies of murine leukemia viruses, p. 296-306. In Unifying concepts of leukemia, International Symposium on Comparative Leukemia Research Proceedings V, Padova, Italy. Karger, New York. 7. Duesberg, P. H., G. S. Martin, and P. K. Vogt. 1970. Glycoprotein components of avian and murine RNA tumor viruses. Virology 41:631-646. 8. Green, R. W., D. P. Bolognesi, W. Schafer, L. Pister, G. Hunsmann, and F. De Noronha. 1973. Polypeptides of mammalian oncornaviruses. I. Isolation and serological analysis of polypeptides from murine and feline C-type viruses. Virology 56:565-579. 9. Hunter, E., R. R. Friis, and P. K. Vogt. 1974. Inhibition of avian sarcoma virus replication by glucosamine. Virology 58:449-456. 9a. Lewandowski, L. J., D. P. Bolognesi, R. E. Smith, and M. Halpern. 1975. Viral glycoprotein synthesis under conditions of glucosamine block in cells transformed by avian sarcoma viruses. Virology 66:347355. 10. Locke, M., N. Krishman, and J. T. McMahon. 1971. A routine method for obtaining high contrast without staining sections. J. Cell Biol. 50:540-544. 11. McLean, J. D., and S. Y. Singer. 1970. A general method for the specific staining of intracellular antigens with ferritin-antibody conjugates. Proc. Natl. Acad. Sci. U.S.A. 65:122-128. 12. Vogt, P. K. 1969. Focus assay of Rous sarcoma virus, p. 198-211. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York.
