- Email: [email protected]
Ultrastructure of the lactic dehydrogenase virus (LDV) and cell-virus relationships
512
DISCUSSION AND PRELIMINARY REPORTS
gen; again, all results were negative. In addition, these isolates did not cause cytopathic effects when inoculated into cultures of G M K cells. Six of these isolates were also tested f o r ability to induce SV40 tumor antigen in H E K cells; none were able :to do SO.
The progeny in 17 plaques from GMK plates were similarly tested, and 13 were positive; all 17 isolates were passed in GMK tube cultures and the resulting virus harvest gave positive results in all eases; representative isolates yielded plaques in both G1VIK and H E K cultures. A number of isolates from both GMK and H E K plaques were tested, and all of these were reidentified as adenovirus type 7 by neutralization tests with serum prepared in rabbits against a recent isolate of human adenovirus type 7. An isolate from GI~IK cells was replated twice i n G M K cultures and grown to a stock harvest by two passages in GMK cells. This virus stock induced the SV40 tumor antigen in 40 % of the cells on GMK coverslip cultures in 24 hours. Ability of the virus to induce the antigen was not neutralized by norreal rabbit serum or anti-SV40 monkey serum, but anti-adenovirus 7 rabbit serum completely prevented the appearance of the .antigen. In contrast, SV40 virus was preVented from inducing the tumor antigen only by the anti-SV40 serum. These serological results rule out the possibility that the induction of the tumor antigen might be due to accidental contamination of the stock by SV40 virus, and are in agreement with similar results obtained with the parent SP2 stock (I-3). From the present data, it appears that plaque passage in GMK cells preserves the determinant of the SV40 tumor antigen in the resultant progeny. In contrast, when the plating is performed in H E K cells, this determinant is lost. The latter observation is perhaps not unexpected in view of the loss of the tumor antigen determinant when a limiting dilution passage was performed in H E K cells (2). The failure to recover the tumor antigen determinant in progeny from H E K cells may be due to the selection in the highly sensitive H E K cells of particles not
carrying the determinant, or to the inability of the determinant to replicate in these cells. REFERENCES 1. HUEBNEn, R. J., CHANOCK, R. M., RIJBIN, B. A., and CASEY, M. J., Proc. Natl. Acad. Sci. U.S. 52, 1333-1340 (1964). 2. Ro~vE, W. P., and BAUM, S. G., Proc. Natl. Acad. Sci. U.S. 52, 1340-1347 (1964). S. RAPP, F., MELNICK, J. L., BUTEL, J. S., and KITAHARA, T., Proc. Natl. Acad. Sci. U.S. 52, 1348-1352 (1964). 4. SABIN, A. B., and KocH, M. A., Proc. Natl. Acad. Sci. U.S. 52, 1131-1138 (1964). 5. RAPP, F., KITAHARA, T., BUTEL, J. S., and MELNICK, J. L., Proc. Natl. Acad. Sci. U.S. 52, 1138-1142 (1964).
6. RAPP, F., BUTEL, J. S., and MELNmK,J. L., Proc. Soc. Ezptl. Biol. Med. 116, 1131-1135 (1964). 7. POPE, J. It., and ROWE,W. P., J. Exptl. Med. 120, 121-128 (1964). 8. WALLIs,C., and MELNICK,J. L., Texas Rept. Biol. Med. 20, 465-475 (1962). ALBERTBOEY~ JOSEPH L. MELNICK FRED RAPP
Department of Virology and Epidemiology Baylor University College of Medicine Houston, Texas 77025 Accepted April 27, 1965
Ultrastructure of the Lactic Dehydrogenase Virus (LDV) and Cell-Virus Relationships
A number of reports have recently appeared concerning the size and shape of the lactic dehydrogenase virus (LDV) (1-7). The present electron microscopic study was undertaken to (a) compare the size and shape of LDV after different methods of preparation, (b) investigate the fine structure of this virus, and (c) determine the site of its replication. CAF-1 and Swiss general purpose N.I.H. mice (4-6 weeks old) were injected intraperitoneally with a stock preparation of LDV. Twenty-four hours later, when the virus titer was approximately 101°'5 IDs0/ml (2), plasma was collected by orbital bleeding with heparinized micropipettes. Pooled plasma (5-10 ml) was diluted with 5-10 ml of 0.36 M potassium citrate, and/or saline
DISCUSSION AND PRELIMINAP~Y REPORTS with and without hyaluronidase (3.0 rag/100 ml). After preliminary clarification at 4000 g and 10,000 g, the resulting superuatant fluid was centrifuged at 105,000 g for 2 hours in the Spineo model L ultracentrifuge. The pellets thus obtained were resuspended in 0.2-0.5 ml of distilled water and stained with 2% phosphotungstie acid (PTA) buffered with KOH at pH 5.3 (8). Other pellets prepared from the same virus pool were fixed either directly with 1% osmium tetroxide buffered at pH 7.4 (9) for 40 minutes, or with 3% glutaraldehyde (10) (buffered at pH 7.4 with sodium cacodylate) for 30 minutes, followed by osmium tetroxide for 20 minutes, and finally embedded in Epon-ArMdite (11). Organs from infected mice (liver, spleen, lung, kidney, lymph nodes, thymus, bone marrow, and mammary gland) were removed and fixed for 60 minutes with 5 % glutaraldehyde (10), followed by osmium tetroxide for 30 minutes and embedded in Epon-Araldite. Peritoneal macrophages were collected from infected mice by washing the peritoneal cavity with 1.0-2.0 ml of Eagle's basal medium. The cells thus obtained were fixed immediately with 2.5 % glutaraldehyde for 15 minutes, centrifuged at 150 g for 10 minutes, and then treated as described above. Thin sections of the pellets and organs were doubly stained with uranyl acetate (12) and lead citrate (13) and examined with a Siemens Elmiskop I electron microscope. The size and shape of LDV was studied by comparing thin sections and PTA-stained resuspended pellets prepared from the same virus pool. As seen in Figs. 1 and 2, thin sections revealed elliptical or oblong particles 36-42 m~ wide and 45-75 m~ long. Rounded particles averaging 40 m~ in diameter were also seen and may represent cross sections of the above particles or spherical virions. In contrast, resuspended negatively stained pellets of unfixed material (Fig. 3) revealed larger and more pleomorphie particles 60-65 m~ in diameter by 70-85 m~ in length. Similar modifications in size and shape following negative staining have been reported with other viruses (14, 15). Further information about the morphological charaeteristies of LDV was obtained from thin sections of peritoneal macrophages which
513
showed particles similar in diameter and length (Figs. 4 and 5) to those found in thin sections of pellets from viremic plasma. The size of LDV obtained herein by thin sections of pellets and tissues is in fair agreement with the estimates made by Gradoeol membrane filtration (2, 3). Plasma pellets and macrophages from uninfected mice failed to reveM virus particles. The fine structure of LDV is seen in Fig. 2. The virus contains a nueleoid 26-29 mt~ in diameter, which consists of a dense shell 7-9 m~ wide and an electron lucent inner core. Surrounding the dense shell of the nudeoid is an outer layer 5-7 m~ wide of low density, limited by what appears to be a thin outer membrane. In all attempt to determine the site of virus replication, liver, kidney, spleen, lymph nodes, thymus, bone marrow, lung, mammary gland, and peritoneal macrophages from infected mice were screened for virus particles. Except for a small percentage of the peritoneal macrophages the virus was not found in any of the organs examined. Thin sections of peritoneal macrophages (Figs. 4 and 5) showed particles in close association with the cell surface and with the membrane of and within cytoplasmic vacuoles. In addition particles resembling LDV were present in cytoplasmic microbodies. Such results suggested that LDV was phagoeytized by the macrophages. It is known that the functional capacity of the riticuloendothelial system is impaired following infection with LDV (16, 17). Physical blockade of the reticuloendothelial system secondary to phagocytosis of virus particles could be responsible for this impairment. On the other hand, some particles gave the appearance of maturation at the cytoplasmic membrane (fig. 5, bottom, right) but definitive evidence of budding has not yet been obtained. In this connection Evans (18) recently found that LDV would replicate in cultures prepared from peritoneM macrophages. These preliminary observations point to the need for further exploration of the relationships between LDV and the reticuloendothelial system.
514
DISCUSSION AND P R E L I M I N A R Y REPORTS
FIGS. 1-3
DISCUSSION AND PRELIMINARY
REPORTS
515
FIG. 4. P e r i t o n e a l macrophage from infected mice. Virus particles (VP a n d arrows) can be seen close to the m e m b r a n e of the cytoplasmic vacuoles. Note the long particle in the u p p e r center. Magnification: X 54,000. FIG. 5. P e r i t o n e a l macrophage from infected mice. Three LDV particles are seen at the cytoplasmic m e m b r a n e . Particles similar in size to LDV can also be seen in cytoplasmic microbodies (rob). Magnification: X 110,000.
The scales in Figs. 1-5 represent 100 mt~. Fie. 1. T h i n section of a n osmium-fixed pellet p r e p a r e d from viremic plasma. Elliptical or oblong particles 36-42 m~ wide a n d 45-75 m~ long and rounded particles averaging 40 mu in d i a m e t e r are present. Magnification: X 60,000. FIG. 2. At higher magnification the fine s t r u c t u r e of LDV can be seen. A p r o m i n e n t ring-shaped nucleoid (nu) 26-29 mu in d i a m e t e r is surrounded b y a less dense m a t e r i a l 5-7 mu thick. There appears to be a t h i n outer m e m b r a n e (m). The nucleoid of a n oblong particle is clearly seen at the upper left. Magnification: X 255,000. FIG. 3. N e g a t i v e l y s t a i n e d p r e p a r a t i o n of unfixed resuspended pellet from the same plasma pool as in Figs. 1 and 2. Elliptical, rounded, a n d pleomorphie particles measuring 60-65 mu in w i d t h and 70-85 m~ in l e n g t h can be seen. Magnification: X 105,000.
516
DISCUSSION AND P R E L I M I N A R Y REPORTS ACKNOWLEDGMENT
The authors wish to thank Dr. Albert J. Dalton for his advice and hospitality during the course of this work. The technical assistance of Mr. B. Elliot, Mrs. M. Smith, and Miss C. Scheele is gratefully acknowledged. REFERENCES 1. BLADEN, I-I. A. JR., and NOTKINS, A. L.,
Virology 21,269-271 (1963). 2. NO•KINS, A. L., and SHOCHAT, S. J., J. Exptl. Med. 117, 735-747 (1963). 3. ROWSON, K. E. K., M A ~ , B. W. J., and SALAMAN,M. H., Life Sci. 7,479-485 (1963). 4. RILEY, V., Proc. Am. Assoc. Cancer Res. 4, 57 (1963). 5. ADAMS,D. I-I., and BOWMAN, B. M., Biochem. J. 90, 477-482 (1964). 6. CRISPENS, C. G., JR., Virology 24, 501-502 (1964). 7. CRISPENS, C. G., JR., and BURNS, T. A., Nature 204, 1302 (1964). 8. BRENNER, S., and HORNE, R. W., Biochim. Biophys. Acta 34, 103-110 (1959). 9. PALADE, G. E., J. Exptl. Med. 95, 285-298 (1952). 10. SAB~t~TINI,D. C., BENSCIt,K. G., and BAR~NETT, R. J., J. Cell Biol. 17, 19-58 (1963). 11. MOLLENHAUER,I-I. H., Stain Teehnol. 39, 111114 (1964).
12. WATSON,M. L., J. Biophys. Biochem. Cytol. 11,729-732 (1958). 13. REY~OT,DS, E. W., J. Cell Biol. 17, 208-212 (1963). 14. BONAR, R. A., HEINE, U., BEARD, D., and BEARD, J. W., J. Natl. Cancer Inst. 30, 949997 (1963). 15. DE HARVEN, E., and FRIEND, C., Virology 23, 119-124 (1964). 16. NOTKINS, A. L., and SCHEELE, C., J. Natl. Cancer Inst. 33, 741-749 (1964). 17. MAHY, B. W. J., ROWSON,K. E. K., SAL&MAN, M. H., and PARR, C. W., Virology 23, 528541 (1964). 18. EvAns, R., J. Gen. Microbial. 37, vii (1964).
Guy DE-TH~1 Macromolecular Biology Section Laboratory of Viral Oncology National Cancer Institute, Bethesda, 14, Maryland and ABNER L. NOTKINS Laboratory of Microbiology National Institute of Dental Research National Institutes of Health Bethesda, Maryland Accepted, April 23, I965
1 A Visiting Scientist in the Laboratory of Viral Oncology.
DISCUSSION AND PRELIMINARY REPORTS
gen; again, all results were negative. In addition, these isolates did not cause cytopathic effects when inoculated into cultures of G M K cells. Six of these isolates were also tested f o r ability to induce SV40 tumor antigen in H E K cells; none were able :to do SO.
The progeny in 17 plaques from GMK plates were similarly tested, and 13 were positive; all 17 isolates were passed in GMK tube cultures and the resulting virus harvest gave positive results in all eases; representative isolates yielded plaques in both G1VIK and H E K cultures. A number of isolates from both GMK and H E K plaques were tested, and all of these were reidentified as adenovirus type 7 by neutralization tests with serum prepared in rabbits against a recent isolate of human adenovirus type 7. An isolate from GI~IK cells was replated twice i n G M K cultures and grown to a stock harvest by two passages in GMK cells. This virus stock induced the SV40 tumor antigen in 40 % of the cells on GMK coverslip cultures in 24 hours. Ability of the virus to induce the antigen was not neutralized by norreal rabbit serum or anti-SV40 monkey serum, but anti-adenovirus 7 rabbit serum completely prevented the appearance of the .antigen. In contrast, SV40 virus was preVented from inducing the tumor antigen only by the anti-SV40 serum. These serological results rule out the possibility that the induction of the tumor antigen might be due to accidental contamination of the stock by SV40 virus, and are in agreement with similar results obtained with the parent SP2 stock (I-3). From the present data, it appears that plaque passage in GMK cells preserves the determinant of the SV40 tumor antigen in the resultant progeny. In contrast, when the plating is performed in H E K cells, this determinant is lost. The latter observation is perhaps not unexpected in view of the loss of the tumor antigen determinant when a limiting dilution passage was performed in H E K cells (2). The failure to recover the tumor antigen determinant in progeny from H E K cells may be due to the selection in the highly sensitive H E K cells of particles not
carrying the determinant, or to the inability of the determinant to replicate in these cells. REFERENCES 1. HUEBNEn, R. J., CHANOCK, R. M., RIJBIN, B. A., and CASEY, M. J., Proc. Natl. Acad. Sci. U.S. 52, 1333-1340 (1964). 2. Ro~vE, W. P., and BAUM, S. G., Proc. Natl. Acad. Sci. U.S. 52, 1340-1347 (1964). S. RAPP, F., MELNICK, J. L., BUTEL, J. S., and KITAHARA, T., Proc. Natl. Acad. Sci. U.S. 52, 1348-1352 (1964). 4. SABIN, A. B., and KocH, M. A., Proc. Natl. Acad. Sci. U.S. 52, 1131-1138 (1964). 5. RAPP, F., KITAHARA, T., BUTEL, J. S., and MELNICK, J. L., Proc. Natl. Acad. Sci. U.S. 52, 1138-1142 (1964).
6. RAPP, F., BUTEL, J. S., and MELNmK,J. L., Proc. Soc. Ezptl. Biol. Med. 116, 1131-1135 (1964). 7. POPE, J. It., and ROWE,W. P., J. Exptl. Med. 120, 121-128 (1964). 8. WALLIs,C., and MELNICK,J. L., Texas Rept. Biol. Med. 20, 465-475 (1962). ALBERTBOEY~ JOSEPH L. MELNICK FRED RAPP
Department of Virology and Epidemiology Baylor University College of Medicine Houston, Texas 77025 Accepted April 27, 1965
Ultrastructure of the Lactic Dehydrogenase Virus (LDV) and Cell-Virus Relationships
A number of reports have recently appeared concerning the size and shape of the lactic dehydrogenase virus (LDV) (1-7). The present electron microscopic study was undertaken to (a) compare the size and shape of LDV after different methods of preparation, (b) investigate the fine structure of this virus, and (c) determine the site of its replication. CAF-1 and Swiss general purpose N.I.H. mice (4-6 weeks old) were injected intraperitoneally with a stock preparation of LDV. Twenty-four hours later, when the virus titer was approximately 101°'5 IDs0/ml (2), plasma was collected by orbital bleeding with heparinized micropipettes. Pooled plasma (5-10 ml) was diluted with 5-10 ml of 0.36 M potassium citrate, and/or saline
DISCUSSION AND PRELIMINAP~Y REPORTS with and without hyaluronidase (3.0 rag/100 ml). After preliminary clarification at 4000 g and 10,000 g, the resulting superuatant fluid was centrifuged at 105,000 g for 2 hours in the Spineo model L ultracentrifuge. The pellets thus obtained were resuspended in 0.2-0.5 ml of distilled water and stained with 2% phosphotungstie acid (PTA) buffered with KOH at pH 5.3 (8). Other pellets prepared from the same virus pool were fixed either directly with 1% osmium tetroxide buffered at pH 7.4 (9) for 40 minutes, or with 3% glutaraldehyde (10) (buffered at pH 7.4 with sodium cacodylate) for 30 minutes, followed by osmium tetroxide for 20 minutes, and finally embedded in Epon-ArMdite (11). Organs from infected mice (liver, spleen, lung, kidney, lymph nodes, thymus, bone marrow, and mammary gland) were removed and fixed for 60 minutes with 5 % glutaraldehyde (10), followed by osmium tetroxide for 30 minutes and embedded in Epon-Araldite. Peritoneal macrophages were collected from infected mice by washing the peritoneal cavity with 1.0-2.0 ml of Eagle's basal medium. The cells thus obtained were fixed immediately with 2.5 % glutaraldehyde for 15 minutes, centrifuged at 150 g for 10 minutes, and then treated as described above. Thin sections of the pellets and organs were doubly stained with uranyl acetate (12) and lead citrate (13) and examined with a Siemens Elmiskop I electron microscope. The size and shape of LDV was studied by comparing thin sections and PTA-stained resuspended pellets prepared from the same virus pool. As seen in Figs. 1 and 2, thin sections revealed elliptical or oblong particles 36-42 m~ wide and 45-75 m~ long. Rounded particles averaging 40 m~ in diameter were also seen and may represent cross sections of the above particles or spherical virions. In contrast, resuspended negatively stained pellets of unfixed material (Fig. 3) revealed larger and more pleomorphie particles 60-65 m~ in diameter by 70-85 m~ in length. Similar modifications in size and shape following negative staining have been reported with other viruses (14, 15). Further information about the morphological charaeteristies of LDV was obtained from thin sections of peritoneal macrophages which
513
showed particles similar in diameter and length (Figs. 4 and 5) to those found in thin sections of pellets from viremic plasma. The size of LDV obtained herein by thin sections of pellets and tissues is in fair agreement with the estimates made by Gradoeol membrane filtration (2, 3). Plasma pellets and macrophages from uninfected mice failed to reveM virus particles. The fine structure of LDV is seen in Fig. 2. The virus contains a nueleoid 26-29 mt~ in diameter, which consists of a dense shell 7-9 m~ wide and an electron lucent inner core. Surrounding the dense shell of the nudeoid is an outer layer 5-7 m~ wide of low density, limited by what appears to be a thin outer membrane. In all attempt to determine the site of virus replication, liver, kidney, spleen, lymph nodes, thymus, bone marrow, lung, mammary gland, and peritoneal macrophages from infected mice were screened for virus particles. Except for a small percentage of the peritoneal macrophages the virus was not found in any of the organs examined. Thin sections of peritoneal macrophages (Figs. 4 and 5) showed particles in close association with the cell surface and with the membrane of and within cytoplasmic vacuoles. In addition particles resembling LDV were present in cytoplasmic microbodies. Such results suggested that LDV was phagoeytized by the macrophages. It is known that the functional capacity of the riticuloendothelial system is impaired following infection with LDV (16, 17). Physical blockade of the reticuloendothelial system secondary to phagocytosis of virus particles could be responsible for this impairment. On the other hand, some particles gave the appearance of maturation at the cytoplasmic membrane (fig. 5, bottom, right) but definitive evidence of budding has not yet been obtained. In this connection Evans (18) recently found that LDV would replicate in cultures prepared from peritoneM macrophages. These preliminary observations point to the need for further exploration of the relationships between LDV and the reticuloendothelial system.
514
DISCUSSION AND P R E L I M I N A R Y REPORTS
FIGS. 1-3
DISCUSSION AND PRELIMINARY
REPORTS
515
FIG. 4. P e r i t o n e a l macrophage from infected mice. Virus particles (VP a n d arrows) can be seen close to the m e m b r a n e of the cytoplasmic vacuoles. Note the long particle in the u p p e r center. Magnification: X 54,000. FIG. 5. P e r i t o n e a l macrophage from infected mice. Three LDV particles are seen at the cytoplasmic m e m b r a n e . Particles similar in size to LDV can also be seen in cytoplasmic microbodies (rob). Magnification: X 110,000.
The scales in Figs. 1-5 represent 100 mt~. Fie. 1. T h i n section of a n osmium-fixed pellet p r e p a r e d from viremic plasma. Elliptical or oblong particles 36-42 m~ wide a n d 45-75 m~ long and rounded particles averaging 40 mu in d i a m e t e r are present. Magnification: X 60,000. FIG. 2. At higher magnification the fine s t r u c t u r e of LDV can be seen. A p r o m i n e n t ring-shaped nucleoid (nu) 26-29 mu in d i a m e t e r is surrounded b y a less dense m a t e r i a l 5-7 mu thick. There appears to be a t h i n outer m e m b r a n e (m). The nucleoid of a n oblong particle is clearly seen at the upper left. Magnification: X 255,000. FIG. 3. N e g a t i v e l y s t a i n e d p r e p a r a t i o n of unfixed resuspended pellet from the same plasma pool as in Figs. 1 and 2. Elliptical, rounded, a n d pleomorphie particles measuring 60-65 mu in w i d t h and 70-85 m~ in l e n g t h can be seen. Magnification: X 105,000.
516
DISCUSSION AND P R E L I M I N A R Y REPORTS ACKNOWLEDGMENT
The authors wish to thank Dr. Albert J. Dalton for his advice and hospitality during the course of this work. The technical assistance of Mr. B. Elliot, Mrs. M. Smith, and Miss C. Scheele is gratefully acknowledged. REFERENCES 1. BLADEN, I-I. A. JR., and NOTKINS, A. L.,
Virology 21,269-271 (1963). 2. NO•KINS, A. L., and SHOCHAT, S. J., J. Exptl. Med. 117, 735-747 (1963). 3. ROWSON, K. E. K., M A ~ , B. W. J., and SALAMAN,M. H., Life Sci. 7,479-485 (1963). 4. RILEY, V., Proc. Am. Assoc. Cancer Res. 4, 57 (1963). 5. ADAMS,D. I-I., and BOWMAN, B. M., Biochem. J. 90, 477-482 (1964). 6. CRISPENS, C. G., JR., Virology 24, 501-502 (1964). 7. CRISPENS, C. G., JR., and BURNS, T. A., Nature 204, 1302 (1964). 8. BRENNER, S., and HORNE, R. W., Biochim. Biophys. Acta 34, 103-110 (1959). 9. PALADE, G. E., J. Exptl. Med. 95, 285-298 (1952). 10. SAB~t~TINI,D. C., BENSCIt,K. G., and BAR~NETT, R. J., J. Cell Biol. 17, 19-58 (1963). 11. MOLLENHAUER,I-I. H., Stain Teehnol. 39, 111114 (1964).
12. WATSON,M. L., J. Biophys. Biochem. Cytol. 11,729-732 (1958). 13. REY~OT,DS, E. W., J. Cell Biol. 17, 208-212 (1963). 14. BONAR, R. A., HEINE, U., BEARD, D., and BEARD, J. W., J. Natl. Cancer Inst. 30, 949997 (1963). 15. DE HARVEN, E., and FRIEND, C., Virology 23, 119-124 (1964). 16. NOTKINS, A. L., and SCHEELE, C., J. Natl. Cancer Inst. 33, 741-749 (1964). 17. MAHY, B. W. J., ROWSON,K. E. K., SAL&MAN, M. H., and PARR, C. W., Virology 23, 528541 (1964). 18. EvAns, R., J. Gen. Microbial. 37, vii (1964).
Guy DE-TH~1 Macromolecular Biology Section Laboratory of Viral Oncology National Cancer Institute, Bethesda, 14, Maryland and ABNER L. NOTKINS Laboratory of Microbiology National Institute of Dental Research National Institutes of Health Bethesda, Maryland Accepted, April 23, I965
1 A Visiting Scientist in the Laboratory of Viral Oncology.