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Study of intracellular SV40 antigens by indirect immunoferritin technique
DISCUSSION
AND
PRELIMINARY
ACKNOWLEDGMENTS The excellent technical assistance of Mrs. Fermina Varacalli is gratefully acknowledged. This investigation was supported by PHS Grant AI 07057-01 VR from the National Institute of Allergy and Infectious Diseases. REFERENCES VILEEH, J., and FREER, J. H.; J. Bacterial. 92, 17161722 (1966). TAYLOR, J., Biochem. Biophys. Res. Commun. 14.447-451 (1964). Ho, M., Virology 17, 262-275 (1962). WAGNER, R. R., and HUANG, A. S., Virology 28, l-10 (1966). J. VILEEB M. H. NG Department o.f Microbiology New York University School of Medicine New York, New York 10016 Accepted November SO, 1966
Study
of Intracellular
Indirect
SV40
lmmunoferritin
Antigens
by
Technique
A number of studies of the intracellular replication cycles of virus in mammalian cells with ferritin-labeled antibody (1) have previously been performed by the direct technique, in which ferritin-labeled specific a&ibody is allowed to react directly with infected cells (2-d). Use of the indirect or sandwich technique, in which infected cells are allowed to react with whole sera containing specific antibody, washed, and then allowed to react with a ferritin-labeled antiglobulin, has been used in the study of cell surface antigens (5). Using the indirect immunoferritin reaction with antihamster and antirabbit globulins labeled with both fluorescein and ferritin, we have previously localized the early antigen (T-antigen, neoantigen) and the structural viral ant’igens of human adenovirus type 12 in infected and transformed cells (6). The present study is concerned with the localizat,ion of t’he early ant,igen (T-antigen, neoantigen), first detected in immunofluorescence by Pope and Rowe in 1964 (7), and of structural viral antigens of SV40 virus in infected and transformed cells. Monolayers of BSC-1 and 1\1,4104 monkey
555
REPORTS
kidney cells and SV40 transformed hamster cells were grown in Eagle’s minimum essential medium with fourfold amino acids and vitamins and 10% fetal calf serum without antibiot’ics. Cultures infectred with SV40 virus were maintained in the same medium with 5 % fetal calf serum until fixation at 24 hours to ;5 days after infection. Cells were infected with SV40 virus using 1 PFU per cell, a mult’iplicit’y of infection that generally produced 30-50 % virus-producing cells within 3 days. The efficiency of infection was monitored by immunofluorescence using standard indirect technique (8). SV40-infected cells grown on coverslips in Leighton tubes were fixed in cold acetone for 10 minutes, dried, and stored in screw cap vials at -70”. With this treatment early and viral antigens remained detectable for several months. The antibody for detection of early SV40 antigen was obtained from sera of hamsters bearing grafted SV40 tumors, and was used at a dilution of 1 :j for both immunofluorescence and immunoferritin. The sera did not neutralize SV40 virus in a plaque reduction test. Structural viral antigens were detect,ed with serum from a rabbit immunized with SV40 stock virus, absorbed t’wice with BSC-1 cells, used at a dilution of I:8 for both immunofluorescence and immunoferritin. Fluorescent rabbit antihamster and sheep antirabbit’ globulins obt’ained through the kindness of Dr. G. Sizaret of t,he Past’eur Institut at Garches, France,’ were ferritin labeled as previously described (6), by means of a t,echnique somewhat modified from that of Rifkind et al., (9). Single-labeled fluorescent antiglobulins gave brilliant staining at a protein concentration of 0.02& 0.1 %I. After the ferrit in label had been added, t’he double-labeled antiglobulins had t*o be used at a protein roncent’ration of 0.5-1.2% when used to detect early and structural viral antigens in immunofluorescence. The indirect immunoferritin reaction was performed on SV40-infected and transformed cells as previously described in the study of adenovirus antigens (8). The monolayer was washed briefly wit’h PBS (0.1 M phosphate buffered saline pH 7.4), allowed to 1 Now commercially Pasteur, Paris.
available
from the Institut
556
DISCUSSION
AND
PRELIMINARY
react for 1 minute with 1.2 X 1O-4 M digitonin in cacodylate buffer pH 7.4, followed immediately by 1 minute of 0.5 formaldehyde in cacodylate. After two lo-minute washes of PBS and a third wash for 10 minutes to as long as 2 weeks at 4”, the cells were scraped, decanted into McNaught protein tubes, and centrifuged; the pellet volume was adjusted to 0.05 ml. Four volumes of diluted whole serum were added, the cells were mixed with a platinum wire and allowed to react with the serum for l-2 hours at room temperature. After three serial washes of PBS during 45 minutes to remove nonspecific globulin, the cells were dispersed in four volumes of double labeled antiglobulin, and allowed to react for l-2 hours at room temperature. After three final washes in PBS, the pellet, usually about 0.02-0.03 ml in volume, was postfixed undisturbed in l-270 osmic acid for 30-45 minutes, dehydrated, and embedded in Epon. Thin sections were stained with uranyl acetate for 5-10 minutes and with lead citrate (IO) for l-5 minutes. Preparations were examined in a Siemens Elmiskop 1 microscope. Controls consisted of normal cells allowed to react with antiviral sera and infected or transformed cells that had reacted with normal sera followed by specific ferrit’in antiglobulin. Cells allowed to react with ferritin antiglobulin alone were also examined. The chief finding as illustrated in Fig. 1 was that SV40 virus could be “stained” by antiviral sera and ferritin-labeled specific antiglobulin, but only when present in the cytoplasm. There was also considerable diffuse and irregularly localized particulate ‘Maining” of material presumed to be struc-
REPORTS
tural viral antigens in nuclei of SV40-infected cells prior to and even after the appearance of the assembled virions, indicating reasonably free entry of antibody. Even when this viral matrix showed extensive “staining,” the virus particles in the nucleus showed no more ferritin than the slight general background nonspecific reaction. Nuclear chromatin and nucleoli were not “stained;” cytoplasmic membranes showed occasional irregular and presumably nonspecific adsorption of ferritin. The findings with respect t,o immunoferritin staining of the SV40 virus particles suggest that virus is altered upon leaving the nucleus, either acquiring antigens detectable by antiviral sera, or losing substances which prevent reaction with antiviral sera in the nucleus.2 The fragile tubules bounded by osmiophilic double membranes, (probably smooth endoplasmic reticulum), through which SV40 virions are excreted from the nucleus (11) were better preserved by double fixation with formaldehyde and osmic acid than by osmic acid alone. Where they remained intact they formed an impenetrable barrier to antibody, so that’ virus in t’hese tubules as well as virus in cytoplasmic vacuoles was not “stained.” Usually these tubules were seen 2 After these findings were observed, we were fortunate in being permitted to see electron micrographs made by Dr. L. Oshiro in an independent study of SV40 virus infection with direct immunoferritin technique. The results obtained with hyperimmune monkey anti SV40 globulin labeled with ferritin and applied to SV40 virus-infected cells agree exactly wit’h our own results, namely, that the virus can be “stained” only upon release into the cytoplasm.
by indirect immunoferritin react,ion, FIG. 1. BSC-1 cells infected with SV40 virus, 3 days, “stained” using rabbit anti SV40 serum and ferritin labeled goat antirabbit globulin. Cytoplasmic virus is “stained” (V+). Nuclear virus and virus in tubules and vacuoles are unstained (V-). Uranyl acetate and lead citrate. Magnification: X 60,000. FIG. 2. BSC-1 cells infected with SV40 virus, 3 days, control for indirect immunoferritin reaction, using normal rabbit serum and ferritin labeled goat antirabbit globulin. Slight random localization of ferritin. Tubules (T) associated with virus release from the nucleus appear to arise from nuclear membrane. chr, chromatin. Uranyl acetate and lead citrate. Magnification: X 60,000. FIG. 3. Hamster SV40 tumor cell grown in vitro and “stained” by indirect immlmoferritin reaction, using serum of tumor-bearing hamsters, and ferritin-labeled rabbit antihamster globulin. Irregular nuclear aggregates (arrows) heavily labeled with ferritin. Chromatin (chr) not stained. Uranyl acetate and lead citrate. Magnification: X 60,000.
I>ISCUSSION
AND PRELIMINARY
REPORTS
557
553
DISCUSSION
AND
PRELIMINARY
REPORTS
as broken fragments with virus adsorbed to ACKNOWLEDGMENTS one wall, but occasional lengths of intact This work was generously supported by a grant tubules were found containing virus like a from the Centre de Recherches sur les Lymphomes row of beads (Fig. 2). They appear to arise Malins and was performed while the senior author was the recipient of a USPHS special fellowship from the outer leaf of the nuclear membrane No. l-F3-CA-4695-01. We wish to thank Doctor but do not appear to cont’ain any virusspecific antigens, since they were never Wilhelm Bernhard for his invaluable aid, encouragement and advice, and for the use of the “stained” in the immunoferritin reaction. facilities of the CNRS Electron Microscopy SerHamster SV40 tumor cells grown in vitro vice. We are grateful to the staff of the Virology and “stained” by indirect immunoferritin Service of Professor Paul Tournier for unfailing technique as illustrated in Fig. 3, showed ir- supplies of virus-infected and normal cell cultures, regular and occasionally rounded small areas and to Mlle Francine Delahaye, for exceptionally of dense accumulation of ferritin in the capable technical help. nucleus. These are believed to represent the REFERENCES early SV40 antigen. Similar small areas, at1. SIKGER, S. J., Kature 183, 1523-1524 (1959). tractive to ferrit’in, were occasionally found 2. MORGAN, C.: RIFKIND, R. A., Hsu, K. C., HOLin the nuclei of normal cells or cells infected DEN, M.; SEEGAL, B. C., and ROSE, H, M., with other viruses, and could not be distinVirology 14, 292-296 (1961). guished morphologically from those found 3. DALES, S., GOM~TOS, P. J., and Hsu, K. C., Virology 25, 193-211 (1965). in the SV40 tumor cells. However, the con4. KBLNINS, V. I., STICH, H. F., and YOHN, 1). S., sistent’ly frequent occurrence of these areas Virology 28, 751-754 (1966). in the SV40 tumor cells and the intensity of 5. BAXENDALL, J., PERLMANN, P., and AFZELIUS, the ferritin “staining” is good evidence for B. A., J. Cell Biol. 14, 144-151 (1962). its specificity. The ability of t’he immuno6. LEVINTHAL, J. D., CEROTTINI, J. C., An~anferritin technique to detect areas of accumuZ~DEH, C., and WIcKEa, R., Intern. J. Cancer. lation of the early SV40 antigen, which 2, (1967). In press. are otherwise unidentifiable on morphologic Y. POPE, J. H., and ROWE, W. P., J. Exptl. Med. grounds in electron microscopy, offers hope 120, 121-128 (1964). that t,his t’echnique will be of value in the 8. CHERRY, W. R., GOLDMAN, II., CIRSKI, T. R., and MOODY, M. D., Public Health Serv. localizat’ion of specific proteins in a variety of Publ. 729. U.S. Government Printing Office, systems. Washington, D. C.: 1961. Use of the indirect technique has obvious 9. RIFKIND, R. A., Hsu, K. C., and MORGAN, C., advantages. Ferritin antiglobulins whose acJ. Histochem. Cytochem. 12, 131-136 (1964). tivit,y had been proved in a previous study 10. REYNOLDS, E. S., J. Cell Biol. 17, 208-212 could be immediately applied to a different (1963). problem by the simple substitution of dif- 11. MAYOR, H. D., STINEBAUGH, S. E., JAMISON, R. M., JoR~~~N, L. E., and MELNICK, J. L., ferent specific rabbit or hamster sera. MoreExptl. Mol. Pathol. 1, 397-416 (1962). over, in view of the loss of activity often associated with ferrit#in labeling, resulting in 12. Hsu, K. C., RIFKIND, R. A., and ZABRISKIE, J. B., Science 142, 1471-1473 (1963). the loss of irreplaceable amounts of valuable JEANA D. LEVINTIUL sera, it is safer to add the ferritin label to RENT? WICKER Institut de Recherches Scientijiques sur le Cancer antiglobulins, which are far easier to obtain and replace. Use of the double label (12) Vilillejuif, France J. C. CER~TTINI allows one t,o test the antiglobulin by Institut de Biochimie immunofluorescence prior to its use in the Universitk de Lausanne more time-consuming immunoferritin re- Lausanne, Switzerland Accepted December 9, 1966 a&ion.
AND
PRELIMINARY
ACKNOWLEDGMENTS The excellent technical assistance of Mrs. Fermina Varacalli is gratefully acknowledged. This investigation was supported by PHS Grant AI 07057-01 VR from the National Institute of Allergy and Infectious Diseases. REFERENCES VILEEH, J., and FREER, J. H.; J. Bacterial. 92, 17161722 (1966). TAYLOR, J., Biochem. Biophys. Res. Commun. 14.447-451 (1964). Ho, M., Virology 17, 262-275 (1962). WAGNER, R. R., and HUANG, A. S., Virology 28, l-10 (1966). J. VILEEB M. H. NG Department o.f Microbiology New York University School of Medicine New York, New York 10016 Accepted November SO, 1966
Study
of Intracellular
Indirect
SV40
lmmunoferritin
Antigens
by
Technique
A number of studies of the intracellular replication cycles of virus in mammalian cells with ferritin-labeled antibody (1) have previously been performed by the direct technique, in which ferritin-labeled specific a&ibody is allowed to react directly with infected cells (2-d). Use of the indirect or sandwich technique, in which infected cells are allowed to react with whole sera containing specific antibody, washed, and then allowed to react with a ferritin-labeled antiglobulin, has been used in the study of cell surface antigens (5). Using the indirect immunoferritin reaction with antihamster and antirabbit globulins labeled with both fluorescein and ferritin, we have previously localized the early antigen (T-antigen, neoantigen) and the structural viral ant’igens of human adenovirus type 12 in infected and transformed cells (6). The present study is concerned with the localizat,ion of t’he early ant,igen (T-antigen, neoantigen), first detected in immunofluorescence by Pope and Rowe in 1964 (7), and of structural viral antigens of SV40 virus in infected and transformed cells. Monolayers of BSC-1 and 1\1,4104 monkey
555
REPORTS
kidney cells and SV40 transformed hamster cells were grown in Eagle’s minimum essential medium with fourfold amino acids and vitamins and 10% fetal calf serum without antibiot’ics. Cultures infectred with SV40 virus were maintained in the same medium with 5 % fetal calf serum until fixation at 24 hours to ;5 days after infection. Cells were infected with SV40 virus using 1 PFU per cell, a mult’iplicit’y of infection that generally produced 30-50 % virus-producing cells within 3 days. The efficiency of infection was monitored by immunofluorescence using standard indirect technique (8). SV40-infected cells grown on coverslips in Leighton tubes were fixed in cold acetone for 10 minutes, dried, and stored in screw cap vials at -70”. With this treatment early and viral antigens remained detectable for several months. The antibody for detection of early SV40 antigen was obtained from sera of hamsters bearing grafted SV40 tumors, and was used at a dilution of 1 :j for both immunofluorescence and immunoferritin. The sera did not neutralize SV40 virus in a plaque reduction test. Structural viral antigens were detect,ed with serum from a rabbit immunized with SV40 stock virus, absorbed t’wice with BSC-1 cells, used at a dilution of I:8 for both immunofluorescence and immunoferritin. Fluorescent rabbit antihamster and sheep antirabbit’ globulins obt’ained through the kindness of Dr. G. Sizaret of t,he Past’eur Institut at Garches, France,’ were ferritin labeled as previously described (6), by means of a t,echnique somewhat modified from that of Rifkind et al., (9). Single-labeled fluorescent antiglobulins gave brilliant staining at a protein concentration of 0.02& 0.1 %I. After the ferrit in label had been added, t’he double-labeled antiglobulins had t*o be used at a protein roncent’ration of 0.5-1.2% when used to detect early and structural viral antigens in immunofluorescence. The indirect immunoferritin reaction was performed on SV40-infected and transformed cells as previously described in the study of adenovirus antigens (8). The monolayer was washed briefly wit’h PBS (0.1 M phosphate buffered saline pH 7.4), allowed to 1 Now commercially Pasteur, Paris.
available
from the Institut
556
DISCUSSION
AND
PRELIMINARY
react for 1 minute with 1.2 X 1O-4 M digitonin in cacodylate buffer pH 7.4, followed immediately by 1 minute of 0.5 formaldehyde in cacodylate. After two lo-minute washes of PBS and a third wash for 10 minutes to as long as 2 weeks at 4”, the cells were scraped, decanted into McNaught protein tubes, and centrifuged; the pellet volume was adjusted to 0.05 ml. Four volumes of diluted whole serum were added, the cells were mixed with a platinum wire and allowed to react with the serum for l-2 hours at room temperature. After three serial washes of PBS during 45 minutes to remove nonspecific globulin, the cells were dispersed in four volumes of double labeled antiglobulin, and allowed to react for l-2 hours at room temperature. After three final washes in PBS, the pellet, usually about 0.02-0.03 ml in volume, was postfixed undisturbed in l-270 osmic acid for 30-45 minutes, dehydrated, and embedded in Epon. Thin sections were stained with uranyl acetate for 5-10 minutes and with lead citrate (IO) for l-5 minutes. Preparations were examined in a Siemens Elmiskop 1 microscope. Controls consisted of normal cells allowed to react with antiviral sera and infected or transformed cells that had reacted with normal sera followed by specific ferrit’in antiglobulin. Cells allowed to react with ferritin antiglobulin alone were also examined. The chief finding as illustrated in Fig. 1 was that SV40 virus could be “stained” by antiviral sera and ferritin-labeled specific antiglobulin, but only when present in the cytoplasm. There was also considerable diffuse and irregularly localized particulate ‘Maining” of material presumed to be struc-
REPORTS
tural viral antigens in nuclei of SV40-infected cells prior to and even after the appearance of the assembled virions, indicating reasonably free entry of antibody. Even when this viral matrix showed extensive “staining,” the virus particles in the nucleus showed no more ferritin than the slight general background nonspecific reaction. Nuclear chromatin and nucleoli were not “stained;” cytoplasmic membranes showed occasional irregular and presumably nonspecific adsorption of ferritin. The findings with respect t,o immunoferritin staining of the SV40 virus particles suggest that virus is altered upon leaving the nucleus, either acquiring antigens detectable by antiviral sera, or losing substances which prevent reaction with antiviral sera in the nucleus.2 The fragile tubules bounded by osmiophilic double membranes, (probably smooth endoplasmic reticulum), through which SV40 virions are excreted from the nucleus (11) were better preserved by double fixation with formaldehyde and osmic acid than by osmic acid alone. Where they remained intact they formed an impenetrable barrier to antibody, so that’ virus in t’hese tubules as well as virus in cytoplasmic vacuoles was not “stained.” Usually these tubules were seen 2 After these findings were observed, we were fortunate in being permitted to see electron micrographs made by Dr. L. Oshiro in an independent study of SV40 virus infection with direct immunoferritin technique. The results obtained with hyperimmune monkey anti SV40 globulin labeled with ferritin and applied to SV40 virus-infected cells agree exactly wit’h our own results, namely, that the virus can be “stained” only upon release into the cytoplasm.
by indirect immunoferritin react,ion, FIG. 1. BSC-1 cells infected with SV40 virus, 3 days, “stained” using rabbit anti SV40 serum and ferritin labeled goat antirabbit globulin. Cytoplasmic virus is “stained” (V+). Nuclear virus and virus in tubules and vacuoles are unstained (V-). Uranyl acetate and lead citrate. Magnification: X 60,000. FIG. 2. BSC-1 cells infected with SV40 virus, 3 days, control for indirect immunoferritin reaction, using normal rabbit serum and ferritin labeled goat antirabbit globulin. Slight random localization of ferritin. Tubules (T) associated with virus release from the nucleus appear to arise from nuclear membrane. chr, chromatin. Uranyl acetate and lead citrate. Magnification: X 60,000. FIG. 3. Hamster SV40 tumor cell grown in vitro and “stained” by indirect immlmoferritin reaction, using serum of tumor-bearing hamsters, and ferritin-labeled rabbit antihamster globulin. Irregular nuclear aggregates (arrows) heavily labeled with ferritin. Chromatin (chr) not stained. Uranyl acetate and lead citrate. Magnification: X 60,000.
I>ISCUSSION
AND PRELIMINARY
REPORTS
557
553
DISCUSSION
AND
PRELIMINARY
REPORTS
as broken fragments with virus adsorbed to ACKNOWLEDGMENTS one wall, but occasional lengths of intact This work was generously supported by a grant tubules were found containing virus like a from the Centre de Recherches sur les Lymphomes row of beads (Fig. 2). They appear to arise Malins and was performed while the senior author was the recipient of a USPHS special fellowship from the outer leaf of the nuclear membrane No. l-F3-CA-4695-01. We wish to thank Doctor but do not appear to cont’ain any virusspecific antigens, since they were never Wilhelm Bernhard for his invaluable aid, encouragement and advice, and for the use of the “stained” in the immunoferritin reaction. facilities of the CNRS Electron Microscopy SerHamster SV40 tumor cells grown in vitro vice. We are grateful to the staff of the Virology and “stained” by indirect immunoferritin Service of Professor Paul Tournier for unfailing technique as illustrated in Fig. 3, showed ir- supplies of virus-infected and normal cell cultures, regular and occasionally rounded small areas and to Mlle Francine Delahaye, for exceptionally of dense accumulation of ferritin in the capable technical help. nucleus. These are believed to represent the REFERENCES early SV40 antigen. Similar small areas, at1. SIKGER, S. J., Kature 183, 1523-1524 (1959). tractive to ferrit’in, were occasionally found 2. MORGAN, C.: RIFKIND, R. A., Hsu, K. C., HOLin the nuclei of normal cells or cells infected DEN, M.; SEEGAL, B. C., and ROSE, H, M., with other viruses, and could not be distinVirology 14, 292-296 (1961). guished morphologically from those found 3. DALES, S., GOM~TOS, P. J., and Hsu, K. C., Virology 25, 193-211 (1965). in the SV40 tumor cells. However, the con4. KBLNINS, V. I., STICH, H. F., and YOHN, 1). S., sistent’ly frequent occurrence of these areas Virology 28, 751-754 (1966). in the SV40 tumor cells and the intensity of 5. BAXENDALL, J., PERLMANN, P., and AFZELIUS, the ferritin “staining” is good evidence for B. A., J. Cell Biol. 14, 144-151 (1962). its specificity. The ability of t’he immuno6. LEVINTHAL, J. D., CEROTTINI, J. C., An~anferritin technique to detect areas of accumuZ~DEH, C., and WIcKEa, R., Intern. J. Cancer. lation of the early SV40 antigen, which 2, (1967). In press. are otherwise unidentifiable on morphologic Y. POPE, J. H., and ROWE, W. P., J. Exptl. Med. grounds in electron microscopy, offers hope 120, 121-128 (1964). that t,his t’echnique will be of value in the 8. CHERRY, W. R., GOLDMAN, II., CIRSKI, T. R., and MOODY, M. D., Public Health Serv. localizat’ion of specific proteins in a variety of Publ. 729. U.S. Government Printing Office, systems. Washington, D. C.: 1961. Use of the indirect technique has obvious 9. RIFKIND, R. A., Hsu, K. C., and MORGAN, C., advantages. Ferritin antiglobulins whose acJ. Histochem. Cytochem. 12, 131-136 (1964). tivit,y had been proved in a previous study 10. REYNOLDS, E. S., J. Cell Biol. 17, 208-212 could be immediately applied to a different (1963). problem by the simple substitution of dif- 11. MAYOR, H. D., STINEBAUGH, S. E., JAMISON, R. M., JoR~~~N, L. E., and MELNICK, J. L., ferent specific rabbit or hamster sera. MoreExptl. Mol. Pathol. 1, 397-416 (1962). over, in view of the loss of activity often associated with ferrit#in labeling, resulting in 12. Hsu, K. C., RIFKIND, R. A., and ZABRISKIE, J. B., Science 142, 1471-1473 (1963). the loss of irreplaceable amounts of valuable JEANA D. LEVINTIUL sera, it is safer to add the ferritin label to RENT? WICKER Institut de Recherches Scientijiques sur le Cancer antiglobulins, which are far easier to obtain and replace. Use of the double label (12) Vilillejuif, France J. C. CER~TTINI allows one t,o test the antiglobulin by Institut de Biochimie immunofluorescence prior to its use in the Universitk de Lausanne more time-consuming immunoferritin re- Lausanne, Switzerland Accepted December 9, 1966 a&ion.