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Electron microscopic localization of intracellular viral antigen by the use of ferritin-conjugated antibody
DISCUSSION
292
AND
PRELIMINARY
T. T., CIECIURA, S. L., and FISHER, H. W., j. Ezptl. Med. 106, 145-158 (1957). 7. KROOTH, R. S., and WEISBERC, A. N., J. Ezptl.
6. PUCK,
Med.
in press
(1961).
H., Science 130, 432-437 (1959). MCQUILKIX, W. T., EVANS, V. T., and EARLE, W. R., J. h’atl. Cancer Inst. 19, 885-896
8. EAGLE, 9.
(1957). 10. DARNELL, J. E., JR., and SAWYER, T. K., Viralogy 11,665-675 (1960). 11. JOECLIK, W. K., and DARSF.LL, J. E., JR., Vi?ology 13,439-447 (1961). 1.2. KROOTH, R. S., and WEIXBERG, A. Iv., Rio&em. Riophys. Research Communs. 3, 518-524
(1960). R. S. J. H. Natiorlal Bcthescla
Electron
KROOTH TJIO
Institutes of Health 14, Maryland
Microscopic Localization Viral Antigen by the
of Intracellular Use of
Ferritin-conjugated Antibody’
The use of ferritin-conjugated antibody globulin (1) enables the sites of antigenantibody interaction to be visualized in the electron microscope (g-4). To apply this method in studies of viral development, infected cells must be treated so that the ferritin-globulin complex gains entrance, while structure and antigenic composition of the virus are preserved. The purpose of this report is to describe a technique that has been used to tag intracellular vaccinia virus with ferritin-conjugated antibody. Antibody globulin, obtained from rabbits immunized with vaccinia virus, was conjugated with ferritin by the method previously described (1, 2). The ferritinconjugated globulin was separated from unconjugated globulin by centrifugation at 100,000 g for 2 hours.2 The resulting pellet was resuspendedin a small volume of phos’ These studies were supported by The National Foundation and by the Office of The Surgeon General, Department of the Army, Washington, D. C., under the auspices of the Commission on Influenza, Armed Forces Epidemiological Board. “A procedure suggested by Dr. S. J. Singer in a personal communication.
REPORTS
phate-buffered isotonic saline at pH 7.5 and adsorbed with acetone-extracted rat and mouse liver tissue or with lyophilized HeLa cells. To ascertain whether specific tagging of the virus occurred, HeLa cells infected with vaccinia virus were immersed in ferritin-conjugated antibody overnight at room temperature. The cells were washed in isotonic saline, fixed in osmium tetroxide, dehydrated, embedded in methacrylate, sectioned, and examined in the electron microscope. Ferritin was found to be localized on the surface of extracellular virus in much the same manner as had been observed with influenza virus (2), but although small amounts of ferritin had been ingested by the cells, most of it remained within cytoplasmic vacuoles and hence failed to tag intracellular virus. To test the effect of fixation on viral antigen, infected cells grown on coverslips were placed in acetone for 15 minutes, fixed in osmium tetroxide, calcium formal, or formalin buffered with phosphate, washed, and subsequently stained with fluoresceinconjugated antibody. Exposure to 5% formalin for several hours had little effect on the brilliance of fluorescence, whereas osmium tetroxide destroyed in less than 1 minute the capacity of antigen to react with antibody.3 However, electron microscopic examination revealed that immersion of intact formalin-fixed cells in ferritin-conjugated antibody globulin did not result in penetration and, further, that graded dilutions of acetone did not render the cell permeable until the concentration of acetone and duration of application had produced severe extraction of cytoplasmic components with disruption of fine structure. Accordingly, it was decided to open the fixed cells by freezing and sectioning. HeLa cells infected with vaccinia virus were fixed in 5% calcium form01 for 5 minutes, scraped from the glass wall of the tissue culture tube into isotonic saline, centrifuged to form a pellet, immersed in 0.88 M ‘This effect of gen differs from obt,aincd by using of antigens (5). structure account be determined.
osmium tetroxide on viral antiSpendlove and Singer’s results the same fixative on other types Whether differences in antigenic for the discrepancy remains to
FIG. 1. An infected cell prepared by fixing, freezing, and sectioning. EarIy forms of vaccinia virus occupy the center of the field. One mature particle is evident at the lower left and another, somewhat distorted, at the cut surface. Ferritin conjugated to nonspecific antibody is scattcrcd through the cytoplasm. Magnification: X 74,000. 293
FIG. 2. Intracellular nification : X 200,000.
vaccinia
virus
tagged
with 294
ferritin-conjugated
specific
antibody.
Mag-
DISCUSSION
AND
PRELIMINARY
sucrose, frozen by placing the centrifuge tube in a carbon dioxide-ethyl alcohol bath, transferred to a gelatin capsule, and cut while still frozen in a cryostat. The sections, 5-8 p thick, were collected in a watchglass, covered with ferritin-conjugated antibody at room temperature, and allowed to stand for 10 minutes. The fluid, which now contained dispersed, transected cells, was transferred to a centrifuge tube, diluted to 15 ml with isotonic saline, and centrifuged at 3500 rpm for 5 minutes. The pellet of sedimented cells was then fixed for 20 minutes in buffered osmium tetroxide, dehydrated in graded dilutions of ethanol, embedded in methacrylate, and thin-sectioned. Examination in the electron microscope revealed that cytoplasmic fine structure was distorted, but that mitochondria, endoplasmic reticulum, and ribonucleoprotein granules could be recognized. The nuclear membrane, chromatin, and nucleoli were clearly delineated. Ferritin particles were scattered throughout the cytoplasm and nucleus. Vaccinia virus withstood this method of preparation remarkably well. Both early developmental forms with an eccentrically placed, dense, oval body and granular, less dense matrix, and also mature viral particles with a biconcave, discoid internal body, could be readily identified. Template sites at which differentiation of the virus is believed to occur (6) were not well preserved, a fact suggesting that viral components at early stages of formation are relatively fragile. Figure 1 shows the cytoplasm of an infected cell treated with ferritin-conjugated globulin obtained from a rabbit immunized with type 2 pneumococcus. The cut surface of the cell traverses the upper third horizontally. Early forms of the virus are apparent, and granules of ferritin are scattered in the cytoplasm. It is noteworthy that there are numerous ferritin granules in the immediate vicinity of the mature viral particle in the lower left corner, but they are not concentrated on the surface of the virus. Above and to the right of this viral particle a mitochondrion with clearly defined cristae has been transected obliquely. Ribonucleoprotein granules are swollen and exhibit diffuse margins.
REPORTS
295
Figure 2 illustrates vacuolated cytoplasm of an infected cell that was exposed to antivaccinia virus globulin conjugated with ferritin. At the upper right is a fragment of endoplasmic reticulum with several swollen ribonucleoprotein granules on its surface. Scattered ferritin particles are also present. Occupying the remainder of the field are mature viral particles containing the characteristic biconcave disk of diminished density. The variation in appearance of these particles can be accounted for by the tngle and level at which each lies with respe:t to the plane of section. The layer of ferritin granules on the surface of the virus marks the sites of antigen-antibody interaction. In several experiments it was shown that tagging could be blocked by exposing the cells to antibody globulin before ferritin-ccnjugated globulin was applied. It should be emphasized that washing failed to renove some of the ferritin which was not spccifitally localized. Therefore the cells tornmonly showed a background of randomly distributed ferritin granules, against vlhich focal concentrations of the granules Good out and revealed the sites of antigen-ntibody reaction. The experimental results indicate that intracellular vaccinia virus can react vith ferritin-conjugated antibody, thereby premitting specific immunologic identifiction of viral particles before release from the host cell. This technique is currently bing used to study other viruses, and metods are being explored to improve preservtion of antigens while retaining cellular fine structure, so that sites of antigen formcion can be identified at early stages of infecion before morphologic differentiation of the virus has occurred. REFERENCES 1. SISGER, S. J., Nature 183, 1523-1524 (195!. 2, RIFKISD, R. A., Hsu, K. C., MORGAN, C., SNXL, B. C., KNOX, A. W., and ROSE, H. M., Nture 187, 1094-1095 (1960). 5. SMITH, C. W., METZGER, J. F., ZACKS, S. Iand KASE, A., Proc. Sot. Exptl. Biol. Med..OP, 336-338 ( 1960 ) 4. LEE, S., Exptl. Cell Research 21, 249-252 (GO). 5. SPENDLOVE, R. S., and SINGER, S. J., Proc. ‘atl. had. Sci. U.S. 47, 14-18 (1961). 6. MORGAN, C., ELLISOX, S. A., ROSE, H. Mand
DISCUSSION
296 MOORE,
D.
H.,
J. Exptl.
AND
100,
Med.
PRELIMINARY
301-310
(1954). COUNCILMAN MORGAN RICHARD A. RIFKIND KONRAD C. Hsu MARGARET HOLDER’ BEATRICE C. SEEcAL HARRY M. ROSE
Departments of Microbiology and Medicine College of Physicians and Surgeons Columbia University New York 32, New York Received March 29,196l
Association
of Infectivity Virus
Bottom
with Component
Alfalfa
Mosaic
Only
Alfalfa mosaic virus (AMV) is composed of at least three serologically similar, differently sedimenting components containing about the same amount of nucleic acid, but differing in infectivity. The top component (73s) is not infectious, but the bottom component (99s) is. No conclusion about the infectivity of the middle component (89s) was reached (1) because when density-gradient tubes were punctured through the sides to collect fractions, contamination of middle component with bottom and top components occurred. An answer has been found by collecting droplets from the bottom of the density-gradient tubes at a regulated rate so that the distribution of nucleoprotein and infectivity curves may be compared. The droplets (ranging from 0.08 to 0.11 ml depending on the experiment) were diluted by the addition of 1 ml of pH 7.0, 0.01 M phosphate buffer, then the optical densities at 260 my were read in microcuvettes with a l-cm light path. Infectivity assays were made as usual on bean (Phaseolus wulgaris L. var. Bountiful) from various points of the absorption curves at a dilution of from one-fourth to one-fifth.
REPORTS
All density-gradient tubes were made of 4, 7, 7, 7 ml of 10, 20, 30, and 40% sucrose in 0.01 M pH 7.0 buffer. Centrifugation was in density-gradient tubes at 23,000 rpm for 2% hours in the SW25.1 swinging-bucket rotor of a Spinco model L preparatory ultracentrifuge. The optical densities of a previously purified preparation of AMV after densitygradient centrifugation formed a trimodal curve (Fig. 1) whereas the infectivity curve was unimodal and coincided with the component which sedimented fastest and is known to correspond to bottom fraction, If both bottom and middle components were infectious, the infectivity curve would be expected to be bimodal. In a further cxperiment, the bottom and middle fractions were separated by needle puncture from densitygradient tubes, concentrated, recycled on density-gradient tubes, and collected in droplets from the tube bottoms. The infectivity curve coincided with the optical-density curve of bottom (Fig. 2) but not with that of middle fraction (Fig. 3) centrifuged in a sister tube. Any infectivity still associated with middle component coincided positionally (see vertical arrow) with bottom fraction. Thus, it appears that bottom is the only infectious component of AMV. ACKNOWLEDGMENTS I wish to thank Mr. ing the purified AMV droplet collections.
J. S. Semancik for and for assisting
preparin the
REFERENCE 1. BANCROFT, Biophys.
J. B., and KAESBERG, P., Biochim. et Acta 39,519-528 (1960). J. B. BAXCROFT Department of Botany and Plant Pathology Purdue University Life Science Building Lafayette, Indiana Received March SO,1961
292
AND
PRELIMINARY
T. T., CIECIURA, S. L., and FISHER, H. W., j. Ezptl. Med. 106, 145-158 (1957). 7. KROOTH, R. S., and WEISBERC, A. N., J. Ezptl.
6. PUCK,
Med.
in press
(1961).
H., Science 130, 432-437 (1959). MCQUILKIX, W. T., EVANS, V. T., and EARLE, W. R., J. h’atl. Cancer Inst. 19, 885-896
8. EAGLE, 9.
(1957). 10. DARNELL, J. E., JR., and SAWYER, T. K., Viralogy 11,665-675 (1960). 11. JOECLIK, W. K., and DARSF.LL, J. E., JR., Vi?ology 13,439-447 (1961). 1.2. KROOTH, R. S., and WEIXBERG, A. Iv., Rio&em. Riophys. Research Communs. 3, 518-524
(1960). R. S. J. H. Natiorlal Bcthescla
Electron
KROOTH TJIO
Institutes of Health 14, Maryland
Microscopic Localization Viral Antigen by the
of Intracellular Use of
Ferritin-conjugated Antibody’
The use of ferritin-conjugated antibody globulin (1) enables the sites of antigenantibody interaction to be visualized in the electron microscope (g-4). To apply this method in studies of viral development, infected cells must be treated so that the ferritin-globulin complex gains entrance, while structure and antigenic composition of the virus are preserved. The purpose of this report is to describe a technique that has been used to tag intracellular vaccinia virus with ferritin-conjugated antibody. Antibody globulin, obtained from rabbits immunized with vaccinia virus, was conjugated with ferritin by the method previously described (1, 2). The ferritinconjugated globulin was separated from unconjugated globulin by centrifugation at 100,000 g for 2 hours.2 The resulting pellet was resuspendedin a small volume of phos’ These studies were supported by The National Foundation and by the Office of The Surgeon General, Department of the Army, Washington, D. C., under the auspices of the Commission on Influenza, Armed Forces Epidemiological Board. “A procedure suggested by Dr. S. J. Singer in a personal communication.
REPORTS
phate-buffered isotonic saline at pH 7.5 and adsorbed with acetone-extracted rat and mouse liver tissue or with lyophilized HeLa cells. To ascertain whether specific tagging of the virus occurred, HeLa cells infected with vaccinia virus were immersed in ferritin-conjugated antibody overnight at room temperature. The cells were washed in isotonic saline, fixed in osmium tetroxide, dehydrated, embedded in methacrylate, sectioned, and examined in the electron microscope. Ferritin was found to be localized on the surface of extracellular virus in much the same manner as had been observed with influenza virus (2), but although small amounts of ferritin had been ingested by the cells, most of it remained within cytoplasmic vacuoles and hence failed to tag intracellular virus. To test the effect of fixation on viral antigen, infected cells grown on coverslips were placed in acetone for 15 minutes, fixed in osmium tetroxide, calcium formal, or formalin buffered with phosphate, washed, and subsequently stained with fluoresceinconjugated antibody. Exposure to 5% formalin for several hours had little effect on the brilliance of fluorescence, whereas osmium tetroxide destroyed in less than 1 minute the capacity of antigen to react with antibody.3 However, electron microscopic examination revealed that immersion of intact formalin-fixed cells in ferritin-conjugated antibody globulin did not result in penetration and, further, that graded dilutions of acetone did not render the cell permeable until the concentration of acetone and duration of application had produced severe extraction of cytoplasmic components with disruption of fine structure. Accordingly, it was decided to open the fixed cells by freezing and sectioning. HeLa cells infected with vaccinia virus were fixed in 5% calcium form01 for 5 minutes, scraped from the glass wall of the tissue culture tube into isotonic saline, centrifuged to form a pellet, immersed in 0.88 M ‘This effect of gen differs from obt,aincd by using of antigens (5). structure account be determined.
osmium tetroxide on viral antiSpendlove and Singer’s results the same fixative on other types Whether differences in antigenic for the discrepancy remains to
FIG. 1. An infected cell prepared by fixing, freezing, and sectioning. EarIy forms of vaccinia virus occupy the center of the field. One mature particle is evident at the lower left and another, somewhat distorted, at the cut surface. Ferritin conjugated to nonspecific antibody is scattcrcd through the cytoplasm. Magnification: X 74,000. 293
FIG. 2. Intracellular nification : X 200,000.
vaccinia
virus
tagged
with 294
ferritin-conjugated
specific
antibody.
Mag-
DISCUSSION
AND
PRELIMINARY
sucrose, frozen by placing the centrifuge tube in a carbon dioxide-ethyl alcohol bath, transferred to a gelatin capsule, and cut while still frozen in a cryostat. The sections, 5-8 p thick, were collected in a watchglass, covered with ferritin-conjugated antibody at room temperature, and allowed to stand for 10 minutes. The fluid, which now contained dispersed, transected cells, was transferred to a centrifuge tube, diluted to 15 ml with isotonic saline, and centrifuged at 3500 rpm for 5 minutes. The pellet of sedimented cells was then fixed for 20 minutes in buffered osmium tetroxide, dehydrated in graded dilutions of ethanol, embedded in methacrylate, and thin-sectioned. Examination in the electron microscope revealed that cytoplasmic fine structure was distorted, but that mitochondria, endoplasmic reticulum, and ribonucleoprotein granules could be recognized. The nuclear membrane, chromatin, and nucleoli were clearly delineated. Ferritin particles were scattered throughout the cytoplasm and nucleus. Vaccinia virus withstood this method of preparation remarkably well. Both early developmental forms with an eccentrically placed, dense, oval body and granular, less dense matrix, and also mature viral particles with a biconcave, discoid internal body, could be readily identified. Template sites at which differentiation of the virus is believed to occur (6) were not well preserved, a fact suggesting that viral components at early stages of formation are relatively fragile. Figure 1 shows the cytoplasm of an infected cell treated with ferritin-conjugated globulin obtained from a rabbit immunized with type 2 pneumococcus. The cut surface of the cell traverses the upper third horizontally. Early forms of the virus are apparent, and granules of ferritin are scattered in the cytoplasm. It is noteworthy that there are numerous ferritin granules in the immediate vicinity of the mature viral particle in the lower left corner, but they are not concentrated on the surface of the virus. Above and to the right of this viral particle a mitochondrion with clearly defined cristae has been transected obliquely. Ribonucleoprotein granules are swollen and exhibit diffuse margins.
REPORTS
295
Figure 2 illustrates vacuolated cytoplasm of an infected cell that was exposed to antivaccinia virus globulin conjugated with ferritin. At the upper right is a fragment of endoplasmic reticulum with several swollen ribonucleoprotein granules on its surface. Scattered ferritin particles are also present. Occupying the remainder of the field are mature viral particles containing the characteristic biconcave disk of diminished density. The variation in appearance of these particles can be accounted for by the tngle and level at which each lies with respe:t to the plane of section. The layer of ferritin granules on the surface of the virus marks the sites of antigen-antibody interaction. In several experiments it was shown that tagging could be blocked by exposing the cells to antibody globulin before ferritin-ccnjugated globulin was applied. It should be emphasized that washing failed to renove some of the ferritin which was not spccifitally localized. Therefore the cells tornmonly showed a background of randomly distributed ferritin granules, against vlhich focal concentrations of the granules Good out and revealed the sites of antigen-ntibody reaction. The experimental results indicate that intracellular vaccinia virus can react vith ferritin-conjugated antibody, thereby premitting specific immunologic identifiction of viral particles before release from the host cell. This technique is currently bing used to study other viruses, and metods are being explored to improve preservtion of antigens while retaining cellular fine structure, so that sites of antigen formcion can be identified at early stages of infecion before morphologic differentiation of the virus has occurred. REFERENCES 1. SISGER, S. J., Nature 183, 1523-1524 (195!. 2, RIFKISD, R. A., Hsu, K. C., MORGAN, C., SNXL, B. C., KNOX, A. W., and ROSE, H. M., Nture 187, 1094-1095 (1960). 5. SMITH, C. W., METZGER, J. F., ZACKS, S. Iand KASE, A., Proc. Sot. Exptl. Biol. Med..OP, 336-338 ( 1960 ) 4. LEE, S., Exptl. Cell Research 21, 249-252 (GO). 5. SPENDLOVE, R. S., and SINGER, S. J., Proc. ‘atl. had. Sci. U.S. 47, 14-18 (1961). 6. MORGAN, C., ELLISOX, S. A., ROSE, H. Mand
DISCUSSION
296 MOORE,
D.
H.,
J. Exptl.
AND
100,
Med.
PRELIMINARY
301-310
(1954). COUNCILMAN MORGAN RICHARD A. RIFKIND KONRAD C. Hsu MARGARET HOLDER’ BEATRICE C. SEEcAL HARRY M. ROSE
Departments of Microbiology and Medicine College of Physicians and Surgeons Columbia University New York 32, New York Received March 29,196l
Association
of Infectivity Virus
Bottom
with Component
Alfalfa
Mosaic
Only
Alfalfa mosaic virus (AMV) is composed of at least three serologically similar, differently sedimenting components containing about the same amount of nucleic acid, but differing in infectivity. The top component (73s) is not infectious, but the bottom component (99s) is. No conclusion about the infectivity of the middle component (89s) was reached (1) because when density-gradient tubes were punctured through the sides to collect fractions, contamination of middle component with bottom and top components occurred. An answer has been found by collecting droplets from the bottom of the density-gradient tubes at a regulated rate so that the distribution of nucleoprotein and infectivity curves may be compared. The droplets (ranging from 0.08 to 0.11 ml depending on the experiment) were diluted by the addition of 1 ml of pH 7.0, 0.01 M phosphate buffer, then the optical densities at 260 my were read in microcuvettes with a l-cm light path. Infectivity assays were made as usual on bean (Phaseolus wulgaris L. var. Bountiful) from various points of the absorption curves at a dilution of from one-fourth to one-fifth.
REPORTS
All density-gradient tubes were made of 4, 7, 7, 7 ml of 10, 20, 30, and 40% sucrose in 0.01 M pH 7.0 buffer. Centrifugation was in density-gradient tubes at 23,000 rpm for 2% hours in the SW25.1 swinging-bucket rotor of a Spinco model L preparatory ultracentrifuge. The optical densities of a previously purified preparation of AMV after densitygradient centrifugation formed a trimodal curve (Fig. 1) whereas the infectivity curve was unimodal and coincided with the component which sedimented fastest and is known to correspond to bottom fraction, If both bottom and middle components were infectious, the infectivity curve would be expected to be bimodal. In a further cxperiment, the bottom and middle fractions were separated by needle puncture from densitygradient tubes, concentrated, recycled on density-gradient tubes, and collected in droplets from the tube bottoms. The infectivity curve coincided with the optical-density curve of bottom (Fig. 2) but not with that of middle fraction (Fig. 3) centrifuged in a sister tube. Any infectivity still associated with middle component coincided positionally (see vertical arrow) with bottom fraction. Thus, it appears that bottom is the only infectious component of AMV. ACKNOWLEDGMENTS I wish to thank Mr. ing the purified AMV droplet collections.
J. S. Semancik for and for assisting
preparin the
REFERENCE 1. BANCROFT, Biophys.
J. B., and KAESBERG, P., Biochim. et Acta 39,519-528 (1960). J. B. BAXCROFT Department of Botany and Plant Pathology Purdue University Life Science Building Lafayette, Indiana Received March SO,1961