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Viral protein in early stages of clover yellow mosaic virus infection of Vicia faba
)‘II~OI,OGY
46, 747-751
Viral
Protein
(1971)
in Early
Stages
Infection D. E. SCHLEGEL Department
of Plant
Pathology,
of Clover of
Vicia
University Accepted
Virus
E. DELISLE
of California, May
Mosaic
f&a
DIANNE
AND
Yellow
Berkeley,
California
94YZO
1.3, 1971
Clover yellow mosaic virus-infected Vicia faba leaf cells contain structures filled with amorphous material in both the cytoplasm and the vacuole. Electron microscope immunoradioautography of such leaf tissue treated with virus-specific lzsIlabeled antibodies showed that these structures contained substantial amounts of virus protein. The structures first appeared about 4-5 days after inoculation of leaves two nodes below those sampled, and were not found in noninoculated tissue. The appearance of such structures coincided with the first recovery of infectivity from these leaves. The leaves sampled were approximately 1 cm long at, the time the plant was inoculated. No virus particles could be distinguished in the amorphous material.
logical observations !ayer
report describes the sequence of development of clover yellow mosaic virus (C Y MV) antigen in young broadbean (Vi&a faba L.) leaves as detected at the electron microscope level, using 1251-labeled antibodies. This study continues a program of investigation of the early stages of virus infection begun with tobacco mosaic virus (TRZV). Earlier cytological studies on the synthesis of new antigen in tobacco cells infected with tobacco mosaic virus indicated that new antigen could be demonstrated in the nucleus within 5 hr after inoculat#ion of the leaves (Langenberg and Schlegel, 1969). These studies involved immunolabeling and light microscope radioatuography. Attempts to extend this study of TMV infections to the electron microscope level have not been successful, due to a number of reasons. Immobilization of ThtV in t,issues by means of the usual neutral fixatives used in electron microscopy has not been successful [acid fixatives mere used in the previous study (Langenberg and Schlegel, 1969)]. Furthermore, the inoculated leaves are of necessity expanding or fully expanded leaves, and as such do not provide good material for cytoThis
around
the
cell
because the cytoplasmic is so thin.
Preliminary studies have shown that, the best met,hods available for immunolabeling of TSIV (Schlegel and Langenberg, 1969) are much more effective on clover yellow mosaic virus. This paper reports t,he al,pearance of antigen and CYXV in young systemically infected Vicia fuba leaves. MATERIALS
AND
METHOIX?
Preparatiov of plant materials. 170ung Vicia jaba L. plants about 10-13 days old were used in all experiments. A single pair of leaves was inoculated on upper and lower surfaces with clover yellow mosaic virus (CYMV) using Carborundum as an abrasive. The inoculum was prepared by grinding approximately 0.5 g of infected leaf tissue in 5 ml of 0.05 N neutral phosphat#e buffer. The leaves chosen for study were < 1 cm long at the time of inoculation and were located two nodes above the inoculated leaves. The plants were inoculated on successive days so that leaves representing 3,4, 5, 6, and 12 days after inoculation could be harvestred simultaneously. Three plants were inoculated at, each time period, thus each 747
748
SCHLEGEL
period studied contained sections from several similar leaves. Each experiment included comparable leaves from healthy plants. The results obtained were consistent in numerous experiments. The samples of diseased and healthy tissues were fixed by vacuum infiltration with a glutaraldehyde-acrolein mixture comprised of equal volumes of a solution containing 3.0 % glutaraldehyde, 3.0 % acrolein, and 6.6 % acetone, and a solution containing 3.2 % gelatin (previously dialyzed against distilled water) in 0.01 M EDTA and 0.02 M phosphate buffer (pH 7.2). After a 2-hr fixation at room temperature, the tissues were washed with 2% dialyzed gelatin in 0.01 M neutral phosphate buffer and 0.005 M EDTA, and presectioned at 300 P in a vibratome (Schlegel and DeLisle, manuscript being submitted). The 300-p slices of tissue were placed in 0.00125 2M boratesaline buffer (pH 8) for application of 125I-yglobulin. Preparation of 1251--y-globulin.The previously described method for labeling antiTMV rabbit serum (Langenberg and Schlegel, 1967) was modified as follows for CYMV. Anti-CYMV rabbit serum was prepared with electrophoretically purified CYMV. The globulin fraction was partially purified by salting out two times with 33 % ammonium sulfate. The anti-CYMV globulin was precipitated with purified CYMV and washed with borate buffer (10.6 g H3B03 and 8 g NaCl per liter, pH 8). The antibodyantigen complex was then split with 0.1 M glycine, pH 2.5, and centrifuged at high speed to sediment the virus. Viral protein was removed from the acidic supernatant by salting out with 14 % sodium chloride and the purified antibody recovered with excess saturated sodium chloride (Shepard and Shalla, 1969). The purified yglobulin was resuspended in 1 ml of borate buffer, pH 8, and centrifuged at slow speed to remove insoluble proteins. The entire yglobulin sample was iodinated with carrierfree 1251 as described by Tsuzuku et al. (1967) and dialyzed against borate buffer to remove excess lz51. Application of 125I-r-globulin to tissue. The r251-r-globulin was preabsorbed with a crude extract of healthy plant tissue prepared by
AND
DELISLE
grinding 8 g of healthy leaf tissue in 12 ml 0.5 M borat’e buffer, pH 7.5, containing 0.01 M sodium diethyldithiocarbamate (DIECA) reducing agent and centrifuged once at slow speed. One milliliter of the supernatant from this healthy tissue extract was mixed winth 1 ml of the lz51-r-globulin and allowed to incubate for 30 min. This preabsorption step was necessary to remove trace amounts of antibodies specific to normal host, constituents. Such antibodies were always present, in spite of the fact that the CYMV used to immunize the rabbits was electrophoretically purified. The mixture of labeled r-globulin and crude healthy extract was then added to the 300-p slices of diseasedtissue and incubated for 6-10 hr at 0”. The 300-p slices contained cells opened by the Vibratome cut on both the top and bottom surfaces; thus either side was accessible to label. The labeled tissue was washed for a minimum of 10 hr in the 0.017 M iodination buffer (pH 8) at 0” with frequent changes. The labeled slices were postfixed in 1% osmium t’etroxide in 0.05 M neutral phosphate buffer, dehydrated in a cold alcohol series, and embedded for precise orientation of a cut-open surface (Schlegel and DeLisle, manuscript being submitted). In as much as the labeled y-globulin utilized in these studies was split off of the antigen (CYMV), controls which involved labeled normal serum were not possible. The specificity of the labeled y-globulin t’o purified CYMV was verified, however, by microprecipitin reactions (van Slogteren, 1955). Radioautography. Radioautograms, both light microscope and electron microscope, were prepared according to the loop method of Caro (1964) using Ilford L4 emulsion. Light microscope radioautography was used to locate areas suitable for further study. Areas thus identified for electron microscope radioautography were sectioned at 70-90 nm and placed on unsupported 75 X 300 mesh, nickel grids. The emulsion was applied directly to the grids and exposed for 116-332 hr. After the grids were developed, the emulsion was removed with 0.2 M NaOH (CO2 free) and the sections st’ained with uranyl acetate in methyl alcohol (Stempak and Ward, 1964). Electron micrographs
VIR,4L
PROTEIN
IN
were made with t,he RCA-EMU-3f microscope on duPont> orthoB-litho film. Radioactive label could appear only in cells which were opened during vibratome presectioning, since r-globulin cannot penetrate intact’ plant cell walls, even though the tissues are fixed. Therefore, during sectioning for electron microscopy the block was oriented to include obviously opened cells. These areas were determined in part by the results of the light, microscope radioautogmphy. The procedure for mount,ing of tissue slices permitted a very accurat#e orientation of t,he tissue pieces, and, routinely, cells 3-4 layers behind the cut edge were open and accessible to ant,ibody. Healthy Gssue controls \vere studied in each experiment, and special care was given to locating open cells when examining these tissues. Several sect,ions were examined from each leaf in each esperimen t . RESULTS
AN11
l>ISCUSSION
Alt,hough the time of initiation of infection in the l-cm leaves varies seasonally, the following seyuence of events has always been observed. At 3 days after inoculation of the lower leaf whorl, the level of label in the young infected leaves and comparable healthy leaves was similar and very low (Pig. 1). Ant’igenic material could not be identified in the cells at t,his t,ime. The general label level on both diseased and healthy &sue was also low on the fourth day (l’ig. 2). However, isolated cells contained moderate to heavily labeled “antigenie inclusions” which had an unresolvable internal structure which resembled neither virus nor cytoplasm. These inclusions were never fourld in heahhy tissue. When present, they always appeared in the cyboplasm at the edge of t,he cytoplasmic membrane or free in the vacuole and were not, themselves, surrounded by a membrane. The general cytoplasmic label in 5-day samples was moderate in the diseased tissue, but only the antigenic inclusions were heavily labeled (E’igs. 3-5). At’ this time after inoculation, inculsions were large and numerous, appearing in mnn.v cells. Viral particles tirst, appeared in 6-day samples (Fig. 6). They were in small packets occurring in the cytoplasm, often in the same
CYMV
TNFIXTIONS
749
cell where ant,igenic inclusions could be found in the vacuolar area. The antigenic inclusions were labeled heavily over their entire structure, while the viral packets labeled heavily only on the edges. By 6 days the size and numbers of the antigrnic inclusions began to decrease. RIassive virus inclusions appeared in t,he 12-day samples, and t’hese were heavily labeled, again at the edges (Fig. 7). In general the cytoplasmic label was reduced at t#his stage and antigenic inclusions were seldom seen. Throughout the experiments inclusion bodies of another type were observed. These resembled the tannin sacs described by Weimraub and Ragetli (1966)) and occurred sporadically (in small numbers) in healthy tissue. The>, nere large and numerous in diseased tissue, especially in 4- and &day samples. They were never labeled; however, they did seem to have some association with a cellular response to infect,ion since they were noticeable in t,he early days of infect,ion (Zig. 2), but, were relatively absent, from healthy tissue am-1 12.day infected tissue. The simplest explanation of the observ:rt,ions report.ed above is that virus moves from the inoculated leaves to young leaves at the t,ip. Kew virus ca11 be detecmd by infectivity assays in l,he young l-cm leaves approximately 3 days after hOcuhti(Jll. Xt this time no evidence for intact, virus can be seen, either by electron microscope observntions or by immunolabeling. Antigenic material first, appears on the fourth day. By the fift’h day large numbers of antigenic inclusions occur in many cells, altliougli intact virus part,icles are not visible. Virus inclu sions tirst appeared in the cytoplasm on t’hc sixth day, but were never seen in the vacuole. By 1% days pcJStiIiOculatiOn, masses of virus were seen, but only a few ant,igenic inclusions remained. Increase in virus infectivit,? paralleled the increase in numbers of antigenic inclusions up to the sixth day wllen virtts particles first appeared. The highly antigenic nature of t,he bodies found in the vacuole suggests that, it is excess virus protein which gradually decreases as the concentration of whole virus mcreases, ahhough some still remained in 12-day samples. The occurrence of excess virus protein in C\XV-infections Iuts been
FIG. 1. Elertron microscope radioautographs of CYMV infection in young leaves of Viciafaba treated with itiI-r-globulin. Healthy tissue control to 12-day samples showing low level of cytoplasmic label. Note that these cells were cut open during vibratome presectioning (arrows). Radioautographic exposure, 116 hr. Scale = 2 p. FIG. 2. CYMV-infected leaf tissue 4 days after inoculation showing general higher level of label in the cytoplasm and small tannin sacs (arrows). No virus particles can be seen. Radioautographic exposure, 332 hr. Scale = 1 p. 750
Fro.
3. Antigenic
inclusions
(ai) in vacuole
(V)
FIG. 4. Antigenic inclusion (ai) in 5-day sample extending int,o vacuole from the tonoplast (1). Note nucleus (N) and nuclear membrane (nm). Radioautographic exposure, 332 hr. Scale = 0.5 P. FIG. 5. Antigenic inclusion (ai) in 5.day sample isolated in vacuole (V). Note apparent inclusion of cytoplasmic materials in antigenic inclusions (arrow). Radioautographic exposure, 332 hr. Scale = 0.5 p. FIG. 6. Virus crystals (UC) in B-day sample. Radioautographic exposure, 144 hr. Scale 0.5 cc.
VIRAL
ll(i
FIG. 7. Heavily labelwl hr. Scale = 0.5 U.
virils
PROTEIN
cryst,als
IN
CYMV
(UC) in cytoplasm
T.-t3
INFECTIOXS
of 12.day
sample.
lladioatltographic
c>sposr~rt’,
754
SCHLEGEL
demonstrated by Purcifull and Shepherd (1964). The antigenic inclusions were always found outside the cytoplasm extending from the tonoplast into the vacuole. Occasionally these antigenic inclusions appeared to be free in the vacuole. However, these apparently unattached antigenic inclusions were associated with cytoplasmic material (Fig. 5), and most likely the electron microscope section did not pass through the area of attachment. No membrane could be detected surrounding any of the antigenic inclusions. This localization could possibly be an artifact of fixation in which the fixation process itself resulted in an extrusion of such material from the cytoplasm into the vacuole. Confirmation of this explanation is not possible, however, since antigenic inclusions were never seen in the cytoplasm itself, a fact which argues against the vacuolar localization being an artifact. Antigenic inclusions have not been reported previously, possibly due to the fact that fixation and embedding methods in general have not included the use of a gelatin infiltration step to stabilize viral materials in cells. However, once we identified these structures, they could be found using normal methods of fixation and embedding. The heavy label over the antigenic inclusions indicates a loose, open arrangement which permits penetration of antibody. This is in strong contrast with the virus inclusions which are labeled only at the edge, suggesting a close packing of virus particles. The impervious nature of viral inclusions to antibodies has been reported previously (Schlegel and Langenberg, 1969). The production of antigen in broadbean leaves infected with clover yellow mosaic virus differs from the production of antigen in tobacco leaves infected with tobacco mosaic virus. The latter do not form the antigenic inclusions distinct from viral inclusions; furthermore, the first antigen appears in the nucleus (Langenberg and Schlegel, 1969) in the case of TMV. Thus it would appear from these results that generalizations with respect to viral protein synthesis are hazardous. The complex and integral relationship of
AND DELISLE
a virus to a living cell has raised many questions about the various stages of virus multiplication. When a sufficient body of information has accumulated, we can begin to put together an overall picture of virus synthesis. ACKNOWLEDGMENT This work was supported in part by USPHS research grant AI03415 from the National Institute of Allergy and Infectious Diseases. REFERENCES L. G. (1964). High-resolution autoradiography. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. I, pp. 327-363. Academic Press, New York. LANGENBERG, W. G., and SCHLEGEL, D. E. (1967). Autoradiography with lz61-labeled antibodies as a means of localizing TMV antigen in plant cells. Virology 32, 167-171. LANGENBERG, W. G., and SCHLEGEL, D. E. (1969). Localization of tobacco mosaic virus protein in tobacco leaf cells during the early stages of infection. Virology 37, 86-93. PURCIFULL, D. E., and SHEPHERD, R. J. (1964). Preparation of the protein fragments of several rod-shaped plant viruses and their use in agargel diffusion tests. Phytopathology 54, 11021108. SCRLEGEL, D. E., and LANGENBERG, W. G. (1969). The prevention of virus redistribution within plant cells during preparation for autoradiogof Diffusible raphy. Zn “Autoradiography Substances” (L. J. Roth and W. E. Stumf, eds.), pp. 131-145. Academic Press, New York. SHEPARD, J. F., and SHALLA, T. A. (1969). Tobacco etch virus cylindrical inclusions: Antigenically unrelated to the causal virus. Virology 38, 185-188. STEMPAK, J. G., and WARD, R. T. (1964). An improved staining method for electron microscopy. J. Cell Biol. 22, 697-701. TSUZUKU, O., YAGI, Y., and PRESSMAN, D. (1967). Preparative purification of lung-localizing rabbit anti-rat lung antibodies in vitro. J. Immunol. 98, 1004-1010. VAN SLOGTEREN, D. H. M. (1955). Serological micro-reactions with plant viruses under paraffin oil. In “Proceedings of the Second Conference on Potato Virus Diseases” (E. Streutgers, A. B. R. Beemster, D. Noordam, and J. P. H. van der Want, eds.), pp. 51-54. H. Veenman and Zonen, Wageningen. WEINTRAUB, M., and RAGETLI, H. W. (1966) Fine structure of ‘inclusion and organelles in Vi& fuba infected with bean yellow mosaic virus. Virology 28, 290-302. CARO,
46, 747-751
Viral
Protein
(1971)
in Early
Stages
Infection D. E. SCHLEGEL Department
of Plant
Pathology,
of Clover of
Vicia
University Accepted
Virus
E. DELISLE
of California, May
Mosaic
f&a
DIANNE
AND
Yellow
Berkeley,
California
94YZO
1.3, 1971
Clover yellow mosaic virus-infected Vicia faba leaf cells contain structures filled with amorphous material in both the cytoplasm and the vacuole. Electron microscope immunoradioautography of such leaf tissue treated with virus-specific lzsIlabeled antibodies showed that these structures contained substantial amounts of virus protein. The structures first appeared about 4-5 days after inoculation of leaves two nodes below those sampled, and were not found in noninoculated tissue. The appearance of such structures coincided with the first recovery of infectivity from these leaves. The leaves sampled were approximately 1 cm long at, the time the plant was inoculated. No virus particles could be distinguished in the amorphous material.
logical observations !ayer
report describes the sequence of development of clover yellow mosaic virus (C Y MV) antigen in young broadbean (Vi&a faba L.) leaves as detected at the electron microscope level, using 1251-labeled antibodies. This study continues a program of investigation of the early stages of virus infection begun with tobacco mosaic virus (TRZV). Earlier cytological studies on the synthesis of new antigen in tobacco cells infected with tobacco mosaic virus indicated that new antigen could be demonstrated in the nucleus within 5 hr after inoculat#ion of the leaves (Langenberg and Schlegel, 1969). These studies involved immunolabeling and light microscope radioatuography. Attempts to extend this study of TMV infections to the electron microscope level have not been successful, due to a number of reasons. Immobilization of ThtV in t,issues by means of the usual neutral fixatives used in electron microscopy has not been successful [acid fixatives mere used in the previous study (Langenberg and Schlegel, 1969)]. Furthermore, the inoculated leaves are of necessity expanding or fully expanded leaves, and as such do not provide good material for cytoThis
around
the
cell
because the cytoplasmic is so thin.
Preliminary studies have shown that, the best met,hods available for immunolabeling of TSIV (Schlegel and Langenberg, 1969) are much more effective on clover yellow mosaic virus. This paper reports t,he al,pearance of antigen and CYXV in young systemically infected Vicia fuba leaves. MATERIALS
AND
METHOIX?
Preparatiov of plant materials. 170ung Vicia jaba L. plants about 10-13 days old were used in all experiments. A single pair of leaves was inoculated on upper and lower surfaces with clover yellow mosaic virus (CYMV) using Carborundum as an abrasive. The inoculum was prepared by grinding approximately 0.5 g of infected leaf tissue in 5 ml of 0.05 N neutral phosphat#e buffer. The leaves chosen for study were < 1 cm long at the time of inoculation and were located two nodes above the inoculated leaves. The plants were inoculated on successive days so that leaves representing 3,4, 5, 6, and 12 days after inoculation could be harvestred simultaneously. Three plants were inoculated at, each time period, thus each 747
748
SCHLEGEL
period studied contained sections from several similar leaves. Each experiment included comparable leaves from healthy plants. The results obtained were consistent in numerous experiments. The samples of diseased and healthy tissues were fixed by vacuum infiltration with a glutaraldehyde-acrolein mixture comprised of equal volumes of a solution containing 3.0 % glutaraldehyde, 3.0 % acrolein, and 6.6 % acetone, and a solution containing 3.2 % gelatin (previously dialyzed against distilled water) in 0.01 M EDTA and 0.02 M phosphate buffer (pH 7.2). After a 2-hr fixation at room temperature, the tissues were washed with 2% dialyzed gelatin in 0.01 M neutral phosphate buffer and 0.005 M EDTA, and presectioned at 300 P in a vibratome (Schlegel and DeLisle, manuscript being submitted). The 300-p slices of tissue were placed in 0.00125 2M boratesaline buffer (pH 8) for application of 125I-yglobulin. Preparation of 1251--y-globulin.The previously described method for labeling antiTMV rabbit serum (Langenberg and Schlegel, 1967) was modified as follows for CYMV. Anti-CYMV rabbit serum was prepared with electrophoretically purified CYMV. The globulin fraction was partially purified by salting out two times with 33 % ammonium sulfate. The anti-CYMV globulin was precipitated with purified CYMV and washed with borate buffer (10.6 g H3B03 and 8 g NaCl per liter, pH 8). The antibodyantigen complex was then split with 0.1 M glycine, pH 2.5, and centrifuged at high speed to sediment the virus. Viral protein was removed from the acidic supernatant by salting out with 14 % sodium chloride and the purified antibody recovered with excess saturated sodium chloride (Shepard and Shalla, 1969). The purified yglobulin was resuspended in 1 ml of borate buffer, pH 8, and centrifuged at slow speed to remove insoluble proteins. The entire yglobulin sample was iodinated with carrierfree 1251 as described by Tsuzuku et al. (1967) and dialyzed against borate buffer to remove excess lz51. Application of 125I-r-globulin to tissue. The r251-r-globulin was preabsorbed with a crude extract of healthy plant tissue prepared by
AND
DELISLE
grinding 8 g of healthy leaf tissue in 12 ml 0.5 M borat’e buffer, pH 7.5, containing 0.01 M sodium diethyldithiocarbamate (DIECA) reducing agent and centrifuged once at slow speed. One milliliter of the supernatant from this healthy tissue extract was mixed winth 1 ml of the lz51-r-globulin and allowed to incubate for 30 min. This preabsorption step was necessary to remove trace amounts of antibodies specific to normal host, constituents. Such antibodies were always present, in spite of the fact that the CYMV used to immunize the rabbits was electrophoretically purified. The mixture of labeled r-globulin and crude healthy extract was then added to the 300-p slices of diseasedtissue and incubated for 6-10 hr at 0”. The 300-p slices contained cells opened by the Vibratome cut on both the top and bottom surfaces; thus either side was accessible to label. The labeled tissue was washed for a minimum of 10 hr in the 0.017 M iodination buffer (pH 8) at 0” with frequent changes. The labeled slices were postfixed in 1% osmium t’etroxide in 0.05 M neutral phosphate buffer, dehydrated in a cold alcohol series, and embedded for precise orientation of a cut-open surface (Schlegel and DeLisle, manuscript being submitted). In as much as the labeled y-globulin utilized in these studies was split off of the antigen (CYMV), controls which involved labeled normal serum were not possible. The specificity of the labeled y-globulin t’o purified CYMV was verified, however, by microprecipitin reactions (van Slogteren, 1955). Radioautography. Radioautograms, both light microscope and electron microscope, were prepared according to the loop method of Caro (1964) using Ilford L4 emulsion. Light microscope radioautography was used to locate areas suitable for further study. Areas thus identified for electron microscope radioautography were sectioned at 70-90 nm and placed on unsupported 75 X 300 mesh, nickel grids. The emulsion was applied directly to the grids and exposed for 116-332 hr. After the grids were developed, the emulsion was removed with 0.2 M NaOH (CO2 free) and the sections st’ained with uranyl acetate in methyl alcohol (Stempak and Ward, 1964). Electron micrographs
VIR,4L
PROTEIN
IN
were made with t,he RCA-EMU-3f microscope on duPont> orthoB-litho film. Radioactive label could appear only in cells which were opened during vibratome presectioning, since r-globulin cannot penetrate intact’ plant cell walls, even though the tissues are fixed. Therefore, during sectioning for electron microscopy the block was oriented to include obviously opened cells. These areas were determined in part by the results of the light, microscope radioautogmphy. The procedure for mount,ing of tissue slices permitted a very accurat#e orientation of t,he tissue pieces, and, routinely, cells 3-4 layers behind the cut edge were open and accessible to ant,ibody. Healthy Gssue controls \vere studied in each experiment, and special care was given to locating open cells when examining these tissues. Several sect,ions were examined from each leaf in each esperimen t . RESULTS
AN11
l>ISCUSSION
Alt,hough the time of initiation of infection in the l-cm leaves varies seasonally, the following seyuence of events has always been observed. At 3 days after inoculation of the lower leaf whorl, the level of label in the young infected leaves and comparable healthy leaves was similar and very low (Pig. 1). Ant’igenic material could not be identified in the cells at t,his t,ime. The general label level on both diseased and healthy &sue was also low on the fourth day (l’ig. 2). However, isolated cells contained moderate to heavily labeled “antigenie inclusions” which had an unresolvable internal structure which resembled neither virus nor cytoplasm. These inclusions were never fourld in heahhy tissue. When present, they always appeared in the cyboplasm at the edge of t,he cytoplasmic membrane or free in the vacuole and were not, themselves, surrounded by a membrane. The general cytoplasmic label in 5-day samples was moderate in the diseased tissue, but only the antigenic inclusions were heavily labeled (E’igs. 3-5). At’ this time after inoculation, inculsions were large and numerous, appearing in mnn.v cells. Viral particles tirst, appeared in 6-day samples (Fig. 6). They were in small packets occurring in the cytoplasm, often in the same
CYMV
TNFIXTIONS
749
cell where ant,igenic inclusions could be found in the vacuolar area. The antigenic inclusions were labeled heavily over their entire structure, while the viral packets labeled heavily only on the edges. By 6 days the size and numbers of the antigrnic inclusions began to decrease. RIassive virus inclusions appeared in t,he 12-day samples, and t’hese were heavily labeled, again at the edges (Fig. 7). In general the cytoplasmic label was reduced at t#his stage and antigenic inclusions were seldom seen. Throughout the experiments inclusion bodies of another type were observed. These resembled the tannin sacs described by Weimraub and Ragetli (1966)) and occurred sporadically (in small numbers) in healthy tissue. The>, nere large and numerous in diseased tissue, especially in 4- and &day samples. They were never labeled; however, they did seem to have some association with a cellular response to infect,ion since they were noticeable in t,he early days of infect,ion (Zig. 2), but, were relatively absent, from healthy tissue am-1 12.day infected tissue. The simplest explanation of the observ:rt,ions report.ed above is that virus moves from the inoculated leaves to young leaves at the t,ip. Kew virus ca11 be detecmd by infectivity assays in l,he young l-cm leaves approximately 3 days after hOcuhti(Jll. Xt this time no evidence for intact, virus can be seen, either by electron microscope observntions or by immunolabeling. Antigenic material first, appears on the fourth day. By the fift’h day large numbers of antigenic inclusions occur in many cells, altliougli intact virus part,icles are not visible. Virus inclu sions tirst appeared in the cytoplasm on t’hc sixth day, but were never seen in the vacuole. By 1% days pcJStiIiOculatiOn, masses of virus were seen, but only a few ant,igenic inclusions remained. Increase in virus infectivit,? paralleled the increase in numbers of antigenic inclusions up to the sixth day wllen virtts particles first appeared. The highly antigenic nature of t,he bodies found in the vacuole suggests that, it is excess virus protein which gradually decreases as the concentration of whole virus mcreases, ahhough some still remained in 12-day samples. The occurrence of excess virus protein in C\XV-infections Iuts been
FIG. 1. Elertron microscope radioautographs of CYMV infection in young leaves of Viciafaba treated with itiI-r-globulin. Healthy tissue control to 12-day samples showing low level of cytoplasmic label. Note that these cells were cut open during vibratome presectioning (arrows). Radioautographic exposure, 116 hr. Scale = 2 p. FIG. 2. CYMV-infected leaf tissue 4 days after inoculation showing general higher level of label in the cytoplasm and small tannin sacs (arrows). No virus particles can be seen. Radioautographic exposure, 332 hr. Scale = 1 p. 750
Fro.
3. Antigenic
inclusions
(ai) in vacuole
(V)
FIG. 4. Antigenic inclusion (ai) in 5-day sample extending int,o vacuole from the tonoplast (1). Note nucleus (N) and nuclear membrane (nm). Radioautographic exposure, 332 hr. Scale = 0.5 P. FIG. 5. Antigenic inclusion (ai) in 5.day sample isolated in vacuole (V). Note apparent inclusion of cytoplasmic materials in antigenic inclusions (arrow). Radioautographic exposure, 332 hr. Scale = 0.5 p. FIG. 6. Virus crystals (UC) in B-day sample. Radioautographic exposure, 144 hr. Scale 0.5 cc.
VIRAL
ll(i
FIG. 7. Heavily labelwl hr. Scale = 0.5 U.
virils
PROTEIN
cryst,als
IN
CYMV
(UC) in cytoplasm
T.-t3
INFECTIOXS
of 12.day
sample.
lladioatltographic
c>sposr~rt’,
754
SCHLEGEL
demonstrated by Purcifull and Shepherd (1964). The antigenic inclusions were always found outside the cytoplasm extending from the tonoplast into the vacuole. Occasionally these antigenic inclusions appeared to be free in the vacuole. However, these apparently unattached antigenic inclusions were associated with cytoplasmic material (Fig. 5), and most likely the electron microscope section did not pass through the area of attachment. No membrane could be detected surrounding any of the antigenic inclusions. This localization could possibly be an artifact of fixation in which the fixation process itself resulted in an extrusion of such material from the cytoplasm into the vacuole. Confirmation of this explanation is not possible, however, since antigenic inclusions were never seen in the cytoplasm itself, a fact which argues against the vacuolar localization being an artifact. Antigenic inclusions have not been reported previously, possibly due to the fact that fixation and embedding methods in general have not included the use of a gelatin infiltration step to stabilize viral materials in cells. However, once we identified these structures, they could be found using normal methods of fixation and embedding. The heavy label over the antigenic inclusions indicates a loose, open arrangement which permits penetration of antibody. This is in strong contrast with the virus inclusions which are labeled only at the edge, suggesting a close packing of virus particles. The impervious nature of viral inclusions to antibodies has been reported previously (Schlegel and Langenberg, 1969). The production of antigen in broadbean leaves infected with clover yellow mosaic virus differs from the production of antigen in tobacco leaves infected with tobacco mosaic virus. The latter do not form the antigenic inclusions distinct from viral inclusions; furthermore, the first antigen appears in the nucleus (Langenberg and Schlegel, 1969) in the case of TMV. Thus it would appear from these results that generalizations with respect to viral protein synthesis are hazardous. The complex and integral relationship of
AND DELISLE
a virus to a living cell has raised many questions about the various stages of virus multiplication. When a sufficient body of information has accumulated, we can begin to put together an overall picture of virus synthesis. ACKNOWLEDGMENT This work was supported in part by USPHS research grant AI03415 from the National Institute of Allergy and Infectious Diseases. REFERENCES L. G. (1964). High-resolution autoradiography. In “Methods in Cell Physiology” (D. M. Prescott, ed.), Vol. I, pp. 327-363. Academic Press, New York. LANGENBERG, W. G., and SCHLEGEL, D. E. (1967). Autoradiography with lz61-labeled antibodies as a means of localizing TMV antigen in plant cells. Virology 32, 167-171. LANGENBERG, W. G., and SCHLEGEL, D. E. (1969). Localization of tobacco mosaic virus protein in tobacco leaf cells during the early stages of infection. Virology 37, 86-93. PURCIFULL, D. E., and SHEPHERD, R. J. (1964). Preparation of the protein fragments of several rod-shaped plant viruses and their use in agargel diffusion tests. Phytopathology 54, 11021108. SCRLEGEL, D. E., and LANGENBERG, W. G. (1969). The prevention of virus redistribution within plant cells during preparation for autoradiogof Diffusible raphy. Zn “Autoradiography Substances” (L. J. Roth and W. E. Stumf, eds.), pp. 131-145. Academic Press, New York. SHEPARD, J. F., and SHALLA, T. A. (1969). Tobacco etch virus cylindrical inclusions: Antigenically unrelated to the causal virus. Virology 38, 185-188. STEMPAK, J. G., and WARD, R. T. (1964). An improved staining method for electron microscopy. J. Cell Biol. 22, 697-701. TSUZUKU, O., YAGI, Y., and PRESSMAN, D. (1967). Preparative purification of lung-localizing rabbit anti-rat lung antibodies in vitro. J. Immunol. 98, 1004-1010. VAN SLOGTEREN, D. H. M. (1955). Serological micro-reactions with plant viruses under paraffin oil. In “Proceedings of the Second Conference on Potato Virus Diseases” (E. Streutgers, A. B. R. Beemster, D. Noordam, and J. P. H. van der Want, eds.), pp. 51-54. H. Veenman and Zonen, Wageningen. WEINTRAUB, M., and RAGETLI, H. W. (1966) Fine structure of ‘inclusion and organelles in Vi& fuba infected with bean yellow mosaic virus. Virology 28, 290-302. CARO,