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Isolation and preliminary studies of soluble protein and infectious nucleic acid from turnip yellow mosaic virus
LETTERS
TO THE
527
EDITORS
Monolayer cultures of L cells were exposed to a mixture of 1 ml of infectious ectromelia and 1 ml of heat-inactivated vaccinia virus which had been added to the growth media. The supernatant fluid obtained after 48 hours revealed the characteristics of vaccinia virus, as shown by the rapid degeneration of the cell culture and the induction of a positive skin reaction in rabbits (Fig. 2). Moreover the specific inclusion bodies of vaccinia were observed by histological examination of the skin lesions. Five separate experiments gave similar results. As the virus preparation thus obtained was a mixture of ectromelia and transformed one, it was passed several times on L cells and purified twice by single-plaque isolations. The plaque-purified virus (lo6 TCID50 per milliliter) was not pathogenic for mice when injected intraperitoneally. Studies on the properties of the transforming agent are now in progress. Details of these experiments will be published elsewhere. REFERENCES 1. BERRY, G. P., and I~EDRICK, H. M., J. Bacterial. 31, 50-51 (1936). 2. GARDENER, R. E., and HYDE, R. R., J. Infectious Diseases 71, 4749 (1942). 3. SMITH, M. H. II., Ann. N. Y. Acad. Sci. 64, 1141-1152 (1952). 4. KILHAM, L., Proc. Sot. Exptl. Biol. Med. 96, 59-62 (1957). 6. BURNET, F. M., and LIND, P. E., ilustralian J. Exptl. Biol. Med. Sci. 32, 133-
143 (1954). 6. GOTLIEB,
T., and
HIRST,
G. K., Virology
2, 235-248 (1956).
Department of Pathology The Research Institute for Microbial Diseases Osaka University Osaka, Japan Received Maich 19, 1969
Isolation
and
Preliminary
Nucleic
Acid
TERUKO HIDESABUR~ JUNTARB
Studies from
Turnip
of Soluble Yellow
Protein Mosaic
and
HANAFUSA HANAFUSA KAMAHORA
Infectious
Virus’
By use of a modified heat denaturation procedure (1) an infectious ribonucleic acid fraction (TYMV-NA) has been isolated from turnip yellow mosaic virus (TYMV) purified by use of the butanol-chloroform procedure (2). The infectivity of this TYMV-NA fraction has been compared with that of whole virus by means of local lesion assays (Table 1) on young leaves of the Wong Bok variety of 1 Aided by grants No. C-2245 and No. CRTY-5028 from The National Cancer Institute, National Institutes of Health, United States Public Health Service, and from The Netherlands Organization for Pure Research.
528
LETTERS
TO THE
TABLE
EDITORS
I
COMPARATIVE INFECTIVITY TESTY ON SAMPLES OF TYMV-NA, TYMV-NA RNAAsE-TREATED, AND TYMV AS DETERMINED BY LOCAI, LESION COUNTS ON YOUNG, RAPIDLY GROWING LEAVES OF THE WONG ROK VARIETY OF CHINESE CABBAGE ISxpt. L 0. Y (Prep. No.) I
Average
numbersa
Control
of local lesions No.
TYMV (2.5 pg/ml) TYMV (0.5 @g/ml) TYMV-NA (100 rg/ml)‘j Untreated i
Sample
II
TYMV
(0.5 pg/ml)
11
TYMV-NA (100 pg/ml) TYMV-NA (100 pg/ml) TYMV-NA (100 pg/ml) RNAaseh treated ! TYMV-NA (50 pg/ml)
III
TYMV
(0.5 fig/ml)
41
TYMV-NA
IV
TYMV (2.5 TYMV (0.5 TYMV (2.5 TYMV (0.5 TYMV (0.5 Untreated TYMV-NA Untreated
pg/ml) pg/ml) pg/ml) pg/ml) rg/ml)\ 1 (500 pg/ml)
a Average of the number 6 RNAase: concentration c RNAase: concentration
i
184 64 88
per half !eaf
37 41 125 27 19 ’ 112 ’
(50 pg/ml)
TYMV-NA (100 pg/ml) TYMV-NA (100 rg/ml) TYMV-NA (500 pg/ml) TYMV-NA (500 pg/ml) TYMV (0.5 a/ml)\ RNAase? treat,ed 1 TYMV-NA (500 pg/ml) RNAaseh treated --
No. 117 90 0 27 67 72 44 167 157 22 0
of lesions from 6 to 8 half leaves. 0.005 fig/ml. 0.05 rg/ml.
Chinese cabbage (Rrassica chinensis L.). This plant proved to be an excellent local lesion host for TYMV when young, rapidly growing leaves were used. It was found that these TYMV-NA preparations contained less than 0.35; protein as determined by the Folk-phenol method. The ultraviolet spectrum resembled that of a nucleic acid, having a maximum at 260 mp and a minimum at 231 mp while the max:min ratio was 2.2 to 2.3. The infectivity was O.lXk5% of that of the same amount of nucleic acid (NA) incorporated in whole virus. This figure compared favorably with the infectivity of NA prepared from other viruses by use of the same and other procedures (1, S-6). Incubation (2 hours at room temperature) of these TYMV-NA preparations at concentrations of 100-500 @g/ml with 0.005 pg/ml ribonuclease (RNAase) resulted in complete loss of infectivity as determined by the failure of appearance of either local lesions or systemic infection (Table 1). Controls consisting of TYMV at a concentration giving approximately equal numbers of local lesions were unaffected when incubated with ten times as much RNAase under the same conditions. Other preparations of
LETTERS
TO
THE
EDITOl-LS
529
FIG. 1. Electron micrograph of part of an air-dried droplet of a mixture of solub!e protein of TYMV with whole TYMV added to demonstrate size difference. Arrows indicate : A: So-i particles or aggregates of smaller particles; B: virus particles; C: row of aggregates of varying size. This row and the row of virus particles represent portions of rings of these components as they dried from the drop. Particles of different sizes tend to form concentric rings with the largest particles innermost as a droplet is air dried. D: outer edge of drop pattern. Magnification: x 190,000. TYMV-NA have been prepared by the alcohol method (7), but these have given extremely irregular results with respect to their infectivity. By dialyzing a suspension of TYMV against a 0.1 M phosphate buffer of pH 11.5 for 2% days at room temperature and subsequently following the purification procedure already described for the isolation of TMV-protein (8) it was possible t,o obtain a preparation of water-soluble TYMV-protein. (See also 9.) This protein is reversibly precipitated below pH 5.3 and is soluble at higher pH values. The
530
LETTERS
TO
THE
EDITOIZY
ultraviolet spectrum was that characteristic of virus protein with a maximum at 280 rnp and a minimum at 250 rnp while the max:min ratio in the best preparations was 2.2. The removal of virtually all nucleic acid was evident from the absence of any detectable phosphorus (less than 0.03%) in these preparations. The number of free amino groups before and after dialysis at pH 11.5 was found to be the same as was determined by ninhydrin tests. From this it can be dedured t,hat, probably no covalent peptide bonds have been broken during t,he preparation of the protein. Electron micrographs (Fig. 1) suggest that the isolated protein consists of veq small particles which have a pronounced tendency to form aggregates approximately 60 il in diameter and larger aggregates of extremely variable size. Studies with the analyt,ical centrifuge showed that these same conditions of aggregation prevailed in suspension and were not merely artifacts of drying the suspension for electron microscopy. Whether or not there is any connection between the two findings, it, is of interest to note that the size of the smaller aggregates, 60 A, corresponds closely with the size of bumps seen on the surface of TYMV particles (10). ilttempts to reconstitute whole virus from a mixture of TYMV-NA and the soluble protein, as has been done with tobacco mosaic virus (a), have so far proved unsuccessful. ACKNOWLEDGMENTS The authors Praenkel-Conrat
are grateful to Drs. W. M. Stanley, for their interest in this work
R.
C. Williams,
and H.
REFERENCES 1.
9. 3. 4. 5. 6. 7. 8, 9. 10.
J. M., and STEERE, R. L., Virology 7, 1277139 (1959). STEERE, R. L., Ph@opatholoy!/ 46, 60-69 (1956). FRAENKEL-CONRAT, H., J. Am. (Them. Sot. 78, 8X2 (1956). GIERER, A., and SCHRAMM, G., Z. Nutu/jofo,sch. llb, 138-142 (1956). RTSHIZHY, G., Ph.D. Thesis, University of California (1958). KNIGHT, C. A., in Methods in Enzy?nolog?/ (S. 1’. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 684. Academic Press, New York, 1957. MARKHA~I, R., and SMITH, K. M., Pamsilolog~] 39, 330-342 (1919). R. C., PrOC. N&l. Ilcad. SCi. u. S. 41, FRAENKEL-CONRAT, and WILLIAMS, 690 (1955). LYTTLETON, J. W., and MATTHEWS, R. E. F., Viroloy!/ 6, 460-471 (1958). STEERE, R. I,., Biochenl. Riophw. Pytol. 3, 45-60 (1958). KAPER,
Virus Laboratory Urbiversity 0,f Cali.fornia
Berkeley, California Received March
J. M. KAPER* R.. L. STEERER
25, 1969
2 Present address: Department of Biochemistry, Leyden State University, Leyden, The Netherlands. 3 Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland.
TO THE
527
EDITORS
Monolayer cultures of L cells were exposed to a mixture of 1 ml of infectious ectromelia and 1 ml of heat-inactivated vaccinia virus which had been added to the growth media. The supernatant fluid obtained after 48 hours revealed the characteristics of vaccinia virus, as shown by the rapid degeneration of the cell culture and the induction of a positive skin reaction in rabbits (Fig. 2). Moreover the specific inclusion bodies of vaccinia were observed by histological examination of the skin lesions. Five separate experiments gave similar results. As the virus preparation thus obtained was a mixture of ectromelia and transformed one, it was passed several times on L cells and purified twice by single-plaque isolations. The plaque-purified virus (lo6 TCID50 per milliliter) was not pathogenic for mice when injected intraperitoneally. Studies on the properties of the transforming agent are now in progress. Details of these experiments will be published elsewhere. REFERENCES 1. BERRY, G. P., and I~EDRICK, H. M., J. Bacterial. 31, 50-51 (1936). 2. GARDENER, R. E., and HYDE, R. R., J. Infectious Diseases 71, 4749 (1942). 3. SMITH, M. H. II., Ann. N. Y. Acad. Sci. 64, 1141-1152 (1952). 4. KILHAM, L., Proc. Sot. Exptl. Biol. Med. 96, 59-62 (1957). 6. BURNET, F. M., and LIND, P. E., ilustralian J. Exptl. Biol. Med. Sci. 32, 133-
143 (1954). 6. GOTLIEB,
T., and
HIRST,
G. K., Virology
2, 235-248 (1956).
Department of Pathology The Research Institute for Microbial Diseases Osaka University Osaka, Japan Received Maich 19, 1969
Isolation
and
Preliminary
Nucleic
Acid
TERUKO HIDESABUR~ JUNTARB
Studies from
Turnip
of Soluble Yellow
Protein Mosaic
and
HANAFUSA HANAFUSA KAMAHORA
Infectious
Virus’
By use of a modified heat denaturation procedure (1) an infectious ribonucleic acid fraction (TYMV-NA) has been isolated from turnip yellow mosaic virus (TYMV) purified by use of the butanol-chloroform procedure (2). The infectivity of this TYMV-NA fraction has been compared with that of whole virus by means of local lesion assays (Table 1) on young leaves of the Wong Bok variety of 1 Aided by grants No. C-2245 and No. CRTY-5028 from The National Cancer Institute, National Institutes of Health, United States Public Health Service, and from The Netherlands Organization for Pure Research.
528
LETTERS
TO THE
TABLE
EDITORS
I
COMPARATIVE INFECTIVITY TESTY ON SAMPLES OF TYMV-NA, TYMV-NA RNAAsE-TREATED, AND TYMV AS DETERMINED BY LOCAI, LESION COUNTS ON YOUNG, RAPIDLY GROWING LEAVES OF THE WONG ROK VARIETY OF CHINESE CABBAGE ISxpt. L 0. Y (Prep. No.) I
Average
numbersa
Control
of local lesions No.
TYMV (2.5 pg/ml) TYMV (0.5 @g/ml) TYMV-NA (100 rg/ml)‘j Untreated i
Sample
II
TYMV
(0.5 pg/ml)
11
TYMV-NA (100 pg/ml) TYMV-NA (100 pg/ml) TYMV-NA (100 pg/ml) RNAaseh treated ! TYMV-NA (50 pg/ml)
III
TYMV
(0.5 fig/ml)
41
TYMV-NA
IV
TYMV (2.5 TYMV (0.5 TYMV (2.5 TYMV (0.5 TYMV (0.5 Untreated TYMV-NA Untreated
pg/ml) pg/ml) pg/ml) pg/ml) rg/ml)\ 1 (500 pg/ml)
a Average of the number 6 RNAase: concentration c RNAase: concentration
i
184 64 88
per half !eaf
37 41 125 27 19 ’ 112 ’
(50 pg/ml)
TYMV-NA (100 pg/ml) TYMV-NA (100 rg/ml) TYMV-NA (500 pg/ml) TYMV-NA (500 pg/ml) TYMV (0.5 a/ml)\ RNAase? treat,ed 1 TYMV-NA (500 pg/ml) RNAaseh treated --
No. 117 90 0 27 67 72 44 167 157 22 0
of lesions from 6 to 8 half leaves. 0.005 fig/ml. 0.05 rg/ml.
Chinese cabbage (Rrassica chinensis L.). This plant proved to be an excellent local lesion host for TYMV when young, rapidly growing leaves were used. It was found that these TYMV-NA preparations contained less than 0.35; protein as determined by the Folk-phenol method. The ultraviolet spectrum resembled that of a nucleic acid, having a maximum at 260 mp and a minimum at 231 mp while the max:min ratio was 2.2 to 2.3. The infectivity was O.lXk5% of that of the same amount of nucleic acid (NA) incorporated in whole virus. This figure compared favorably with the infectivity of NA prepared from other viruses by use of the same and other procedures (1, S-6). Incubation (2 hours at room temperature) of these TYMV-NA preparations at concentrations of 100-500 @g/ml with 0.005 pg/ml ribonuclease (RNAase) resulted in complete loss of infectivity as determined by the failure of appearance of either local lesions or systemic infection (Table 1). Controls consisting of TYMV at a concentration giving approximately equal numbers of local lesions were unaffected when incubated with ten times as much RNAase under the same conditions. Other preparations of
LETTERS
TO
THE
EDITOl-LS
529
FIG. 1. Electron micrograph of part of an air-dried droplet of a mixture of solub!e protein of TYMV with whole TYMV added to demonstrate size difference. Arrows indicate : A: So-i particles or aggregates of smaller particles; B: virus particles; C: row of aggregates of varying size. This row and the row of virus particles represent portions of rings of these components as they dried from the drop. Particles of different sizes tend to form concentric rings with the largest particles innermost as a droplet is air dried. D: outer edge of drop pattern. Magnification: x 190,000. TYMV-NA have been prepared by the alcohol method (7), but these have given extremely irregular results with respect to their infectivity. By dialyzing a suspension of TYMV against a 0.1 M phosphate buffer of pH 11.5 for 2% days at room temperature and subsequently following the purification procedure already described for the isolation of TMV-protein (8) it was possible t,o obtain a preparation of water-soluble TYMV-protein. (See also 9.) This protein is reversibly precipitated below pH 5.3 and is soluble at higher pH values. The
530
LETTERS
TO
THE
EDITOIZY
ultraviolet spectrum was that characteristic of virus protein with a maximum at 280 rnp and a minimum at 250 rnp while the max:min ratio in the best preparations was 2.2. The removal of virtually all nucleic acid was evident from the absence of any detectable phosphorus (less than 0.03%) in these preparations. The number of free amino groups before and after dialysis at pH 11.5 was found to be the same as was determined by ninhydrin tests. From this it can be dedured t,hat, probably no covalent peptide bonds have been broken during t,he preparation of the protein. Electron micrographs (Fig. 1) suggest that the isolated protein consists of veq small particles which have a pronounced tendency to form aggregates approximately 60 il in diameter and larger aggregates of extremely variable size. Studies with the analyt,ical centrifuge showed that these same conditions of aggregation prevailed in suspension and were not merely artifacts of drying the suspension for electron microscopy. Whether or not there is any connection between the two findings, it, is of interest to note that the size of the smaller aggregates, 60 A, corresponds closely with the size of bumps seen on the surface of TYMV particles (10). ilttempts to reconstitute whole virus from a mixture of TYMV-NA and the soluble protein, as has been done with tobacco mosaic virus (a), have so far proved unsuccessful. ACKNOWLEDGMENTS The authors Praenkel-Conrat
are grateful to Drs. W. M. Stanley, for their interest in this work
R.
C. Williams,
and H.
REFERENCES 1.
9. 3. 4. 5. 6. 7. 8, 9. 10.
J. M., and STEERE, R. L., Virology 7, 1277139 (1959). STEERE, R. L., Ph@opatholoy!/ 46, 60-69 (1956). FRAENKEL-CONRAT, H., J. Am. (Them. Sot. 78, 8X2 (1956). GIERER, A., and SCHRAMM, G., Z. Nutu/jofo,sch. llb, 138-142 (1956). RTSHIZHY, G., Ph.D. Thesis, University of California (1958). KNIGHT, C. A., in Methods in Enzy?nolog?/ (S. 1’. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 684. Academic Press, New York, 1957. MARKHA~I, R., and SMITH, K. M., Pamsilolog~] 39, 330-342 (1919). R. C., PrOC. N&l. Ilcad. SCi. u. S. 41, FRAENKEL-CONRAT, and WILLIAMS, 690 (1955). LYTTLETON, J. W., and MATTHEWS, R. E. F., Viroloy!/ 6, 460-471 (1958). STEERE, R. I,., Biochenl. Riophw. Pytol. 3, 45-60 (1958). KAPER,
Virus Laboratory Urbiversity 0,f Cali.fornia
Berkeley, California Received March
J. M. KAPER* R.. L. STEERER
25, 1969
2 Present address: Department of Biochemistry, Leyden State University, Leyden, The Netherlands. 3 Crops Research Division, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland.