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Influence of assay conditions on infectivity of heated poliovirus
DISCUSSION
AND
PRELIMIXARY
601
REPORTS
From these observations it seems plausible to conclude that the tumorproducing factor obtained by phenolic extraction of the cottontail papilloma tissue does not consist of intact virus particles surviving the procedure. It appears to be definitely associated with the nucleic acid fraction and is probably of DNA nature. ACKNOWLEDGMENTS The author wishes to express his deepest appreciation to Dr. Charles A. Evans for his thoughtful guidance and encouragement during the study, and to Dr. Milton Gordon of the Department of Biochemistry for valuable advice. REFERENCES 1. GIERER, A.? and SCHRAMM, G., 2. Naturforsch. llb, 138-142 (1956). 2. FRAENKEL-CONRAT, H., SINGER, B., and WILI,IAMS, R. C., Biochim. et Biophys. Acta 26, 87-96 (1957). S. FRANKLIN, R. M., WECKER, E., and HENRY, C., Virology 7, 220-235 (1959). 4. BROWN, F., and STEWART, D. L., Virology 7, 408418 (1959). 5. ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., SPRUNT, K., and VAN DAMME, O., J. Exptl. Med. 108, 493306 (1958). 6. HOLLAND, J. J., MCLAREN, L. C., and SYVERTON, J. T., J. Exptl. Med. 110, 65-80 (1959). 7. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUNTER, W. S., FRIEND, C., and BENDICH, A., Proc. Natl. Acad. Sci. CT. S. 46, 180551808 (1959). 8. DMOCHOWSKI, L., PEARSON, L. O., SYKES, J. A., GREY, C. E., and GRIFFIN, A. C., Proc. Am. Assoc. Cancer Research, 3, 107 (Abstract) (1960). 9. SHOPE, R. E., J. Exptl. Med. 68, 607-624 (1933). 10. WECKER, E., Virology 7, 241-243 (1959).
YOHEI 1~05
Department of Microbiology School of Medicine University of Washington Seattle, Washington Received September 21, 1960 5 On leave Japan.
Influence
from
Department
of Assay
of Hygiene,
Conditions
Nara
on Infectivity
Gakugei
of Heated
University,
Nara,
Poliovirus
Since heating poliovirus abolishes its ability to attach readily to host cells (1) and destroys its antibody combining capacity (2), this form of inactivation may be the result of denaturation of surface protein units. It seemed conceivable that the ribonucleic acid (RNA) in such particles was still unaffected and could be shown to be active if it could enter the
602
DISCUSSION
1
I
10
AND
I
I
20
PRELIMINARY
I
I
30
REPORTS
I
I
40
I
I
50
I
I
60
MINUTES 1. Rate of loss of infectivity of highly purified poliovirus (type 1, strain Mahoney) in physiological salt solution at 54” C. Virus survival, measured as PFU on amnion cell monolayers seeded with virus, in 1 M salt solution pH 8.0 (curve 1) and in physiological salt solution pH 7.4 (curve 2). Curve 3: cell monolayers seeded with virus in 1 M salt solution after exposure to RNAase. FIG.
cell. Hypertonic salt solut,ion facilitates the entry of poliovirus RKA into a cell (3) in such a manner that it can initiate infection, and we have tried to facilitate the entry of heat-inactivated virus by the same method. The results are shown in Fig. 1. Type 1 poliovirus, strain Mahoney, was grown in amnion cells, purified as previously reported (4), diluted in saline, and heated at 54” C. The initial titer was 5 X log PFU per milliliter. Samples were removed periodically and assayed on amnion cell monolayers. When the dilutions were made in physiological salt solution at pH 7.4 the titer showed a rapid decline down to a survival of about 10p6, after which the slope became more gradual (curve 2). When the dilutions were made in hypertonic saline, 1 M at pH 8.0, curve 1 was obtained, which deviates widely from curve 2 below a survival of 10p4. Beyond this point curve 1 assumes the same slope as the toe of curve 2, but at a level roughly 100 times higher. When heated specimens were treated with RNAase (9 rg/ml, 10 minutes at room temperature) and then assayed after dilution in hypertonic saline, the results given in curve 3 were obtained. The explanation of these results which seems most simple and direct is as follows: Heating at 54” C denatures the viral protein coat and thus
DISCUSSION
AND
PRELIMINARY
603
REPORTS
prevents virus attachment but leaves the virus Rn’A largely intact. Cells can be induced to take up these “nonadsorbing” particles at an increased rate and with an efficiency such that about 1 in lo4 particles enters and infects a cell. This low efficiency of activation prevents any demonstration of a significant hypertonic saline effect until heat’ has reduced the background of active virus below 104. RSAase cannot be shown to have an effect until infection under the influence of hypertonic saline becomes significant. The final slope of curves 1 and 2 represents the inactivation of RKA.
Information on methods of preparation of highly purified poliovirus by Drs. Levintow and Darnell is highly appreciated. I am indebted to Mrs. G. Lange for capable technical assistance. This work was supported in part by the Deutsche Forschungsgemeinschaft
A. F., fkteriol. Revs. 23, 224-231 (1959). J. E., and BENSETT, B. L., cited by YOUNGNER, J., J. I~t~ntuno/. 78, 282-290 (1957). 3. KOCH, G., KOENIG, S., and ALESANDER, H. E. Vidogy 10, 329-343 (1960). 4. KOCH, 6. Z. Natwrforsch. in press. I. GRAHAM,
2.
SALK,
Laboratorium der Stijtung zur Erforschung der spinalen Kinderltihmutcg und der Multiplen Sklerose Hamburg 20, Germany lieceivcd August 1, 1960
The Infection
GEBHARD
of Apical Meristems of Tomato Roots with Tobacco Mosaic after Treatment with Ethylenediaminetetraacetic Acid
KocH
Virus
There is much evidence that, many plant, viruses do not infect the meristematic cells of plants regardless of whether t#he meristems be shoot, flower, or root’. Holmes (6, 7) has used this fact, to obtain virusfree clones of sweet potatoes [Ipomoea batatas (L.) Lam.] by grafting shoot meristems to virus-free seedlings. The inability of viruses to infect floral meristems was postulated by Nelson (10) and by Crowley (2) as the reason for the lack of seed transmission of highly infectious viruses such as tobacco mosaic virus (TMV). White (13) obtained evidence that TMV does not infect the apical meristems of tomato roots, and more recent work by Bergmann (1) would seem to substantiate this conclusion.
AND
PRELIMIXARY
601
REPORTS
From these observations it seems plausible to conclude that the tumorproducing factor obtained by phenolic extraction of the cottontail papilloma tissue does not consist of intact virus particles surviving the procedure. It appears to be definitely associated with the nucleic acid fraction and is probably of DNA nature. ACKNOWLEDGMENTS The author wishes to express his deepest appreciation to Dr. Charles A. Evans for his thoughtful guidance and encouragement during the study, and to Dr. Milton Gordon of the Department of Biochemistry for valuable advice. REFERENCES 1. GIERER, A.? and SCHRAMM, G., 2. Naturforsch. llb, 138-142 (1956). 2. FRAENKEL-CONRAT, H., SINGER, B., and WILI,IAMS, R. C., Biochim. et Biophys. Acta 26, 87-96 (1957). S. FRANKLIN, R. M., WECKER, E., and HENRY, C., Virology 7, 220-235 (1959). 4. BROWN, F., and STEWART, D. L., Virology 7, 408418 (1959). 5. ALEXANDER, H. E., KOCH, G., MOUNTAIN, I. M., SPRUNT, K., and VAN DAMME, O., J. Exptl. Med. 108, 493306 (1958). 6. HOLLAND, J. J., MCLAREN, L. C., and SYVERTON, J. T., J. Exptl. Med. 110, 65-80 (1959). 7. DIMAYORCA, G. A., EDDY, B. E., STEWART, S. E., HUNTER, W. S., FRIEND, C., and BENDICH, A., Proc. Natl. Acad. Sci. CT. S. 46, 180551808 (1959). 8. DMOCHOWSKI, L., PEARSON, L. O., SYKES, J. A., GREY, C. E., and GRIFFIN, A. C., Proc. Am. Assoc. Cancer Research, 3, 107 (Abstract) (1960). 9. SHOPE, R. E., J. Exptl. Med. 68, 607-624 (1933). 10. WECKER, E., Virology 7, 241-243 (1959).
YOHEI 1~05
Department of Microbiology School of Medicine University of Washington Seattle, Washington Received September 21, 1960 5 On leave Japan.
Influence
from
Department
of Assay
of Hygiene,
Conditions
Nara
on Infectivity
Gakugei
of Heated
University,
Nara,
Poliovirus
Since heating poliovirus abolishes its ability to attach readily to host cells (1) and destroys its antibody combining capacity (2), this form of inactivation may be the result of denaturation of surface protein units. It seemed conceivable that the ribonucleic acid (RNA) in such particles was still unaffected and could be shown to be active if it could enter the
602
DISCUSSION
1
I
10
AND
I
I
20
PRELIMINARY
I
I
30
REPORTS
I
I
40
I
I
50
I
I
60
MINUTES 1. Rate of loss of infectivity of highly purified poliovirus (type 1, strain Mahoney) in physiological salt solution at 54” C. Virus survival, measured as PFU on amnion cell monolayers seeded with virus, in 1 M salt solution pH 8.0 (curve 1) and in physiological salt solution pH 7.4 (curve 2). Curve 3: cell monolayers seeded with virus in 1 M salt solution after exposure to RNAase. FIG.
cell. Hypertonic salt solut,ion facilitates the entry of poliovirus RKA into a cell (3) in such a manner that it can initiate infection, and we have tried to facilitate the entry of heat-inactivated virus by the same method. The results are shown in Fig. 1. Type 1 poliovirus, strain Mahoney, was grown in amnion cells, purified as previously reported (4), diluted in saline, and heated at 54” C. The initial titer was 5 X log PFU per milliliter. Samples were removed periodically and assayed on amnion cell monolayers. When the dilutions were made in physiological salt solution at pH 7.4 the titer showed a rapid decline down to a survival of about 10p6, after which the slope became more gradual (curve 2). When the dilutions were made in hypertonic saline, 1 M at pH 8.0, curve 1 was obtained, which deviates widely from curve 2 below a survival of 10p4. Beyond this point curve 1 assumes the same slope as the toe of curve 2, but at a level roughly 100 times higher. When heated specimens were treated with RNAase (9 rg/ml, 10 minutes at room temperature) and then assayed after dilution in hypertonic saline, the results given in curve 3 were obtained. The explanation of these results which seems most simple and direct is as follows: Heating at 54” C denatures the viral protein coat and thus
DISCUSSION
AND
PRELIMINARY
603
REPORTS
prevents virus attachment but leaves the virus Rn’A largely intact. Cells can be induced to take up these “nonadsorbing” particles at an increased rate and with an efficiency such that about 1 in lo4 particles enters and infects a cell. This low efficiency of activation prevents any demonstration of a significant hypertonic saline effect until heat’ has reduced the background of active virus below 104. RSAase cannot be shown to have an effect until infection under the influence of hypertonic saline becomes significant. The final slope of curves 1 and 2 represents the inactivation of RKA.
Information on methods of preparation of highly purified poliovirus by Drs. Levintow and Darnell is highly appreciated. I am indebted to Mrs. G. Lange for capable technical assistance. This work was supported in part by the Deutsche Forschungsgemeinschaft
A. F., fkteriol. Revs. 23, 224-231 (1959). J. E., and BENSETT, B. L., cited by YOUNGNER, J., J. I~t~ntuno/. 78, 282-290 (1957). 3. KOCH, G., KOENIG, S., and ALESANDER, H. E. Vidogy 10, 329-343 (1960). 4. KOCH, 6. Z. Natwrforsch. in press. I. GRAHAM,
2.
SALK,
Laboratorium der Stijtung zur Erforschung der spinalen Kinderltihmutcg und der Multiplen Sklerose Hamburg 20, Germany lieceivcd August 1, 1960
The Infection
GEBHARD
of Apical Meristems of Tomato Roots with Tobacco Mosaic after Treatment with Ethylenediaminetetraacetic Acid
KocH
Virus
There is much evidence that, many plant, viruses do not infect the meristematic cells of plants regardless of whether t#he meristems be shoot, flower, or root’. Holmes (6, 7) has used this fact, to obtain virusfree clones of sweet potatoes [Ipomoea batatas (L.) Lam.] by grafting shoot meristems to virus-free seedlings. The inability of viruses to infect floral meristems was postulated by Nelson (10) and by Crowley (2) as the reason for the lack of seed transmission of highly infectious viruses such as tobacco mosaic virus (TMV). White (13) obtained evidence that TMV does not infect the apical meristems of tomato roots, and more recent work by Bergmann (1) would seem to substantiate this conclusion.