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Infectious nucleic acid in plants with tobacco mosaic
890
LETTERS
TO THE
EDITORS
The relationship of the tissue culture virus to the original cat spleen virus was established in two ways. (1) Antisera prepared in rabbits against the original cat spleen PL virus neutralized 500-l ,000 TCI& of the sixteenth passage tissue culture virus establishing a serological relationship between the two agents. (2) PL virus at t.he sixteenth passage was inoculated into six cats. Temperatures, total white blood cell counts, serology, and mortality are shown in Table 1. Temperature elevations of 104.0” F or higher occurred 24-96 hours after virus inoculation, persisted for l-7 days and then decreased. The four cats dying of the disease had temperatures from 100” F to 103.0” F except cat number 49 which had a temperature of 107.0” F. The cats that died of PL had white blood cell count of 2,000 per mm3 or less. Cat number 49 at the time of death had fifty cells per mm3 in the heart blood. The bone marrow of this cat showed aplasia of the leucoblastie elements.2 The sera of cats which recovered from the disease and those which died later than the fifteenth day neutralized at least 1,000 TCDa of virus. It is evident from the results presented t.hat the panleucopeniavirus of cats was established in tissue culture and serially passaged, producing a characteristic cytopathogenic eflect. The tissue culture adapted virus reproduced clinical panleucopenia in cats. Division of Microbiological Research *Jensen-Salsbery Laboratories, Inc. Kansas City, Missorcri Received July 19, 1957 Infectious
Nucleic
V.S.
BOLIN
Acid in Plants with Tobacco Mosaic*
During the development of chromatographic techniques for the separation of components in extracts from Turkish tobacco plants infected with tobacco mosaic virus (TMV) the effluent from ion exchange cellulose columns was passed through a quartz absorption cell of a recording spectrophotometer. Ten-ml fractions were collected and assayed for infectivity on local lesion hosts. Both cation (carboxymethyl) and anion (diethylaminoethyl) exchanging cellulose adsorbents (1) were used and both separated two types of infectious components that gave typical TMV symptoms in susceptible hosts. The first infectious component moved through either type of cellulose without any appreciable adsorption under t.he conditions of the experiments. This component appeared to be a nucleic acid. It absorbed strongly in the ultraviolet at 260 rnp and its infectivity was completely lost when incubated with crystalline ribonuclease at 36” for 24 hours. Simiiar control fractions incubated under the same conditions without ribonuclease retained some infectivity. TMV rods could not be found when these infectious nucleic acid fractions were examined with the electron microscope. 2 Histopathology by F. C. Helwig, Pathologist, Department of Pathology and Research, St. Luke’s Hospit.al, Kansas City, Missouri. 1 This investigation was supported, in part, by research grant No. AT(ll-l)80 Proj. 3 from the United States Atomic Energy Commission.
LETTERS
TO THE
EDITORS
391
The second infective component appeared 24 to 48 hours later after the passage of 800 to 1000 ml of developing solvent which carried no infectivity. This component was identified as the TMV nucleoprotein. It was not eluted unt,il the pH of the developing solvent approached a value of 3.8 to 4.0 (near the isoelectric point of TMV nucleoprotein). The infectivity of t,hese preparations was not lost when they were incubated with ribonuclease. Many typical TMV rods could be seen when these fractions were studied with the electron microscope. Fraenkel-Conrat (Z-4) and Gierer and Schram (5) have shown that infectious nucleic acid can be obtained b.y degrading virus nucleoprotein. In the experiments reported here the plant tissue was ground in liquid nitrogen and t,he chromatography was carried out using phosphate buffer systems and an anaerobic technique that is reported elsewhere (6). It, is believed that the mild extraction and chromatographic techniques used would not result in the degradation of nucleoprotein to release nucleic acid. The relative infectivity of the nucleic acid and nucleoprotein components appeared to vary with the age of the infections. In one experiment where an extract from a plant with a g-day-old infection was chromatographed, the amount of infectivity in the nucleic acid fractions appeared to be several times that in the nucleoprotein fractions. In several experiments with extracts from 6-month-old infections the infectivity in the nucleoprotein fractions appeared to be many hundredfold greater. Further studies are needed to establish these relations but the evidence reported here suggests that the nucleic acid is a precursor of the nucleoprotein. These experiments indicate the need for caution in the development of virus assay techniques because both types of infectious entities produce typical TMV symptoms following infection. Yet they are different physically and chemically, and extraction procedures may selectively favor one over the other. But most important of all, the research reported here suggests that we need to re-examine our concept of viruses for it may be that many other viruses occur naturally in two infective forms. Or possibly some may exist only as nucleic acids and never as nucleoproteins. REFERENCES 1. PETERSON, E. A., and SOBER, H. A. J. Am. Chem. Sot. 78, 751-755 (1956). 2. FRAENKEL-CONRAT, H., J. Am. Chem. Sot. 78, 882-883 (1956). 8. FRAENKEL-CONRAT, H., SINGER, B., and WILLIAMS, R. C. Biochim. et Biophys. Acta In press (1957). 4. FRAENKEL-CONRAT, H., Ann. N. Y. Acad. Sci. In press (1957). 5. GIERER, A.,and SCHRAMM,G.NCL~UT~ 177,702 (1956) 6. COCHRAN, G. W., and CHIDESTER, J. L. Submitted to the editors of Science in (1957). Department of Botany and Plant Pathology Utah State University Logan, Utah Received July SO, 1957
G. W. COCHRAN J. L. CHIDESTER
LETTERS
TO THE
EDITORS
The relationship of the tissue culture virus to the original cat spleen virus was established in two ways. (1) Antisera prepared in rabbits against the original cat spleen PL virus neutralized 500-l ,000 TCI& of the sixteenth passage tissue culture virus establishing a serological relationship between the two agents. (2) PL virus at t.he sixteenth passage was inoculated into six cats. Temperatures, total white blood cell counts, serology, and mortality are shown in Table 1. Temperature elevations of 104.0” F or higher occurred 24-96 hours after virus inoculation, persisted for l-7 days and then decreased. The four cats dying of the disease had temperatures from 100” F to 103.0” F except cat number 49 which had a temperature of 107.0” F. The cats that died of PL had white blood cell count of 2,000 per mm3 or less. Cat number 49 at the time of death had fifty cells per mm3 in the heart blood. The bone marrow of this cat showed aplasia of the leucoblastie elements.2 The sera of cats which recovered from the disease and those which died later than the fifteenth day neutralized at least 1,000 TCDa of virus. It is evident from the results presented t.hat the panleucopeniavirus of cats was established in tissue culture and serially passaged, producing a characteristic cytopathogenic eflect. The tissue culture adapted virus reproduced clinical panleucopenia in cats. Division of Microbiological Research *Jensen-Salsbery Laboratories, Inc. Kansas City, Missorcri Received July 19, 1957 Infectious
Nucleic
V.S.
BOLIN
Acid in Plants with Tobacco Mosaic*
During the development of chromatographic techniques for the separation of components in extracts from Turkish tobacco plants infected with tobacco mosaic virus (TMV) the effluent from ion exchange cellulose columns was passed through a quartz absorption cell of a recording spectrophotometer. Ten-ml fractions were collected and assayed for infectivity on local lesion hosts. Both cation (carboxymethyl) and anion (diethylaminoethyl) exchanging cellulose adsorbents (1) were used and both separated two types of infectious components that gave typical TMV symptoms in susceptible hosts. The first infectious component moved through either type of cellulose without any appreciable adsorption under t.he conditions of the experiments. This component appeared to be a nucleic acid. It absorbed strongly in the ultraviolet at 260 rnp and its infectivity was completely lost when incubated with crystalline ribonuclease at 36” for 24 hours. Simiiar control fractions incubated under the same conditions without ribonuclease retained some infectivity. TMV rods could not be found when these infectious nucleic acid fractions were examined with the electron microscope. 2 Histopathology by F. C. Helwig, Pathologist, Department of Pathology and Research, St. Luke’s Hospit.al, Kansas City, Missouri. 1 This investigation was supported, in part, by research grant No. AT(ll-l)80 Proj. 3 from the United States Atomic Energy Commission.
LETTERS
TO THE
EDITORS
391
The second infective component appeared 24 to 48 hours later after the passage of 800 to 1000 ml of developing solvent which carried no infectivity. This component was identified as the TMV nucleoprotein. It was not eluted unt,il the pH of the developing solvent approached a value of 3.8 to 4.0 (near the isoelectric point of TMV nucleoprotein). The infectivity of t,hese preparations was not lost when they were incubated with ribonuclease. Many typical TMV rods could be seen when these fractions were studied with the electron microscope. Fraenkel-Conrat (Z-4) and Gierer and Schram (5) have shown that infectious nucleic acid can be obtained b.y degrading virus nucleoprotein. In the experiments reported here the plant tissue was ground in liquid nitrogen and t,he chromatography was carried out using phosphate buffer systems and an anaerobic technique that is reported elsewhere (6). It, is believed that the mild extraction and chromatographic techniques used would not result in the degradation of nucleoprotein to release nucleic acid. The relative infectivity of the nucleic acid and nucleoprotein components appeared to vary with the age of the infections. In one experiment where an extract from a plant with a g-day-old infection was chromatographed, the amount of infectivity in the nucleic acid fractions appeared to be several times that in the nucleoprotein fractions. In several experiments with extracts from 6-month-old infections the infectivity in the nucleoprotein fractions appeared to be many hundredfold greater. Further studies are needed to establish these relations but the evidence reported here suggests that the nucleic acid is a precursor of the nucleoprotein. These experiments indicate the need for caution in the development of virus assay techniques because both types of infectious entities produce typical TMV symptoms following infection. Yet they are different physically and chemically, and extraction procedures may selectively favor one over the other. But most important of all, the research reported here suggests that we need to re-examine our concept of viruses for it may be that many other viruses occur naturally in two infective forms. Or possibly some may exist only as nucleic acids and never as nucleoproteins. REFERENCES 1. PETERSON, E. A., and SOBER, H. A. J. Am. Chem. Sot. 78, 751-755 (1956). 2. FRAENKEL-CONRAT, H., J. Am. Chem. Sot. 78, 882-883 (1956). 8. FRAENKEL-CONRAT, H., SINGER, B., and WILLIAMS, R. C. Biochim. et Biophys. Acta In press (1957). 4. FRAENKEL-CONRAT, H., Ann. N. Y. Acad. Sci. In press (1957). 5. GIERER, A.,and SCHRAMM,G.NCL~UT~ 177,702 (1956) 6. COCHRAN, G. W., and CHIDESTER, J. L. Submitted to the editors of Science in (1957). Department of Botany and Plant Pathology Utah State University Logan, Utah Received July SO, 1957
G. W. COCHRAN J. L. CHIDESTER