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The effect of tetrachloro-o-benzoquinone on southern bean mosaic virus and on its nucleic acid
VIROLOGY
46, 764-768
(1971)
Short
Communications
The Effect of Tetrachloro-o-benzoquinone Virus
on Southern
and on Its Nucleic
Plant viruses differ in their sensitivity to o-quinones. Some viruses are totally inactivated by short exposure to quinone concentrations below 0.1 mg quinone/mg virus (2, 5). Viruses inactivated at low quinone concentrations undergo few, if any, major changes in particle structure (W, 3,5). Recent results suggest that these viruses are inactivated by quinone “tanning” of the viral protein which produces virus incapable of being uncoated (5). All other viruses studied thus far tolerate low quinone concentrations without appreciable effect, but are totally inactivated by exposure to quinone concentrations between 1 and 10 mg quinone/mg virus. Loss of infectivity in this latter group of viruses is accompanied by particle disruption (5). Experiments reported here indicate that southern bean mosaic virus (SBMV) may represent yet another group of plant viruses that are virtually immune to quinone inactivation. SBMV remains highly infectious and essentially intact in the presence of 125 mg quinone/mg virus. SBMV was purified and infectious RNA was extracted by methods previously described (I). Both SBMV and SBMV-RNA were suspended in 0.02 M neutral phosphate buffer and were used at final concentrations of 2 pg/ml and 10 fig/ml, respectively. Tetrachloro-o-benzoquinone (TCQ) was used as the test quinone for reasons discussed earlier (4). The TCQ was dissolved in 0.3-0.5 ml of ethyl alcohol and was diluted to the desired concentrations in 0.02 M neutral phosphate buffer immediately before use. Low concentrations of virus and nucleic acid were used in these tests to reduce the anomalous results that often occur due to aggregation of concentrated virus exposed to high quinone concentrations. However, these low concentrations made it necessary to use carriers to quantitatively recover virus and 764
Bean Mosaic
Acid
nucleic acid during repurification following quinone treatment. Tobacco mosaic virus (TMV), inactivated by ultraviolet irradiation, was used to pellet SBMV by high-speed centrifugation. Yeast RNA (1% final concentration) was added to SBMV-RNA solutions prior to precipitation with cold ethyl alcohol and sodium acetate. A solution containing 10 pg/ml of SBMV-RNA produced 151 lesions per half-leaf before, and 179 lesions per half-leaf after, repurification with 1% yeast RNA. Solutions containing SBMV and SBMVRNA were incubated for 15 min at room temperature at final concentrations of 1.25, 12.5, and 125 mg TCQ/mg of virus or RNA. After repurification, each sample was suspended in phosphate buffer and assayed on Pinto beans. Each sample treated was compared on opposite half-leaves with virus or nucleic acid similarly treated, but not exposed to TCQ. TABLE PERCENT RNA
Q&one ratio (mg
TCQ/mg
1
INACTIVATION OF SBMV AND SBMVEXPOSED FOR 15 MIN TO VARIOUS CONCENTRATIONS OF TCQ 70 Inactivationa Whole
virus
virus or RNA)
10 rg/ml
2 pgg/ml
125 12.5 1.25
38 38 27
29 0 0
RNA 10 pg/ml 100 99 89
2 pg/ml 100 98 90
a Each sample was inoculated on 6 half-leaves of Phaseolus vulgaris ‘Pinto’; opposite half-leaves were inoculated with untreated virus or RNA solution of equal concentrations. Average lesion numbers on control half -leaves were : whole virus, 10 rg/ml 131; 2 pg/ml 65; RNA, 10 Kg/ml 152; 2 rg/ml 121.
SHORT
7fi5
COMMUNICATIONS
a 0.1-
Depth
in cm
1. Absorbance profile of centrifuged density-gradient column containing 80 pg of SBMV-RKA: (a) untreated; (b) treated with 1.3 mg of TCQ per mg of RNA; (c) treat,ed with 13 mg of TCQ per mg of RNA. Linear sucrose gradients (0.243 M, in 0.02 M phosphate buffer, pH 7). centrifrlgation for lfi hr at, 24,000 rpm, Spinro’ Model L Centrifuge, SW 25.1 rotor. FIG.
Little, if any, inactivation occurred when SBMV was exposed to either 1.25 or 12.5 mg TCQ/mg virus (Table 1). Only slight inactivation occurred at 125 mg TCQ/mg virus. Approximately 90 % of the SBMVRNA was inactivated at, 1.25 mg TCQ/mg RNA. Essentially complete inactivation of t’he RNA occurred at 12.5 mg TCQ/mg RNA. Solutions containing SO pg of SBMVRNA/ml were incubated wit’h 0, 1.3, and 13 mg of TCQ/mg RX-\. These were repurified without carrier and were then analyzed by density gradient, centrifugation. The results (E’ig. 1) indicate Ohat noninfectious RNA remained essent,ially intact,. A broadening of the RNA peak and some aggregation occurred at both quinone concentrations, but ’ Mention of specific equipment, trade products, or a commercial company does not constitute its endorsement by the U. S. (+overnment over similar prodllcts or companies not named.
no evidence of disrupbion of phosphodiest,er bonds was found. The result#s suggest, that, inactivation of SBMV-RNA did not, result from major breaks in the nucleic acid molccule. Evidently SB:\IV, unlike other viruses st’udied thus far, remains essent’ially intact even in the presence of very high concent,rations of TCQ. It, appears t,hat, t’he intact, capsid effectively prevents TCQ inactiv:rt,ion of the viral nucleic acid. The fact, that, SB:\IV-RKA is inact8ivnted by approximately the same TCQ concentrations that, were necessary to ir1activat.e tobacco ringspot virus, pot’ato virus X and ThlV (6) suggestIs t,hat inactivation of these viruses may occur when the RKA of each is exposed during capsid disrupt.ion by TCQ. This hypothesis is supported by unpublished data (H. S. Aldwinkle, personal correspondence), indicat,ing that Ti\,IV-RX.4 is inactivnt et1 iu the range of 1 mg TCQ/mg RT\‘A.
766
SHORT
COMMUNICATIONS
Knowledge that SBMV can withstand high quinone concentrations will make this virus a valuable tool in further investigations. Comparative studies of three isometric viruses (peanut stunt virus, tobacco ringspot virus, and SBMV) that exhibit a range of sensitivities to quinones should provide useful information on the mechanism of quinone inactivation. ACKNOWLEDGMENT Portions of this work were supported by the National Science Foundation Grant GB-5639. REFERENCES T. O., Virology 27,425-428 (1965). R. E., and FULTON, R. W., Virology 13, 44-52 (1961).
1. DIENER, 8. HAMPTON,
3. MINK, G. I., Virology 26, 700-707 (1965). HUISMAN, O., and SAKSENA, K. N., Virology 29t437-443 (1966). 6. MINK, G. I., and SAKSENA, K. N., 00, 00-00. G. I. MINK T. 0. DIENER Department of Plant Pathology, Irrigated Agrim&are Research and Extension Center Washington State University Prosser, Washington 99360 Plant Virology Laboratory, Plant Science Research Division Agricultural Research Service, U. S. Department of Agriculture Beltsville, Maryland 80706 Accepted April 2.2, 1971
4. MINK, G. I.,
46, 764-768
(1971)
Short
Communications
The Effect of Tetrachloro-o-benzoquinone Virus
on Southern
and on Its Nucleic
Plant viruses differ in their sensitivity to o-quinones. Some viruses are totally inactivated by short exposure to quinone concentrations below 0.1 mg quinone/mg virus (2, 5). Viruses inactivated at low quinone concentrations undergo few, if any, major changes in particle structure (W, 3,5). Recent results suggest that these viruses are inactivated by quinone “tanning” of the viral protein which produces virus incapable of being uncoated (5). All other viruses studied thus far tolerate low quinone concentrations without appreciable effect, but are totally inactivated by exposure to quinone concentrations between 1 and 10 mg quinone/mg virus. Loss of infectivity in this latter group of viruses is accompanied by particle disruption (5). Experiments reported here indicate that southern bean mosaic virus (SBMV) may represent yet another group of plant viruses that are virtually immune to quinone inactivation. SBMV remains highly infectious and essentially intact in the presence of 125 mg quinone/mg virus. SBMV was purified and infectious RNA was extracted by methods previously described (I). Both SBMV and SBMV-RNA were suspended in 0.02 M neutral phosphate buffer and were used at final concentrations of 2 pg/ml and 10 fig/ml, respectively. Tetrachloro-o-benzoquinone (TCQ) was used as the test quinone for reasons discussed earlier (4). The TCQ was dissolved in 0.3-0.5 ml of ethyl alcohol and was diluted to the desired concentrations in 0.02 M neutral phosphate buffer immediately before use. Low concentrations of virus and nucleic acid were used in these tests to reduce the anomalous results that often occur due to aggregation of concentrated virus exposed to high quinone concentrations. However, these low concentrations made it necessary to use carriers to quantitatively recover virus and 764
Bean Mosaic
Acid
nucleic acid during repurification following quinone treatment. Tobacco mosaic virus (TMV), inactivated by ultraviolet irradiation, was used to pellet SBMV by high-speed centrifugation. Yeast RNA (1% final concentration) was added to SBMV-RNA solutions prior to precipitation with cold ethyl alcohol and sodium acetate. A solution containing 10 pg/ml of SBMV-RNA produced 151 lesions per half-leaf before, and 179 lesions per half-leaf after, repurification with 1% yeast RNA. Solutions containing SBMV and SBMVRNA were incubated for 15 min at room temperature at final concentrations of 1.25, 12.5, and 125 mg TCQ/mg of virus or RNA. After repurification, each sample was suspended in phosphate buffer and assayed on Pinto beans. Each sample treated was compared on opposite half-leaves with virus or nucleic acid similarly treated, but not exposed to TCQ. TABLE PERCENT RNA
Q&one ratio (mg
TCQ/mg
1
INACTIVATION OF SBMV AND SBMVEXPOSED FOR 15 MIN TO VARIOUS CONCENTRATIONS OF TCQ 70 Inactivationa Whole
virus
virus or RNA)
10 rg/ml
2 pgg/ml
125 12.5 1.25
38 38 27
29 0 0
RNA 10 pg/ml 100 99 89
2 pg/ml 100 98 90
a Each sample was inoculated on 6 half-leaves of Phaseolus vulgaris ‘Pinto’; opposite half-leaves were inoculated with untreated virus or RNA solution of equal concentrations. Average lesion numbers on control half -leaves were : whole virus, 10 rg/ml 131; 2 pg/ml 65; RNA, 10 Kg/ml 152; 2 rg/ml 121.
SHORT
7fi5
COMMUNICATIONS
a 0.1-
Depth
in cm
1. Absorbance profile of centrifuged density-gradient column containing 80 pg of SBMV-RKA: (a) untreated; (b) treated with 1.3 mg of TCQ per mg of RNA; (c) treat,ed with 13 mg of TCQ per mg of RNA. Linear sucrose gradients (0.243 M, in 0.02 M phosphate buffer, pH 7). centrifrlgation for lfi hr at, 24,000 rpm, Spinro’ Model L Centrifuge, SW 25.1 rotor. FIG.
Little, if any, inactivation occurred when SBMV was exposed to either 1.25 or 12.5 mg TCQ/mg virus (Table 1). Only slight inactivation occurred at 125 mg TCQ/mg virus. Approximately 90 % of the SBMVRNA was inactivated at, 1.25 mg TCQ/mg RNA. Essentially complete inactivation of t’he RNA occurred at 12.5 mg TCQ/mg RNA. Solutions containing SO pg of SBMVRNA/ml were incubated wit’h 0, 1.3, and 13 mg of TCQ/mg RX-\. These were repurified without carrier and were then analyzed by density gradient, centrifugation. The results (E’ig. 1) indicate Ohat noninfectious RNA remained essent,ially intact,. A broadening of the RNA peak and some aggregation occurred at both quinone concentrations, but ’ Mention of specific equipment, trade products, or a commercial company does not constitute its endorsement by the U. S. (+overnment over similar prodllcts or companies not named.
no evidence of disrupbion of phosphodiest,er bonds was found. The result#s suggest, that, inactivation of SBMV-RNA did not, result from major breaks in the nucleic acid molccule. Evidently SB:\IV, unlike other viruses st’udied thus far, remains essent’ially intact even in the presence of very high concent,rations of TCQ. It, appears t,hat, t’he intact, capsid effectively prevents TCQ inactiv:rt,ion of the viral nucleic acid. The fact, that, SB:\IV-RKA is inact8ivnted by approximately the same TCQ concentrations that, were necessary to ir1activat.e tobacco ringspot virus, pot’ato virus X and ThlV (6) suggestIs t,hat inactivation of these viruses may occur when the RKA of each is exposed during capsid disrupt.ion by TCQ. This hypothesis is supported by unpublished data (H. S. Aldwinkle, personal correspondence), indicat,ing that Ti\,IV-RX.4 is inactivnt et1 iu the range of 1 mg TCQ/mg RT\‘A.
766
SHORT
COMMUNICATIONS
Knowledge that SBMV can withstand high quinone concentrations will make this virus a valuable tool in further investigations. Comparative studies of three isometric viruses (peanut stunt virus, tobacco ringspot virus, and SBMV) that exhibit a range of sensitivities to quinones should provide useful information on the mechanism of quinone inactivation. ACKNOWLEDGMENT Portions of this work were supported by the National Science Foundation Grant GB-5639. REFERENCES T. O., Virology 27,425-428 (1965). R. E., and FULTON, R. W., Virology 13, 44-52 (1961).
1. DIENER, 8. HAMPTON,
3. MINK, G. I., Virology 26, 700-707 (1965). HUISMAN, O., and SAKSENA, K. N., Virology 29t437-443 (1966). 6. MINK, G. I., and SAKSENA, K. N., 00, 00-00. G. I. MINK T. 0. DIENER Department of Plant Pathology, Irrigated Agrim&are Research and Extension Center Washington State University Prosser, Washington 99360 Plant Virology Laboratory, Plant Science Research Division Agricultural Research Service, U. S. Department of Agriculture Beltsville, Maryland 80706 Accepted April 2.2, 1971
4. MINK, G. I.,