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Release of nucleic acid from turnip yellow mosaic virus under mild conditions
VIROLOGY 6: 460-471 (1958)
Release
of Nucleic Acid from Turnip Yellow Virus under Mild Conditions J. W. LYTTLETON
Plant
Chemistry
AND
Mosaic
R. E. F. MATTHEWS
Laboratory, D.S.I.R., Palmerston North, and Plant Division, D.S.I.R., Auckland, New Zealand
Diseases
Accepted June 16, 1968 The ribonucleic acid (RNA) of turnip yellow mosaic virus (TYMV) has been released from the protein under milder conditions than those previously employed. For example 66% of the RNA was released from a virus preparation buffered at pH 7.6 after 10 minutes’ heating at 45”. RNA obtained by this procedure had a sedimentation coefficient of 4.3-4.7 S. This release occurred without obvious depolymerization or gross denaturation of the virus protein shell. The virus particles ina preparation showed a continuous spectrum of stabilities with respect to heating in buffered solutions. INTRODUCTION
TYMV is generally considered to be one of the more stable plant viruses. The virus in expressed plant sap loses its infectivity only after 10 minutes’ heating at 70-75” (Markham and Smith, 1949). Two methods have been used for isolation of the RNA from this virustreatment with 33% ethanol at room temperature and heating to temperatures near 100” in aqueous solution for short periods (Cohen and Schachman, 1957). In both these methods the virus protein is denatured. In the experiments described here we have found that the RNA can be released from the virus by heating between 37” and 50” in aqueous solutions buffered near pH 7. The RNA is released without depolymeriaation of the virus protein. The protein appears to undergo little denaturation since it remains soluble in water. MATERIALS
AND
METHODS
The type strain of TYMV (Markham and Smith, 1949) was cultured in Chinese cabbage (Brassica &inensis) var. Wong Bok grown under glass. Crystalline virus was isolated by the ammonium sulfate procedure of Markham and Smith (1949). Such preparations contain a mixture of 460
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nucleoprotein particles containing about 38 % RNA and protein particles apparently identical in size and composition but with no RNA, in a proportion of two particles of nucleoprotein to about one of protein. These two types of particle can be separated by differential centrifugation. For our experiments these preparations were used unfractionated. Antisera were prepared in rabbits by the intravenous injection of purified virus. In testing the effect of temperature on the release of RNA from the virus nucleoprotein in various media, solutions were heated in small tubes in a water bath controlled to +I”. Duration of heating was measured from the time tubes were placed in the bath. On removal from the bath the tubes were immediately cooled in tap water. The amount of nucleic acid remaining bound to virus protein was then determined by the serological-chromatographic procedure (Matthews, 1954). After the heat treatment a slight excess of a TYMV antiserum was added. The mixtures were incubated for 2 hours at room temperature and then overnight at 4”. The specific precipitates were centrifuged down, washed twice with 2.0 ml. of 0.14 M saline, dried, hydrolyzed with alkali, and the amount of RNA present estimated by chromatographic isolation of the ribonucleotides, as described previously (Matthews, 1954, 1957). The components produced from TYMV by heating in the presence of alkaline buffers were studied in an air-driven ultracentrifuge of the Bauer-Pickels type (Bauer and Pickels, 1937) equipped with a PhilpotSvensson optical system (Svensson, 1940). At the time of these experiments, the diagrams produced by the optical system were not of sufficiently high quality to provide an accurate measure of the area of the very narrow peaks produced by sedimenting virus; hence quantitative studies of the relative concentrations of components with differing sedimentation coefficients produced by virus breakdown have not been possible. RESULTS
Efect of pH on Release of RNA Figure 1 shows the percentage of RNA released from the virus in 0.025 &l phosphate buffer between pH 6 and pH 8 following heating for 30 minutes at 45”. Under these conditions about half the RNA was released at pH 6.8. Similar curves were obtained with borate and tris(hydroxymethyl)aminomethane buffers.
LYTTLETON
462
100
AND
MATTHEWS
I
I
I
I
I
6.0
6.4
6.8
7.2
7.6
-
80-
8.0
PH 1. Release of RNA from TYMV at various pH values after heating at 45” for 30 minutes. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.025 M phosphate buffer in 0.1 M NaCl. FIG.
Phosphate buffer was effecOive down to about 0.001 M in causing release of RNA. For example when 0.3 mg of virus in 2 ml of 0.1 M NaCl plus phosphate buffer at pH 7.6 of varying molarity was heated at 46” for 10 minutes the release of RNA occurred as follows: 0.075 M-63% ; 0.0025 M-62yo ; Saline-l. 6%
0.025 M-65% 0. ooo75 M-45’Y ,o
0.0075 M-66% 0.00025 M-8. So/,
No significant amount of RNA was released when the virus was heated at 50” for 60 minutes in 0.14 M saline or distilled water. However, the buffering capacity of rabbit serum at a dilution in the reacting mixture of l/200 was sufficient to allow escape of some nucleic acid from the virus on mild heating. Rate of Loss qf RNA at Various Temperatures and pH Values We have not examined the release of RNA from the virus between room temperature (about 20”) and 37”.
80 ,H 7.60
60 B $ 7
50
“z
40
u z
30
s
20
,H 6.72
)H 6.55
IO
20 Time
30 of
40
heating
50
60
(minutes)
Fro. 2. Rate of release of RNA from TYMV at various pH values. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.025 M phosphate buffer in 0.1 J1 N&l.
46” 42”
90 80 70 60
a z 50 u 6 40 s 30
37O
20 IO I
2
3
4
5
Time of heoting (hours) FIG. 3. Release of RNA from TYMV after heating for various times and temperatures. For explanation of curves see text. Each tube contained 0.68 mg of a virus preparation in 1 ml. of 0.1 ICI NaCl plus 0.025 M phosphate buffer at pH 7.0. 4.63
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With increasing temperature between 37” and 50” or increasing pH between about 6.4 and 8, the initial rate of loss of RNA from the virus increased. However, for any particular conditions the initial rate was not maintained and after a certain time there was little further loss of RNA if conditions remained unaltered. Figure 2 shows the release of RNA with time at 45” for three pH values. Figure 3 shows release with time at pH 7.0 for three temperatures. In this experiment, all points on the 37” curve were obtained from tubes heated at 37” for the time indicated. For the 42” curve all tubes were heated first at 37” for 60 minutes and then at 42”. The 46’ tubes were heated 60 minutes at 37”, followed by 60 minutes at 42”, and then at 46”.
Other Factors Affecting Loss of RNA Different preparations of virus varied in their stability to heating in buffered solution. This variation does not appear to be connected with the age of a preparation, and its causes are not known. Changes in concentration of the virus over the range 0.08-4.0 mg/ml had little effect on the rate of release of RNA on heating at 44” for 10 minutes in 0.1 M saline and 0.25 M phosphate buffer pH 7.6.
0
I IO
I 20 % Sucrose
I 30
FIG. 4. Effect of sucrose on release of RNA from TYMV. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.14 M NaCl and 0.075 M phosphate buffer at pH 7.6. Tubes were heated at 45” for 12 minutes.
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The amount of RNA released from the virus under similar conditions of heating was reduced when sucrose was added to the incubation mixture. Figure 4 shows the relationship between concentration of sucrose and release of RNA. Characterization of Components after Release of RNA from Virus Prior to dissociation, analysis of the TYMV preparation in the ultracentrifuge at 42,000 rpm revealed two components with sedimentation coefficients of 108 S and 50 S at infinite dilution. These components correspond to the complete virus particle (the “bottom” component) and the TTMV protein without R?JA (the ‘%op” component) (Mark-
cc
FIG. 5. Ultracentrifuge diagrams of unheated and heated TYMV. Sedimentation from left to right. In each test 1 volume of TYMV (20 mg/ml in 0.15 M NaCl) was added to 0.25 volume 0.3 M phosphate buffer pH 7.5. a. Unheated; slow peak = virus protein, fast peak = virus nucleoprotein. b. Heated 50” for 10 seconds; virus nucleoprotein peak reduced in amount. Two new components: the slower being free RNA; the faster is the disrupted protein shell. c. Heated 50” for 60 seconds and stood 4 hours at 18”. Slight breakdown of the RNA peak is apparent. All photographs were taken after 12 minutes’ centrifugation at 42,000 rpm.
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ham, 1951). After dissociation, two new components are observed with sedimentation coefficients of 33-36 S and 4.44.6 S, respectively. When the degree of dissociation of TYMV is increased, either by prolonging the heating time or raising the temperature, the concentrations of these two new components increase together, while the concentration of the “bottom” component drops. In phosphate buffer (pH 7.5, 4.06 J1) the degree of dissociation after heating at 45” for 3 minutes was approximately 50 %, while at 50” for 30 seconds the dissociation was practically complete. The “top” component, on the other hand, was unaffect,ed by these treatments. Figure 5 shows the patterns obtained from heated and unheated samples of TYMV. In contrast to free RNA, the RNA in the intact virus particle is not susceptible to attack by pancreatic ribonuclease. Incubation with ribonuclease, therefore, should depolymerize only those ultracentrifugal components containing free RNA. A 2-ml sample of a virus preparation containing 14 mg/ml in 0.15 M NaCl was mixed with 0.5 ml phosphate buffer (pH 7.5, 0.3 M) and heated to 50” for 30 seconds. An aliquot (0.7 ml) of this heat-treated solution was incubated with 0.1 ml pancreatic ribonuclease solution (1 mg/ml in water) for 3 hours at 18”. A control incubation contained no ribonuclease. The ultracentrifuge patterns produced by the enzyme-treated sample and the control showed that ribonuclease had degraded the 4.5 S component to a form which did not leave the meniscus of the cell at 60,000 rpm. The other components remained unaltered. This indication that the RNA was contained in the 4.5 S component was confirmed by partial separation of the products of the dissociation in the ultracentrifuge cell, and analyzing the fractions obtained for RNA. The cell was fitted with two layers of filter paper at its base to minimize redissolving of material which sedimented completely. Two ultracentrifuge runs were then carried out with virus dissociated as in the previous experiment. In the first, at, the conclusion of the run, the 33 S component sedimented halfway across the cell, while in the second it sedimented completely. In a third run, undissociated virus was centrifuged until both “top” and “bottom” components had sedimented completely. The supernatants were withdrawn as rapidly as possible, and the sediment in the filter papers at the base of the cell was extracted with 0.15 M NaCl. Ultraviolet absorption curves were prepared for the various supernatant fluids and redissolved sediments. All showed a maximum at 260 rnp, so that comparison of optical densities at this
NUCLEIC
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TABLE DISTRIBUTION
OF RNA AFTER DISSOCIATED
467
VIRUS
1
ULTRACENTRIFUGE RUNS ON CELLS AND UN~ISSOCIATED TYMV Supernatant fluid RNA in
Material
MOSAIC
CONTAINING
Redissolved sediment - --___ RNA. in
Total RNA, in
analyzed
1. I)issociat,ed TYMV, 33 S sedimented 2. I>issociated TYMV, 33 S pletely sedimented 3. Undissociated TYMV, bot.h ponents sedimented 4. Dissociated TTMV, not, t rifuged
half
71
7.8
11
19
x5
27
com-
73
4
16
21
89
28
com-
5
5
66
101
71
106
rerl-
106
3-l
-
106
31
a Total opt.ical densit.y at, 260 rnp in the sample standardized to a volume of 1 ml. !’ Expressed as a percentage of the t,otal RNA in an equivalent sample of undissociated unsedimented virus.
wavelength will reflect primarily the concentration of RNA, although absorption due to protein will make a minor contribution, for which no correction has been made. In addition to these measurements the RNA still bound to protein was estimated directly for each fraction by the serological-chromatogrsphic method. Results arc given in Table 1. These show that removal of the 33 S component from the supernatant of dissociated TYMV does not remove the RNA; while in the undissociated system the same amount of centrifuging will remove almost all the RNA from solution. It appears, then, that the 33 S component contains no RNA and is presumably protein in nature, while the 4.5 S component contains all the RNA liberated from the dissociated virus molecule. This 4.5 X component, which is liberated by heating only when the pH of the TYMV solution is greater than about 6.4, shows no change in sedimentation behavior when the pH of the solution is lowered to 4.5 after dissociation has been carried out. On standing at pH 7.5 for 2-3 days at 2”, the component breaks up into smaller fragments which are not resolved from the meniscus of the ultracentrifuge cell at 60,000 rpm. This breakdown is accelerated at, higher pH values, and at pH 9.5 it occurs within a few hours.
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TABLE
2
NUMBER OF LOCAL LESIONS PRODUCED ON CHINESE CABBAGE BY TYMV HEATING FOR THREE MINUTES AT 48” IN 0.14 M SALINE, AND
IN
0.05 M PH 7.5
PHOSPHATE
o/u RNA remaining in virusa
Treatment
AFTER
BUFFER No. of local lesions (total on 20 half-leaves)
Heated in saline, cooled to 18”
96
354
Heated in phosphate, cooled to 18”
25
65)
Heated in saline, cooled to 1”
97
Heated in phosphate, cooled to lo
30
804) i
YOInfectivity rem&h@
18
a Amount of RNA remaining compared by serological chromatographic with virus held at 18” in saline. b Total lesions from phosphate preparation x 100. Total lesions from saline preparation
Infectivity
of TYMV
10
84 method
Preparations after Heating in Bu$ered Solution
Infectivity tests made on virus solutions after heating showed that in phosphate buffer at pH 7.5 much of the infectivity is lost (Table 2). Cooling the heated preparations to 1" between heating treatment and inoculation gave no increase in the residual infectivity. DISCUSSION
The methods used to prepare infectious nucleic acid from intact tobacco mosaic virus in vitro involve conditions which could not prevail in the living leaf cell. Thus Gierer and Schramm (1956) treated tobacco mosaic virus with water-saturated phenol for 8 minutes at 5’, while Fraenkel-Conrat et al. (1957) used a 1% solution of sodium dodecyl sulfate at pH 8.5 and 50” for 5 minutes. The methods previously used to prepare RNA from TYMV are likewise fairly severe compared with possible conditions in viva. Two methods have been used: treatment with 33 % ethanol at room temperature and heating for short periods at temperatures near 100” (Cohen and Schachman, 1957). In contrast to these procedures, the conditions under which we have obtained release of RNA from a proportion of the TYMV particles are such as might prevail in the living cell-namely pH 7 at) 37”. The results given in Table 2 show that most of the RNA released is noninfectious. To determine whether any small amount of infectivity
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is associated with the RNA released under our conditions, it will be necessary to carry out tests on a larger scale with RNA freed from remaining intact virus. Cohen and Schachman (1957) have made a detailed ultracentrifuge study of the properties of RNA obtained from TYMV by heating to 100” and by ethanol denaturation of the virus. Their products were not homogeneous with respect to molecular weight. The weight average calculated from the viscosity and the highest, sedimentation coefficient observed (4.4 S) gave a molecular weight of 76,000. Fresh preparations of RNA obtained by our method have always given sedimentation coefficients in the range 4.4-4.6 S. Presumably, therefore, the weight average molecular weight of our material is about 75,000. This result supports t,heir suggestion that there may be about 25-50 RNA molecules per TYMV particle. TYMV consists of a roughly spherical shell of protein surrounding a central core of nucleic acid (Markham, 1951; Schmidt et al., 1954). The naturally occurring nucleic acid-free “top” component is an apparently identical protein shell without nucleic acid. The X-ray evidence suggests that the protein shell is built up from 60 identical subunits (Klug el al., 1957). When we first found that the RNA could be released from TYMV under conditions such that the remaining protein was not denatured (to the extent that it was still water-soluble), we had hoped that the protein would either be depolymerized to the small subunits indicated by the X-ray evidence or that it would be identical with the “top” component. The ultracentrifuge data show clearly that neither of these possibilities can be true. The 33 S protein was less homogeneous than the “top” component which had a sedimentation coefficient of 45 X under the same concentration conditions. The heterogeneity was not nearly as great as would be expected from random fragmentation of the protein shell. This sedimentation behavior is consistent with the idea that the RNA, in escaping from the protein ruptures the shell, thereby lowering its symmetry and decreasing the sedimentation coefficient. The data in Fig. 3 on the release of RNA from TYMV at various temperatures might be interpreted as suggesting that a portion of the RNA was lost from each of the particles at any given temperature. The sedimentation data make this possibility unlikely since they show, within the limits of the sensitivity of the method, that the loss of RKA from a particle is an all-or-none event. Thus the particles in a prepara-
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tion of TYMV appear to have a continuous range of stabilities to heat at pH values near 7. This variation in stability makes very difficult any interpretation of the curve in Fig. 1 in terms of possible molecular change within the virus particle. Little is known about the nature of the bonds between protein and RNA in virus nucleoproteins. At the pH at which RNA begins to escape from a proportion of the particles a substantial proportion of the secondary phosphate groups on the RNA are becoming ionized. This is consistent with the idea that nonionized phosphate groups play a role in the bonding between RNA and protein in intact virus. On this basis it would be reasonable to assume that rupture of the TYMV particle is caused by thermal agitation of RNA freed by pH change from its normal binding to the inner surface of the protein shell. Rupture of the virus is suppressed by the presence of sucrose in the medium. The degree of suppression is related linearly to concentration and hence to. the osmotic pressure of the sucrose solution. This relationship can be explained if we assume that the protein shell is impermeable to sucrose. Then the increased osmotic pressure in the external medium would increase the resistance of the protein shell to rupture from within. It is now known that a range of viruses can be inactivated in plants by heat, and the plants completely freed of virus (Kassanis, 1957). In such treatments infected plants or parts of plants are heated for periods of days or weeks at temperatures between about 36” and 40” or they may be heated a.t higher temperatures (usually 50-55’) for a few minutes. Little is known about the mechanism of such inactivation. The in vitro results with TYMV described here suggest a possible mechanism for heat inactivation of viruses in viva. It is not unreasonable to assume that pH values in the living cell may lie in the range 6.6-7.0. If conditions of pH and temperature in the cell were such that the virus RNA was forced from the protein coat under conditions unfavorable for further multiplication the virus would be inactivated. Nothing is known about the structure of most of the viruses that can be inactivated by heating in viao. Those whose shape is definitely known are roughly spherical (tomato bushy stunt, tobacco necrosis, carnation ringspot, tobacco ringspot viruses), while those that are more stable to heat in vivo are rods (TMV, potato viruses X and Y). When plants syst,emically infected with tomato bushy stunt virus are kept at 36” both infectivity of the sap and amount of specific virus
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antigen decrease, but infectivity decreases much more rapidly than specific antigen content (Kassanis, 1952). This sequence would agree with a mechanism where RNA escapedfrom the protein and was rapidly inactivated, while the more stable protein shell was degraded more slowly. No tests on the in vivo inactivation of TYMV by heat have yet been made, but this virus should provide good material for testing the hypothesis suggested here. The naturally occurring “top” component, is relatively heat-stable and should provide a good yardstick for measuring changes in the virus nucleoprotein induced by heat in IGO. REFEREXCES
RAUER, J. M., and PICKELS, E. G. (1937). Improved
air-driven type of ult,racentrifuge for molecular sedimentation. J. Exptl. Med. 66, 565-586. COHEN, S. S., and SCHACHMAN,H. K. (1957). Physical studies on the ribonucleic acid of turnip yellow mosaic virus. Virology 3, 575-586. FRAEXKEL-CONRAT, H., SINGER, B., and WILLIAMS, R. C. (1957). Infectivity of viral nucleic acid. Biochim. et Biophys. Acta 26, 87-96. GIERER, A., and SCHRAMM,G. (1956). Infectivit,y of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. KASSANIS, B. (1952). Some effects of high temperature on the suscept)ibility of plants to infection with viruses. Ann. Appt. Biol. 39, 358-369. KASSANIS, B. (1957). Effects of changing temperature on plant, virus diseases. Advances in Virus Research 4, 221-241. KLIJG, A., FINCH, J. T., and FRAPU'KLINR. E. (1957). The structure of turnip yellow mosaic virus: x-ray diffraction studies. Biochim. et Biophys. Acta 26, 242-252. MARKHAM, R. (1951). Physicochemical studies on the turnip yellow mosaic virus. Discussions Faraday Sot. No. 11, 221-227. MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. MATTHEWS, R. E. F. (1954). Effect of some purine analogues on tobacco mosaic virus. J. Gen. Microbial. 10, 521-532. MATTHEWS, R. E. F. (1957). Pla,nt Virlts Serology, 128 pp. Cambridge Univ. Press, London and sew York. SCHMIDT,P., KAESBERG, P., and BEEMAN, W. W. (1954). Small angle x-ray scattering from turnip yellow mosaic virus. Biochim. et Biophys. Acta 14, l-11. SVENSSON, H. (1940). IXrekte photographische Aufnahme von Elektrophoresediagrammen. Kolloid-2.87, 181-189.
Release
of Nucleic Acid from Turnip Yellow Virus under Mild Conditions J. W. LYTTLETON
Plant
Chemistry
AND
Mosaic
R. E. F. MATTHEWS
Laboratory, D.S.I.R., Palmerston North, and Plant Division, D.S.I.R., Auckland, New Zealand
Diseases
Accepted June 16, 1968 The ribonucleic acid (RNA) of turnip yellow mosaic virus (TYMV) has been released from the protein under milder conditions than those previously employed. For example 66% of the RNA was released from a virus preparation buffered at pH 7.6 after 10 minutes’ heating at 45”. RNA obtained by this procedure had a sedimentation coefficient of 4.3-4.7 S. This release occurred without obvious depolymerization or gross denaturation of the virus protein shell. The virus particles ina preparation showed a continuous spectrum of stabilities with respect to heating in buffered solutions. INTRODUCTION
TYMV is generally considered to be one of the more stable plant viruses. The virus in expressed plant sap loses its infectivity only after 10 minutes’ heating at 70-75” (Markham and Smith, 1949). Two methods have been used for isolation of the RNA from this virustreatment with 33% ethanol at room temperature and heating to temperatures near 100” in aqueous solution for short periods (Cohen and Schachman, 1957). In both these methods the virus protein is denatured. In the experiments described here we have found that the RNA can be released from the virus by heating between 37” and 50” in aqueous solutions buffered near pH 7. The RNA is released without depolymeriaation of the virus protein. The protein appears to undergo little denaturation since it remains soluble in water. MATERIALS
AND
METHODS
The type strain of TYMV (Markham and Smith, 1949) was cultured in Chinese cabbage (Brassica &inensis) var. Wong Bok grown under glass. Crystalline virus was isolated by the ammonium sulfate procedure of Markham and Smith (1949). Such preparations contain a mixture of 460
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nucleoprotein particles containing about 38 % RNA and protein particles apparently identical in size and composition but with no RNA, in a proportion of two particles of nucleoprotein to about one of protein. These two types of particle can be separated by differential centrifugation. For our experiments these preparations were used unfractionated. Antisera were prepared in rabbits by the intravenous injection of purified virus. In testing the effect of temperature on the release of RNA from the virus nucleoprotein in various media, solutions were heated in small tubes in a water bath controlled to +I”. Duration of heating was measured from the time tubes were placed in the bath. On removal from the bath the tubes were immediately cooled in tap water. The amount of nucleic acid remaining bound to virus protein was then determined by the serological-chromatographic procedure (Matthews, 1954). After the heat treatment a slight excess of a TYMV antiserum was added. The mixtures were incubated for 2 hours at room temperature and then overnight at 4”. The specific precipitates were centrifuged down, washed twice with 2.0 ml. of 0.14 M saline, dried, hydrolyzed with alkali, and the amount of RNA present estimated by chromatographic isolation of the ribonucleotides, as described previously (Matthews, 1954, 1957). The components produced from TYMV by heating in the presence of alkaline buffers were studied in an air-driven ultracentrifuge of the Bauer-Pickels type (Bauer and Pickels, 1937) equipped with a PhilpotSvensson optical system (Svensson, 1940). At the time of these experiments, the diagrams produced by the optical system were not of sufficiently high quality to provide an accurate measure of the area of the very narrow peaks produced by sedimenting virus; hence quantitative studies of the relative concentrations of components with differing sedimentation coefficients produced by virus breakdown have not been possible. RESULTS
Efect of pH on Release of RNA Figure 1 shows the percentage of RNA released from the virus in 0.025 &l phosphate buffer between pH 6 and pH 8 following heating for 30 minutes at 45”. Under these conditions about half the RNA was released at pH 6.8. Similar curves were obtained with borate and tris(hydroxymethyl)aminomethane buffers.
LYTTLETON
462
100
AND
MATTHEWS
I
I
I
I
I
6.0
6.4
6.8
7.2
7.6
-
80-
8.0
PH 1. Release of RNA from TYMV at various pH values after heating at 45” for 30 minutes. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.025 M phosphate buffer in 0.1 M NaCl. FIG.
Phosphate buffer was effecOive down to about 0.001 M in causing release of RNA. For example when 0.3 mg of virus in 2 ml of 0.1 M NaCl plus phosphate buffer at pH 7.6 of varying molarity was heated at 46” for 10 minutes the release of RNA occurred as follows: 0.075 M-63% ; 0.0025 M-62yo ; Saline-l. 6%
0.025 M-65% 0. ooo75 M-45’Y ,o
0.0075 M-66% 0.00025 M-8. So/,
No significant amount of RNA was released when the virus was heated at 50” for 60 minutes in 0.14 M saline or distilled water. However, the buffering capacity of rabbit serum at a dilution in the reacting mixture of l/200 was sufficient to allow escape of some nucleic acid from the virus on mild heating. Rate of Loss qf RNA at Various Temperatures and pH Values We have not examined the release of RNA from the virus between room temperature (about 20”) and 37”.
80 ,H 7.60
60 B $ 7
50
“z
40
u z
30
s
20
,H 6.72
)H 6.55
IO
20 Time
30 of
40
heating
50
60
(minutes)
Fro. 2. Rate of release of RNA from TYMV at various pH values. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.025 M phosphate buffer in 0.1 J1 N&l.
46” 42”
90 80 70 60
a z 50 u 6 40 s 30
37O
20 IO I
2
3
4
5
Time of heoting (hours) FIG. 3. Release of RNA from TYMV after heating for various times and temperatures. For explanation of curves see text. Each tube contained 0.68 mg of a virus preparation in 1 ml. of 0.1 ICI NaCl plus 0.025 M phosphate buffer at pH 7.0. 4.63
464
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With increasing temperature between 37” and 50” or increasing pH between about 6.4 and 8, the initial rate of loss of RNA from the virus increased. However, for any particular conditions the initial rate was not maintained and after a certain time there was little further loss of RNA if conditions remained unaltered. Figure 2 shows the release of RNA with time at 45” for three pH values. Figure 3 shows release with time at pH 7.0 for three temperatures. In this experiment, all points on the 37” curve were obtained from tubes heated at 37” for the time indicated. For the 42” curve all tubes were heated first at 37” for 60 minutes and then at 42”. The 46’ tubes were heated 60 minutes at 37”, followed by 60 minutes at 42”, and then at 46”.
Other Factors Affecting Loss of RNA Different preparations of virus varied in their stability to heating in buffered solution. This variation does not appear to be connected with the age of a preparation, and its causes are not known. Changes in concentration of the virus over the range 0.08-4.0 mg/ml had little effect on the rate of release of RNA on heating at 44” for 10 minutes in 0.1 M saline and 0.25 M phosphate buffer pH 7.6.
0
I IO
I 20 % Sucrose
I 30
FIG. 4. Effect of sucrose on release of RNA from TYMV. Each tube contained 0.3 mg of a virus preparation in 2 ml of 0.14 M NaCl and 0.075 M phosphate buffer at pH 7.6. Tubes were heated at 45” for 12 minutes.
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The amount of RNA released from the virus under similar conditions of heating was reduced when sucrose was added to the incubation mixture. Figure 4 shows the relationship between concentration of sucrose and release of RNA. Characterization of Components after Release of RNA from Virus Prior to dissociation, analysis of the TYMV preparation in the ultracentrifuge at 42,000 rpm revealed two components with sedimentation coefficients of 108 S and 50 S at infinite dilution. These components correspond to the complete virus particle (the “bottom” component) and the TTMV protein without R?JA (the ‘%op” component) (Mark-
cc
FIG. 5. Ultracentrifuge diagrams of unheated and heated TYMV. Sedimentation from left to right. In each test 1 volume of TYMV (20 mg/ml in 0.15 M NaCl) was added to 0.25 volume 0.3 M phosphate buffer pH 7.5. a. Unheated; slow peak = virus protein, fast peak = virus nucleoprotein. b. Heated 50” for 10 seconds; virus nucleoprotein peak reduced in amount. Two new components: the slower being free RNA; the faster is the disrupted protein shell. c. Heated 50” for 60 seconds and stood 4 hours at 18”. Slight breakdown of the RNA peak is apparent. All photographs were taken after 12 minutes’ centrifugation at 42,000 rpm.
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ham, 1951). After dissociation, two new components are observed with sedimentation coefficients of 33-36 S and 4.44.6 S, respectively. When the degree of dissociation of TYMV is increased, either by prolonging the heating time or raising the temperature, the concentrations of these two new components increase together, while the concentration of the “bottom” component drops. In phosphate buffer (pH 7.5, 4.06 J1) the degree of dissociation after heating at 45” for 3 minutes was approximately 50 %, while at 50” for 30 seconds the dissociation was practically complete. The “top” component, on the other hand, was unaffect,ed by these treatments. Figure 5 shows the patterns obtained from heated and unheated samples of TYMV. In contrast to free RNA, the RNA in the intact virus particle is not susceptible to attack by pancreatic ribonuclease. Incubation with ribonuclease, therefore, should depolymerize only those ultracentrifugal components containing free RNA. A 2-ml sample of a virus preparation containing 14 mg/ml in 0.15 M NaCl was mixed with 0.5 ml phosphate buffer (pH 7.5, 0.3 M) and heated to 50” for 30 seconds. An aliquot (0.7 ml) of this heat-treated solution was incubated with 0.1 ml pancreatic ribonuclease solution (1 mg/ml in water) for 3 hours at 18”. A control incubation contained no ribonuclease. The ultracentrifuge patterns produced by the enzyme-treated sample and the control showed that ribonuclease had degraded the 4.5 S component to a form which did not leave the meniscus of the cell at 60,000 rpm. The other components remained unaltered. This indication that the RNA was contained in the 4.5 S component was confirmed by partial separation of the products of the dissociation in the ultracentrifuge cell, and analyzing the fractions obtained for RNA. The cell was fitted with two layers of filter paper at its base to minimize redissolving of material which sedimented completely. Two ultracentrifuge runs were then carried out with virus dissociated as in the previous experiment. In the first, at, the conclusion of the run, the 33 S component sedimented halfway across the cell, while in the second it sedimented completely. In a third run, undissociated virus was centrifuged until both “top” and “bottom” components had sedimented completely. The supernatants were withdrawn as rapidly as possible, and the sediment in the filter papers at the base of the cell was extracted with 0.15 M NaCl. Ultraviolet absorption curves were prepared for the various supernatant fluids and redissolved sediments. All showed a maximum at 260 rnp, so that comparison of optical densities at this
NUCLEIC
ACID
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YELLOW
TABLE DISTRIBUTION
OF RNA AFTER DISSOCIATED
467
VIRUS
1
ULTRACENTRIFUGE RUNS ON CELLS AND UN~ISSOCIATED TYMV Supernatant fluid RNA in
Material
MOSAIC
CONTAINING
Redissolved sediment - --___ RNA. in
Total RNA, in
analyzed
1. I)issociat,ed TYMV, 33 S sedimented 2. I>issociated TYMV, 33 S pletely sedimented 3. Undissociated TYMV, bot.h ponents sedimented 4. Dissociated TTMV, not, t rifuged
half
71
7.8
11
19
x5
27
com-
73
4
16
21
89
28
com-
5
5
66
101
71
106
rerl-
106
3-l
-
106
31
a Total opt.ical densit.y at, 260 rnp in the sample standardized to a volume of 1 ml. !’ Expressed as a percentage of the t,otal RNA in an equivalent sample of undissociated unsedimented virus.
wavelength will reflect primarily the concentration of RNA, although absorption due to protein will make a minor contribution, for which no correction has been made. In addition to these measurements the RNA still bound to protein was estimated directly for each fraction by the serological-chromatogrsphic method. Results arc given in Table 1. These show that removal of the 33 S component from the supernatant of dissociated TYMV does not remove the RNA; while in the undissociated system the same amount of centrifuging will remove almost all the RNA from solution. It appears, then, that the 33 S component contains no RNA and is presumably protein in nature, while the 4.5 S component contains all the RNA liberated from the dissociated virus molecule. This 4.5 X component, which is liberated by heating only when the pH of the TYMV solution is greater than about 6.4, shows no change in sedimentation behavior when the pH of the solution is lowered to 4.5 after dissociation has been carried out. On standing at pH 7.5 for 2-3 days at 2”, the component breaks up into smaller fragments which are not resolved from the meniscus of the ultracentrifuge cell at 60,000 rpm. This breakdown is accelerated at, higher pH values, and at pH 9.5 it occurs within a few hours.
468
LYTTLETON
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TABLE
2
NUMBER OF LOCAL LESIONS PRODUCED ON CHINESE CABBAGE BY TYMV HEATING FOR THREE MINUTES AT 48” IN 0.14 M SALINE, AND
IN
0.05 M PH 7.5
PHOSPHATE
o/u RNA remaining in virusa
Treatment
AFTER
BUFFER No. of local lesions (total on 20 half-leaves)
Heated in saline, cooled to 18”
96
354
Heated in phosphate, cooled to 18”
25
65)
Heated in saline, cooled to 1”
97
Heated in phosphate, cooled to lo
30
804) i
YOInfectivity rem&h@
18
a Amount of RNA remaining compared by serological chromatographic with virus held at 18” in saline. b Total lesions from phosphate preparation x 100. Total lesions from saline preparation
Infectivity
of TYMV
10
84 method
Preparations after Heating in Bu$ered Solution
Infectivity tests made on virus solutions after heating showed that in phosphate buffer at pH 7.5 much of the infectivity is lost (Table 2). Cooling the heated preparations to 1" between heating treatment and inoculation gave no increase in the residual infectivity. DISCUSSION
The methods used to prepare infectious nucleic acid from intact tobacco mosaic virus in vitro involve conditions which could not prevail in the living leaf cell. Thus Gierer and Schramm (1956) treated tobacco mosaic virus with water-saturated phenol for 8 minutes at 5’, while Fraenkel-Conrat et al. (1957) used a 1% solution of sodium dodecyl sulfate at pH 8.5 and 50” for 5 minutes. The methods previously used to prepare RNA from TYMV are likewise fairly severe compared with possible conditions in viva. Two methods have been used: treatment with 33 % ethanol at room temperature and heating for short periods at temperatures near 100” (Cohen and Schachman, 1957). In contrast to these procedures, the conditions under which we have obtained release of RNA from a proportion of the TYMV particles are such as might prevail in the living cell-namely pH 7 at) 37”. The results given in Table 2 show that most of the RNA released is noninfectious. To determine whether any small amount of infectivity
NUCLEIC
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YELLOW
MOSAIC
VIRUS
469
is associated with the RNA released under our conditions, it will be necessary to carry out tests on a larger scale with RNA freed from remaining intact virus. Cohen and Schachman (1957) have made a detailed ultracentrifuge study of the properties of RNA obtained from TYMV by heating to 100” and by ethanol denaturation of the virus. Their products were not homogeneous with respect to molecular weight. The weight average calculated from the viscosity and the highest, sedimentation coefficient observed (4.4 S) gave a molecular weight of 76,000. Fresh preparations of RNA obtained by our method have always given sedimentation coefficients in the range 4.4-4.6 S. Presumably, therefore, the weight average molecular weight of our material is about 75,000. This result supports t,heir suggestion that there may be about 25-50 RNA molecules per TYMV particle. TYMV consists of a roughly spherical shell of protein surrounding a central core of nucleic acid (Markham, 1951; Schmidt et al., 1954). The naturally occurring nucleic acid-free “top” component is an apparently identical protein shell without nucleic acid. The X-ray evidence suggests that the protein shell is built up from 60 identical subunits (Klug el al., 1957). When we first found that the RNA could be released from TYMV under conditions such that the remaining protein was not denatured (to the extent that it was still water-soluble), we had hoped that the protein would either be depolymerized to the small subunits indicated by the X-ray evidence or that it would be identical with the “top” component. The ultracentrifuge data show clearly that neither of these possibilities can be true. The 33 S protein was less homogeneous than the “top” component which had a sedimentation coefficient of 45 X under the same concentration conditions. The heterogeneity was not nearly as great as would be expected from random fragmentation of the protein shell. This sedimentation behavior is consistent with the idea that the RNA, in escaping from the protein ruptures the shell, thereby lowering its symmetry and decreasing the sedimentation coefficient. The data in Fig. 3 on the release of RNA from TYMV at various temperatures might be interpreted as suggesting that a portion of the RNA was lost from each of the particles at any given temperature. The sedimentation data make this possibility unlikely since they show, within the limits of the sensitivity of the method, that the loss of RKA from a particle is an all-or-none event. Thus the particles in a prepara-
470
LYTTLETON
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tion of TYMV appear to have a continuous range of stabilities to heat at pH values near 7. This variation in stability makes very difficult any interpretation of the curve in Fig. 1 in terms of possible molecular change within the virus particle. Little is known about the nature of the bonds between protein and RNA in virus nucleoproteins. At the pH at which RNA begins to escape from a proportion of the particles a substantial proportion of the secondary phosphate groups on the RNA are becoming ionized. This is consistent with the idea that nonionized phosphate groups play a role in the bonding between RNA and protein in intact virus. On this basis it would be reasonable to assume that rupture of the TYMV particle is caused by thermal agitation of RNA freed by pH change from its normal binding to the inner surface of the protein shell. Rupture of the virus is suppressed by the presence of sucrose in the medium. The degree of suppression is related linearly to concentration and hence to. the osmotic pressure of the sucrose solution. This relationship can be explained if we assume that the protein shell is impermeable to sucrose. Then the increased osmotic pressure in the external medium would increase the resistance of the protein shell to rupture from within. It is now known that a range of viruses can be inactivated in plants by heat, and the plants completely freed of virus (Kassanis, 1957). In such treatments infected plants or parts of plants are heated for periods of days or weeks at temperatures between about 36” and 40” or they may be heated a.t higher temperatures (usually 50-55’) for a few minutes. Little is known about the mechanism of such inactivation. The in vitro results with TYMV described here suggest a possible mechanism for heat inactivation of viruses in viva. It is not unreasonable to assume that pH values in the living cell may lie in the range 6.6-7.0. If conditions of pH and temperature in the cell were such that the virus RNA was forced from the protein coat under conditions unfavorable for further multiplication the virus would be inactivated. Nothing is known about the structure of most of the viruses that can be inactivated by heating in viao. Those whose shape is definitely known are roughly spherical (tomato bushy stunt, tobacco necrosis, carnation ringspot, tobacco ringspot viruses), while those that are more stable to heat in vivo are rods (TMV, potato viruses X and Y). When plants syst,emically infected with tomato bushy stunt virus are kept at 36” both infectivity of the sap and amount of specific virus
NUCLEIC
ACID
AND
TURNIP
YELLOW
MOSAIC
VIRUS
471
antigen decrease, but infectivity decreases much more rapidly than specific antigen content (Kassanis, 1952). This sequence would agree with a mechanism where RNA escapedfrom the protein and was rapidly inactivated, while the more stable protein shell was degraded more slowly. No tests on the in vivo inactivation of TYMV by heat have yet been made, but this virus should provide good material for testing the hypothesis suggested here. The naturally occurring “top” component, is relatively heat-stable and should provide a good yardstick for measuring changes in the virus nucleoprotein induced by heat in IGO. REFEREXCES
RAUER, J. M., and PICKELS, E. G. (1937). Improved
air-driven type of ult,racentrifuge for molecular sedimentation. J. Exptl. Med. 66, 565-586. COHEN, S. S., and SCHACHMAN,H. K. (1957). Physical studies on the ribonucleic acid of turnip yellow mosaic virus. Virology 3, 575-586. FRAEXKEL-CONRAT, H., SINGER, B., and WILLIAMS, R. C. (1957). Infectivity of viral nucleic acid. Biochim. et Biophys. Acta 26, 87-96. GIERER, A., and SCHRAMM,G. (1956). Infectivit,y of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. KASSANIS, B. (1952). Some effects of high temperature on the suscept)ibility of plants to infection with viruses. Ann. Appt. Biol. 39, 358-369. KASSANIS, B. (1957). Effects of changing temperature on plant, virus diseases. Advances in Virus Research 4, 221-241. KLIJG, A., FINCH, J. T., and FRAPU'KLINR. E. (1957). The structure of turnip yellow mosaic virus: x-ray diffraction studies. Biochim. et Biophys. Acta 26, 242-252. MARKHAM, R. (1951). Physicochemical studies on the turnip yellow mosaic virus. Discussions Faraday Sot. No. 11, 221-227. MARKHAM, R., and SMITH, K. M. (1949). Studies on the virus of turnip yellow mosaic. Parasitology 39, 330-342. MATTHEWS, R. E. F. (1954). Effect of some purine analogues on tobacco mosaic virus. J. Gen. Microbial. 10, 521-532. MATTHEWS, R. E. F. (1957). Pla,nt Virlts Serology, 128 pp. Cambridge Univ. Press, London and sew York. SCHMIDT,P., KAESBERG, P., and BEEMAN, W. W. (1954). Small angle x-ray scattering from turnip yellow mosaic virus. Biochim. et Biophys. Acta 14, l-11. SVENSSON, H. (1940). IXrekte photographische Aufnahme von Elektrophoresediagrammen. Kolloid-2.87, 181-189.