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Some properties of decomposition products of potato virus X
VIROLOGY
7,37&384
Some
(1959)
Properties
F. C. Rothamsted
of Decomposition Potato Virus X BAWDEN
Experimental
AND
Station, Accepted
A.
of
KLECZKOWSKI
Harper&en,
December
Products
Hertjordshire,
England
IY, 1958
The small surface potential of potato virus X (PVX) probably accounts for its tendency to become insoluble during purification. The protein produced by splitting the virus with alkali (at about pH 10) has a higher surface potential than the virus: electrophoretic mobility of the protein in M/15 pH 7.0 phosphate buffer is -0.15 &ec/volt/cm, compared with -0.04 for that of the virus. The nucleic acid, mobility -1.7, is not infective when it is separated electrophoretically from alkali-split virus. The protein, but not the nucleic acid, precipitates with PVX antiserum. Material with the same electrophoretic mobility as protein produced by splitting the virus with alkali is somet,imes detectable in sap from infected plants. Treatments that aggregate protein fragments from tobacco mosaic virus into viruslike rods produce amorphous precipitates of PVX protein that do not redissolve; this difference may reflect, the different ways in which surface charges are distributed on the protein from the two viruses. The nucleic acid seems not to contribute to the surface pot,ential of either virus. Disrupting PVX with phenol gives aatersoluble material consisting predominantly of nucleic acid; freshly made preparat,ions have some infectivity, which is not conferred by residual intact virus particles. INTRODUCTION
There is now much information on the properties of fragments of tobacco mosaic virus (TMV), on the anomalous protein that remains in the supernatant fluid when sap from infected plants is centrifuged at high speed, and on the infectivity of the virus nucleic acid, but there is no comparable information on any other virus. This paper describes preliminary experiments to see how far the results obtained with TMV apply to pot,ato virus X (PVX). MATERIALS
AND
The strain of virus X used produces systemic
symptoms
of
a mottle
with 375
METHODS
white necrotic
necrotic local lesions and spots in White Burley
376
BAWDEN
AND
KLECZKOWSKI
tobacco (Nicotiuna tabacum L. var. Judy’s Pride) and Nicotiana glutinosa L. Virus preparations were made from fresh sap of systemically infected tobacco or N. glutinosa plants by a series of three alternate highand low-speed centrifugations in which the pellets were suspended in distilled water. Antisera with precipitin titers of l/2000 were prepared by injecting rabbits intravenously with purified virus in 0.9 % NaCl solution. Ten were injected twice a week for 3 weeks, and the animals were bled 10 days after the last injection. Serological tests were made by adding 1 ml of antigen solutions at different concentrations to a series of tubes each containing 1 ml of the antiserum at a dilution of l/100; the mixtures were incubated for 3 hours at JO”, during which time several readings were made, and then left overnight at room temperature for a final reading. All the dilutions were made in 0.9 % NaCl, and for controls antigen solutions were incubated with 0.9 % NaCl. Electrophoretic analyses and separations were done in a PerkinElmer model 38A electrophoresis apparatus using the 2-ml Tiselius cell. Fluids were first dialyzed against M/15 pH 7.0 phosphate buffer (Na2HP04-KH2POd), and the potential gradient was 9.5 voltsicm. The virus was split with alkali by incubating 1 % solutions, adjusted to pH 10.0-10.3 with 0.1 N NaOH, for 20 hours at 2”, after which they were dialyzed for 24 hours against M/l5 pH 7.0 phosphate buffer, also at 2”. Nucleic acid was isolated by treat’ing 1% virus solutions with phenol. One volume of M/15 pH 7.0 phosphate buffer was added to 4 volumes of a virus solution (in HzO), cooled to O”, mixed with 5 volumes of cold watersaturated phenol, and then treated as described by Gierer and Schramm (1956) for TMV. Infectivity tests were made by the local-lesion method in tobacco trimmed to three leaves per plant. At least eight plants were used for each test, and the preparations to be compared were inoculated t’o equal numbers of left- and right-hand half-leaves, and to equal numbers at each leaf position. Inoculations were made by rubbing the surface of the leaves with the forefinger wet with the inoculum. The relative infectivities of tested preparations were computed by graphic interpolation, using curves obtained by plotting numbers of lesions against logs of concentrations of a reference virus preparation. Spectrophotometric examinations were made with a Unicam quartz spectrophotometer.
DECOMPOSITIOK
PRODUCTS
OF
POTATO
-. _.,. ...--”
VIRUS
377
X
” -
Direction
of movement
FIG. 1. Ascending pattern formed taken aft,er (a) 20 and (b) 60 minutes’
by
(useending)
a 0.47, solution electrophoresis.
of PVX.
Photographs
RESULTS
Decomposition
by Alkali
Purified preparations of PVX give a single boundary that is almost stationary in the electrophoretic cell in M/15 pH 7 phosphate buffer and a potential gradient of 9.5 volts/cm (Fig. 1). The mobility is too slight to measure accurately but is about -0.04 p/set/volt/cm. This means that the surface potential of the virus particles is very low, which may account for the tendency of purified or partially purified preparat’ions of the virus to become insoluble and for individual filaments to associat’e laterally and produce entangled masses (Kleczkowski and Nixon, 1950). Incubation at pH values 10.0-10.3 for 20 hours at 2” splits the virus into two components: Nu, with mobility of - 1.7 p/set/volt/ cm, and P, with mobility about -0.15 H/set/volt/cm (Figs. 2 and 3). After separating one from the other by electrophoresis, the component,s Su and P give, respectively, ultraviolet (UV) absorption spectra typical for a nucleic acid and for a protein (Fig. 4). After incubation at pH 10.3 the virus is entirely decomposed into the components P and Nu (Fig. 3), but after incubation at pH 10.0 there is still a component, R (Fig. 2) with the electrophoretic mobility of about’ -0.04 p/set/volt/cm. Com-
378
BAWDEN
AND
KLECZKOWSKI
(,_(
Direction
of movement
FIG. 2. Ascending pattern formed by incubation at pH 10 for u) hours 30 minutes’ electrophoresis.
(S_ 1
_s.(: i_.
a.
(aac&dingl
by a 1.3% solution at 2”. Photographs
FIG. 3. Ascending pattern formed by a 0.57, solution by incubation at pH 10.3 for 20 hours at 2”. Photographs and (c) 60 minutes’ erectrophoresis.
of PVX partially split taken after (a) 5 and (b)
of PVX completely split taken after (a) 6, (b) 30,
DECOMPOSITION
PRODUCTS
Wavelength
FIG. 4. UV absorption
OF
POTATO
VIRUS
379
X
(in mp)
spectra of PVX and of its decomposition
products.
ponent R sedimentsinto a pellet during 2 hours’ centrifugation at 70,000 Q, whereas the components P and Nu remain in the supernatant fluid. Component R has a UV absorption spectrum identical wit,h that of PVX, precipitates with PVX antiserum in the sameway as PVX, gives an electron micrograph indistinguishable from that of PVX, and, when inoculated to N. tabacum at the same concentrations as PVX, produces only slight,ly fewer lesions than untreated PVX. Component R, therefore, is probably unchanged residual virus. This behavior contrasts with that of TMV which, when most of a preparation is decomposedby alkali, leaves a proportion that has little infectivity but has the original serological behavior, morphology, electrophoretic mobility, and UV absorption spectrum (Bawden and Pirie, 1957; Kleczkowski, 1959). Neither the nucleic acid (component Nu) nor the protein (component P) seemsto be infective. In none of our tests did Nu produce any lesions;
380
BAWDEN
AND
KLECZKOWSKI
P had less than 0.5% of the infectivity of the original PVX, and this could have come from contamination with a small amount of residual virus. From the behavior of nucleic acid preparations made with phenol, which are described later, it is reasonable to suspect that when first split from the virus by alkali the nucleic acid may be infective, but that it loses this property during the time that elapses before it can be separated and tested. Nu does not precipitate with PVX antiserum. Component P precipitates, but much more slowly than does PVX, and it forms precipitates of a dense granular type, in striking contrast to the cloudy, transparent floccules produced by PVX; it gives a precipitation end point at about 0.015 g/l in conditions in which normal virus preparations precipitate to about 0.0005 g/l. This difference in precipitation behavior between component P and intact virus parallels the difference between the alkali-produced protein of TMV and intact TMV (Bawden and Pirie, 1957). Like the corresponding protein of TMV, the protein from PVX has small particles; it gives water-clear solutions up to a concentration of 0.5 %, it does not sediment into a pellet during 2 hours’ centrifugation at 70,000 g, and it is not resolved by the electron microscope. The two most striking differences between results obtained with PVX and those obtained with TMV are (1) that at pH 7 PVX moves in the electric field only very slowly in comparison with TMV, and (2) that the protein from PVX moves faster than intact PVX, whereas the protein from TMV moves more slowly than the intact TMV (Schramm et al., 1955; Kleczkowski, 1959). The nucleic acid of TMV seems not to contribute to the surface potential of the virus (Kramer and Wittmann, 1958; Kleczkowski, 1959). The fact, that the negative surface potential of the protein from PVX is greater than that of the original PVX strongly suggests that the nucleic acid of PVX also contributes nothing to the surface potential of this virus, for if it did, it should increase the potential, i.e., make it more negatively charged. The results can be explained by assuming that the negative charge is distributed unevenly on the surface of the particles of PVX prot,ein, as was assumed by Kramer and Wittmann (1958) for particles of unaggregated protein of TMV. However, whereas the behavior of TMV protein makes it necessary to assume that the parts of their surface that formed the outer wall of the original virus particle are more negatively charged than the parts of their surface that were in the interior of the
DECOMPOSITION
PRODUCTS
OF
POTBTO
VIRUS
X
381
original virus particle, the opposite assumption has to be made for the protein fragments from PVX. Treatments that make TMV protein aggregate and form cylinders resembling virus particles do not similarly aggregate the protein from alkali-split PVX. For instance, when precipitated from pH 7.0 phosphate buffer by adding saturated (NHJ 2SO.t solution slowly to give 0.4 saturation, conditions that make preparations of TMV protein show anisotropy of flow and produce microscopic birefringent needles, PVX protein produces no elongated particles but it precipitates as amorphous material that does not redissolve when it is centrifuged down and resuspended in water or buffer. This difference in behavior between the proteins from the two viruses is probably explicable by the differences in the distribution of their surface charges. Kramer and Wittmann (1958) argue that the distribution they postulate for the charges on TMV protein on the alkaline side of the isoelectric point favors it)s orderly aggregation to produce cylinders. If their argument is valid, the opposite distribution of charge on PVX protein would tell against such an orderly aggregation. dggregation into viruslike threads might be expected were the protein precipit,ated on the acid instead of the alkaline side of its isoelectric point, but we have not tested this. When sap from plants infected with TMV is centrifuged at high speed, l-5 % of the mat’erial serologically related to the virus fails to sediment into a pellet. This, the X-protein of Taknhashi and Ishii (1952), has the sameelectrophoretic mobility as the protein produced by disrupting the virus with alkali and resemblesit in many other ways. Small componeiits, with mobilities of -0.2 and -0.4 p/set/volt/cm, are sometimes detectable electrophoret.ically in sap from plants infected with PVX (Bnwden and Kleczkowski, 1957), and it seemedthat these might be analogous to the unsedimentable material in sap from plants infected with TMV. We have some results suggesting this to be so, but the anomalous material in sap from plants infected with PVX occurs in too small amounts and its behavior is t,oo erratic for any exact, work on its properties or composition. The amount of such material varies greatly in sap from different lots of plants; none has been detectable in many samples, and in some samples there has been only one small peak with mobility of -0.15 to -0.2 p/set/volt/cm. As this is similar to the mobility of protein produced by disrupting PVX with alkali, it seemsreasonable to assumethat the component is also virus protein,
382
BAWDEN
AND TABLE
INFECTIVITY
OF
PVX MADE
PVX PVX PVX
acid acid acid
undiluted diluted diluted
1
AND OF A NUCLEIC ACID FROM IT WITH PHENOL” Nondialyzable cleic acid hdl)
Inoculum
Nucleic Nucleic Nucleic
KLECZKOWSKI
1:5 1:25
at 200 mg/l at 40 mg/l at 8 mg/l
PREPARATION
nu-
Numbers of local lesions on 8 half-leaves
35 7 1.4
132 15 2
10 2 0.4
1390 530 64
a The nucleic acid was prepared from a 1% solution of the PVX used as inoculum in the experiment. By plotting the numbers of lesions formed by PVX against logs of its concentration, interpolation shows that infectivity of nucleic acid at 35 mg/l equals that of about 10 mg/l PVX, which corresponds to about 0.5 mg of virus nucleic acid per liter. Thus, considering infectivity in relation to nucleic acid contents, the isolated nucleic acid was about 1.47, as infective as the intact virus.
Nucleic Acid Prepared with Phenol Nucleic acid preparations freshly made by treating PVX with phenol are, as Table 1 shows, often infective. Indeed, the infectivity of some preparations compares favorably per unit weight of phosphorus with that of similarly made nucleic acid from TMV, but, as in much other work with PVX, results are more variable. The greater difficulty of drawing any firm conclusions about the infectivity of nucleic acid preparations from PVX comes partly from the greater difficulty of making reliable infectivity tests, partly because it is impossible to start with such concentrated virus preparations as with TMV, and partly because the yield of nucleic acid that fails to pass through a dialysis sack is very much smaller. With tobacco mosaic virus, almost all the original nucleic acid of the virus can be obtained in a form that remains in the sack after 24hr dialysis at 2” against M/15 pH 7.0 phosphate buffer, whereas with PVX only 5-7 % remains. On the assumption that the infectivity of the freshly made preparations lies in the undialyzable material, the nucleic acid preparations from PVX had infectivities of up to about 1.5 % of infectivity of the intact virus per unit weight of nucleic acid. This is slightly higher than the corresponding figure with nucleic acid from
DECOMPOSITION
PRODUCTS
OF
POTATO
VIRUS
X
383
TMV, which is usually below 1%. The significance of statements about the relative infectiviCy of nucleic acid preparaCons and of intact virus, however, is uncertain. With TMV the results of such comparisons depend not only on the relative concentrations at which the two inocula are used, but also on the physiological state of the plants inoculated (Bawden and Pirie, 1957), and this may be equally true wit,h PVX. The UV absorption spectra of t’he phenol-made preparations are t’ypical for nucleic acid and resemble those given by nucleic acid separat,ed electrophoretically from PVX split by alkali (Fig. 1). This shows that the preparations are predominantly nucleic acid, but does not exclude the possibility that t,heir infectivity lies in residual virus, for contamination with enough virus t,o confer their infectivit,y would not noticeably distort the absorption spectrum. Evidence from other sources, however, seems ho exclude this possibility. First’, t’he infectivity of preparations made from 1% PVX solutions are about as infective as the original virus preparations at, 5 mg/l, at which concentration the characteristic threadlike virus particles are readily detectable in the electron microscope, whereas none was seen in the many phenol-made preparations examined. Secondly, but less conclusive, the nucleic acid preparations give no visible precipitate with PVX antiserum, whereas intact virus preparations precipitate down to concentrat’ions of 0.5 mg/‘l or less. Thirdly, and perhaps the best evidence bhat t’he infectivity resides in material other than residual virus particles, is its instability; incubation for 20 minutes at 20” at pH 7 with beef pancreatic ribonuclease at 0.05 mg/l, a treatment without effect on the infectivity of intact PVX, destroys the infectivity of t’he phenol-made preparations; also, bheir infectivity always falls great,ly and is usually abolished by keeping preparations for a day at 20” and pH 7. There is adequate evidence to conclude t’hat material other than intact PVX is responsible for the infectivity of these preparations and that the nucleic acid is an essential part of this material, but the evidence does not prove that the nucleic acid alone is infective. This is the simplest, and a plausible conclusion, but it is equally possible that infectivity is carried by particles that contain nucleic acid combined with something else, perhaps a small amount of protein or a polypeptide. However, this degree of uncertainty also still surrounds the infectivity possessedby preparations of TMV nucleic acid, which is a more favorable subject for experiments to decide the precise chemical identity of the minimal infective unit.
384
BAWDEN
AND
KLECZKOWSKI
ACKNOWLEDGMENTS We thank Mr. H. L. Nixon scope examinations.
and Mr.
R. D. Woods
for making
the electron
micro-
REFERENCES BAWDEN, F. C., and KLECZKOWSKI, A. (1957). An electrophoretic study of sap from uninfected and virus-infected tobacco plants. Virology 4, 26-40. BAWDEN, F. C., and PIRIE, N. W. (1957). The activity of fragmented and reassembled tobacco mosaic virus. J. Gen. Microbial. 17, 80-95. GIERER, A., and SCHRAMM, G. (1956). Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. KLECZKOWSKI, A. (1959). Aggregation of the protein of tobacco mosaic virus with and without combination with the virus nucleic acid. Virology 7, 385 (1959). KLECZKOWSKI, A., and NIXON, H. L. (1950). An electron-microscope study of potato virus X in different states of aggregation. J. Gen. Microbial. 4,220-224. KRAMER, E., and WITTMAN’N, H. G. (1958). Elektrophoretische Untersuchungen der A-Proteine dreier Tabakmosaikvirus-Stimme. Z. Naturjorsch. 13b, 30-33. SCHRAMM, G., SCHUMACHER, G., and ZILLIG, W. (1955). tfber die Struktur des Tabakmosaikvirus. III. Mitt.: Der Zerfall in alkalischer Liisung. Z. Naturjorsch. lob, 481-492. TAKAHASHI, W. N., and ISHII, M. (1952). An abnormal protein associated with tobacco mosaic virus infection. Nature 169, 419420.
7,37&384
Some
(1959)
Properties
F. C. Rothamsted
of Decomposition Potato Virus X BAWDEN
Experimental
AND
Station, Accepted
A.
of
KLECZKOWSKI
Harper&en,
December
Products
Hertjordshire,
England
IY, 1958
The small surface potential of potato virus X (PVX) probably accounts for its tendency to become insoluble during purification. The protein produced by splitting the virus with alkali (at about pH 10) has a higher surface potential than the virus: electrophoretic mobility of the protein in M/15 pH 7.0 phosphate buffer is -0.15 &ec/volt/cm, compared with -0.04 for that of the virus. The nucleic acid, mobility -1.7, is not infective when it is separated electrophoretically from alkali-split virus. The protein, but not the nucleic acid, precipitates with PVX antiserum. Material with the same electrophoretic mobility as protein produced by splitting the virus with alkali is somet,imes detectable in sap from infected plants. Treatments that aggregate protein fragments from tobacco mosaic virus into viruslike rods produce amorphous precipitates of PVX protein that do not redissolve; this difference may reflect, the different ways in which surface charges are distributed on the protein from the two viruses. The nucleic acid seems not to contribute to the surface pot,ential of either virus. Disrupting PVX with phenol gives aatersoluble material consisting predominantly of nucleic acid; freshly made preparat,ions have some infectivity, which is not conferred by residual intact virus particles. INTRODUCTION
There is now much information on the properties of fragments of tobacco mosaic virus (TMV), on the anomalous protein that remains in the supernatant fluid when sap from infected plants is centrifuged at high speed, and on the infectivity of the virus nucleic acid, but there is no comparable information on any other virus. This paper describes preliminary experiments to see how far the results obtained with TMV apply to pot,ato virus X (PVX). MATERIALS
AND
The strain of virus X used produces systemic
symptoms
of
a mottle
with 375
METHODS
white necrotic
necrotic local lesions and spots in White Burley
376
BAWDEN
AND
KLECZKOWSKI
tobacco (Nicotiuna tabacum L. var. Judy’s Pride) and Nicotiana glutinosa L. Virus preparations were made from fresh sap of systemically infected tobacco or N. glutinosa plants by a series of three alternate highand low-speed centrifugations in which the pellets were suspended in distilled water. Antisera with precipitin titers of l/2000 were prepared by injecting rabbits intravenously with purified virus in 0.9 % NaCl solution. Ten were injected twice a week for 3 weeks, and the animals were bled 10 days after the last injection. Serological tests were made by adding 1 ml of antigen solutions at different concentrations to a series of tubes each containing 1 ml of the antiserum at a dilution of l/100; the mixtures were incubated for 3 hours at JO”, during which time several readings were made, and then left overnight at room temperature for a final reading. All the dilutions were made in 0.9 % NaCl, and for controls antigen solutions were incubated with 0.9 % NaCl. Electrophoretic analyses and separations were done in a PerkinElmer model 38A electrophoresis apparatus using the 2-ml Tiselius cell. Fluids were first dialyzed against M/15 pH 7.0 phosphate buffer (Na2HP04-KH2POd), and the potential gradient was 9.5 voltsicm. The virus was split with alkali by incubating 1 % solutions, adjusted to pH 10.0-10.3 with 0.1 N NaOH, for 20 hours at 2”, after which they were dialyzed for 24 hours against M/l5 pH 7.0 phosphate buffer, also at 2”. Nucleic acid was isolated by treat’ing 1% virus solutions with phenol. One volume of M/15 pH 7.0 phosphate buffer was added to 4 volumes of a virus solution (in HzO), cooled to O”, mixed with 5 volumes of cold watersaturated phenol, and then treated as described by Gierer and Schramm (1956) for TMV. Infectivity tests were made by the local-lesion method in tobacco trimmed to three leaves per plant. At least eight plants were used for each test, and the preparations to be compared were inoculated t’o equal numbers of left- and right-hand half-leaves, and to equal numbers at each leaf position. Inoculations were made by rubbing the surface of the leaves with the forefinger wet with the inoculum. The relative infectivities of tested preparations were computed by graphic interpolation, using curves obtained by plotting numbers of lesions against logs of concentrations of a reference virus preparation. Spectrophotometric examinations were made with a Unicam quartz spectrophotometer.
DECOMPOSITIOK
PRODUCTS
OF
POTATO
-. _.,. ...--”
VIRUS
377
X
” -
Direction
of movement
FIG. 1. Ascending pattern formed taken aft,er (a) 20 and (b) 60 minutes’
by
(useending)
a 0.47, solution electrophoresis.
of PVX.
Photographs
RESULTS
Decomposition
by Alkali
Purified preparations of PVX give a single boundary that is almost stationary in the electrophoretic cell in M/15 pH 7 phosphate buffer and a potential gradient of 9.5 volts/cm (Fig. 1). The mobility is too slight to measure accurately but is about -0.04 p/set/volt/cm. This means that the surface potential of the virus particles is very low, which may account for the tendency of purified or partially purified preparat’ions of the virus to become insoluble and for individual filaments to associat’e laterally and produce entangled masses (Kleczkowski and Nixon, 1950). Incubation at pH values 10.0-10.3 for 20 hours at 2” splits the virus into two components: Nu, with mobility of - 1.7 p/set/volt/ cm, and P, with mobility about -0.15 H/set/volt/cm (Figs. 2 and 3). After separating one from the other by electrophoresis, the component,s Su and P give, respectively, ultraviolet (UV) absorption spectra typical for a nucleic acid and for a protein (Fig. 4). After incubation at pH 10.3 the virus is entirely decomposed into the components P and Nu (Fig. 3), but after incubation at pH 10.0 there is still a component, R (Fig. 2) with the electrophoretic mobility of about’ -0.04 p/set/volt/cm. Com-
378
BAWDEN
AND
KLECZKOWSKI
(,_(
Direction
of movement
FIG. 2. Ascending pattern formed by incubation at pH 10 for u) hours 30 minutes’ electrophoresis.
(S_ 1
_s.(: i_.
a.
(aac&dingl
by a 1.3% solution at 2”. Photographs
FIG. 3. Ascending pattern formed by a 0.57, solution by incubation at pH 10.3 for 20 hours at 2”. Photographs and (c) 60 minutes’ erectrophoresis.
of PVX partially split taken after (a) 5 and (b)
of PVX completely split taken after (a) 6, (b) 30,
DECOMPOSITION
PRODUCTS
Wavelength
FIG. 4. UV absorption
OF
POTATO
VIRUS
379
X
(in mp)
spectra of PVX and of its decomposition
products.
ponent R sedimentsinto a pellet during 2 hours’ centrifugation at 70,000 Q, whereas the components P and Nu remain in the supernatant fluid. Component R has a UV absorption spectrum identical wit,h that of PVX, precipitates with PVX antiserum in the sameway as PVX, gives an electron micrograph indistinguishable from that of PVX, and, when inoculated to N. tabacum at the same concentrations as PVX, produces only slight,ly fewer lesions than untreated PVX. Component R, therefore, is probably unchanged residual virus. This behavior contrasts with that of TMV which, when most of a preparation is decomposedby alkali, leaves a proportion that has little infectivity but has the original serological behavior, morphology, electrophoretic mobility, and UV absorption spectrum (Bawden and Pirie, 1957; Kleczkowski, 1959). Neither the nucleic acid (component Nu) nor the protein (component P) seemsto be infective. In none of our tests did Nu produce any lesions;
380
BAWDEN
AND
KLECZKOWSKI
P had less than 0.5% of the infectivity of the original PVX, and this could have come from contamination with a small amount of residual virus. From the behavior of nucleic acid preparations made with phenol, which are described later, it is reasonable to suspect that when first split from the virus by alkali the nucleic acid may be infective, but that it loses this property during the time that elapses before it can be separated and tested. Nu does not precipitate with PVX antiserum. Component P precipitates, but much more slowly than does PVX, and it forms precipitates of a dense granular type, in striking contrast to the cloudy, transparent floccules produced by PVX; it gives a precipitation end point at about 0.015 g/l in conditions in which normal virus preparations precipitate to about 0.0005 g/l. This difference in precipitation behavior between component P and intact virus parallels the difference between the alkali-produced protein of TMV and intact TMV (Bawden and Pirie, 1957). Like the corresponding protein of TMV, the protein from PVX has small particles; it gives water-clear solutions up to a concentration of 0.5 %, it does not sediment into a pellet during 2 hours’ centrifugation at 70,000 g, and it is not resolved by the electron microscope. The two most striking differences between results obtained with PVX and those obtained with TMV are (1) that at pH 7 PVX moves in the electric field only very slowly in comparison with TMV, and (2) that the protein from PVX moves faster than intact PVX, whereas the protein from TMV moves more slowly than the intact TMV (Schramm et al., 1955; Kleczkowski, 1959). The nucleic acid of TMV seems not to contribute to the surface potential of the virus (Kramer and Wittmann, 1958; Kleczkowski, 1959). The fact, that the negative surface potential of the protein from PVX is greater than that of the original PVX strongly suggests that the nucleic acid of PVX also contributes nothing to the surface potential of this virus, for if it did, it should increase the potential, i.e., make it more negatively charged. The results can be explained by assuming that the negative charge is distributed unevenly on the surface of the particles of PVX prot,ein, as was assumed by Kramer and Wittmann (1958) for particles of unaggregated protein of TMV. However, whereas the behavior of TMV protein makes it necessary to assume that the parts of their surface that formed the outer wall of the original virus particle are more negatively charged than the parts of their surface that were in the interior of the
DECOMPOSITION
PRODUCTS
OF
POTBTO
VIRUS
X
381
original virus particle, the opposite assumption has to be made for the protein fragments from PVX. Treatments that make TMV protein aggregate and form cylinders resembling virus particles do not similarly aggregate the protein from alkali-split PVX. For instance, when precipitated from pH 7.0 phosphate buffer by adding saturated (NHJ 2SO.t solution slowly to give 0.4 saturation, conditions that make preparations of TMV protein show anisotropy of flow and produce microscopic birefringent needles, PVX protein produces no elongated particles but it precipitates as amorphous material that does not redissolve when it is centrifuged down and resuspended in water or buffer. This difference in behavior between the proteins from the two viruses is probably explicable by the differences in the distribution of their surface charges. Kramer and Wittmann (1958) argue that the distribution they postulate for the charges on TMV protein on the alkaline side of the isoelectric point favors it)s orderly aggregation to produce cylinders. If their argument is valid, the opposite distribution of charge on PVX protein would tell against such an orderly aggregation. dggregation into viruslike threads might be expected were the protein precipit,ated on the acid instead of the alkaline side of its isoelectric point, but we have not tested this. When sap from plants infected with TMV is centrifuged at high speed, l-5 % of the mat’erial serologically related to the virus fails to sediment into a pellet. This, the X-protein of Taknhashi and Ishii (1952), has the sameelectrophoretic mobility as the protein produced by disrupting the virus with alkali and resemblesit in many other ways. Small componeiits, with mobilities of -0.2 and -0.4 p/set/volt/cm, are sometimes detectable electrophoret.ically in sap from plants infected with PVX (Bnwden and Kleczkowski, 1957), and it seemedthat these might be analogous to the unsedimentable material in sap from plants infected with TMV. We have some results suggesting this to be so, but the anomalous material in sap from plants infected with PVX occurs in too small amounts and its behavior is t,oo erratic for any exact, work on its properties or composition. The amount of such material varies greatly in sap from different lots of plants; none has been detectable in many samples, and in some samples there has been only one small peak with mobility of -0.15 to -0.2 p/set/volt/cm. As this is similar to the mobility of protein produced by disrupting PVX with alkali, it seemsreasonable to assumethat the component is also virus protein,
382
BAWDEN
AND TABLE
INFECTIVITY
OF
PVX MADE
PVX PVX PVX
acid acid acid
undiluted diluted diluted
1
AND OF A NUCLEIC ACID FROM IT WITH PHENOL” Nondialyzable cleic acid hdl)
Inoculum
Nucleic Nucleic Nucleic
KLECZKOWSKI
1:5 1:25
at 200 mg/l at 40 mg/l at 8 mg/l
PREPARATION
nu-
Numbers of local lesions on 8 half-leaves
35 7 1.4
132 15 2
10 2 0.4
1390 530 64
a The nucleic acid was prepared from a 1% solution of the PVX used as inoculum in the experiment. By plotting the numbers of lesions formed by PVX against logs of its concentration, interpolation shows that infectivity of nucleic acid at 35 mg/l equals that of about 10 mg/l PVX, which corresponds to about 0.5 mg of virus nucleic acid per liter. Thus, considering infectivity in relation to nucleic acid contents, the isolated nucleic acid was about 1.47, as infective as the intact virus.
Nucleic Acid Prepared with Phenol Nucleic acid preparations freshly made by treating PVX with phenol are, as Table 1 shows, often infective. Indeed, the infectivity of some preparations compares favorably per unit weight of phosphorus with that of similarly made nucleic acid from TMV, but, as in much other work with PVX, results are more variable. The greater difficulty of drawing any firm conclusions about the infectivity of nucleic acid preparations from PVX comes partly from the greater difficulty of making reliable infectivity tests, partly because it is impossible to start with such concentrated virus preparations as with TMV, and partly because the yield of nucleic acid that fails to pass through a dialysis sack is very much smaller. With tobacco mosaic virus, almost all the original nucleic acid of the virus can be obtained in a form that remains in the sack after 24hr dialysis at 2” against M/15 pH 7.0 phosphate buffer, whereas with PVX only 5-7 % remains. On the assumption that the infectivity of the freshly made preparations lies in the undialyzable material, the nucleic acid preparations from PVX had infectivities of up to about 1.5 % of infectivity of the intact virus per unit weight of nucleic acid. This is slightly higher than the corresponding figure with nucleic acid from
DECOMPOSITION
PRODUCTS
OF
POTATO
VIRUS
X
383
TMV, which is usually below 1%. The significance of statements about the relative infectiviCy of nucleic acid preparaCons and of intact virus, however, is uncertain. With TMV the results of such comparisons depend not only on the relative concentrations at which the two inocula are used, but also on the physiological state of the plants inoculated (Bawden and Pirie, 1957), and this may be equally true wit,h PVX. The UV absorption spectra of t’he phenol-made preparations are t’ypical for nucleic acid and resemble those given by nucleic acid separat,ed electrophoretically from PVX split by alkali (Fig. 1). This shows that the preparations are predominantly nucleic acid, but does not exclude the possibility that t,heir infectivity lies in residual virus, for contamination with enough virus t,o confer their infectivit,y would not noticeably distort the absorption spectrum. Evidence from other sources, however, seems ho exclude this possibility. First’, t’he infectivity of preparations made from 1% PVX solutions are about as infective as the original virus preparations at, 5 mg/l, at which concentration the characteristic threadlike virus particles are readily detectable in the electron microscope, whereas none was seen in the many phenol-made preparations examined. Secondly, but less conclusive, the nucleic acid preparations give no visible precipitate with PVX antiserum, whereas intact virus preparations precipitate down to concentrat’ions of 0.5 mg/‘l or less. Thirdly, and perhaps the best evidence bhat t’he infectivity resides in material other than residual virus particles, is its instability; incubation for 20 minutes at 20” at pH 7 with beef pancreatic ribonuclease at 0.05 mg/l, a treatment without effect on the infectivity of intact PVX, destroys the infectivity of t’he phenol-made preparations; also, bheir infectivity always falls great,ly and is usually abolished by keeping preparations for a day at 20” and pH 7. There is adequate evidence to conclude t’hat material other than intact PVX is responsible for the infectivity of these preparations and that the nucleic acid is an essential part of this material, but the evidence does not prove that the nucleic acid alone is infective. This is the simplest, and a plausible conclusion, but it is equally possible that infectivity is carried by particles that contain nucleic acid combined with something else, perhaps a small amount of protein or a polypeptide. However, this degree of uncertainty also still surrounds the infectivity possessedby preparations of TMV nucleic acid, which is a more favorable subject for experiments to decide the precise chemical identity of the minimal infective unit.
384
BAWDEN
AND
KLECZKOWSKI
ACKNOWLEDGMENTS We thank Mr. H. L. Nixon scope examinations.
and Mr.
R. D. Woods
for making
the electron
micro-
REFERENCES BAWDEN, F. C., and KLECZKOWSKI, A. (1957). An electrophoretic study of sap from uninfected and virus-infected tobacco plants. Virology 4, 26-40. BAWDEN, F. C., and PIRIE, N. W. (1957). The activity of fragmented and reassembled tobacco mosaic virus. J. Gen. Microbial. 17, 80-95. GIERER, A., and SCHRAMM, G. (1956). Infectivity of ribonucleic acid from tobacco mosaic virus. Nature 177, 702-703. KLECZKOWSKI, A. (1959). Aggregation of the protein of tobacco mosaic virus with and without combination with the virus nucleic acid. Virology 7, 385 (1959). KLECZKOWSKI, A., and NIXON, H. L. (1950). An electron-microscope study of potato virus X in different states of aggregation. J. Gen. Microbial. 4,220-224. KRAMER, E., and WITTMAN’N, H. G. (1958). Elektrophoretische Untersuchungen der A-Proteine dreier Tabakmosaikvirus-Stimme. Z. Naturjorsch. 13b, 30-33. SCHRAMM, G., SCHUMACHER, G., and ZILLIG, W. (1955). tfber die Struktur des Tabakmosaikvirus. III. Mitt.: Der Zerfall in alkalischer Liisung. Z. Naturjorsch. lob, 481-492. TAKAHASHI, W. N., and ISHII, M. (1952). An abnormal protein associated with tobacco mosaic virus infection. Nature 169, 419420.