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Observation on negatively stained tobacco ringspot virus and its two RNA-deficient components
VIROLOGY
39,
it%-239
(1969)
Observation Virus
on Negatively and
of Plant
Tobacco
Its Two RNA-Deficient
E. M. DAVISON1 Department
Stained
Ringspot
Components
R. I. B. FRANCKI
AND
Pathology, Waite Agricultural Research Institute, Adelaide, South Australia
University
of
Accepted May 26, 1969
Unpenetrated, partly penetrated, penetrated, and disrupted particles were observed in preparations of tobacco ringspot virus and its two RNA-deficient components after negative staining with either phosphotungstic acid (PTA) or many1 acetate (UAc). The proportion of particles penetrated by PTA increased with increasing pH of the stain, but time of staining had no effect. RNA-containing particles were positively stained after long periods in UAc, followed by thorough washing. These observations show that unpenetrated particles observed in PTA stained preparations of TRSV do not necessarily represent particles devoid of RNA. INTRODUCTION
Since Brenner and Horne (1959) introduced negative staining, it has been used extensively to elucidate the structure of viruses. Phosphotungstic acid (PTA), at neutral pH, has been most commonly used; this outlines virus particles and may penetrate areas not occupied by structural elements (Horne and Wildy, 1963). That the stain penetrates particles in preparations of small polyhedral viruses has sometimes been interpreted as evidence that these are devoid of nucleic acid (Brenner and Horne, 1959). But virus preparations shown by examination in the analytical ultracentrifuge to be free of such “empty” particles may contain numerous particles penetrated by PTA (Markham, 1962). Thus, penetrated particles may be artifacts produced during preparation of virus for electron microscopy. Tobacco ringspot virus (TRSV) B component contains about 35% RNA, and the RI component about 21%, but the T component contains no RNA (State-Smith et al., 1965; Randles and Francki, 1965); however, 1 Present address: Botanic Gardens, Adelaide, S. Australia.
all three components appear to have cal protein shells (State-Smith et al., Here we report on the appearance of and its RNA-deficient components stained with PTA and uranyl acetate under various conditions. MATERIALS
identi1965). TRSV when (UAc)
AND METHODS
PuriJication and separation of components. The isolate of TRSV obtained from Gladiolus and purified as described by Randles and Francki (1965) was used throughout this study. The three components T, M, and B were separated by centrifugation in linear density-gradients containing 5-25 % (w/v) sucrose in 0.02 M phosphate buffer, pH 7.2, for 2.5 hours at 23,000 rpm in a Spinco Model L ultracentrifuge using an SW 25.1 rotor. The components were isolated from the centrifuged gradients with an ISCO density
gradient
fractionator.
The fractions
used for electron microscopy were dialysed against 0.005 M borate buffer, pH 9, and concentrated
by centrifugation
at 226,000
g for 1.5 hours. The level of contamination of the separated components was found, by recentrifugation in density gradients, to be less than 5% by weight. Electron microscopy. Copper grids covered 235
236
DAVISON
AND
with carbon-stabilized Formvar membrsnes were floated on a drop of a component preparation and transferred to a drop of 2% (w/v) PTA or UAc for 30 set or longer. The pH of the PTA was adjusted with KOH solution; the UAc was used as an aqueous solution, pH about 4.5. With staining periods longer than 5 min, the grids were prepared in a humid chamber. To wash out the stain, the grids were transferred through a series of 10 drops of distilled water on a glass slide. The grids were examined in a Siemens Elmiskop 1 electron microscope. In each experiment, one field from each of two replicate treatments was photographed at a magnification of X 80,000. The numbers of unpenetrated, partly penetrated and penetrated particles were counted and analyzed by linear regression. The diameters of particles in the centres of photographs were measured. RESULTS
Preparations Stained with PTA Electron micrographs of T, M, and B components stained with PTA, pH 7 for 30 set, are presented in Figs. l-3; these show unpenetrated (A), partially penetrated (B),
FRANCKI
and penetrated particles (C), together with disrupted particles (D). With T and B components the proportion of unpenet,rabed and penetrated particles depended on the pH of the stain (Fig. 4). At pH 8 and 9 staining was very uneven and particles were difficult to recognize, thus these results are not included in Fig. 4. The uneven staining above pH 7 may possibly be due to the hydrolysis of PTA which is only stable under acid or neutral conditions (Hiickel, 1950). Analyses of the data (Fig. 4) show that although the effect of stain was similar in T and B components the proportion of particles susceptible to penetration was about 20 % higher in T than in B component. Both T and B differed significantly from M component. The proportion of particles which were partially penetrated did not differ significantly when preparations were stained at different pH’s, being less than 10 % in preparations of M and B components and lo-20% in preparations of T component. No increase in the proportions of unpenetrated, partly penetrated or penetrated particles in any of the components could be detected in preparations stained for 30 see to 30 min.
FIGS. l-3. TRSV components negatively stained with PTA, pH 7, for 30 sec. Fig. 1-B component; Fig. 2-M component; and Fig. 3-T component. A, unpenetrated; B, partly penetrated; C, penetrated; and D, disrupted particles.
NEGATIVE
STAINING
OF TOBACCO
RINGSPOT
VIRUS
B
237
T
-A!
4’ as108.2 b= -6.6
? 5.30 ? 1.02
a= 96.1: b= -1.7:
5.40 1.04
a= 95.9 t 7.16 b= -6.9 t 1.36
I a= b=
-l&.2? 3.60 6.2 + 0.69
a,
a=-0.7t2.99 b= 1.3:
10.5:
b=
0.57
4.75
4.7t0.91
I
0
-I 3
4
5
6
7
f
l
:
T-y-
3
4
5
6
7
3
4
5
6
7
PH FIG. 4. Effect of pH of PTA on the proportion of unpenetrated and penetrated particles in the B, M, and T, components of TRSV. These graphs combine the results from two virus preparations, each point representing the mean of two experiments.
When a preparation of B component was stained in PTA, pH 4 or 7, for 4 hours and then washed in water, all the stain was removed from the particles. The mean diameter of particles from all three components, when stained between pH 4 and 7, was about 27 rnp. In PTA at pH 9 the particles appeared swollen, showing a 10% increase in diameter. However, these figures were based on relatively few measurements as at both pH 8 and 9, in addition to the uneven staining already mentioned, many particles were disrupted. Stability
of TRSV
Components in
PTA
Although no differences in the proportions of unpenetrated, partly penetrated and penetrated particles were observed with increasing staining time, this could have been due to steady disruption of the particles, partly penetrated, and penetrated particles being
envisaged as stages of degradation. Disrupted particles were always observed in micrographs but no quantitative assessment of their proportion was made as only those with relatively little damage could be identified with certainty. To test the stability of the B component in PTA, samples of virus were diluted with an equal volume of 2 % (w/v) phosphotungstic acid at pH 6,7,8, and 9 or with distilled water. These mixtures were incubated at 25” for 2.5 hours and then subjected to density gradient centrifugation as described above. As judged by the position, shape, and area under the neaks obtained from the density gradient fraitionator, the virus was not affected by incubation with PTA between pH 6 and 8. However, virus (B component) incubated at pH 9 produced a peak of area only half that of the control, indieating a loss of normal virus particles.
DAVISON
238
AND FRANCKI
FIG. 5-7. TRSV-B component stained with UAc. Fig. s-stained for 30 sec.; Fig. 6-stained for 4 hours (not washed); and Fig. ‘I-stained for 2 hours and washed. A, unpenetrated; B, partly penetrated; C, penetrated; and D, disrupted particles. TABLE 1 PENETRATION OF TRSV COMPONENTS BY NEGATIVE STAINS
Penetration
B component y0 PTA (pH7)
Unpenetrated Partlypenetrated Penetrated
T component y0
M component y.
UAc &$j
UAc CDR’i
UAc
62
67
84
75
38
30
9
13
7
17
18
30
29
20
9
8
44
40
Effect of pH on the Solubility ponents
of TRSV Com-
Preparations of B, M, and T components were incubated for 1 hour at room temperature in 0.1 M acetate buffers ranging from pH 3.8 to 7. Spectrophotometric analysis at 230-300 rnp (Francki, 196S), showed greatly increased light scattering between pH 4.2 and 6.1. This was probably due to precipitation of the components and indicated that the isoelectric point of all TRSV components lies in this pH range. Preparations Stained with UAc Unpenetrated, partly penetrated, and penetrated particles were also observed in electron micrographs of all three components of TRSV stained with aqueous UAc, pH 4.5 (Fig. 5). Short staining times, between 30 set and 10 min, had no effect on
the proportions of the various particles, which were similar to the proportions of pa.rticles observed after PTA staining at pH 7 (Table 1). Unpenetrated and penetrated particles were also seen after staining with UAc for 2 to 4 hours (Fig. 6). Although stain surrounding the particles could be washed away, Fig. 7 shows that UAc which had penetrated the capsid could not be removed. This is unlike PTA, which could be completely removed by washing. DISCUSSION
All three components of TRSV were stained by negative contrast with PTA,i rrespective of the pH of the stain and staining time. However, whereas UAc stained by negative contrast after short times, the RNA cores could be positively stained by longer staining followed by thorough washing. Penetrated, partly penetrated and unpenetrated particles were observed in negatively stained preparations of all three components. Thus, our results differ from the uniform penetration or nonpenetration of virus preparations after negative staining with PTA and UAc reported by Breese (1968), Murant et al. (1968), Halperen et aE. (1964) but are similar to those of Markham (1962), who observed irregular staining of B component particles in turnip yellow mosaic virus. As unpenetrated particles in T were not due to contamination with B or M com-
NEGATIVE
STAINING
OF TOBACCO
ponents, unpenetrated particles do not neccessarily contain RNA. If penetrated particles in B and M components are devoid of RNA, loss of RNA must occur while the stain dries, as no liberation earlier in the staining procedure could be detected below pH 9. Penetrated particles stained with UAc are not necessarily devoid of RNA because when B or M components stained for 2 hours or more are washed, the particles have positively stained cores (Fig. 7). The heterogeneity of staining of TRSV components may possibly arise from slight damage during extraction, purification, or preparation for electron microscopy. The increased proportion of penetrated particles in preparations stained with PTA at higher pH may be due to a slight swelling of the capsid which then allows stain to penetrate between the capsomeres. Swelling of virus with increasing pH has been observed with bromegrass mosaic virus (Incardona and Kaesberg, 1964) and cowpea chlorotic mottle virus (Bancroft et al., 1967). Finlay and Teakle (1969), found that several viruses unstable in phosphotungstic acid at pH 7 will remain intact at a lower pH, and they suggested that viruses are least distorted near their isoelectric points. ACKNOWLEDGMENTS We wish to thank Dr. A. C. Jennings for helpful discussions, Mr. J. R. Finlay and Dr. D. S. Teakle for letting us see their manuscript prior to publication, Mrs. Margaret Atkinson for help with statistical analyses, Messrs. D. Coleman and C. Grivell for technical assistance, and Mrs. L. Wichman for preparation of the line diagram. This work was supported by a grant from the Rural Credits Development Fund of the Reserve Bank of Australia. REFERENCES BANCROFT, J. B., HILLS, G. J., and MARKHAM,
R. (1967). A study of the self-assembly process in a
RINGSPOT
VIRUS
239
small spherical virus. Formation of organized structure from protein subunits in vitro. ViroZogy 31, 354-379. BREESE, S. S. (1968). Observations on complete and empty capsids of foot-and-mouth disease virus. J. Gen. Viral. 2, 46.5468. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103-110. FINLAY, J. R., and TEAKLE, D. S. (1969). The effect of pH on the particle stability of a phosphotungstate-stained tobacco necrosis virus. J.Gen. Microbial. (in press). FRANCKI, R. I. B. (1968). Inactivation of cucumber mosaic virus (Q strain) nucleoprotein by pancreatic ribonuclease. Virology 34, 694-700. HALPEREN, S., EGGERS,H. J., and TAMM, I. (1964). Complete and coreless hemagglutinating particles produced in ECHO 12 virus-infected cells. Virology 23, 81-89. HORNE, R. W., and WILDY, P. (1963). Virus structure revealed by negative staining. Advan. Virus Res. 10, 101-170. H~~CKEL, W. (1950). “Structural Chemistry of Inorganic Compounds,” Vol. 1, p. 183. Elsevier, Amsterdam. INCARDONA, N. L., and KAESBERG, P. (1964). A pH-induced structural change in bromegrass mosaic virus. Biophys. J. 4, 11-12. MARKHAM, R. (1962). The analytical ultracentrifuge as a tool for the investigation of plant viruses. Advan. Virus Res. 9, 24-270. MURANT, A. F., TAYLOR, C. E., and CHAMBERS,J. (1968). Properties, relationships and transmission of a strain of raspberry ringspot virus infecting raspberry cultivars immune to the common Scottish strain. Ann. Appl. Biol. 61, 175 186. RANDLES, J. W., and FRANCKI, R. I. B. (1965). Some properties of a tobacco ringspot virus isolate from South Australia. Australian J. Biol. Sci. 18. 979-986. STACE-SMITH, R., REICHMANN, M. E., andWRIGHT, N. S. (1965). Purification and properties of tobacco ringspot virus and two RNA-deficient components. Virology 25, 487-494.
39,
it%-239
(1969)
Observation Virus
on Negatively and
of Plant
Tobacco
Its Two RNA-Deficient
E. M. DAVISON1 Department
Stained
Ringspot
Components
R. I. B. FRANCKI
AND
Pathology, Waite Agricultural Research Institute, Adelaide, South Australia
University
of
Accepted May 26, 1969
Unpenetrated, partly penetrated, penetrated, and disrupted particles were observed in preparations of tobacco ringspot virus and its two RNA-deficient components after negative staining with either phosphotungstic acid (PTA) or many1 acetate (UAc). The proportion of particles penetrated by PTA increased with increasing pH of the stain, but time of staining had no effect. RNA-containing particles were positively stained after long periods in UAc, followed by thorough washing. These observations show that unpenetrated particles observed in PTA stained preparations of TRSV do not necessarily represent particles devoid of RNA. INTRODUCTION
Since Brenner and Horne (1959) introduced negative staining, it has been used extensively to elucidate the structure of viruses. Phosphotungstic acid (PTA), at neutral pH, has been most commonly used; this outlines virus particles and may penetrate areas not occupied by structural elements (Horne and Wildy, 1963). That the stain penetrates particles in preparations of small polyhedral viruses has sometimes been interpreted as evidence that these are devoid of nucleic acid (Brenner and Horne, 1959). But virus preparations shown by examination in the analytical ultracentrifuge to be free of such “empty” particles may contain numerous particles penetrated by PTA (Markham, 1962). Thus, penetrated particles may be artifacts produced during preparation of virus for electron microscopy. Tobacco ringspot virus (TRSV) B component contains about 35% RNA, and the RI component about 21%, but the T component contains no RNA (State-Smith et al., 1965; Randles and Francki, 1965); however, 1 Present address: Botanic Gardens, Adelaide, S. Australia.
all three components appear to have cal protein shells (State-Smith et al., Here we report on the appearance of and its RNA-deficient components stained with PTA and uranyl acetate under various conditions. MATERIALS
identi1965). TRSV when (UAc)
AND METHODS
PuriJication and separation of components. The isolate of TRSV obtained from Gladiolus and purified as described by Randles and Francki (1965) was used throughout this study. The three components T, M, and B were separated by centrifugation in linear density-gradients containing 5-25 % (w/v) sucrose in 0.02 M phosphate buffer, pH 7.2, for 2.5 hours at 23,000 rpm in a Spinco Model L ultracentrifuge using an SW 25.1 rotor. The components were isolated from the centrifuged gradients with an ISCO density
gradient
fractionator.
The fractions
used for electron microscopy were dialysed against 0.005 M borate buffer, pH 9, and concentrated
by centrifugation
at 226,000
g for 1.5 hours. The level of contamination of the separated components was found, by recentrifugation in density gradients, to be less than 5% by weight. Electron microscopy. Copper grids covered 235
236
DAVISON
AND
with carbon-stabilized Formvar membrsnes were floated on a drop of a component preparation and transferred to a drop of 2% (w/v) PTA or UAc for 30 set or longer. The pH of the PTA was adjusted with KOH solution; the UAc was used as an aqueous solution, pH about 4.5. With staining periods longer than 5 min, the grids were prepared in a humid chamber. To wash out the stain, the grids were transferred through a series of 10 drops of distilled water on a glass slide. The grids were examined in a Siemens Elmiskop 1 electron microscope. In each experiment, one field from each of two replicate treatments was photographed at a magnification of X 80,000. The numbers of unpenetrated, partly penetrated and penetrated particles were counted and analyzed by linear regression. The diameters of particles in the centres of photographs were measured. RESULTS
Preparations Stained with PTA Electron micrographs of T, M, and B components stained with PTA, pH 7 for 30 set, are presented in Figs. l-3; these show unpenetrated (A), partially penetrated (B),
FRANCKI
and penetrated particles (C), together with disrupted particles (D). With T and B components the proportion of unpenet,rabed and penetrated particles depended on the pH of the stain (Fig. 4). At pH 8 and 9 staining was very uneven and particles were difficult to recognize, thus these results are not included in Fig. 4. The uneven staining above pH 7 may possibly be due to the hydrolysis of PTA which is only stable under acid or neutral conditions (Hiickel, 1950). Analyses of the data (Fig. 4) show that although the effect of stain was similar in T and B components the proportion of particles susceptible to penetration was about 20 % higher in T than in B component. Both T and B differed significantly from M component. The proportion of particles which were partially penetrated did not differ significantly when preparations were stained at different pH’s, being less than 10 % in preparations of M and B components and lo-20% in preparations of T component. No increase in the proportions of unpenetrated, partly penetrated or penetrated particles in any of the components could be detected in preparations stained for 30 see to 30 min.
FIGS. l-3. TRSV components negatively stained with PTA, pH 7, for 30 sec. Fig. 1-B component; Fig. 2-M component; and Fig. 3-T component. A, unpenetrated; B, partly penetrated; C, penetrated; and D, disrupted particles.
NEGATIVE
STAINING
OF TOBACCO
RINGSPOT
VIRUS
B
237
T
-A!
4’ as108.2 b= -6.6
? 5.30 ? 1.02
a= 96.1: b= -1.7:
5.40 1.04
a= 95.9 t 7.16 b= -6.9 t 1.36
I a= b=
-l&.2? 3.60 6.2 + 0.69
a,
a=-0.7t2.99 b= 1.3:
10.5:
b=
0.57
4.75
4.7t0.91
I
0
-I 3
4
5
6
7
f
l
:
T-y-
3
4
5
6
7
3
4
5
6
7
PH FIG. 4. Effect of pH of PTA on the proportion of unpenetrated and penetrated particles in the B, M, and T, components of TRSV. These graphs combine the results from two virus preparations, each point representing the mean of two experiments.
When a preparation of B component was stained in PTA, pH 4 or 7, for 4 hours and then washed in water, all the stain was removed from the particles. The mean diameter of particles from all three components, when stained between pH 4 and 7, was about 27 rnp. In PTA at pH 9 the particles appeared swollen, showing a 10% increase in diameter. However, these figures were based on relatively few measurements as at both pH 8 and 9, in addition to the uneven staining already mentioned, many particles were disrupted. Stability
of TRSV
Components in
PTA
Although no differences in the proportions of unpenetrated, partly penetrated and penetrated particles were observed with increasing staining time, this could have been due to steady disruption of the particles, partly penetrated, and penetrated particles being
envisaged as stages of degradation. Disrupted particles were always observed in micrographs but no quantitative assessment of their proportion was made as only those with relatively little damage could be identified with certainty. To test the stability of the B component in PTA, samples of virus were diluted with an equal volume of 2 % (w/v) phosphotungstic acid at pH 6,7,8, and 9 or with distilled water. These mixtures were incubated at 25” for 2.5 hours and then subjected to density gradient centrifugation as described above. As judged by the position, shape, and area under the neaks obtained from the density gradient fraitionator, the virus was not affected by incubation with PTA between pH 6 and 8. However, virus (B component) incubated at pH 9 produced a peak of area only half that of the control, indieating a loss of normal virus particles.
DAVISON
238
AND FRANCKI
FIG. 5-7. TRSV-B component stained with UAc. Fig. s-stained for 30 sec.; Fig. 6-stained for 4 hours (not washed); and Fig. ‘I-stained for 2 hours and washed. A, unpenetrated; B, partly penetrated; C, penetrated; and D, disrupted particles. TABLE 1 PENETRATION OF TRSV COMPONENTS BY NEGATIVE STAINS
Penetration
B component y0 PTA (pH7)
Unpenetrated Partlypenetrated Penetrated
T component y0
M component y.
UAc &$j
UAc CDR’i
UAc
62
67
84
75
38
30
9
13
7
17
18
30
29
20
9
8
44
40
Effect of pH on the Solubility ponents
of TRSV Com-
Preparations of B, M, and T components were incubated for 1 hour at room temperature in 0.1 M acetate buffers ranging from pH 3.8 to 7. Spectrophotometric analysis at 230-300 rnp (Francki, 196S), showed greatly increased light scattering between pH 4.2 and 6.1. This was probably due to precipitation of the components and indicated that the isoelectric point of all TRSV components lies in this pH range. Preparations Stained with UAc Unpenetrated, partly penetrated, and penetrated particles were also observed in electron micrographs of all three components of TRSV stained with aqueous UAc, pH 4.5 (Fig. 5). Short staining times, between 30 set and 10 min, had no effect on
the proportions of the various particles, which were similar to the proportions of pa.rticles observed after PTA staining at pH 7 (Table 1). Unpenetrated and penetrated particles were also seen after staining with UAc for 2 to 4 hours (Fig. 6). Although stain surrounding the particles could be washed away, Fig. 7 shows that UAc which had penetrated the capsid could not be removed. This is unlike PTA, which could be completely removed by washing. DISCUSSION
All three components of TRSV were stained by negative contrast with PTA,i rrespective of the pH of the stain and staining time. However, whereas UAc stained by negative contrast after short times, the RNA cores could be positively stained by longer staining followed by thorough washing. Penetrated, partly penetrated and unpenetrated particles were observed in negatively stained preparations of all three components. Thus, our results differ from the uniform penetration or nonpenetration of virus preparations after negative staining with PTA and UAc reported by Breese (1968), Murant et al. (1968), Halperen et aE. (1964) but are similar to those of Markham (1962), who observed irregular staining of B component particles in turnip yellow mosaic virus. As unpenetrated particles in T were not due to contamination with B or M com-
NEGATIVE
STAINING
OF TOBACCO
ponents, unpenetrated particles do not neccessarily contain RNA. If penetrated particles in B and M components are devoid of RNA, loss of RNA must occur while the stain dries, as no liberation earlier in the staining procedure could be detected below pH 9. Penetrated particles stained with UAc are not necessarily devoid of RNA because when B or M components stained for 2 hours or more are washed, the particles have positively stained cores (Fig. 7). The heterogeneity of staining of TRSV components may possibly arise from slight damage during extraction, purification, or preparation for electron microscopy. The increased proportion of penetrated particles in preparations stained with PTA at higher pH may be due to a slight swelling of the capsid which then allows stain to penetrate between the capsomeres. Swelling of virus with increasing pH has been observed with bromegrass mosaic virus (Incardona and Kaesberg, 1964) and cowpea chlorotic mottle virus (Bancroft et al., 1967). Finlay and Teakle (1969), found that several viruses unstable in phosphotungstic acid at pH 7 will remain intact at a lower pH, and they suggested that viruses are least distorted near their isoelectric points. ACKNOWLEDGMENTS We wish to thank Dr. A. C. Jennings for helpful discussions, Mr. J. R. Finlay and Dr. D. S. Teakle for letting us see their manuscript prior to publication, Mrs. Margaret Atkinson for help with statistical analyses, Messrs. D. Coleman and C. Grivell for technical assistance, and Mrs. L. Wichman for preparation of the line diagram. This work was supported by a grant from the Rural Credits Development Fund of the Reserve Bank of Australia. REFERENCES BANCROFT, J. B., HILLS, G. J., and MARKHAM,
R. (1967). A study of the self-assembly process in a
RINGSPOT
VIRUS
239
small spherical virus. Formation of organized structure from protein subunits in vitro. ViroZogy 31, 354-379. BREESE, S. S. (1968). Observations on complete and empty capsids of foot-and-mouth disease virus. J. Gen. Viral. 2, 46.5468. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of viruses. Biochim. Biophys. Acta 34, 103-110. FINLAY, J. R., and TEAKLE, D. S. (1969). The effect of pH on the particle stability of a phosphotungstate-stained tobacco necrosis virus. J.Gen. Microbial. (in press). FRANCKI, R. I. B. (1968). Inactivation of cucumber mosaic virus (Q strain) nucleoprotein by pancreatic ribonuclease. Virology 34, 694-700. HALPEREN, S., EGGERS,H. J., and TAMM, I. (1964). Complete and coreless hemagglutinating particles produced in ECHO 12 virus-infected cells. Virology 23, 81-89. HORNE, R. W., and WILDY, P. (1963). Virus structure revealed by negative staining. Advan. Virus Res. 10, 101-170. H~~CKEL, W. (1950). “Structural Chemistry of Inorganic Compounds,” Vol. 1, p. 183. Elsevier, Amsterdam. INCARDONA, N. L., and KAESBERG, P. (1964). A pH-induced structural change in bromegrass mosaic virus. Biophys. J. 4, 11-12. MARKHAM, R. (1962). The analytical ultracentrifuge as a tool for the investigation of plant viruses. Advan. Virus Res. 9, 24-270. MURANT, A. F., TAYLOR, C. E., and CHAMBERS,J. (1968). Properties, relationships and transmission of a strain of raspberry ringspot virus infecting raspberry cultivars immune to the common Scottish strain. Ann. Appl. Biol. 61, 175 186. RANDLES, J. W., and FRANCKI, R. I. B. (1965). Some properties of a tobacco ringspot virus isolate from South Australia. Australian J. Biol. Sci. 18. 979-986. STACE-SMITH, R., REICHMANN, M. E., andWRIGHT, N. S. (1965). Purification and properties of tobacco ringspot virus and two RNA-deficient components. Virology 25, 487-494.