J. gen. Virol. (I978), 4o, 379-389
379
Printed in Great Britain
Physical and Chemical Properties of Cynosurus Mottle Virus
By N. A. M O H A M E D
Plant Diseases Division, D.S.LR., Private Bag, Auckland, New Zealand
(Accepted I March I978)
SUMMARY
Particles of cynosurus mottle virus (CyMV) purified from systemically infected
oat leaves have a sedimentation coefficient of I I3S, buoyant density of 1.399 g [ml
in CsC1 and I'332 in Cs~SO4, diffusion coefficient of o'I3OX IO-l°m2/s and
E0.1%
260 of 7"3. Virus particles are isometric with a diameter of 27 to 29 nm in
negatively stained preparations; the hydrodynamic diameter was calculated from
the D20. w as 32"8 nm. The particle mol. wt. was calculated as 6.86 x io G. CyMV
particles contain 22. 5 °/o single-stranded'RNA of mol. wt. approx. 1"5 × IO~ and
a molar nucleotide composition of: A, 2 4 " 2 % ; G , 24"3%; C, 26"2~o and U,
25.3%; R N A purified by the phenol-SDS method was infectious. The melting
profile of native CyMV-RNA was typical of a single stranded R N A with secondary
structure; the hyperchromicity was 33 % and the Tm 6I °C. The protein mol. wt.
was 3o5oo. The completed cryptogram for CyMV is R ] I : I'5 [22"5 : S/S : S / V e [
Ap.
CyMV particles were degraded by o.02 to o'o5 ~o SDS at p H 7.0 into a component with a sedimentation coefficient of 83S and by o.1% SDS into the R N A and
protein constituents; virus dissociation was enhanced by EDTA and reduced by
MgC12. At pH 5"o, the virus was not affected by SDS concentrations of up to 8 %.
The virus was unstable in E D T A and NaC1 at pH 8"25.
INTRODUCTION
A virus, originally described by A'Brook (I972) as lolium mottle virus, was later renamed
cynosurus mottle virus (CyMV) by Catherall et al. (I 977)- This virus has isometric particles
28 nm in diameter, is widespread in grasses in New Zealand, and is transmitted in a nonpersistent manner by Rhopalosiphum padi L. (Mohamed, I978). It is a potential cause of
economically significant losses in grasses and cereals. This paper describes the physical and
chemical properties of CyMV particles.
METHODS
Source of virus. Two virus isolates were used in this study, one collected from short
rotation ryegrass (Lolium perenne L. x multiflorum L.) and the other from crested dogstail
(Cynosurus cristatus L.); both isolates were propagated in Arena sativa L. cv. Mapua 7o.
Virus was transmitted by inoculation with sap as described by Mohamed (I 978).
Purification. Batches of 20 to 30 g of frozen, systematically infected leaf tissue were
homogenized in a Waring Blendor in IOO ml of a buffer containing o.I M-potassium
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380
N. A. MOHAMED
phosphate, o.ooI M-EDTA and o'5 ~o mercaptoethanol, pH 7"o. The extract was clarified by
emulsification with chloroform and the virus particles concentrated by two cycles of
differential centrifugation. The concentrated virus was further purified by centrifugation
through I o to 40 °/o sucrose density gradients and re-concentrated by high speed centrifugation. The final virus pellet was re-suspended in distilled water to give a virus concentration
of about 2 mg/ml. For calculation of the u.v. extinction coefficient, samples containing
about 2 to 3 mg of virus were dried to constant weight in an oven at lO5 °C. For controls,
equivalent amounts of distilled water were treated in the same way.
Sedimentation coefficient and buoyant density determinations. The sedimentation coefficient
of virus particles was determined in a Beckman Model E analytical ultracentrifuge using
Schlieren optics; the s20. w value was calculated using the graphical method of Markham
(I96O).
The buoyant density of virus particles in caesium chloride was determined by equilibrium
banding in the analytical ultracentrifuge. Virus, at a concentration of o'I mg [ml, was
centrifuged in a solution of CsC1, of starting density I "40 g/ml, in o.oI M-phosphate buffer,
p H 7"o, for 24 h at 29 5oo rev/rain. The buoyant density was calculated by the method o f
Ifft et al. 0961). The buoyant density was also determined in CsC1 and Cs2SO4 in a preparative rotor using a two-step gradient (Brunk & Leick, 1969). A CsC1 solution of density
•3o g/ml was mixed with about I m g of virus and layered on to a CsC1 solution of density
1"5o g/ml. For the Cs2SO4 gradient, a solution of density 1"25 g/ml was mixed with I m g
of virus and layered on to a solution of density 1.45 g/ml. The gradients were then centrifuged for z 4 h at 23 50o rev/min in the swing-out rotor of the MSE z 5 centrifuge.
Diffusion coeffÉcient measurements. Purified virus preparations were dialysed against
distilled water and centrifuged to pellet the virus, and the pellets were re-suspended in
distilled water. The virus suspension was then filtered through millipore filters (pore size
o'45 #m) to remove any dust particles. The diffusion coefficient was determined by laser
self-beating spectroscopy (Harvey, 1973); measurements were taken as described by Farrell
et al. (1974).
Nucleic acid studies. Nucleic acid was extracted from purified virus by the single-phase
phenol-SDS method of Diener & Schneider (1968); virus protein was removed as described
by Rezaian & Francki (I973). Marker RNA species from tobacco leaf and E. coli were
extracted as described by Randles & Coleman (I97O).
Virus and marker nucleic acids were treated at zo °C for ~o min with 5 #g/ml of ribonuclease which had previously been boiled for 5 min. The nucleic acids were also treated
with deoxyribonuclease (2o #g/ml) at 2o °C for lO min.
For infectivity assays, the virus nucleic acid was diluted with Yarwood's buffer (Yarwood,
1972) to give a final concentration of 50o #g [ml and inoculated to oat plants using sterile
gauze pads.
For nucleotide analyses, the nucleic acid preparations were hydrolysed by boiling in
o.I N-HCI in a sealed glass vial for i h. The nucleotides were separated on cellulose-coated
thin-layer plates using a solvent containing isopropanol :HC1 :water ( 7 o : 2 4 : 6 ) and
their ratios determined as described by Markham 0955).
Formylation of virus and E. coli nucleic acid was carried out by heating purified nucleic
acid in o'I5 M-sodium chloride- o'oi5 M-sodium citrate (SSC) buffer with I M-formaldehyde at 63 °C for 15 min (Boedtker, i97I ).
Thermal denaturation profiles were obtained on a Pye-Unicam SP~8oo spectrophotometer with the SPI8o5 temperature programmer. Readings were corrected for thermal
expansion.
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Properties of C y M V
38I
The sedimentation coefficient was determined on linear-log sucrose density gradients
(Brakke & Van Pelt, 197o) containing SSC, and using E. coli 23S and I6S ribosomal R N A
as markers. After layering the nucleic acid on the gradients, they were centrifuged for
16"5 h at 23000 rev/min in the Spinco SW25 rotor. The gradients were then scanned at
254 nm.
Molecular weight of the nucleic acid was determined by polyacrylamide gel electrophoresis using either a slab-gel apparatus (Reid & Bieleski, 1968) and the tris-borateE D T A buffer of Peacock & Dingman (1968), or in cylindrical gels, 0"5 cm diam. × 8 cm,
and the sodium phosphate-SDS buffer of Loening (1968). Gels containing 2.4% acrylamide-o'5~o agarose were used. Nucleic acid samples containing 5 ~o sucrose were layered
on the gels and electrophoresed at 20o V for 2 h (slab gels) or at 3 mA/gel at 42 V for 2 h
(cylindrical gels). After electrophoresis, gels were fixed in lO% trichloroacetic acid for
IO min, rinsed in water and stained in o'2~o toluidine blue for I5 min. Gels were de-stained
overnight in distilled water. The following R N A species were used as mol. wt. markers:
E. coli ribosomal RNA, I.o8 × lO6 and o'56 × lO6 (Loening, I968) and tobacco leaf ribosomal RNA, I'28 × 10 6, I'08 X IO 6, 0"7 x io 6 and 0"56 x lO6 (Loening, 1968).
The phosphorus content of purified virus was estimated by the method of Letham (I 969).
Estimation of protein molecular weight. Virus preparations in buffer containing 5M-urea,
I °/o SDS, 0"5 % mercaptoethanol and 0"005 M-sodium phosphate, p H 7"2, were heated at
Ioo°C for I, 2 or 5 min, or at 37°C for 2 h. Marker proteins, bovine serum albumin
(mol. wt. 68ooo), ovalbumin (43 ooo), lactic dehydrogenase (36ooo), papain (23 ooo) and
cytochrome c (I 16oo), were boiled in the buffer for 2 rain. Virus protein was carboxymethylated as described by Matthews (1974). Five to ten #1 of protein preparation containing 5 % sucrose and approx. I m g / m l protein were layered on top of cylindrical 6 % or
lO% polyacrylamide gels and electrophoresed at 5 mA/gel at 3o V for 6 h. The electrophoresis buffer contained o'5 K-urea, o.1% SDS and o-1 M-sodium phosphate, p H 7-2.
Gels were stained in o.o2 % Coomassie blue in a solution of methanol : acetic acid : water
(I5 : 2 : 33) for 2 h at 37 °C and de-stained in the solvent overnight at 37 °C.
Electron microscopy. Virus preparations were negatively stained for 2 min with 2 % PTA
at pH 7"o, z % PTA at phi 4"8, 2 % uranyl acetate or 2 % uranyl formate; after staining,
grids were examined in a JEOL IooB electron microscope.
Effect of SDS on virus stability. Virus preparations in distilled water were mixed with
buffer (sodium acetate at pH 5"o or sodium phosphate at p H 7"o) to a final concentration
o f o-I M-buffer, and SDS was added to give the required concentration. The mixture was
left at 2o °C for I h before centrifuging at 295oo rev/min in the analytical ultracentrifuge;
a control at the appropriate pH, but without SDS, was used in every run.
RESULTS
Properties of the virus particles
Purified virus preparations in o.oi M-phosphate, pH 7"o, or in distilled water showed a
single peak in sucrose density gradients and in the analytical ultracentrifuge. No host
contaminants were detected by examination of purified preparations in the electron microscope, by electrophoresis of virus protein on polyacrylamide gels, or by serology. Yields
of virus were: I m g per 5 g leaf tissue for an isolate causing a severe mottle in oats, and
I mg per 3 g leaf for an isolate causing a mild mottle in oats.
In distilled water, purified virus had an absorption spectrum with a maximum at 258 nm
and a minimum at 239 nm. The Amx/A=i~ ratio was 1"31 __0"02 and the A~oo/A~8o ratio
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382
N.A. MOHAMED
Fig. ~. Electron micrograph of CyMV particles stained with uranyl formate.
was 1.6~ _+0.02. The absorption coefficient at 260 nm (T mg/ml, i cm light path), determined
on four different preparations, was 7"28 + o'I 1 (not corrected for light scattering).
The sedimentation coefficient of CyMV in either distilled water, o.o~ M-phosphate at
p H 7"0, or o.I M-sodium acetate buffer at p H 5'0, was II3S. This value was calculated
using turnip yellow mosaic virus (~ I4S) as a marker. Sedimentation rates were also measured in distilled water using virus concentrations of 0.8, 0'6, 0"4 and o.2 mg/ml; the s20.,,,
extrapolated to infinite dilution, was I ~oS. There was some effect of virus concentration on
sedimentation rate and the rates ranged from 116S for virus at o.8 m g / m l to I IzS for virus
at o.2 mg/ml.
The buoyant density of CyMV in CsC1, determined by analytical ultracentrifugation,
was 1-399 + o'oo4 g / m l (average of 4 replicates). When virus was fixed in 4 % formaldehyde
before centrifugation, the density was not affected. The buoyant density in CsC1, determined
in a preparative run using a two-step gradient, was I-4oo g / m l both for untreated virus and
for virus previously fixed in 1% formaldehyde. In Cs2SO4, CyMV formed a single band with
a buoyant density of 1.332 g/ml.
Electron microscopy
Particle structure was best shown when virus preparations were stained with uranyl
formate (Fig. I). Virus particles were isometric with a diam. of 28 nm; some particles were
penetrated by stain and others not. More penetrated particles were observed in the uranyl
salt stains than with P T A at p H 7"o or 4"8.
Diffusion coefficient
The diffusion coefficient of CyMV in distilled water, was o"13o4 +0"0039 x io -l° m2/s.
These are the average values for 2 runs, 20 measurements being made each time; the statistical error in each run was 3 %- The coefficient of the quadratic terms was less than o.2 %
of that of the linear term, indicating that the suspension of virus was monodisperse and free
of contaminating materials (Harvey, I973).
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Properties of C y M V
40
I
383
I
I
60
Temperature CC)
80
30
E
.o
20
K
10
0 t"'--<~'-e~'v----------&
2O
40
! 00
Fig. 2. Thermal denaturation profiles of CyMV-RNA in SSC buffer at pH 7"0 for:
0 - - - 0 , native R N A and © - - O , formylated RNA.
Properties of virus nucleic acid
When virus nucleic acid was treated with nucleases and electrophoresed in polyacrylamide
gels, the nucleic acid was digested by ribonuclease but not by deoxyribonuclease in SSC
buffer, indicating that it was a single-stranded RNA. Virus R N A purified by the phenol-SDS
method was used to inoculate 28 oat plants; I4 of these developed systemic symptoms
typical of CyMV, indicating that the nucleic acid was infectious.
The molar base composition of the virus nucleic acid, determined for four different
preparations, was: adenine 24"2 + P3 %, guanine 24"3 + 1.5 %, cytosine 26.2 + 3"I % and
uracil 25"3 + 2"7 %. Phosphorus determinations on eight different virus preparations gave
an average value of 2.I6 % phosphorus in the virus. Assuming a phosphorus content o f
9.6 % in the RNA, the nucleic acid content of the virus was calculated as 22"5 + ['2 %.
Melting profile of CyMV-RNA
The melting curve of native CyMV-RNA in SSC buffer was characteristic of a singlestranded RNA with some secondary structure (Fig. 2) as denaturation occurred over
a broad temperature range (40 to 7o °C) and at a lower temperature than for a doublestranded RNA. The T,,~ value for the R N A was 6~ °C and the hyperchromicity was 33 %.
As a completely base-paired polynucleotide would have about 5o% hyperchromicity
(Doty et al. I959), this figure suggests that 66% of the bases in CyMV-RNA may be involved in base pairing in SSC buffer.
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384
N.A. MOHAMED
2.5
16S
I
23S
•
27S
~,
t
0
l
%%
(b)
17S
0.5
V
,:\
,
I
•
,
I
,~
I
t
\
Sedimentation
Fig. 3. lJ.v. absorption profiles of virus and E. coli marker RNA species centrifuged in Iinear log
sucrose density gradients. (a) Native CyMV (27S) and E. coli RNA; (b) Formaldehyde-treated
CyMV-RNA - note the u.v. absorbing material in top third of gradient.
The melting curve of formaldehyde-treated C y M V - R N A (Fig. 2) indicates that most of
the base-pairing was removed by formylation, the residual hyperchromicity being less than
4%.
Determination o f molecular weights o f nucleic acid
When native virus RNA, prepared by the phenol-SDS method, was electrophoresed on
2"4 % polyacrylamide-agarose gels, only one band was observed. The mol. wt. of the R N A ,
determined on four different virus preparations and six runs, was I'5t x IO6 (_+ 0"o2 x Io~).
C y M V - R N A sedimented as a single peak in linear-log sucrose density gradients (Fig. 3 a)
with a sedimentation coefficient of 27-5S. The rnol. wt. (MW) of the R N A , calculated using
the formula M W = I557 S 2'°r (Hull et al. r969), was P49 × roe; this figure is similar to that
obtained by gel electrophoresis.
Formaldehyde-treated C y M V - R N A sedimented as a single peak but the u.v. scan showed
that there was a considerable amount of u.v.-absorbing material between the virus R N A
peak and the top of the gradient (Fig. 3 b). This suggests that there may be degradation of
the virus R N A on formaldehyde treatment at 63 °C or that hidden nicks in the R N A are
exposed. The sedimentation coefficient was calculated as 17" I S (average of three experiments)
using both native and formaldehyde-treated E. coli r R N A as markers. The mol. wt. (M),
calculated from the formula S = 0-083 M °'as (Brakke & Van Pelt, I97O), was I-2 × IO6,
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Properties of C y M V
385
1
v
V
vv
Electrophoresis
--
Fig. 4. Absorbance scan of formaldehyde-treated CyMV-RNA after electrophoresis on 2
polyacrylamide - o'5 ~ agarose gel and staining. Note main virus RNA band (t) plus minor bands
(arrowed).
On gel electrophoresis in 2 % polyacrylamide gels, formylated virus R N A formed one
major band and a number of minor bands (Fig. 4). The mol. wt. of the major band, calculated using formylated E. coli r R N A markers, was ~'33 × Io~; minor bands had tool. wt.
ranging from o'67 × Io 6 to oq8 × io 6. These results indicate that there is some breakage in
the C y M V - R N A molecule on heating with formaldehyde. The E. coli r R N A markers were
not degraded by formaldehyde treatment. The u.v.-absorbing material at the top of the
gradient in Fig. 3(b) could be the small pieces of R N A resolved on the polyacrylamide gels.
Molecular weight of virus protein
When CyMV particles were treated with urea-SDS-mercaptoethanol and the denatured
proteins electrophoresed on polyacrylamide gels, only one band was present. N o other
bands were detected even when the gels were loaded with large amounts of virus protein.
The mol. wt. was within the range 3o 5oo + 3oo, calculated from ten runs on four different
preparations and two different gel concentrations. No extra peaks or differences in mol. wt.
were evident when virus protein was prepared by heating at Ioo °C for I, z or 5 min, or
at 37 °C for 2 h, or by carboxymethylation.
Virus stability
The effects of high salt concentrations and E D T A on the stability of CyMV particles
were examined at p H 7"0 and p H 8"25. Preparations of virus, in o.I M-sodium phosphate at
p H 7'o or in o.o~ M-tris-HC1, o.~ M-NaC1 at p H 8"25, were treated with o.ooI M-EDTA a n d / o r
I-O M-NaC! at zo °C for ~ h. The preparations were then centrifuged in the analytical
ultracentrifuge at 29 5o0 rev/min. Virus particles were stable in the presence of E D T A or
I "o M-NaCI at p H 7"0 and 8"25 and in E D T A in the presence of NaCI at p H 7"o. At p H 8'25,
however, virus particles were degraded by E D T A in the presence of NaC1.
The stability of the virus particles was further examined by treating them with varying
concentrations of SDS at p H 7"o and 5"o. CyMV in o.I M-sodium phosphate buffer, p H 7"0,
was treated with SDS for r h at 20 °C before centrifugation in the analytical ultracentrifuge.
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386
N.A. MOHAMED
(a)
(d)
Virus
(b)
(e)
',
•
¢
(c)
"..
__.
(f)
83S
[
"-.
26S
",..~. 2 ,
°r
~.
.. /':
i!
;'
r
u~ jf
Sedimentation-~,~
Fig. 5. Tracings of Schlieren patterns of SDS-treated CyMV in o.t M-sodium phosphate buffer,
PH7'o, after 12rain at 295oo rev/min. SDS concentration was (a) o, (b) o'oo5 %, (e) o'oI ~,
(d) 0.02 Voo,(e) 0"05 Yoo,(f) o'1%.
Virus in buffer only was used as a control on every run (Fig. 5a). A low concentration of SDS
(o'oo5%) had no effect on virus stability (Fig. 5b) but at o-oI % SDS, some of the virus
particles were affected resulting in the formation of a slower sedimenting component
(Fig. 5 c). At o.o2 % SDS, about 50 % of the virus was degraded to the slower sedimenting
component (Fig. 5 d) and at o'o5 % SDS, no virus was detected but the slower sedimenting
component and a virus R N A peak were present (Fig. 5 e). At higher concentrations of SDS
( o q % ) , the slower sedimenting component was not detected (Fig. 5 f ) and virus was
degraded completely into the component R N A and protein. The sedimentation coefficient
of the slower sedimenting component was determined as 83S using intact virus 013S) as
a standard; that of the R N A component was 26S which is in fair agreement with the value
determined in sucrose gradients.
CyMV was also treated with SDS in o q M-sodium acetate buffer, p H 5"0 for I h at
2o °C and examined in the analytical ultracentrifuge using virus in buffer as a control.
Increasing concentrations of SDS from o. 1% to 8 % had no effect on virus stability. CyMV
was also incubated at p H 5.o with 2. 5 % SDS for ~ h, 24 h and 48 h and with 2. 5 % SDS
plus o-oI M-EDTA for I h; no degradation of virus particles occurred under any of these
conditions.
Further experiments were carried out to determine the effect of MgCI~ and E D T A on
the stability of CyMV at p H 7"o. As a concentration of o.oz % SDS was found to degrade
5o % of the virus particles to form the 83S component, virus preparations were treated with
o.o2 % SDS in the presence of o.oI M-EDTA or o'o5 M-MgC12. In the presence of EDTA,
virus was completely degraded by o.02 % SDS into the 83S component and R N A . In the
presence of MgCI~, however, the effects of SDS were reduced and only a small fraction of
the virus was degraded. These results show that MgCI 2 stabilizes the virus particles at
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Properties of CyMV
387
pH 7"o whilst EDTA increases virus degradation in the presence of SDS at pH 7'o (Ronald
& Tremaine, I976 ).
DISCUSSION
The object of this study was to determine the physical and chemical properties of CyMV
particles. Using the data presented here, the completed cryptogram for CyMV is R [I :
I'5/22" 5 : S/S : S/Ve/Ap.
Molecular weight determinations of native CyMV RNA gave comparable results by
gel electrophoresis (I'5I x io °) and sedimentation analysis 0"49 x ~o6). This would indicate
that the secondary structure of the RNA did not affect its mobility in gels or its sedimentation in sucrose gradients (Loening, x967). The melting profile, however, showed that there
was considerable secondary structure in the molecule (Fig. 2) which would affect the tool. wt.
determination (Boedtker, I971). Formylation of CyMV-RNA removed almost all the
secondary structure (Fig. 2) and the sedimentation coefficient was reduced by 38 % from
27.5S to I7.1S. The mol. wt. estimates of formylated RNA were 1.2 x IO6 by sedimentation
and ~'33 x io 6 by gel electrophoresis, decreases of 2o % and I2 %, respectively, when compared to the values for native RNA. These estimates, however, cannot be considered reliable
as there is evidence that formalin treatment combined with heating caused breakages in
CyMV-RNA resulting in the formation of a number of lower mol. wt. species (Fig. 3 b
and 4)- Therefore the value for native CyMV-RNA is the most reliable estimate of mol. wt.
available at present.
Using the diffusion coefficient determined for CyMV particles and assuming a value of
o'692 for the partial specific volume (calculated from the protein and nucleic acid composition), the mol. wt. of the virus particle was calculated by the Svedberg equation to be
6.86 x io 6. The tool. wt. of virus RNA, calculated from this value and the RNA content of
the virus (22.5 %), was I'54 x io6; the tool. wt. of the virus protein (assuming 18o subunits)
was 29 5oo. These figures are in agreement with those determined by gel electrophoresis for
the protein (3o 5oo) and from gel electrophoresis and sedimentation analysis for native RNA
(I "5 × 106). Therefore these values seem reliable estimates of mol. wt.
The hydrodynamic diam. of the virus particle, calculated from the diffusion coefficient
using the Stokes-Einstein equation (Harvey, I973), was 32-8 + 0"5 nm. This compares with
a value of 28 nm determined by measurement of negatively-stained particles in the electron
microscope. The discrepancy between these values is probably due to the distortion caused
by staining and drying of particles for examination in the electron microscope. Harvey
(i 973) found similar differences with particles of turnip yellow mosaic virus when measured
in the electron microscope (27 nm) and when calculated from the diffusion coefficient (29"5
rim).
The effects of SDS on CyMV at pH 7"o and 5"o indicate that at the higher pH, virus
particles were SDS sensitive. The slow-sedimenting 83S component, formed as an intermediate in the degradation of virus particles at low SDS concentrations at pH 7"o, may be
a result of the swelling of virus particles caused by binding of SDS to the virion (Ronald &
Tremaine, T976). At the lower pH, CyMV was resistant to SDS even at concentrations of
8 % for I h or 2"5 % for 48 h; this stability was not affected by treatment with EDTA.
The results of these experiments show that CyMV particles are stabilized by different types
of pH dependent bonds. At pH 5"o, particles are stabilized by protein-protein interactions
which are not sensitive to SDS or EDTA. At pH 7"o, however, these protein-protein bonds
do not play a role in virus stability and the particles are stabilized primarily by RNAprotein interactions which are sensitive to SDS. Divalent ions are necessar3 for virus
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N.A.
MOHAMED
stability at p H 7.o in the presence of SDS because E D T A increased the sensitivity of virus to
degradation by SDS while MgC12 enhanced virus stability. The virus particles were not
affected by E D T A - N a C I in the absence of SDS, indicating that bonds involving divalent
cations are of secondary importance at p H 7. At p H 8"25, however, virus particles were
degraded by EDTA-NaC1 indicating that bonds involving divalent cations are of primary
importance in virus stability at that pH.
Boatman & Kaper (1976) and Ronald & Tremaine (1976) attempted to classify simple
isometric viruses by the sensitivity of their particles to SDS at p H 7'o. In their classifications,
CyMV would fall into the first group, which includes viruses sensitive to low concentrations
of SDS. Unlike the majority of viruses in this group, CyMV is totally resistant to SDS at
p H 5"0 and resembles the A strain of cowpea chlorotic mottle virus in this respect (Ronald
& Tremaine, 1976).
On the basis of its physical, chemical and biological properties, CyMV does not belong
to any of the recognized plant virus groups (Fenner, I976 ) but appears to be another of
a heterogeneous.group of small isometric R N A viruses. Although attempts have been made
to classify these viruses into distinct groups (Nelson & Tremaine, 1975; Hull, 1977), there are
as yet insufficient data available for a reliable classification. Hull (1977) classified these
viruses into three groups on the basis of protein mol. wt., sedimentation coefficient, banding
behaviour in Cs2SOa, virus stability, and in vivo particle distribution. He classified lolium
mottle virus (A'Brook, I972) in the phleum mottle virus ( P M ¥ ) group but his data differ
sufficiently from those reported for CyMV in this paper to indicate that they may be
different viruses. CyMV has properties from each of the first two groups of Hull's classification - it forms a single band in Cs2SO4 gradients like members of the P M V group but the
protein mol. wt. is nearer to that of the southern bean mosaic virus (SBMV) group and,
like the latter group, it is also unstable in NaC1-EDTA at p H 8"25. CyMV is also serologically related to cocksfoot mottle virus (Mohamed, I978), a member of the SBMV group
(Hull, I977), but differs in its reaction to SDS at p H 7"0 (Ronald & Tremaine, 1976). The
properties of the viruses classified by Hull (1977) into the P M V and SBMV groups are not
well studied at present and much more information is needed before any worthwhile
classification can be made.
I thank P. D. Scotti, Entomology Division, D.S.I.R., for buoyant density calculations
and Professor E. Collins, Physics Dept, Auckland University for determination of diffusion
coefficients.
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