Stability of Belladonna Mottle Virus Particles: the Role of Polyamines

J. gen. Virol. (1987), 68, 1533-1542. Printed in Great Britain
1533
Key words: BDMV/capsid stability/polyamines
Stability of Belladonna Mottle Virus Particles: the Role of Polyamines
and Calcium
By H A N D A N A H A L S U B B A R A O
S A V I T H R I , 1.
SANJEEV KUMAR MUNSHI,-" SANKURATRI SURYANARAYANA,
SOUNDARDIVAKAR
2 AND M A T H U R R .
N. M U R T H Y 2
t
1Department of Biochemistry and 2Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560 012, India
(Accepted II March 1987)
SUMMARY
The stability of belladonna mottle virus (BDMV) has been studied with respect to
elevated pH and to freezing and thawing. BDMV, purified by a modified procedure,
was stable at alkaline pH, in contradiction to earlier reports. This difference in the
stability could be attributed to the presence of 90 to 140 molecules of spermidine, 20 to
50 molecules of putrescine and 500 to 900 calcium ions in each virus particle. The
polyamines could be easily exchanged with other cations such as potassium or caesium
and this resulted in a loss of particle stability. These cations may therefore play a role in
maintaining the integrity of particle structure. The formation of empty protein shells as
a result of freezing and thawing BDMV particles parallels earlier observations on
turnip yellow mosaic virus particles.
INTRODUCTION
Belladonna mottle virus (BDMV, Moline & Fries, 1974) belongs to the tymovirus group of
monopartite R N A plant viruses. The nucleic acid in these viruses is encapsidated in a T = 3
icosahedral shell of protein subunits with an approximate Mr of 20 000. The type member of this
group, turnip yellow mosaic virus (TYMV) has been extensively studied in terms of the forces
that stabilize the particle structure. The virions of TYMV are stabilized by strong hydrophobic
interactions between the protein subunits (Kaper, 1971, 1975). Neutron scattering experiments
revealed that there is little or no penetration of nucleic acid into the densely packed protein coat
of TYMV (Jacrot et al., 1977). A variety of conditions, such as pH above 11-5, freezing and
subsequent thawing, elevated temperatures around 45 °C or exposure to certain chemical
agents, result in the release of nucleic acid and formation of empty shells from the virions of
TYMV (Kaper, 1960, 1971, 1975; Katouzian-Safadi et al., 1980; Katouzian-Safadi & BerthetColominas, 1983; Keeling & Matthews, 1982). While TYMV has been the subject of many
detailed studies, other members of this group of viruses have been examined in less detail.
Recently, it has been shown that RNA is released from BDMV at alkaline pH but that the
addition of polyamines and cations prevents this release (Virudachalam et al., 1983a, b).
However, BDMV purified in our laboratory by a modified procedure was stable at alkaline pH.
This observation prompted a detailed examination of the stability of BDMV particles. The
results presented in this paper highlight the role of polyamines and other cations in stabilizing
the particle structure and provide an explanation for the instability of BDMV isolated earlier
(Virudachalam et al., 1983a).
METHODS
Isolation ofBDMV. BDMV was propagated in either Nicotiana glutinosa or N. clevelandff or the hybrid N.
clevelandii × N. glutinosa(Christie, 1969)plants. Infected leaves were harvested 10 to 14 days after inoculation and
homogenized in either 0-05 M-sodiumcitrate or 0-05 M-sodiumacetate buffer at pH 5.5. The virus was purified by
0000-7572 © 1987 SGM
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1534
H.S.
SAVITHR! AND OTHERS
differential centrifugation. Empty capsids formed in vivo were separated from intact particles by centrifugation in
a Beckman model L-8 ultracentrifuge for 4 h at 25000 r.p.m, in an SW27 rotor using a 15 to 4 0 ~ (w/v) sucrose
gradient. Subsequently the virus was processed through one more cycle of differential centrifugation and
resuspended in 0-05 M-sodium citrate (or acetate) buffer pH 5-5. Only trace amounts of empty capsids were
detected when the purified virus was examined using a Beckman model-E analytical ultracentrifuge equipped
with Schlieren optics (Fig. 3a, top trace). This virus sample was found to be serologically identical with purified
BDMV kindly supplied by Dr P. Argos, Purdue University, West Lafayette, Ind., U.S.A.
Stability of BDMV at alkaline pH. BDMV (2 to 10 mg/ml) was dialysed against 500 ml of 0.05 M-sodium citrate
pH 5.5 for 18 h at 4 °C for equilibration. The virus solution was then dialysed separately against 500 ml of 0.01 Msodium acetate pH 5-0 or 0.01 M-Tri~HC1, at either pH 7.0 or pH 9-0 for another 48 h with four changes of 500 ml
of the corresponding buffers. The virus samples (400 ktg in 400 p.1)were layered on 10 to 45 ~o (w/v) sucrose density
gradients in the respective buffers prepared for a Beckman SW50.1 rotor and centrifuged at 45000 r.p.m, for 1 h.
The fractions (nine drops collected manually) were analysed by estimating the protein concentration by the Lowry
method (Lowry et al., 195 l), using bovine serum albumin as standard, and by centrifugation in either a Beckman
model E analytical ultracentrifuge equipped with Schlieren optics or a Beckman model L8-70 ultracentrifuge with
a u.v. preparation scanner.
Estimation of calcium and polyamines. A suspension of virus (50 mg/ml) in 0.05 M-sodium acetate pH 5-5 was
dialysed at 4 °C for 48 h against 50 ml of the same buffer with four changes. The virus concentration was reestimated after dialysis by the Lowry procedure (Lowry et al., 1951). The calcium content of these samples was
estimated by the fluorometric method of Kepner & Hercules (1963). Protein was precipitated by the addition of
trichloroacetic acid to 10~ (w/v) and the mixture was centrifuged for I min in an Eppendorf centrifuge. To a 20 I-tl
sample of this clear supernatant fraction, 1 ml of calcein (Sigma) (16 mg/ml) and 5 ml of 0.4 M-KOH were added.
The fluorescence intensity at 520 nm, when the excitation wavelength was set to 470 nm was recorded immediately
after the addition of the dye. The calcium content was estimated from the observed intensity data by comparison
with that obtained from known amounts of calcium.
The polyamine content of samples of BDMV (1.5 mg) was estimated essentially by the method of Nickerson &
Lane 0977). The dansylated polyamine derivatives were spotted on to a silica gel 60 F254 plate (E. Merck) and
developed for 3 h using cyclohexane : ethyl acetate (3 : 2, v/v) as the solvent. The intensity of fluorescence was
enhanced by spraying the plates with l0 ml of triethanolamine : isopropanol (1:4, v/v) and subsequently drying the
plates overnight in a vacuum. The plates were viewed and photographed under a u.v. lamp. Dansylated
spermidine, spermine, putrescine and cadaverine were used as standards. The photographs were digitized using a
Joyce-Loebl film scanner attached to a PDP 11/44 computer. The peaks were integrated after subtracting the
average intensity in the background. The polyamines were estimated by comparison with the integrated
intensities for a set of standards of authentic spermine, spermidine and putrescine.
Exchangeability of viral polyamines. Exchangeability of viral polyamines with other cations was tested by
dialysing BDMV (5 rag) in 0.05 M-sodium citrate pH 5.5 against 100 ml of 0.05 M-sodium citrate containing 3 MCsCl, 3 M-KC1 or 3 M-NaC1 for 48 h at 4 °C with two changes of the same buffer (100 ml) followed by dialysis
against 0.05 M-sodium citrate pH 5.5, to decrease the concentration of CsC1, KC1 or NaC1. Similarly, the
exchangeability of polyamines at alkaline pH was tested by dialysing BDMV (5 rag) against 100 ml of 0.01 M-TrisHC1 pH 9.0 containing 3 M-CsC1 with two changes of the same buffer followed by dialysis against 0-01 M-Tris-HC1
pH 9-0. This was followed by dialysis against 0-01 M-sodium citrate pH 5.5 to remove Tris-HCl which otherwise
gave additional spots on thin layer chromatography (t.l.c.) plates. The stability of BDMV particles after removal
of polyamines was examined by sucrose density gradient centrifugation.
Effect offreezing and thawing. BDMV (500 ~tg) in 10 to 200 ~tl of 0.01 M-Tris-HC1 pH 7.5 was immersed in liquid
nitrogen for 2 min and subsequently thawed at room temperature (approx. 20 °C). The samples were diluted to 400
~tg in 400 p.l and analysed by centrifugation in 10 to 45 ~ (w/v) sucrose density gradients. The amount of empty
capsid formed ~vas calculated as a percentage of the 'total area under the peaks corresponding to intact particles
and empty shells. The estimates had a root mean square error of approximately 5%.
Electron microscopy. Virus samples were negatively stained with 2 ~ uranyl acetate and examined in a Philips
301 electron microscope operating at 60 to 80 kV using formvar-coated grids.
RESULTS
Calcium and polyamine content of BDMV
The calcium contents of particles in five different preparations of the virus were in the range
500 to 900 molecules per virus particle. Approximately 90 to 140 molecules of spermidine and 20
to 50 molecules of putrescine were present in each virus particle (Fig. 1). The amount of
spermine could not be reliably estimated as it was present at a relatively low concentration. The
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Stability of B D M V
0
A B CDE
•
o
GH
t
•
•
O
•
O
I
O
I
!
80
e
0
E
= 40
O0
0~
/
,
0
Fig. 1
I0
Fraction number
2O
Fig. 2
Fig. 1. Estimation of polyamines. Samples of 1 to 3 ~tl of dansylated polyamines obtained from 1.5 mg
of virus were spotted on silica gel plates along with 0.75 nmol of standard dansylated polyamines. Lane
1, spermine (A); lane 2, spermidine (B); lane 3, putrescine (C); lane 4, cadaverine (D); lanes 5, 6 and 7,
samples of viral polyamine derivatives (1, 2, 3 ~tl respectively) of BDMV isolated from N. glutinosa;
lanes 8 and 9, polyamine derivatives of BDMV purified from the Nicotiana hybrid and N. clevelandii
respectively (3 ~1). The plates were developed and photographed as described in the text. E, Dansyl
derivative of NH3; F and G byproducts; H, dansoic acid. O, origin.
Fig. 2. Effect of pH on the stability of BDMV. BDMV (2 to 10 mg/ml) was dialysed against 0.01 Msodium acetate pH 5.0 or 0-01 M-Tris-HC1pH 7 or pH 9 for 48 h and analysed on 10 to 45 % (w/v) sucrose
density gradients prepared in the respective buffers. The arrow indicates the anticipated position of
empty capsids. Sedimentation was from right to left. O, pH 5; A, pH 7; D, pH 9.
polyamines extracted from virus samples purified using different species of Nicotiana exhibited
identical patterns on t.l.c, plates (Fig. 1) indicating that the nature and content of polyamines in
BDMV is independent of the propagation host.
Effect of pH on the stability of B D M V
BDMV was dialysed against 0.01 M-sodium citrate p H 5.0, or 0.0t M-Tris-HC1 p H 7 or 9, and
analysed by sucrose gradient centrifugation (Fig. 2). Only 5 to 10% of a slower sedimenting
component was found at pH 9 in contrast to the formation of 100K empty capsid reported by
Virudachalam et al. (1983a). Similar results were obtained when the samples at pH 5, 7 or 9 were
centrifuged in a Model E analytical ultracentrifuge equipped with Schlieren optics (Fig. 3, top
trace) indicating that there was little or no effect of pH on the stability of the virus particles.
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1536
H.S. SAVITHRI AND OTHERS
Fig. 3. Analytical ultracentrifugation of BDMV (5 to 8 mg/ml) dialysed against: (a) top trace, 0.05 Msodium citrate pH 5.0; (a) bottom trace, 0.05 M-sodiumcitrate pH 5.0 containing 0.1 M-CsC1;(b) top
trace, 0.05 M-Tris-HC1pH 9-0; (b) bottom trace, 0-05 M-Tris-HC1 pH 9.0 containing 0.1 M-CsC1.
Centrifugation was performed at 24630 r.p.m, and the pictures were recorded 24 rain (a) or 16 min (b)
after attaining full speed. The bar angle was 60°. Sedimentation was from right to left.
Exchangeability of polyamines with cations
Dialysis of virus samples against 3 M-CsC1 at pH 5.5 or pH 9.0 resulted in a complete loss of
polyamines (Fig. 4). Similar results were obtained when the samples were dialysed against 3 MKCI or 3 M-NaC1, although, 3 M-NaC1 was less effective in exchanging with polyamines (data
not shown). Dialysis of these BDMV samples devoid of polyamines against 0.01 M-Tris-HC1 pH
9.0 resulted in the formation of empty protein shells (Fig. 5) clearly demonstrating that the
exchange of polyamines with monovalent cations results in the loss of stability at alkaline pH.
In a separate experiment, BDMV samples were dialysed against 0.1 M-CsC1 at different pH
values. At this concentration of CsC1, there was no observable loss of polyamines. However, the
sedimentation coefficient of the virus increased by about 2 0 ~ above pH 7, while no such
increase was observed for empty capsids (Fig. 3).
Formation of empty capsids by freezing and thawing
Although significant amounts of the slower sedimenting components were not observed by
dialysis against pH 5, 7 or 9 (Fig. 2), they were readily formed by freezing and subsequent
thawing of the virus solution at each of the three pH values (Fig. 6). Electron microscopy of the
intact virus (Fig. 7a) and the slowly sedimenting component obtained by freezing and thawing
indicated that the latter consisted predominantly of empty shells (particles stained at centre ;
Fig. 7b). Preliminary results indicate that the empty capsids formed by freezing and thawing
have approximately the same sedimentation coefficient (54S) as the empty capsids formed in
vivo (data not shown). However, the empty capsids produced upon freezing and thawing TYMV
lack between five and seven coat protein molecules (Katouzian-Safadi & Berthet-Colominas,
1983). A similar loss of some coat protein could occur in BDMV. The formation of empty
capsids upon freezing and thawing was found to be dependent on virus concentration, buffer
molarity, and presence or absence of different anions or cations.
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Stability o f B D M V
1537
H
G
F
E
D
C
B
A
O
1
2
3
4
5
6
7
8
9
10
Fig. 4. Exchangeabilityof polyamines. Samples of BDMV were dialysed against 3 M-CsCIpH 5.5 or 9.0
prior to extraction and dansylation of polyamines. The derivatives were spotted and analysed as
described in Fig. 1. Lanes 1, 2, 3 and 4, standard dansylated polyamines: spermine, spermidine,
putrescine and cadaverine, respectively. Lanes 5 and 6, 1 and 2 gl of BDMV dialysed against 0.05 Msodium citrate pH 5.5. Lanes 7 and 8, 1 and 2 ~tl of BDMV samples dialysed against 3 M-CsC1pH 5.5.
Lanes 9 and 10, 1 and 2 gl of BDMV samples dialysed against 3 M-CsC1pH 9.0. O, A to H are as in
Fig. 1.
To examine the effect of virus concentration on the formation of empty capsids by freezing
and thawing, B D M V was frozen and thawed at concentrations indicated in Fig. 8 and then
diluted to the same final concentration prior to analysis on sucrose density gradients. It was
apparent from Fig. 8 that 5 0 ~ of empty capsids were formed at approximately 2,5 to 3 mg/ml in
0.01 M-Tris-HC1 p H 7.5. However, complete formation of empty shells could be observed even
at 0.5 mg/ml in 5 mM buffer, indicating that the ionic strength of the buffer plays a crucial role in
the release of R N A . Hence, at a fixed concentration of virus (9.3 mg/ml), the effect of ionic
strength on the formation of empty shells was monitored. Increase in ionic strength resulted in
the protection of virus against release of R N A (Table 1).
In addition to the protection observed by an increase in buffer ionic strength, certain anions
and cations also prevented the release of R N A . Table 2 shows the effect of chloride salts on the
formation of empty capsids. The order of effectiveness in preventing the formation of empty
shells was Mg 2+ > N a + > Ca 2+ ~_ Li + ~ Cs ÷ > K +. As is apparent from Table 3, none of the K +
salts tested was able to protect the virus particles. Even with sodium salts, there was no
correlation between the degree of protection and the Hofmeister series o f electrolytes. Similar
results have been obtained with T Y M V and with chicory yellow mottle virus (Katouzian-Safadi
et al., 1980; Quacquarelli et al., 1972).
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H. S. SAVITHRI AND OTHERS
1538
zl.
.~ 25
e
e~
i'\o 1
8°1I
.~ 40
O
I
10
Fraction number
Fig. 5
20
0
I
I
10
Fraction number
20
Fig. 6
Fig. 5. Stability of BDMV particles after removal of polyamines. BDMV (5 mg/ml) was dialysed
against 0.01 M-sodium citrate pH 5.5 containing 3 M-CsC1for 48 h and then transferred to 0.01 M-sodium
citrate pH 5.5 (©) or 0.01 M-Tris-HC1 pH 9.0 (I-7). II, BDMV dialysed against 0.01 M-Tris-HCI pH 9.0
containing 3 M-CsCI for 48 h followed by dialysis against buffer not containing CsCI. O, Undialysed
BDMV. The samples were analysed as described for Fig. 2.
Fig. 6. Effect of freezing and thawing on BDMV. Samples of BDMV (9.25 mg/ml) in 0.01 M-sodium
acetate pH 5, or 0.01 M-Tris-HCI pH 7 or pH 9 were frozen for 2 min in liquid nitrogen and allowed to
thaw at room temperature. The samples (400 ~tg in 400 p.1)were layered on 10 to 45 ~o sucrose gradients
in 0.05 M-sodium phosphate buffer pH 6.2 and analysed as described in Fig. 2. Arrow indicates the
anticipated position of intact particles. ©, pH 5; A, pH 7; I-q, pH 9.
T a b l e 1.
Effect of buffer ionic strength on freezing and thawing of BDMV*
Buffer concentration
5 mM
10 mM
50 mM
100 mM
Empty capsids (~)
100
92
56
20
* BDMV (9"25 mg/ml) in Tris-HC1 pH 7.5 at different molarities was frozen and thawed. Percentage of empty
capsids was determined as described for Fig. 8.
T a b l e 2.
Protection of intact particles during freezing and thawing by the addition of different
chloride salts*
Salt
None
NaCI
KC1
LiC1
CsCI
CaClz
MgC1z
Empty capsids (~)
100
25
85
40
48
44
15
* Chloride salts (40 mM) were added to BDMV (8.75 mg/ml) in 0.01 M-Tris-HCI pH 7.5. The analysis was
performed as described for Table 1 and Fig. 8.
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"
10
I
"
Fig. 8
BDMV (mg/ml)
I
1
Fig. 8. Effect of virus concentration on the formation of empty capsids obtained by freezing and thawing BDMV. Samples of BDMV at concentrations of 2 to 20
mg/ml in 0.01 M-Tris-HCI pH 7.5 were frozen and thawed. Analysis followed the procedure described for Fig. 2. The fraction of virus converted to empty shells was
calculated as a percent~tge of the total area under the two peaks corresponding to intact particles and empty shells.
Fig. 7. Electron micrograph of intact BDMV (a) and top component (b) obtained by freezing and thawing and subsequent separation as described in Fig. 6. The
samples were stained with 2 ~ uranyl acetate. Bar marker represents 50 nm.
Fig. 7
0
~ 5o
,100
I
20
1540
H.s.
SAVITHRI AND OTHERS
Table 3. Effect of different salts of sodium and potassium on the freezing and thawing of B D M V *
Empty capsids (~)
A
Anion
Potassium salt
Sodium salt
Chloride
Nitrate
Iodide
Chlorate
Thiocyanate
Acetate
82
90
100
ND
100
ND
25
60
NDt
50
ND
17
* The concentrations of the virus and salts during freezing and thawing were 8.75 mg/ml in 0.01 m Tris-HC1 pH
7.5 and 40 mM respectively.
]" ND, Not determined.
DISCUSSION
BDMV has been studied recently using nuclear magnetic resonance (NMR) spectroscopy.
These studies indicated that, unlike TYMV, it lacks polyamines (Virudachalam et al., 1983 a, b).
It has been proposed that this lack of polyamines results in the release of RNA at alkaline pH.
However, BDMV particles purified in our laboratory by a modified procedure do not release
RNA at pH 9 and, moreover, contain polyamines and Ca 2÷. There are about 500 to 900 Ca 2+,
approximately 90 to 140 spermidine, 20 to 50 putrescine molecules and traces of spermine per
virion, amounts similar to those in TYMV except that TYMV particles also contain trace
amounts of cadaverine (Cohen & Greenberg, 1981).
The use of 4 0 ~ (w/w) CsC1 density gradient centrifugation in the purification of BDMV by
earlier investigators (Virudachalam et al., 1983a) could have resulted in the replacement of
polyamines by Cs ÷, an idea that is supported by the observation that polyamines in BDMV
particles prepared in our laboratory are lost upon dialysis against 3 M-CsC1. The loss of
polyamines during isolation would therefore reduce the stability of virus particles and result in
consequent release of RNA at alkaline pH. On the other hand, purification of TYMV, a related
virus, using caesium chloride density gradient centrifugation does not result in loss of
polyamines as evidenced by ~3C and 1H NMR spectroscopic studies (Virudachalam et al.,
1983b). Also Ca 2+ is not easily exchanged with protons when TYMV is titrated to neutral pH
(Durham et al., 1977). These results suggest that the polyamines and calcium are intrinsic
components of TYMV (Cohen & Greenberg, 1981) and are not easily exchanged for other
cations. However, the data of Noort et al. (1982) indicate that the exchange between polyamines
of TYMV and caesium ions occurs in more drastic conditions (3.6 M-CsC1, 37 °C for 30 rain). In
contrast, the bound cations can be easily exchanged in the capsid of BDMV. However, there are
interesting differences between the exchangeabilities of polyamines with different cations. K +
and Cs + ions replace the polyamines more readily than Na +. Also, the exchange of polyamines
for caesium is concentration-dependent. These results suggest that the process of exchange of
ions is probably not an all or none phenomenon. For TYMV, the exchange is slow and more
difficult than for BDMV. The bottom component B1~ of TYMV is converted to a more dense
component B2, upon incubation of the virus with CsC1 at elevated temperatures (Noort et al.,
1982). This treatment results in partial degradation of RNA suggesting some conformational
change and reduced stability of the virion (Noort et al., 1982). Further, the conversion of Bxa to
B2~ is accompanied by a loss of polyamines and Mg e+ which might be responsible for the
sensitivity of the B2a component to ribonuclease. The nature of the viral capsids, the cation
binding site or its access channel, could account for the observed differences in the
exchangeability of polyamines by different cations, in TYMV and BDMV. These results suggest
a correlation between the presence of polyamines and viral stability in tymoviruses.
Dialysis of BDMV against CsCI (0.1 M) above pH 7 results in an increase in the sedimentation
coefficient by 20~. Although the sedimentation coefficient of BDMV does not increase upon
dialysis against 0.1 M-CsCI at pH 5.5, the polyamines are exchanged with caesium ions when
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Stability o f B D M V
1541
particles are dialysed against 3 M-CsC1 pH 5.5. Hence, apart from the caesium ions replacing
polyamines, many more ions bind to the virus particle at alkaline pH values. This is
understandable in terms of the recent laser Raman spectroscopic studies (Prescott et al., 1985) in
which it was shown that the degree of protonation of RNA bases in BDMV is reduced by raising
the pH from 5-0 to 8.0. The increased charge on RNA can then support a large number of
caesium ions resulting in an increase in apparent sedimentation coefficient.
Although BDMV prepared by the present procedure does not form empty capsids at alkaline
pH, it does form such capsids upon freezing and thawing. RNA was completely released from
the virus at 0.5 mg/ml in 5 m i buffer suggesting that BDMV particles are more sensitive to
freezing and thawing than those of TYMV. The effects of freezing and thawing in the presence
of electrolytes are similar to those observed for TYMV (Katouzian-Safadi et al., 1980).
Freezing and thawing is a very complex phenomenon involving interactions between the
water molecules, ions and virus particles (Katouzian-Safadi et al., 1980; Katouzian-Safadi &
Haenni, 1986). Katouzian-Safadi et al. (1980) have discussed the concentration dependence of
RNA release in terms of inter-particle interactions brought about during freezing and thawing.
The protection by increased ionic strength of buffer and the electrolytes has been explained in
terms of an increased dielectric constant of the medium resulting in decreased aggregation
(Katouzian-Safadi et al., 1980). However, the lack of protection by potassium salts is difficult to
explain on the basis of such a mechanism. It is also possible that incubation of virus with high
concentrations of cations may release bound polyamines which might result in modified
electrostatic interactions between particles.
The results presented in this paper clearly indicate that polyamines and calcium are inherent
components of BDMV particles and their loss results in the instability of the virus.
We are grateful to Professor N. Appaji Rao for helpful discussions and encouragement. We would like to thank
Dr V. Prakash, C.F.T.R.I., Mysore, Dr Vidya Shivaswamy, Dr A. Basu and Dr K. Varalakshmi, I.I.Sc., for their
help in carrying out some experiments. We thank T. U. Sahana for excellent technical assistance. H.S.S. was
supported by the University Grants Commission, India.
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