ABSTRACT Newcastle disease virus (NDV) is an enveloped RNA virus. Certain biological activities of the NDV are determined by two surface glycoproteins -haemaglutinin-neuraminidase (HN) and fusion-protein (F). These proteins play an active role in the viru

L Doumanova

N Terezova

V Neitchev

Y Abashev

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Biotechnol. & Biotechnol. Eq. 19/2005/1 155 
EFFECTS OF THE NDV-GLYCOPROTEINS ON THE  
ELECTROPERMEABILIZATION OF LIPID BILAYERS 
 
L. Doumanova1, V. Neitchev2, N. Terezova2, Y. Abashev1 
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia1 
Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria2 
 
ABSTRACT 
Newcastle disease virus (NDV) is an enveloped RNA virus. Certain biological activities of 
the NDV are determined by two surface glycoproteins –haemaglutinin-neuraminidase 
(HN) and fusion-protein (F). These proteins play an active role in the virus-cell interac-
tion altering the lipid order in the virus membrane. Liposomes from L-α-phosphatidyl-
choline with embedded viral components, HN and F, were exposed to an alternating elec-
tric field of 120 V peak to peak square electric field waves at three different frequencies of 
0.01, 1 and 100 kHz to perturb the liposome permeation. Two independent sets of experi-
ments were conducted: liposomes without viral components, and liposomes with embed-
ded viral components. To monitor the changes in permeation of liposome particles, the 
leakage of calcium ions from liposomes was measured by recording of the fluorescence 
emission due to the complex formation reaction with fluorescence dye in the medium so-
lution. The increase of temperature of the solution in the experimental chamber for elec-
tropermeabilization was also registered. The frequency dependence of both the leakage of 
Ca ions and temperature increments was obtained. It was observed that the efficiency of 
the Ca ions leakage in the both cases has a minimum at 1 kHz. The percentage of leakage 
in the second experiment increased by a factor of 25% in comparison to the same data 
valid in the first experiment. The values of temperature increment followed similar fre-
quency dependencies as those for Ca ions leakage with some varieties. There is accumu-
lating evidence to show that higher values of temperature increment correspond to higher 
values in Ca ions leakage in the presence of HN and F. Experimental similarity between 
both, as a function of frequency, suggests that the energy dissipation of electric field by 
the sample is the main reason for these phenomena. 
 
Introduction 
Recently numerous studies have been de-
voted to the effects of electric field (EF) on 
cell suspensions and artificial lipid mem-
branes (1, 2, 3, 4). It is well known that a 
bilayer lipid membrane is a good barrier for 
ions and hydrophilic molecules. Such a 
permeation barrier is readily modified by 
imposing a transmembrane electric poten-
tial exceeding the dielectric strength of the 
membrane. In this case the membrane spe-
cific conductance increased dramatically, 
sometimes to values as high as 1 S cm -1 in 
microseconds. This effect is reversible: 
repetitive voltage scans did not alter the V-
I characteristics of the membrane. Never-
theless the information which has already 
been obtained in these studies concerning 
the effects of EF on membrane permeabi-
lity, the process of electroporation of cell 
membranes as well as related phenomena 
remain unclear. 
Of all such phenomena, by far the most 
important is the membrane contact which 
may activate and regulate the membrane 
"docking" process. It is an essential prere-
quisite to fusion , but it does not necessar-
ily lead to fusion. How ever, the role of EF 
 
Biotechnol. & Biotechnol. Eq. 19/2005/1 156 
in such phenomena is still obscure. Here 
we review some data which demonstrate 
the role of virus components of NDV on 
the electropermabilization of assembled 
lipid bilayers to Ca ions. The data show the 
existence of frequency dependence window 
at 1 kHz. Furthermore, the data were inter-
preted according to the theory of interac-
tions of electromagnetic fields with highly 
polarized and deformed materials such as 
lipid vesicles (5). The frequency depen-
dence in the presence of embedded virus 
components in the lipid bilayer, could be 
also explained as a decrease of the mem-
brane binding rigidity and disordering ef-
fect of these proteins (6). 
Materials and Methods 
Virus. Newcastle disease virus, vaccinal 
strain La Sota, was grown in 10- day old 
embryonated chicken eggs. The eggs were 
incubated at 37 °C for 72 h and the allan-
toic fluid was harvested. After sedimenta-
tion of the cellular debris at 1000 x g for 20 
min at 4 °C, the virus in the supernatant 
was pelleted at 28 000 rpm/h for 30 min. 
The viral pellet was dispersed in PBS (pH 
7.2 ) and applied to a 5-60% (w/w) sucrose 
lineal gradient in TNE buffer (pH 7.2) at 
28 000 rpm/h for 3 h at 4 °C in Beckman 
SW 28 rotor. The virus band was collected, 
dialyzed against PBS and pelleted at 28 
000 rpm/h for 90 min at 4 °C. 
Purification of the NDV-glycoproteins. 
The purified virions were suspended in 
PBS, pH 7.4 containing 2% octyl-glucoside 
(OG) at room temperature (RT) during 1 h 
to solubilize glycoproteins. The solubilized 
glycoproteins were separated from mem-
brane (M)-protein by centrifugation at 30 
000 rpm/min for 45 min. 
Preparation of samples. For preparation 
of lipid vesicles liposomes, commercial 
product of Sigma L-α-phosphatidylcholine 
from frozen egg yolk was used. Fluores-
cence marker Quin-2 from Dijondo (Japan) 
with molecular weight of 693.8 was pur-
chased product. Lipid, 100 mg/ml in 9:1 
solution of chloroform/methanol was dried 
under nitrogen gas flow in a round-bot-
tomed tube. Then required amounts of 
CaCl2 were added to the dried lipid in 10 
ml dH2O and the mixture was vortexed for 
60 min at room temperature. These 
liposomes were used as controls. The sam-
ples of reconstituted lipid assemblies with 
embedded virus HN and F proteins were 
formed by solubilization of lipid dried film 
in 10 ml. 2% octylglycoside solution con-
taining the required amounts of mixture 
HN and F (total average molecular weight 
of 132 kD) at molar ratio lipid/virus com-
ponents 100:1. The samples were then 
dialyzed against 2 mM CaCl2 for several 
days to remove the detergent. The residue 
was resolved in 10 ml destilled water and 
the mixture was vortexed as above. Control 
liposomes and such containing virus pro-
teins were characterized for particle size 
distribution using a dynamic light scatte-
ring size meter (Photal Otsuka Electronics 
LPA 3000m, Japan). 
Instruments and electric field treatment. 
The apparatus for application of EF was 
described in details in previous paper 
(6).The voltage applied between the elec-
trodes had an effective value of 80 V peak 
to peak. The chamber was from a round 
glass tube with diameter 1 cm and two wire 
Pt electrodes at a distance of 1 mm. The 
enhancement of temperature due to the EF 
was measured before and after EF applica-
tion by a sensitive miniature thermocouple. 
EF of 30 seconds duration was applied to 
the sample in the chamber. The electro-
permeabilization protocol was as follows: 
to a part (1.5 ml) of liposomes suspension 
or reconstituted assemblies respectively, a 
solution of 1.5ml. of fluorescence dye Quin 
2 with concentration 1.10-3 M in water was 
added. The mixture was incubated for se-
veral minutes before, just prior to the EF 
application. 
Fluorescence intensities and increments 
of temperature were then measured for dif-
ferent  frequencies  of   EF.  The  excitation  
 
Biotechnol. & Biotechnol. Eq. 19/2005/1 157 
Fig. 1.        
% Ca+2 leakage
0
5
10
15
20
25
controls 0,01 1 100
Frequency,kHz
F
lu
or
es
ce
nc
e 
in
te
ns
it
y,
a.
u.
pure lipozomes
lipozomes+[HN-F]
140
157
117
142
170
185
 
 
wavelength was at 339 nm and detection at 
492 nm. 
Results and Discussion 
Ca ions leakage and effect of frequency 
All date related to the leakage of Ca ions 
were obtained as an increase of fluores-
cence dye intensity after EF treatment of 
controls and samples in the chamber at 
three different frequencies. The histograms 
at Fig. 1 showed permanent increase of Ca 
ions leakage in comparison to controls 
without embedded virus proteins. The 
maximal Ca ions release in the individual 
samples was achieved within 2-3 minutes 
after the EF application. The frequency 
dependence of Ca ions leakage at Fig. 1 
demonstrates a gradual decrease at fre-
quency of 0.01 and 1 kHz, and then an in-
crease was at 100 kHz. This tendency was 
obvious in both cases, pure liposomes and 
liposomes with embedded virus proteins. 
The data above confirm that the applied EF 
may affect the process of liposome leakage 
under the conditions of our experiment. 
The presence of virus components in the 
liposomes undoubtedely play an additional 
role in this phenomenon. Two conclusions 
can be drawn in this case - the effect of 
frequency is connected with polarization of 
liposome particle and this effect is more 
pronounced in the presence of HN and F. 
Effects of HN and F virus components 
on Ca ions leakage 
The maximum changes in the leakage and 
temperature increment of Ca ions were 
obtained with reconstituted lipid assemblies 
containing embedded virus proteins, HN 
and F, and perturbed with EF at 100 kHz. 
The significance of the differences between 
these particles and free liposomes, and 
electric field frequency may be evaluated 
as reported firstly in the theory of lipid- 
protein interactions (7,8) and secondly in 
the theory of interactions of electromag-
netic fields with high polarized and de-
formed materials as liposome particles. (5). 
In this paper we shall restrict only to the 
first, since our chief conclusion is that the 
leakage of Ca ions from liposomes is a re-
sult of the EF- induced transmembrane po-
tential. The heating of the solution in the 
chamber is due to the high electroconduc-
tance of the medium.The leakage of Ca 
ions through the membrane into chamber 
increased the ionic strength and osmolarity 
of the medium. Experimental similarity 
between the fluorescence measurements 
characterizing the Ca ions leakage and 
those of increment of temperature as a 
function of frequency, suggests that the 
energy of dissipation of EF by the sample 
 
Biotechnol. & Biotechnol. Eq. 19/2005/1 158 
is the main reason for those phenomenon. 
Then, we will explain the differences in the 
data in the presence of HN and F in the 
liposomes. It is quite conceivable that the 
organization of lipid assemblies may be 
affected by the presence of HN and F in the 
bilayer to a significant extent. A number of 
theoretical models and explanations have 
put forward to provide a consistent inter-
pretation of similar results. An adapted ex-
planation for the case when external EF is 
applied concerns the appearance of fusion 
pores and channels during the application 
of EF. They open instantaneously and have 
strictly defined levels of conductance. 
Further, it is well known that lipid bilayer 
fusion is very sensitive to the spontaneous 
curvature and deformation of lipid mem-
brane. Proteins can interact with lipids to 
change membrane spontaneous curvature 
and some membrane functions as mem-
brane permeability. In the course of EF 
application these processes are to the high-
est degree. Assimilating the body of 
knowledge on possible role of HN and F in 
this phenomenon, we may add that mem-
brane surface proteins increase signifi-
cantly the pore stability so that they can be 
stable for seconds and longer. Taking in 
account this fact we kept the time of EF 
application within seconds. The proteins in 
one membrane can also promote a topo-
logical rearrangement of all membrane 
components and structures, influencing the 
membrane functions. Interpretation of such 
phenomena in terms of protein-membrane 
interactions leads naturally to certain impli-
cations and consequences. Thus the role of 
HN and F on the electropermeabilization of 
liposome particles probably contribute po-
tentially to an increase of new research in-
terests. At molecular level these depen-
dences involve the changes in membrane 
curvature and deformation of lipid bilayer 
due to the EF. The last may be significantly 
influenced in the presence of HN and F in 
the bilayer. The well known hypothesis 
regarding the possible role of frequency in 
membrane electropermeabilization is that at 
lower values the EF dependent processes 
on the lipid membrane are not so intensi-
fied. In our experiment it is valid for fre-
quencies of 0.01 and 1 kHz as the electro-
poration has not yet reached its maximum 
value. At values above 1 kHz the EF de-
pendent processes on the membrane sur-
face and inside in the particle will perturb 
the membrane either through interaction 
with surface charges, dielectric forces or 
local heating due to the highly promoted 
membrane currents. In the case of local 
heating a breakdown of the membrane may 
be observed which leads to a significant 
increase in liposome leakage. 
Joule-heating of the chamber solution 
A rapid thermal heating from 5-7 °C of the 
solution in the chamber was recorded after 
the EF application. In Fig. 2 dependence of 
temperature increment ∆T = T2 – T1 are 
given as a function of frequency in two 
separate experiments 1 and 2, where T2 is 
the measured temperature after EF applica-
tion and T1 is the initial temperature in the 
chamber. As can be seen there is a correla-
tion with the results found for Ca ions 
leakage at different frequencies. In Fig. 2 
the values of temperature increment for the 
sample (experiment 2) are higher than 
those for control (experiment 1) in all 
cases, but the shape of both dependencies 
is similar. The values of ∆T decreased 
gradually with increasing the frequency 
from 0.01 to 1 kHz. The changes are negli-
jible (about 1-1.5 °C or less). More obvious 
changes were registered at a frequency of 
100 kHz. The values of increment in ex-
periment 2 are about 4 °C higher than those 
in experiment 1. These results show a 
similarity with the results for Ca ions lea-
kage measured by fluorescence intensity 
changes as a function of EF frequency. 
There is accumulating evidence to show 
that the higher values of temperature in-
crement correspond to higher values of Ca 
ions leakage, as can be seen from Figs 1 
and 2., but  could  not  found  a  correlation  
 
Biotechnol. & Biotechnol. Eq. 19/2005/1 159 
Fig. 2         
0
2
4
6
8
10
0,01 1 100
Frequency,kHz
In
cr
em
en
t 
of
 T
o  
C
control
HN+F
2
1
 
 
between the percentage of Ca ions leakage 
and temperature increment. 
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EFFECTS OF THE NDV-GLYCOPROTEINS ON THE ELECTROPERMEABILIZATION OF LIPID BILAYERS

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EFFECTS OF THE NDV-GLYCOPROTEINS ON THE ELECTROPERMEABILIZATION OF LIPID BILAYERS

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