Infectious bursal disease virus changes the potassium current properties of chicken embryo fibroblasts.
ABSTRACT Infectious bursal disease virus (IBDV) is the causative agent of an economically significant poultry disease. IBDV infection leads to apoptosis in chicken embryos and cell cultures. Since changes in cellular ion fluxes during apoptosis have been reported, we investigated the membrane ion currents of chicken embryo fibroblasts (CEFs) inoculated with the Cu-1 strain of IBDV using the patch-clamp recording technique. Incubation of CEFs with IBDV led to marked changes in their K+ outward current properties, with respect to both the kinetics of activation and inactivation and the Ca2+ dependence of the activation. The changes occurred in a time-dependent manner and were complete after 8 h. UV-treated noninfectious virions induced the same K+ current changes as live IBDV. When CEFs were inoculated with IBDV after pretreatment with a neutralizing antibody, about 30% of the cells showed a normal K+ current, whereas the rest exhibited K+ current properties identical to or closely resembling those of IBDV-infected cells. Incubation of CEFs with culture supernatant from IBDV-infected cells from which the virus particles were removed had no influence on the K+ current. Our data strongly suggest that the K+ current changes induced by IBDV are not due to virus replication, but are the result of attachment and/or membrane penetration. Possibly, the altered K+ current may delay the apoptotic process in CEFs after IBDV infection.
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Article: Ion homeostasis and apoptosis[Show abstract] [Hide abstract]
ABSTRACT: Alterations in the transmembrane gradients of several physiological ions may influence programmed cell death. In particular, recent data suggest that increases in intracellular calcium may either promote or inhibit apoptosis, depending on the level, timing and location, whereas loss of intracellular potassium promotes apoptosis.Current Opinion in Cell Biology 09/2001; · 11.41 Impact Factor
Article: Granulysin-Induced Cell Death
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ABSTRACT: Abstract : We studied the novel hypothesis that an upmodulation of channels for outward delayed rectifier K+ current (Iκ) plays a key role in ceramide-induced neuronal apoptosis. Exposure for 6-10 h to the membrane-permeable C2-ceramide (25 μM) or to sphingomyelinase (0.2 unit/ml), but not to the inactive ceramide analogue C2-dihydroceramide (25 μM), enhanced the whole-cell Iκ current without affecting the transient A-type K+ current and increased caspase activity, followed by neuronal apoptosis 24 h after exposure onset. Tetraethylammonium (TEA) or 4-chloro-N,N-diethyl-N-heptylbenzenebutanaminium tosylate (clofilium), at concentrations inhibiting Iκ, attenuated the C2-ceramide-induced caspase-3-like activation as well as neuronal apoptosis. Raising extracellular K+ to 25 mM similarly blocked the C2-ceramide-induced cell death ; the neuroprotection by 25 mM K+ or TEA was not eliminated by blocking voltage-gated Ca2+ channels. An inhibitor of tyrosine kinases, herbimycin A (10 nM) or lavendustin A (0.1-1 μM), suppressed Iκ enhancement and/or apoptosis induced by C2-ceramide. It is suggested that ceramide-induced Iκ current enhancement is mediated by tyrosine phosphorylation and plays a critical role in neuronal apoptosis.Journal of Neurochemistry 08/1999; 73(3):933 - 941. · 3.97 Impact Factor
Infectious Bursal Disease Virus Changes the Potassium Current Properties
of Chicken Embryo Fibroblasts
Holger Repp,*Hermann Nieper,† Henning J. Draheim,*,1Andreas Koschinski,*
Hermann Mu ¨ller,† and Florian Dreyer*,2
*Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-University, D-35392 Giessen; and †Institute of Virology,
University of Leipzig, D-04103 Leipzig, Germany
Received February 11, 1997; returned to author for revision March 20, 1997; accepted April 13, 1998
Infectious bursal disease virus (IBDV) is the causative agent of an economically significant poultry disease. IBDV infection
leads to apoptosis in chicken embryos and cell cultures. Since changes in cellular ion fluxes during apoptosis have been
reported, we investigated the membrane ion currents of chicken embryo fibroblasts (CEFs) inoculated with the Cu-1 strain
of IBDV using the patch-clamp recording technique. Incubationof CEFs with IBDV led to marked changes intheir K?outward
current properties, with respect to both the kinetics of activationand inactivationand the Ca2?dependence of the activation.
The changes occurred in a time-dependent manner and were complete after 8 h. UV-treated noninfectious virions induced
the same K?current changes as live IBDV. When CEFs were inoculated with IBDV after pretreatment with a neutralizing
antibody, about 30%of the cells showed a normal K?current, whereas the rest exhibited K?current properties identical to
or closely resembling those of IBDV-infectedcells.Incubationof CEFs withculture supernatant fromIBDV-infectedcells from
which the virus particles were removed had no influence on the K?current. Our data strongly suggest that the K?current
changes induced by IBDV are not due to virus replication, but are the result of attachment and/or membrane penetration.
Possibly, the altered K?current may delay the apoptotic process in CEFs after IBDV infection.
© 1998 Academic Press
Several viruses are known to cause alterations in
plasma membrane permeability, which can affect intra-
cellular ion homeostasis and contribute to cytolysis and
the death of the infected cells (Carrasco, 1995). The
nature of virus-induced cell permeability changes, how-
ever, is only roughly understood. Viral proteins such as
influenza A virus M2protein, NB glycoproteinofinfluenza
B virus, Vpu and Vpr of human immunodeficiency virus
type 1 (HIV-1) (Lamb and Pinto, 1997), and E2 protein of
Semliki Forest virus (Schlegel et al., 1991) were reported
to formion channel-like pores in the plasma membrane.
On the other hand, infection of cells with human cyto-
megalovirus (Bakhramov et al., 1995), transformation by
Rous sarcoma virus (Repp et al., 1993; Draheim et al.,
1994) and simian virus 40 (Teulon et al., 1992, 1994), or
exposure to the HIV-1 proteins Nef (Werner et al., 1991)
and gp120 (Bubien et al., 1995) modulate existing ion
channels with selectivity for potassium (K?channels).
K?channels are the mostubiquitous ionchannels and
are present in every eukaryotic cell, with various K?
channel types often being found in the same cell. K?
channels contribute to a wide range of cellular pro-
cesses. They play an important role in the maintenance
of the membrane resting potential, are involved in elec-
trical excitability, e.g., of neurons and myocardial cells,
andcontribute to the regulationofcellularionhomeosta-
sis (Rudy, 1988; Cook, 1990; Breitwieser, 1996; Ackerman
and Clapham, 1997). In addition to these classical phys-
iological functions, K?channels have been implicated in
cellular proliferation (Deutsch et al., 1986; Amigorena et
al., 1990; Nilius and Wohlrab, 1992) and also in the
transformation process (Rane, 1991; Teulon et al., 1992;
Repp et al., 1993). Recently, a new function of K?chan-
nels became apparent when it was reported that they
may be critically involved inthe alterations ofcellularion
homeostasis during the process of apoptosis (Beauvais
et al., 1995; Deluca et al., 1996; Szabo et al., 1996).
Infectious bursal disease virus (IBDV), a member of
the family Birnaviridae (Dobos et al., 1979), is the etio-
logical agent of Gumboro disease (Cheville, 1967), an
immunodeficiency that causes severe economical loss
in poultry industries. Infections of young chickens result
in high mortality rates, due either to the acute course of
the disease or to the consequences of a B-cell-depen-
dent immunodeficiency (Becht, 1980; Kibenge et al.,
1988; Mu ¨ller et al., 1992). IBDV infection induces apopto-
sis in chicken peripheral blood lymphocytes, which may
account for the immunosuppressive effect (Vasconcelos
1Present address: Boehringer-Ingelheim KG, D-55218 Ingelheim,
2To whomcorrespondence and reprint requests should be addressed
at Rudolf-Buchheim-Institut fu ¨r Pharmakologie der Justus-Liebig-Univer-
sita ¨t Giessen, Frankfurter Strasse 107, D-35392 Giessen, Germany. Fax:
?49 641 99 47650. E-mail: Florian.Dreyer@pharma.med.uni-giessen.de.
VIROLOGY 246, 362–369 (1998)
ARTICLE NO. VY989187
Copyright © 1998 by Academic Press
All rights of reproduction in any form reserved.
and Lam, 1994). IBDV-induced apoptosis was also ob-
served in chicken embryo fibroblasts (CEFs) (Tham and
Moon, 1996). The purpose of the present work was to
determine whetherIBDV infectionmayleadtoalterations
in the K?current properties of this cell type. Normal
CEFs exhibit a delayed activating, Ca2?- and voltage-
dependent K?outward current that requires only 1 nM
free intracellular Ca2?for full activation (Repp et al.,
1993; Draheimetal., 1994). We observed thatIBDV mark-
edly changedthe kinetic properties andthe Ca2?depen-
dence ofthe K?currentinthese cells.The changes were
notdependentonvirus replicationbutare mostlikely the
resultofvirus attachmentand/ormembrane penetration.
Kinetics of IBDV replication in CEFs
Prior to the electrophysiological experiments, the time
course ofIBDV replication in CEFs was studied by direct
IBDV-specific antigens were detected in only 10%of the
infected cells (Figs. 1b and 2); at 16 and 32 h postinfec-
tion the numbers increased to 30 and 45%, respectively
(Figs. 1c, 1d, and 2). Those CEFs found negative forvirus
antigens were still viable (Fig. 2).
Changes in membrane ion current properties after
Ion channel activity can be detected by measuring
changes in cell membrane currents, of either whole
cells or excised membrane patches. For the electro-
physiological measurements of the present study, we
used the whole-cell recording configuration of the
patch-clamp technique. In normal CEFs a voltage-
dependent K?outward current with a kinetics of de-
layed activation is activated at membrane potentials
more positive than ?30 mV (Fig. 3a) with an activation
time constant ?a? 20.2 ? 3.8 ms (SD, n ? 14) and a
FIG. 1. Detection of IBDV antigens in IBDV-infected CEFs (twice subcultured) by direct immunofluorescence using fluorescein-conjugated chicken
anti-IBDV serum. Uninfected cells (a) and those at 8 (b), 16 (c), and 32 h (d) after infection with IBDV strain Cu-1 at a m.o.i. of 5. The bar represents
363CHANGES OF POTASSIUM CURRENT BY IBDV
mean amplitude of 371 ? 93 pA (SD, n ? 12) at ?50
mV (Repp et al., 1993). During prolonged membrane
depolarization to ?30 mV, the amplitude of the K?
outward current of normal CEFs shows a time-depen-
dent decrease (Fig. 3b) with an inactivation time con-
stant ?i? 9.7 ? 2.3 s (SD, n ? 14) and a complete
inactivation within about 30 s.
Two hours after IBDV infection, the ion currents
were identical to those of normal CEFs (Fig. 3c).
Changes were firstobserved 4 h afterinfection: the ion
currents showed stronger fluctuations (Fig. 3d) and
became only partially inactivated during prolonged
membrane depolarization (not shown). Eight hours af-
ter infection, the membrane current properties were
profoundly altered. While the ion current was still ac-
tivated at potentials more positive than ?30 mV and
possessed almost the same amplitude (436 ? 104 pA
at ?50 mV; SD, n ? 14) as that of normal cells, the
activation was more rapid (?a? 5.2 ? 2.8 ms; SD, n ?
5) and the ion current was again characterized by
strong fluctuations (Fig. 3e). Furthermore, the ampli-
tude of the ion current showed no time-dependent
decrease during prolonged membrane depolarization
(Fig. 3f). The characteristics observed 8 h after IBDV
infection persisted up to 16 h postinfection.
Since the proportion of viable CEFs declined after
IBDV infection (see Fig. 2), itmay be assumed thatsome
measurements were made on ‘‘dead’’ cells. This possi-
bility, however, can be excluded. Immediately after the
establishment of the whole-cell recording configuration,
the resting membrane potential of the cells was con-
trolled in the current-clamp mode. Only those CEFs
which exhibited the normal resting membrane potential
of about ?30 mV (Repp et al., 1993) were further inves-
tigated, whereas ‘‘dead’’ cells with 0 membrane potential
were not used.
In normal CEFs, the ion channels that are respon-
sible for the membrane outward current are highly
selective for potassium ions (Repp et al., 1993; Dra-
heim et al., 1994). In IBDV-infected CEFs, current–
voltage curves that were measured in the presence of
identical concentrations of either NaCl (150 mM) or
potassium glutamate (150 mM) in the intracellular and
extracellular solution yielded a membrane current ra-
tio INa/IKof 0.04 at ?50 mV membrane potential. This
confirmed that in IBDV-infected CEFs the ion channels
that underly the membrane outward current are also
highly selective for K?.
In the whole-cell recording configuration of the patch-
clamptechnique the solutioninthe recording pipette has
direct access to the cytoplasm of the cell. The cell vol-
ume (usually 1–10 pl) is very small compared to the
volume of the solution in the recording pipette (about 10
?l). For this reason, the soluble components of the cyto-
plasm are exchanged completely for the recording pi-
pette solution within a few seconds after the establish-
ment of the whole-cell recording configuration. This
makes it possible to control the intracellular ion compo-
sition by using defined solutions in the recording pipette
(? intracellular solution). When Ca2?-free intracellular
solution buffered with the Ca2?-chelating agent BAPTA
(5 mM) was used, no K?currents were detectedeitherin
normal or in IBDV-infected CEFs (Fig. 4), which clearly
demonstrates theirCa2?dependence. The K?current of
normal CEFs requires only traces of free intracellular
Ca2?([Ca2?]i) to become fully activated, i.e., free [Ca2?]i
of 1 nM leads to almost complete activation (Fig. 4;
Draheim et al., 1994). IBDV-infected cells, however, re-
quired about 100-fold higher free [Ca2?]i: their mem-
brane outward current required an intracellular Ca2?
concentration ofmore than 200 nM forfull activation and
became half-maximallyactivatedata concentrationof20
nM (Fig. 4).
The Ca2?-dependent K?outward current of IBDV-in-
fected CEFs was blocked by the classical K?channel
blocker tetraethylammoniumion (TEA) with a concentra-
tion of 2 mM for half-maximal inhibition. It was blocked
completely by charybdotoxin (300 nM), a component of
the venom of the scorpion Leiurus quinquestriatus he-
braeus, which has been shown to block various types of
Ca2?- and voltage-dependent K?channels (Dreyer,
IBDV replication is not responsible for K?current
The marked changes in the K?current properties
described above were found in all viable CEFs (n ? 72)
investigated 8 h after IBDV infection. At this time, how-
ever, only 10% of the CEFs were expressing detectable
viral antigens (see Fig. 2). This implied that the changes
in K?current properties may not be the result of virus
FIG. 2. Percentage of IBDV antigen-positive cells and viable cells in
cultures of CEFs (twice subcultured) at different times after IBDV
infection at a m.o.i. of 5. IBDV antigens were detected by direct immu-
nofluorescence using fluorescein-conjugatedchickenanti-IBDV serum.
Viability of the cells was verified by fluorescein diacetate/ethidium
364REPP ET AL.
replication. To address this question, CEFs were incu-
bated with IBDV that had been inactivated by UV irradi-
ation. Eight hours after incubation with UV-treated non-
infectious virions, all of the CEFs investigated (n ? 6)
exhibited the same changes in K?current properties as
observed in cells infected with live IBDV (see Figs. 3e
and3f),indicating thatvirus replicationis notresponsible
for these changes.
To test whether the K?current alterations may be
caused by a diffusible mediator, possibly released into
the culture medium by those CEFs already replicating
IBDV, we aspirated culture supernatant from CEFs 8 h
after IBDV infection, removed the virus particles by ultra-
centrifugation and ultrafiltration, and incubated normal
CEFs 8 h in this medium at 37°C. None of the investi-
gated CEFs (n ? 7) showed K?current alterations.
FIG.3.Whole-cell membrane currents ofnormal CEFs (a, b) andofCEFs atdifferenttimes afterinfectionwithIBDV strainCu-1 (c–f) inthe presence
of 1 ?M free [Ca2?]i. Each of the families of membrane currents (a, c–e) was elicited by membrane potential steps of 400 ms duration from?70 to
?50 mV in20-mV increments starting froma holding potential of?50 mV.Single traces (b, f) were recordedduring a 30-s depolarizing testpulse from
a holding potential of ?50 mV to ?30 mV. The membrane currents are not corrected for capacitive and leakage components. Note the different time
scale in (b) and (f). p.i., postinfection.
365CHANGES OF POTASSIUM CURRENT BY IBDV
Effect of a neutralizing antibody on the IBDV-induced
We tested different IBDV-specific antibodies and
found that the mAb B1 that recognizes the VP2 protein
of IBDV Cu-1 most potently neutralized IBDV infectivity.
Consequently, this antibody was used to investigate
the effect of a neutralizing antibody on the IBDV-in-
duced K?current changes. IBDV particles (2 ? 106
PFU in 1 ml) were incubated with mAb B1 at a dilution
of 1:25 for 30 min at 37°C. The antibody-pretreated
IBDV particles were applied to CEFs (2 ? 105cells) for
30 min at 37°C, and after 8 h the ion currents were
measured. Surprisingly, three of nine cells exhibited
K?current properties, as observed in CEFs infected
with live IBDV (see Figs. 3e and 3f), three cells showed
normal K?currents (see Figs. 3a and 3b), and three
cells had K?currents with the typical activation kinet-
ics found in normal cells but with fluctuations (see Fig.
3d) and a partial inactivation during prolonged depo-
larization. Since we used a dilution of the mAb B1 of
1:25, which is aboutsevenfold more concentrated than
the dilution that completely prevented the appearance
of plaques in a plaque assay using 2 ? 106PFU (see
Materials and Methods), this result could not be ex-
plained by assuming an insufficient neutralization.
Therefore, we investigated the effect of antibody pre-
treatment on the binding of IBDV to CEFs. We found
that even at the low dilution of 1:10 mAb B1, the
binding of [35S]methionine-labeled IBDV particles to
CEFs was reduced only to 50% of the value observed
inthe absence ofthis antibody. As a control, IBDV Cu-1
was incubated with an irrelevant mAb specific for the
hemagglutinin of influenza A virus. This treatment did
not alter the binding to CEFs. Altogether, these data
indicate thatthe binding and/ormembrane penetration
but not the replication of IBDV is critical for the K?
current changes in CEFs.
The present study shows a new biological effect of
IBDV. Infection of CEFs with IBDV leads to marked
changes in their K?outward current properties, with
respect to the kinetics of activation and inactivation and
the Ca2?dependence of the activation. IBDV is the first
aviancytolytic virus describedas modulating K?channel
functions. For mammalian cytolytic viruses, an effect on
K?channels is already known. Infection of human fibro-
blasts with human cytomegalovirus leads to the activa-
tion of a voltage-dependent K?outward current with
delayed activation kinetics (Bakhramov et al., 1995). Ex-
posure of chicken dorsal root ganglion cells to the HIV-1
protein Nef (Werner et al., 1991) and of rat astrocytes to
gp120ofHIV-1(Bubienetal., 1995) increases K?channel
activity, and it has been speculated that this effect may
contribute to the CNS pathophysiology associated with
The K?current changes induced by IBDV were com-
plete 8 h after inoculation with the virus and were ob-
served in all IBDV-infected CEFs investigated, implying
that virus/cell interactions occurred on each cell. This
idea is in accordance with the existence of specific
binding sites forIBDV onthe surface ofCEFs, whichwas
confirmed recently (Nieper and Mu ¨ller, 1996). When we
studied the time course ofIBDV replication in CEFs, viral
antigens were detected 8 h after infection in only 10%of
the cells, as determined by direct immunofluorescence.
As we useda m.o.i.of5, this low proportionis surprising.
It is in agreement, however, with previous data concern-
ing the growth characteristics of IBDV that showed the
earliest increase in virus titers after 6 h postinfection
(Nick et al., 1976).
The observation that only a fraction of the CEFs was
IBDV antigen-positive but that all cells exhibited K?
current alterations 8 h after IBDV infection suggested
that virus replication is not responsible for the K?
current changes. This concept was confirmed by the
findings that UV-treated noninfectious virions caused
the same K?current alterations as observed with live
virus and that pretreatment of IBDV with a neutralizing
antibody was notable to preventthe appearance ofK?
current properties typical of IBDV-infected cells. In
contrast, incubation of CEFs with culture supernatant
taken from IBDV-infected cells from which the virus
particles were removed did not induce K?current
FIG. 4. Dependence of the K?outward currents on free [Ca2?]iof
normal CEFs (squares) and of CEFs 8 h after IBDV infection (circles).
The K?current amplitudes I were determined at the end of 400 ms
pulses to ?50 mV and are corrected for leakage current. The data are
means ? SD foratleastfourdifferentcells foreachconcentration. The
curves were fitted to the data using the equation I/Imax? 1/[1 ?
(EC50/c)n], where I is the K?current amplitude, Imaxthe K?current
amplitude at 10?6M free [Ca2?]i, EC50the concentration at which the
half-maximal effect is observed, and c the free [Ca2?]i. For IBDV-
infected CEFs, the best fit was obtained with a Hill coefficient n ? 1.6
and an EC50value of 20 nM for activation by intracellular Ca2?.
366REPP ET AL.
changes, indicating that the changes are not mediated
by a diffusible component possibly released into the
culture medium by CEFs replicating IBDV. Altogether,
these data indicate that the K?current changes in-
duced by IBDV are not linked to virus replication but
most likely the result of virus binding and/or mem-
It may be surprising that it takes 4 h from the time of
inoculation of CEFs with IBDV to the appearance of K?
current changes. With regard to this delay, a direct inter-
action ofthe membrane receptorforIBDV, which has not
yet been identified, and the K?channels seems less
probable. A possible explanation is that IBDV binding
and/or membrane penetration triggers intracellular sig-
nals in CEFs that have no immediate effect on K?chan-
nels.This idea is inaccordance withourobservationthat
upon stimulation of CEFs with certain growth factors,
effects on K?channels are notobserved until 2 h follow-
ing application (Repp et al., 1995). Interestingly, stimula-
tion with insulin or platelet-derived growth factor evokes
changes inthe K?currentproperties ofCEFs verysimilar
to those observed with IBDV, suggesting similarities be-
tween virus-triggered and growth factor receptor-medi-
ated signal transduction to K?channels in these cells.
Many viruses interfere with the apoptosis of infected
cells, presumably retarding cellularapoptosis in orderto
prolong the life of the cell and thereby to maximize the
number of virions. The host cell, on the other hand,
should promote apoptosis as a direct response to viral
infection, thereby inhibiting viral growth and blocking
viral spread (Krakauer and Payne, 1997). In response to
IBDV infection, apoptosis has also been observed in
different cell types (Vasconcelos and Lam, 1994, 1995;
Tham and Moon, 1996; Fernandez-Arias et al., 1997;
Ojeda et al., 1997). Several groups have reported data
supporting the idea that K?channels may play a pivotal
role in the cell death program (Beauvais et al., 1995;
Deluca et al., 1996; Szabo et al., 1996, 1997; McCarthy
and Cotter, 1997). Recently, a direct correlation between
apoptosis and the activity of K?channels was estab-
lished. Attenuation of the K?outward current in neocor-
tex neurons (Yu et al., 1997) and inhibition of K?efflux in
lymphocytes reduced apoptosis (Bortner et al., 1997).
Since the Ca2?dependence ofthe K?outwardcurrentin
CEFs is shifted by 2 orders of magnitude to higher
concentrations upon infection with IBDV, the K?channel
open state probability is reduced compared to nonin-
fected cells. Thus, the reduction of K?channel activity
may be a mechanismused by IBDV to delay the apopto-
tic process, at least in CEFs. Further studies investigat-
ing the effects ofK?channel blockers and activators will
help to define the role of K?channels in the IBDV infec-
tion process and in apoptosis as a cellular response to
MATERIALS AND METHODS
Cells and virus
Preparation of CEFs was performed as described
(Repp et al., 1993). Cells were maintained at 37°C in a
humidified, 5% CO2/95% air atmosphere in Dulbecco’s
modified Eagle’s medium (DMEM) containing 5% new-
born calf serum, 2 mM L-glutamine, and antibiotics.
Serotype 1 strain Cu-1 of IBDV (Nick et al., 1976) was
adapted to CEFs and remained pathogenic for chicken
(Lange etal., 1987). Viruses were propagated in CEFs as
described (Cursiefen et al., 1979). Confluent CEFs were
infected with IBDV at a multiplicity of infection (m.o.i.) of
5, and virus replication was determined by direct immu-
nofluorescence using fluorescein-conjugated chicken
anti-IBDV serum(Mu ¨ller, 1986). Viability of IBDV-infected
CEFs was verified by fluoresceindiacetate/ethidiumbro-
mide staining (Thiel and Burkhardt, 1984).
Inactivation of IBDV particles with UV light was per-
formed ata wavelengthof254 nm. UV irradiationwas for
30 min using a distance of 5 cm between the source of
light and the tissue culture dishes that contained IBDV.
Inactivation of viral replication was verified by a plaque
assay (Becht et al., 1988).
Removal ofvirus particles fromthe culture supernatant
of IBDV-infected CEFs was achieved as follows. Eight
hours after IBDV infection of CEFs, the culture superna-
tant was aspirated and centrifuged 2.5 h at 270,000 g.
The supernatant was ultrafiltered through centrifugal
concentrators (Macrosep 50-kDa cutoff, Pall Filtron, Karl-
stein, Germany). The filtrate was free of infectious parti-
cles and soluble viral proteins, as proven by a plaque
assay and Western blot analysis, respectively (Mu ¨ller,
1986; Becht et al., 1988).
Neutralization of viral infectivity and binding studies
Preparation and characterization of the neutralizing
mouse monoclonal antibody (mAb) B1 that specifically
recognizes the VP2 proteinofIBDV Cu-1 were performed
according to protocols described previously (Bechtetal.,
1988). To test the capacity of the mAb B1 for neutraliza-
tionofviral infectivity inCEFs, IBDV particles were mixed
with serial dilutions of the antibody (stock concentration
5 mg/ml) and incubated for 30 min at 37°C. In a neutral-
izationassay using the standardprocedure describedby
Becht et al. (1988), mAb B1 showed a neutralization titer
of 1:32,000 when 100 plaque-forming units/ml (PFU) was
used. The neutralizationtiterrepresents the reciprocal of
the antibody dilution at which plaque counts were re-
In the electrophysiological experiments, 2 ? 105CEFs
were infected with 2 ? 106PFU. When 2 ? 106PFU of
IBDV Cu-1 were incubated with the mAb B1, no plaques
were visible in the plaque assay at antibody dilutions of
up to 1:160.
367 CHANGES OF POTASSIUM CURRENT BY IBDV
The binding of IBDV to CEFs was investigated using
[35S]methionine-labeled virus particles (Nieper and Mu ¨l-
ler, 1996). We used the protocol described in this report
to measure the influence of antibodies on the binding of
IBDV Cu-1 to CEFs. Prior to the binding assay, IBDV
particles were incubated for 30 min at 37°C either with
the neutralizing mAb B1 or, as a control, with a mAb that
specifically recognizes the hemagglutinin of influenza A
Membrane ion current measurements
Twice subcultured confluent cultures of CEFs were
trypsinized and cells were plated at a density of 2 ? 105
cells per 35-mm dish and recultured for 12–24 h. There-
after, the cells were infected with IBDV at a m.o.i. of 10,
using dilutions of culture supernatant of IBDV-infected
CEFs that was taken 24 h postinfection and centrifuged
to separate off the included cells. Since the PFU:particle
ratio of this culture supernatant is about 1:100 (Mu ¨ller et
al., 1986), it can be estimated that each CEF was inocu-
lated with about1000 virus particles. Afteradsorption for
30 min at 37°C (this was taken as 0 time) cells were
washed twice with DMEM and reincubated at 37°C for
various time periods prior to the membrane current re-
cordings.We usedthe whole-cellrecording configuration
of the patch-clamp technique (Hamill et al., 1981), as
applied recently to normal and Rous sarcoma virus-
transformed CEFs (Repp et al., 1993). For optimal patch-
clamp conditions, normal CEFs and CEFs inoculated
with IBDV were made to assume a rounded morphology
by incubation with trypsin (0.25 mg/ml) for 1 min at room
temperature in Ca2?-free bath solution and subsequent
washing with bath solution. The recordings were made
between 15 min and 2 h after trypsin treatment. As a
control, membrane currents of cells without trypsin pre-
treatment were measured; in this case, the same mem-
brane current properties were observed as those in
Whole-cell membrane currents were recorded using
an EPC-7 patch-clamp amplifier (List Electronics, Darm-
stadt, Germany). Data acquisition and off-line analysis
were performed with an Atari Mega ST4 computer with
M2LAB Atari Data software (Instrutech Corp., Elmont,
NY). Extracellularsolution contained (mM) NaCl 140, KCl
3, MgCl22, CaCl22, glucose 10, HEPES (N-2-hydroxyeth-
ylpiperazine-N?-2-ethanesulfonic acid) 10, pH 7.4, ad-
justed with NaOH. The recording pipette contained in-
tracellularsolution composed of(mM) K?glutamate 140,
NaCl 20, MgCl22, HEPES 10, pH 7.3, adjusted with KOH.
For recordings in the absence of free intracellular Ca2?,
the Ca2?-chelating agent BAPTA [1,2-bis(2-aminophe-
noxy)ethane-N,N,N?,N?-tetraacetic acid] at a concentra-
tion of 5 mM was added to the pipette solution. Free
intracellular Ca2?concentrations ([Ca2?]i) of 10?9, 10?8,
10?7, or 10?6M were obtained using 5 mM of the Ca2?
chelator EGTA [ethylene glycol bis(?-aminoethyl ether)
N,N,N?,N?-tetraacetic acid] and total Ca2?concentra-
tions of0.03, 0.31, 2.0, or4.35mM, assuming anapparent
dissociation constant KDof 0.15 ?M (pH 7.3) for the
Ca2?–EGTA complex. Recording pipettes had resis-
tances of5–10 M? whenfilled withintracellularsolution.
Initial cell-attached seal resistances were approximately
25 G?. The data were corrected for the liquid junction
potential between the pipette and bath solutions, which
was ?10 mV for the standard K?glutamate internal
solution. The electrophysiological measurements were
performed at 20–22°C.
We are indebted to C. Zibuschka forexperttechnical assistance and
to J. Dickson, W. H. Gerlich, E. Martinson, and H.-J. Thiel for their criti-
cal comments on the manuscript. This work was supported by the
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369CHANGES OF POTASSIUM CURRENT BY IBDV