Journal of General Virology (2010), 91, 2762–2772 DOI 10.1099/vir.0.

025650-0

Correspondence
J. Schneider-Schaulies
jss@vim.uni-wuerzburg.de
N-(3-Cyanophenyl)-2-phenylacetamide, an
effective inhibitor of morbillivirus-induced
membrane fusion with low cytotoxicity
K. Singethan,1 G. Hiltensperger,2 S. Kendl,1 J. Wohlfahrt,1 P. Plattet,3
U. Holzgrabe2 and J. Schneider-Schaulies1
1
Institut für Virologie und Immunbiologie, University of Würzburg, Germany
2
Institut für Pharmazie und Lebensmittelchemie, University of Würzburg, Germany
3
Department für klinische Veterinärmedizin, University of Bern, Switzerland

Received 21 July 2010
Accepted 31 July 2010
Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we
tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit
measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane
fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity
to inhibit MV- and CDV-induced (IC5053 mM), but not NiV-induced, membrane fusion. This
compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is
low [50 % cytotoxic concentration (CC50)¢300 mM], leading to a CC50/IC50 ratio of
approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog
brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human
PBMC with recombinant wild-type MV is inhibited by an IC50 of approximately 20 mM. The cell-to-
cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely
inhibited by compound 1 at 50 mM, indicating that the virus spread between brain cells is
dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound
is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture
experiments including highly sensitive primary cells.

INTRODUCTION complications are acute post-infectious measles enceph-

alitis and, based on a persistent infection, subacute
The genus of closely related viruses Morbillivirus, contains
sclerosing pan-encephalitis (SSPE), which is always lethal.
measles virus (MV) and canine distemper virus (CDV),
SSPE does not occur after measles vaccination (Duclos &
which cause devastating diseases in their hosts. MV, one of
Ward, 1998).
the most contagious aerosol-transmitted viruses, still
causes more than 100 000 deaths each year. After entering CDV causes systemic infections similar to but distinct from
the upper respiratory tract, MV exhibits a pronounced human measles in carnivores such as canines, felids, ferrets,
tropism for mono- and lymphocytic cells and soon viral raccoons and seals, with lethality rates, depending on the
replication is detected in draining lymph nodes (Pfeuffer host, of up to 100 % (Appel et al., 1972; Harder &
et al., 2003; de Swart et al., 2007; Ferreira et al., 2010). Osterhaus, 1997; von Messling et al., 2003). In a high
During measles, patients develop a pronounced leukopenia percentage of animals the CNS is infected, causing severe
that may be due to enhanced adhesion of lymphocytes in complications with infiltration of inflammatory cells and
secondary lymphoid organs (Nanan et al., 1999; Dittmar demyelination (Summers & Appel, 1994; Vandevelde &
et al., 2008). In immunocompetent patients, MV infection Zurbriggen, 1995). Encephalomyelitis is the most common
is usually cleared by the virus-specific immune response, cause of death of CDV-infected animals, meaning that
while the general immune response to other antigens is CDV is much more neurotropic than MV. Live attenuated
suppressed for several weeks after the rash (reviewed vaccines against CDV have existed since the 1950s and their
by Schneider-Schaulies & Schneider-Schaulies, 2009). application led to a drastic reduction in CDV epidemics.
Following replication in lymphoid tissues, virus spreads However, between 1991 and 1995 there were several big
to various organs and can be detected in the skin, the outbreaks in France, Japan, Germany and Scandinavia
gastrointestinal tract, the lungs and the eyes. It may also which especially affected dogs with a high vaccination rate
enter the brain (reviewed by Ludlow et al., 2009). CNS (Blixenkrone-Moller et al., 1993; Gemma et al., 1996). The
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 Morbillivirus fusion inhibitor

reason is probably antigenic drift of the wild-type virus. RESULTS

This generates genetically different strains and makes the
vaccine inefficient (Harder & Osterhaus, 1997). Compounds 1 and 5 block morbillivirus envelope
Zoonotic outbreaks of respiratory diseases and encephalitis protein-induced, but not NiV-induced, cell fusion
affecting humans, horses and pigs in Australia, Malaysia We synthesized five acetanilide derivatives (Fig. 1), includ-
and Singapore have led to the isolation of two novel ing the MV-induced fusion inhibitor AS-48 (compound 5;
paramyxoviruses, Hendra and Nipah (NiV) viruses, which
have been assigned to the genus Henipavirus in the family
Paramyxoviridae (Murray et al., 1995; Chua, 2003). The
natural hosts of both viruses are fruit bats of the genus
Pteropus. While causing a respiratory disease with low
mortality rates in pigs, a severe febrile encephalitis with
high mortality rates was observed in NiV-infected humans
(estimated case fatality rate, 40–50 %). Recent outbreaks of
NiV infection in Bangladesh and India have led to further
human deaths and may have been a result of both direct
bat–human and human–human transmissions (Butler,
2004; Chadha et al., 2006). EphrinB2-positive cells,
including endothelial cells, are the major cellular targets
for NiV and syncytia of endothelial cells in blood vessels
are recognized as a characteristic feature of NiV infection
(Wong et al., 2002; Negrete et al., 2005). A late-onset
encephalitis may follow months after the acute infection,
indicating virus persistence in the brain (Tan et al., 2002).
Membrane fusion induced by paramyxoviruses is an essential
step in the viral infection cycle that is mediated by
cooperation of both viral envelope glycoproteins, the
attachment protein (H or G) and the fusion protein (F).
After cleavage of the precursor F protein into F1 and F2
subunits, these subunits form an active H–F1/2 complex
mediating pH independent virus–cell and cell–cell fusion.
Small-molecule inhibitors, fitting into a pocket of the MV F
protein and preventing membrane fusion by blocking the
natural interaction between the two essential heptad-repeat
regions within the F protein, have been designed. They
interfere with the conformational changes of the F protein at
the beginning of the fusion process (Plemper et al., 2004,
2005; Sun et al., 2006). Given the structural similarity of
paramyxovirus F proteins, we tested some of these small-
molecule inhibitors and several derivatives in a NiV F protein-
based fusion assay (Niedermeier et al., 2009). Molecular
modelling indicated that one compound fitted well into a
cavity present in the NiV F protein. This compound inhibited
NiV-induced cell fusion but not MV-induced cell fusion, thus
underlining specific differences between morbillivirus and
henipavirus F proteins (Niedermeier et al., 2009).
We investigated other small-molecule fusion inhibitors,
including N-(3-cyanophenyl)-2-phenylacetamide (com-
pound 1; Sun et al., 2006), and describe its high capacity
to inhibit MV- and CDV-induced, but not NiV-induced, Fig. 1. Compounds and synthesis pathways used in this study. (a)
membrane fusion. Compound 1 is of outstanding interest Compounds 1–5. (b) Scheme 1, synthesis pathway of compounds
because it can be synthesized easily and its cytotoxicity is 1–3. Reagents: (i) NaHCO3, CHCl3, room temperature; (ii) KOH,
low. Even primary human peripheral blood lymphocytes H2O, 100 6C; (iii) SOCl2, MeOH, 70 6C. (c) Scheme 2, synthesis
(PBL) and primary dog brain cells (DBC) tolerated virus- pathway of compounds 4 and 5. Reagents: (i) ethyl chloroformate,
inhibitory concentrations of this compound without signs NEt3, (NH4)2CO3, absolute acetone, room temperature; (ii)
of cytotoxicity. pyridine, phenylacetyl chloride, THF, room temperature.
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K. Singethan and others

compound 11f in Sun et al., 2006), using a modified method

as described by Sun et al. (2006) (see Methods). These
compounds were tested in a cell–cell fusion assay based on the
transfection of viral envelope proteins in the absence of other
viral genes. The envelope proteins of MV (strain Edmonston),
CDV (strain Onderstepoort) and NiV (Malaysia brain isolate)
were used. In this fusion assay the mean sizes of the syncytia
(number of nuclei in a syncytium) are presented as a
percentage of the control versus increasing concentrations of
the compounds (Fig. 2). In this assay, MV-induced cell fusion
was most efficiently inhibited with compound 1 (IC5053 mM;
identical to compound 3g in Sun et al., 2006) followed by
compound 5 (IC5058 mM; identical to compound AS-48),
and less efficiently by compounds 3, 2 and 4. CDV-induced
cell fusion was also efficiently inhibited by compounds 1 and
5 (IC5056 and 4 mM, respectively), whereas NiV-induced cell
fusion was not inhibited in this concentration range. Thus in
our hands, compound 1 was slightly better at inhibiting MV
envelope protein-induced cell fusion than compound 5. Both
substances were similarly active for CDV envelope protein-
induced fusion. Since the synthesis of compound 1 is
straightforward and easy, this compound was an interesting
candidate for further investigation.

Compound 1 efficiently inhibits MV and CDV

uptake and virus-induced cell fusion
In order to assess the ability of compound 1 to inhibit
syncytium formation induced by infectious wild-type MV
and CDV, we used the recombinant wild-type viruses rMV–
IC323–eGFP and rCDV–A75/17red, which express eGFP
and tomato red, respectively. As target cells in this infection-
inhibition assay we used Vero cells expressing the appro-
priate receptors, human and canine CD150 (Vero–hSLAM
and Vero–dogSLAM). In the first series of experiments virus
and inhibitors were mixed, added to the cells and the
mixture incubated for 48 h at 37 uC. Live cells were analysed
using a UV microscope and photomicrographs were taken
(Fig. 3). Syncytium formation was inhibited in a dose-
dependent manner by compound 1 and only few single Fig. 2. Dose-dependent inhibition of envelope protein-induced cell
infected cells were observed in the presence of .10 mM fusion. Vero cells were transfected with plasmids expressing the F
compound 1. Similar results were observed using compound and H proteins of MV strain Edmonston (a), F and H proteins of
5 (data not shown). CDV strain Onderstepoort (b) and F and G proteins of NiV (c).
Compounds were added after transfection at the concentrations
To investigate the effect of the inhibitors separately on virus–
indicated and the cells were incubated for 24 h at 37 6C. Mean
cell and cell–cell fusion, we either pre-incubated virus with
numbers of nuclei in at least 50 syncytia per concentration per
the inhibitors or added the inhibitors after infection of the compound were quantified by microscopy. Relative fusion is
cells (Fig. 4). In Fig. 4(a), rMV–IC323–eGFP and rCDV– presented as a percentage relative to the untreated control cells
A75/17red were mixed with compound 1 and incubated for (100 %). Mean values and SD of three independent experiments
15 min at room temperature prior to infection of Vero– are presented. SD for compounds 1 and 5 are also shown.
hSLAM and Vero–dogSLAM cells for 16 h. Under these
conditions the number of infected cells deceased with
increasing concentrations of the inhibitors. In Fig. 4(b), the results as are shown for compound 1 were obtained with
cells were first infected, then washed and further incubated compound 5 (data not shown). In addition, pre-incubation
in the presence of compound 1 for 24 h at 37 uC. This of cells with compounds 1 or 5, and subsequent infection in
treatment reduced the size of syncitia in an inhibitor the absence of these compounds did not affect infection or
concentration-dependent manner, but did not reduce the syncytium formation (data not shown). Thus both virus–cell
number of infected single cells (foci of infection). Similar and cell–cell fusions were inhibited by compounds 1 or 5.
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 Morbillivirus fusion inhibitor

Fig. 3. Inhibition of syncytium formation by compound 1 in recombinant wild-type MV- and CDV-infected Vero–hSLAM and
Vero–dogSLAM cells. (a) Vero–hSLAM cells were infected with rMV–IC323–eGFP. (b) Vero–dogSLAM cells were infected
with rCDV–A75/17red at an m.o.i. of 0.1. Compound 1 was mixed with virus prior to infection in increasing concentrations (0–
30 mM). Virus and inhibitor were left in the cell culture and incubated for 48 h at 37 6C. Fluorescence and bright-field images
were taken using a 20¾ objective lens.

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K. Singethan and others

Fig. 4. Virus–cell fusion and cell–cell fusion are inhibited by compound 1. (a) The viruses (rMV–IC323–eGFP and rCDV–A75/
17red) were mixed with compound 1 and incubated for 15 min at room temperature prior to infection of cells. Vero–hSLAM and
Vero–dogSLAM cells, upper and lower panels respectively, were incubated at 37 6C for 16 h. (b) The cells were first infected
with the viruses for 1 h at 37 6C, then washed with PBS and further incubated in medium in the presence of compound 1 for
24 h at 37 6C. Images were taken using a 20¾ objective lens.

As a consequence of the inhibition of virus-induced membrane were determined using Vero–hSLAM and Vero–dogSLAM
fusion, the titres of infectious viral particles were reduced by cells for virus production and titration, respectively. Inhibition
compounds 1 and 5 in a concentration-dependent manner (Fig. of viral titres of more than 2 logs (.99.5 %) was obtained
5). Viral titres of rMV–IC323–eGFP and rCDV–A75/17red with concentrations of 30 mM for compounds 1 and 5.
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 Morbillivirus fusion inhibitor

assessed the toxicity of compound 1 using phytohaemag-

glutinin (PHA)-stimulated primary human peripheral blood
lymphocytes (PBL). In this case we used the DNA-
intercalating substance 7-amino-actinomycin D conjugated
to APC-Alexa Fluor 750 (7AAD), which is incorporated into
damaged cells, and evaluated the result by flow cytometry.
Compound 1 was non-toxic in PBL at concentrations
¡100 mM, whereas compound 5 was non-toxic only at
concentrations ¡30 mM (Fig. 6b). The CC50 values of
compounds 1 and 5 in PBL were approximately 300 and
100 mM, respectively.

Fig. 5. Reduction of virus titres by treatment with compounds 1

and 5 in MV- and CDV-infected Vero cells. Vero–hSLAM and
Vero–dogSLAM cells (1¾105), were infected with rMV–
IC323eGFP (a) and rCDV–A75/17red (b), respectively, at an
m.o.i. of 0.1 in the presence of increasing concentrations of
compounds 1 or 5, as indicated, and incubated for 3 days. As in
Fig. 4, the compounds were mixed with virus prior to infection and
left in the culture. Total virus (cell-bound and released) was
prepared and titrated using Vero–hSLAM and Vero–dogSLAM
cells (results with SD of three independent experiments are
presented).

Fig. 6. Determination of cell viability in the presence of compounds

using Vero cells and PBL. (a) Vero cells (1¾105 per well) were
Cytotoxicity of the compounds incubated for 48 h with increasing concentrations of compounds 1
Cytotoxicity in Vero cells was determined using the or 5. After removal of the medium, cells were incubated for 2 h at
tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 37 6C with MTT solution, then for 45 min at room temperature with
extraction solution; finally, aliquots were evaluated using an ELISA
tetrazolium bromide (MTT), which is incorporated in living
reader. Results are presented as percentage viability relative to
cells and converted into a violet formazan product by
DMSO-treated control cells. (b) PBL (2¾105) were treated with
mitochondrial dehydrogenases. After treatment of Vero the compounds 1 or 5 for 48 h and processed for flow cytometry.
cells, no considerable cytotoxic effects of compounds 1 or 5 Before measurement, the cells were incubated with 7AAD for
were observed at concentrations ¡100 mM (Fig. 6a), and at 10 min, then washed with FACS buffer and the incorporation of
concentrations of 300 mM 46 and 28 % of cells remained 7AAD measured using the far-red channel of the flow cytometer.
vital, respectively. Compound 5 was always slightly more Results are the mean values from three independent experiments
toxic than compound 1. Since Vero cells are quite robust and are presented as the percentage of viable cells relative to
and may not reflect the sensitivity of primary cells, we also untreated healthy cells.
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K. Singethan and others
Compound 1 can be used in sensitive primary
cultures of lymphocytes and brain cells
The above findings encouraged us to test the capacity of
compound 1 to inhibit the infection of primary human
PBL with recombinant wild-type MV and DBC with
recombinant wild-type CDV. PBL were stimulated with
PHA for 48 h prior to infection to stimulate the expression
of the MV receptor CD150 (not shown; Tatsuo et al., 2000;
Erlenhoefer et al., 2001). The cells were then infected at an
m.o.i. of 0.5 with rMV–IC323–eGFP in the absence or
presence of increasing concentrations of compound 1 and
48 h later the infection of CD3+ T and CD19+ B
lymphocytes was analysed by flow cytometry (Fig. 7). A
higher percentage of B than T lymphocytes was infected
(approximately 50 and 30 %, respectively) with the
recombinant wild-type MV (Fig. 7b). In addition, the
mean fluorescence intensity (MFI) of eGFP, which directly
reflects the level of virus replication, was three- to fourfold
higher in B than in T lymphocytes, and was 50 % inhibited
by compound 1 (and compound 5, data not shown) at
concentrations of approximately 20 mM in both cell types
(Fig. 7c).
DBC were infected with rCDV–A75/17red at an m.o.i. of
0.01 and cultivated for 12 days at 37 uC in the presence or
absence of concentrations of 50 mM compound 1 (Fig. 8).
The number of foci and the cell-to-cell spread were
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drastically reduced in the presence of compound 1,
indicating that the activity of the F protein is required
for cell-to-cell spread of CDV in persistently infected DBC.
Morphologically, there was no sign of toxicity detectable in
compound 1-treated DBC cultures, thus demonstrating
that this concentration is well tolerated by these sensitive
primary brain cells.
DISCUSSION
We investigated the capacity of two previously described
MV inhibitors and three new derivatives to inhibit MV-,
CDV- and NiV-induced membrane fusion. MV and CDV
envelope protein-mediated membrane fusion was selec-
tively inhibited by compounds 1 and 5, whereas NiV F/G
protein-mediated cell fusion was not affected. Compound
1 [N-(3-cyanophenyl)-2-phenylacetamide] was found to be
of special interest because it can be synthesized easily and
its cytotoxicity is low. Even primary human PBL and
primary DBC tolerated virus-inhibitory concentrations of
compound 1 without signs of cytotoxicity. The finding that
a higher percentage of B than T lymphocytes is infected by
wild-type MV and that B cells express more viral protein,
as indicated by eGFP, reflects a finding in lymphatic tissue
by Condack et al. (2007). The infection of both B and T
cells was inhibited by compounds 1 or 5 by 50 % at
Fig. 7. Inhibition of MV infection of primary
human PBL. PBL (1¾105 per well) were
infected in the absence or presence of
compound 1 with rMV–IC323–eGFP at an
m.o.i. of 0.5. The cells were incubated for 48 h
at 37 6C and then processed for flow cyto-
metry with PE-conjugated antibodies to CD3
and CD19 to allow separate analysis of T and
B lymphocytes. (a) Examples of dot plots
showing infection (eGFP expression) of
CD3+ T cells and CD19+ B cells.
Percentages of cells are given in the quad-
rants. (b) The percentages of MV-infected
(eGFP-positive) B and T cells of the total
number of gated B and T cells in the presence
of increasing concentrations of compound 1.
(c) The eGFP expression (MFI) of infected B
and T cells in the presence of increasing
concentrations of compound 1. Results and SD
of five independent experiments are given.
Journal of General Virology 91

Fig. 8. Inhibition of the spread of CDV in dog brain cultures by
compound 1. DBC were infected with rCDV–A75/17red at an
m.o.i. of 0.01 and further cultivated in the presence (50 mM; a, b)
or absence (c, d) of compound 1 for 12 days at 37 6C. The red
fluorescence shows infected cells (a, c) in comparison with the
bright-field photographs demonstrating that the primary brain cell
cultures are intact (b, d).
concentrations of approximately 20 mM. The spread of
recombinant wild-type CDV in DBC was nearly completely
inhibited by concentrations of 50 mM compound 1,
suggesting that infection and virus spread in these
persistently infected cultures are dependent on the activity
of the viral fusion protein. Thus, compound 1 is a most
applicable inhibitor for tissue culture experiments with
highly sensitive primary cells and may be a good candidate
for application in an animal model of MV or CDV
infections.
Compound 1 was assessed earlier as a derivative of an
effective inhibitor of MV-induced membrane fusion (AS-
48, compound 5) (Plemper et al., 2004, 2005; Sun et al.,
2006). Sun et al. (2006) tested the activity of compound 1
(compound 3g in Sun et al., 2006) using Vero cells and an
attenuated vaccine-like MV strain (Edmonston), which
uses the receptor CD46 on Vero cells (Erlenhoefer et al.,
2002). In this publication the most efficient compound was
compound 5, which carries nitro and amido groups meta
and ortho to the anilide nitrogen, respectively. This
compound was also tested and found to be active against
various wild-type MV strains with IC50 values of 0.6–3 mM
(Plemper et al., 2005). Importantly, compounds 1 and 5
interact, presumably, with the same region in the viral F
protein, since compound 5-resistant strains, with a
mutated aa 462 in the C-terminal heptad-repeat domain,
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Morbillivirus fusion inhibitor
were also much less effectively inhibited by compound 1
(compare IC50 of 6.5 mM with IC50 of .600 mM for resistant
strains) (Doyle et al., 2006; Sun et al., 2006). The IC50
concentration of compound 1, obtained in our fusion-
inhibition assay using transfection of the viral envelope
proteins of MV strain Edmonston (IC50 of approximately
3 mM), coincided quite well with the described value for MV
Edmonston (6.5 mM). In addition, we obtained similar results
for the CDV envelope proteins of strain Onderstepoort (IC50
of approximately 6 mM). These data suggest that compound 1
interacts at a similar site for the CDV-F protein and for the
MV-F protein site determined earlier.
In comparison with certain peptides derived from heptad-
repeat regions (HR) of the F protein, which imitate the
natural interaction between HR-A and HR-B necessary for
the conformational change of the F protein, small-molecule
inhibitors are not immunogenic, are more soluble, are easier
to synthesize and show better gastrointestinal adsorption
(Meanwell & Krystal, 2000). In tissue culture experiments
compound 1 may replace the fusion-inhibiting peptide (FIP;
Z-D-Phe-Phe-Gly where Z is a benzyloxycarbonyl protection
group; Richardson & Choppin, 1983). FIP is widely used for
inhibition of morbillivirus-induced cell fusion but its
mechanism of action is not clear. In contrast to compound
1, FIP is supposed to interact with the cell membrane and to
inhibit the conversion of phospholipid bilayers to the
hexagonal phase (Epand, 1986).
Binding of morbillivirus attachment protein H to its host
cell receptor simultaneously activates protein F, which will
then undergo a structural rearrangement leading to virus–
cell or cell–cell membrane fusion. These processes are not
only important during acute infections, but may also
influence viral spread during persistent infections in the
brain. It has been demonstrated that the F protein of wild-
type CDV is a major determinant of persistent infection
(Plattet et al., 2005, 2009). CDV-A75/17, a virulent
demyelinating viral strain isolated from the lymphoid
tissues of an experimentally infected dog, spreads efficiently
in DBC cultures with extremely limited cell–cell fusion and
a low level of cytolysis, whereas the attenuated laboratory
strain Onderstepoort induces ample syncytium formation
and cell lysis. However, non-cytolytic cell-to-cell spread
promotes viral persistence and the development of chronic
disease (Vandevelde & Zurbriggen, 2005). In line with
these results, it has been documented that haemagglutinin
was strictly required to allow efficient CDV-A75/17 spread
in DBC (Wyss-Fluehmann et al., 2010). However, in the
latter study the absence of protein H may have influenced
protein F transport competence and/or proper folding of
protein F. Here we now extend these results further using
one of the fusion inhibitors (compound 1) binding to a
cavity in protein F by clearly demonstrating that, in the
presence of protein H, protein F is also essential for
controlling CDV cell-to-cell lateral spread in brain cell
cultures. These data support the notion that cell-to-cell
transmission of wild-type CDV in brain cells lacking
signalling lymphocyte activation molecule (SLAM) expres-
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K. Singethan and others
sion is driven by a classical mechanism of fusion, thereby
presumably implicating an as yet unidentified cellular
receptor, protein H attachment and subsequent triggering
of protein F.
Ribavirin (RBV) is the only approved antiviral that is in use
against MV infections of the CNS (SSPE) and NiV
infections. It is a water-soluble synthetic nucleoside with
broad-spectrum antiviral properties that has been shown to
be active in vitro against many viruses (Huffman et al.,
1973; Gururangan et al., 1990; Honda et al., 1994; Ishii
et al., 1996; Chong et al., 2001; Graci & Cameron, 2006;
Elia et al., 2008). Although RBV was used as an antiviral
agent during the first NiV outbreak in 1999 in Malaysia, its
antiviral properties could not be confirmed in the generally
accepted hamster model (Georges-Courbot et al., 2006). In
the case of SSPE the treatment with inosiplex (isoprino-
sine), intraventricular alpha interferon and RBV, which is
applied in many cases, may at best prolong the disease
course (Weissbrich et al., 2003). Unfortunately, the high
concentrations of RBV required have undesirable side
effects, e.g. haemolytic anaemia (De Franceschi et al.,
2000). Future work will show whether compounds with
lower toxicity based on phenyl-2-phenylacetamide may be
suitable for application in vivo.
METHODS
Cells, viruses and antibodies. Vero cells (African green monkey;
ATCC CRL 6318), Vero cells expressing human CD150 (Vero–
hSLAM) and Vero cells expressing canine CD150 (Vero–dogSLAM;
Vero–hSLAM and Vero–dogSLAM were gifts of Dr Y.Yanagi, Kyushu
University, Fukuoka, Japan) were cultured in Eagle’s minimal
essential medium (MEM) containing 5 % FCS, 100 U penicillin ml21
and 100 mg streptomycin ml21. Recombinant MV wild-type strain
IC323–eGFP [rMV–IC323–eGFP (Hashimoto et al., 2002)] was
propagated using Vero–hSLAM. CDV strain Onderstepoort large
plaque was propagated in Vero cells and recombinant CDV wild-type
strain A75/17, expressing tomato red [rCDV–A75/17red, (Plattet et al.,
2004)] was propagated in Vero–dogSLAM cells.
PBMC obtained from buffy coats of healthy adult volunteers were
diluted 1 : 6 in Versene (GIBCO) and enriched with lymphocyte
separation medium LSM 1077 (PAA Laboratories). Fresh PBMC were
washed three times with PBS and suspended in RPMI 1640 (GIBCO)
medium containing 10 % FCS. PBMC were plated and incubated for
2 h at 37 uC to separate non-adherent PBL from adherent cells. PBL
were stimulated with 2.5 mg PHA-L ml21 (PHA, Roche Diagnostics)
for 48 h before infection. Stimulation of the cells was assessed by
measuring CD69 and CD150 expression by flow cytometry. DBC
(dissociated brain tissue cultures derived from neonatal dogs) were
prepared and maintained on poly-L-lysine-coated dishes as described
by Zurbriggen et al. (1984, 1995).
Polyclonal dog-anti-CDV hyperimmune serum was a kind gift from
M. Appel, Cornell University, Ithaca, NY, USA, and was used at a
1 : 1500 dilution. MV-specific hyperimmune serum from a patient
with SSPE was used at a 1 : 6000 dilution. The secondary antibody,
FITC-conjugated rabbit-anti-dog, was obtained from DAKO and
Alexa Fluor488-conjugated anti-human secondary antibody was
obtained from Molecular Probes. A mAb against CD150 (5C6) was
generated in our laboratory (Erlenhoefer et al., 2001) and purified
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using a protein G column. Phycoerythrin PE-labelled antibodies to
CD3, CD14, CD19 and CD69 were obtained from Becton Dickinson.
Synthesis of small-molecule inhibitors. Compounds 1–5 (Fig. 1)
were synthesized in a similar way to that which has been described for
compound 5, a lead structure of the third generation of the substances
oxazole 1 and acetamide 4 (Plemper et al., 2004, 2005; Sun et al.,
2006). Compound 1 was synthesized according to Sun et al. (2006),
using equimolar amounts of 3-aminobenzonitrile and phenylacetyl
chloride in the presence of sodium carbonate. The resulting product
was suspended in an aqueous solution of sodium hydroxide (2 M)
and refluxed for 30 h. Compound 2 was precipitated by the addition
of concentrated hydrochloric acid. For esterification, 1 equiv. of
compound 2 and 10 equiv. of thionyl chloride were dissolved in
methanol and refluxed for 17 h. The solvent was removed in vacuo
and the residue was recrystallized from methanol to yield product 3
(Fig. 1b). Compounds 4 and 5 were synthesized by a two-step
procedure, starting off with the amidation of the corresponding
benzoic acids. Therefore, 1 equiv. of the corresponding benzoic acid,
5 equiv. of ethyl chloroformate and 7 equiv. of triethylamine were
dissolved in absolute acetone under argon atmosphere. The in situ-
generated anhydrides were converted into the corresponding primary
amides using ammonium carbonate. The final amidation was carried
out in tetrahydrofuran (THF), using equimolar amounts of the
corresponding amide and phenylacetyl chloride in the presence of
3 equiv. of pyridine to yield compounds 4 and 5. The crude products
were purified and isolated by column chromatography (Fig. 1c).
Expression plasmids, transfection and quantification of cell–
cell fusion. NiV glycoprotein genes (F and G; GenBank accession no.
AF212302; Malaysian isolate from human brain tissue kindly
provided by Pierre Rollin and Tom Ksiazek, Centers for Disease
Control and Prevention, Atlanta, GA, USA) were cloned into vector
pczCFG, a derivative of the replication-deficient murine leukemia
virus, yielding pczCF5–NiVFtag2 and pczCF5–NiVG (Moll et al.,
2004). The CDV strain OND-LP protein H and protein F expressing
plasmids used for transfection were pCG–CDV–H and pCG–CDV–F.
The MV strain Edmonston protein H- and protein F-expressing
plasmids were pCG–H5 and pCG–F1. For transient expression cells
were transfected using polyethyleneimine (PEI 25 kDa; Polyscience).
Briefly, a monolayer of 46105 Vero cells per well of a six-well plate
were washed with pre-warmed medium. Mixtures of 2 mg DNA in
50 ml serum-free medium and 3 ml PEI (1 mg PEI ml21 in serum-free
medium) in 50 ml serum-free medium (incubated for 30 min at room
temperature) were added to 1 ml medium per well. Syncytia were
quantified after 24 or 48 h of incubation at 37 uC. Compounds were
added to the media prior to infection at the indicated concentrations.
Phase-contrast photomicrographs were taken of random regions by
using a 206 objective lens with a digital camera (Leica) and the mean
number of nuclei per syncytium in each well was calculated from
several experiments.
Cell viability assays. MTT (Sigma) is incorporated into living cells
and converted into a violet formazan product by mitochondrial
dehydrogenases. Only living vital cells and cells in the very early phase
of apoptosis can transform MTT to crystals. Vero cells (2.56105) in a
six-well plate were pre-incubated for 20 h with the chemical
compounds. Medium was removed and the cells incubated for 2 h
with 1 ml fresh medium per well (MEM with 5 % FCS). After removal
of the medium, cells were incubated for 2 h at 37 uC with 750 ml MTT
solution per well. The solution was removed and the cells incubated
for 45 min at room temperature with 750 ml extraction solution.
Twelve 50 ml aliquots were transferred into a 96-well plate and A570
evaluated using an ELISA plate reader. Results (mean values) were
presented as percentage viability relative to control cells that were
treated with amounts of DMSO equivalent to those used for the
compounds.
Journal of General Virology 91

To determine the toxicity of the compounds in cells growing in
suspension (PBMC), incorporation of 7AAD (7AAD staining
solution; eBioscience) into damaged cells was measured by flow
cytometry. Fluorescence was detected in the far-red range of the
spectrum (650 nm long-pass filter).
ACKNOWLEDGEMENTS
We thank the Deutsche Forschungsgemeinschaft and the Elite
Netzwerk Bayern for financial support.
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