role of double-stranded rna and n of classical swine fever ... · infection. first, sk-6 cells were...
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lsevier.com/locate/yviro
Virology 343 (20
Role of double-stranded RNA and Npro of classical swine fever
virus in the activation of monocyte-derived dendritic cells
Oliver Bauhofer, Artur Summerfield, Kenneth C. McCullough, Nicolas Ruggli *
Institute of Virology and Immunoprophylaxis (IVI), Sensemattstrasse 293, CH-3147 Mittelhausern, Switzerland
Received 13 May 2005; returned to author for revision 25 June 2005; accepted 12 August 2005
Available online 8 September 2005
Abstract
Classical swine fever virus (CSFV) is a noncytopathogenic (ncp) positive-sense RNA virus that replicates in myeloid cells including
macrophages and dendritic cells (DC). The virus does not induce type I interferon (IFN-a/h), which in macrophages has been related to the
presence of the viral Npro gene. In the present work, the role of viral double-stranded (ds)RNA and Npro in the virus–host cell interaction has been
analyzed. Higher levels of detectable dsRNAwere produced by a genetically engineered cytopathogenic (cp) CSFV compared with ncp CSFV, and
cp CSFV induced IFN-a/h in PK-15 cells. With DC, there was only a small difference in the levels of dsRNA between the cp and ncp viruses, and
no IFN-a/h was produced. However, the cp virus induced a higher degree of DC maturation, in terms of CD80/86 and MHC II expression. Npro
deletion mutants induced an increase in DC maturation and IFN-a/h production–for both ncp and cp viruses–despite reduced replication
efficiency in the DC. Deletion of Npro did not influence dsRNA levels, indicating that the interference was downstream of dsRNA turnover
regulation. In conclusion, the capacity of CSFV to replicate in myeloid DC, and prevent IFN-a/h induction and DC maturation, requires both
regulated dsRNA levels and the presence of viral Npro.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Pestivirus; Classical swine fever virus; Npro; Double-stranded RNA; Dendritic cell; Interferon-a/h; CD80/86; MHC II; Innate immunity
Introduction
Classical swine fever virus (CSFV) is an enveloped positive-
sense RNAvirus that belongs to the genus Pestivirus within the
Flaviviridae family (Fauquet et al., 2005). The virus is highly
contagious for pigs and wild boar and has a worldwide
distribution. Depending on the CSFV strain, the outcome of
an infection can vary from severe hemorrhagic symptoms with
high mortality rate to a mild and almost unapparent disease. The
other members of the Pestivirus genus are the bovine viral
diarrhea virus (BVDV) and the border disease virus (BDV) that
cause disease in cattle and sheep, respectively. Pestiviruses are
closely related to the flaviviruses and hepaciviruses, two
important groups of human pathogens of the Flaviviridae
family (Fauquet et al., 2005). The genome of the pestiviruses
0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2005.08.016
* Corresponding author. Fax: +41 31 848 9222.
E-mail addresses: [email protected] (O. Bauhofer),
[email protected] (A. Summerfield),
[email protected] (K.C. McCullough),
[email protected] (N. Ruggli).
consists of a single RNA molecule of positive polarity. It
contains one open reading frame (ORF) flanked by two non-
translated regions (NTR). An internal ribosome entry site
(IRES) located in the 5V NTR mediates cap-independent
translation initiation of a polyprotein. Co- and post-translational
processing by cellular and viral proteases leads to the proteins
Npro, C, Erns, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and
NS5B (for review, see Lindenbach and Rice, 2001). Npro is
unique to pestiviruses within the Flaviviridae and exhibits
autoprotease activity resulting in rapid cleavage from the
nascent polyprotein (Rumenapf et al., 1998; Stark et al.,
1993; Wiskerchen et al., 1991). So far, Npro is the only gene
known to be non-essential for the pestivirus life cycle (Lai et al.,
2000; Tratschin et al., 1998). While mutant CSFV lacking the
Npro gene (DNpro CSFV) are replication-competent in cell
culture, they have lost their capacity to interfere with cellular
antiviral defense mechanisms, i.e. double-stranded (ds) RNA-
mediated apoptosis and interferon (IFN)-a/h induction (Ruggli
et al., 2003). In fact, DNpro CSFV, unlike wild-type virus,
induce IFN-a/h in macrophages and PK-15 cells (Ruggli et al.,
2003). DNpro CSFV are also avirulent in pigs but can induce a
05) 93 – 105
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O. Bauhofer et al. / Virology 343 (2005) 93–10594
protective immune response (Mayer et al., 2004). Npro of CSFV
is an antagonist of dsRNA-mediated IFN-a/h induction
independently of the CSFV context (La Rocca et al., 2005;
Ruggli et al., in press). A recent report suggests that Npro of
BVDV is also involved in suppressing interferon-dependent
antiviral responses (Horscroft et al., 2005).
CSFV is a monocytotropic virus that replicates in dendritic
cells (DC) without inducing cell activation (Carrasco et al.,
2004). This is important considering that DC are central players
in the induction of early immune responses against pathogens.
One process employed by DC is activation triggered by
pathogen associated molecular patterns (PAMP). For RNA
viruses, an important PAMP inducing host response is the
dsRNA of replicative intermediates and replicative forms
produced during genome replication (for review, see Good-
bourn et al., 2000; Jacobs and Langland, 1996). Such dsRNA is
recognized in the cell by receptors such as toll-like receptor
(TLR)3 (Alexopoulou et al., 2001; Matsumoto et al., 2004), the
retinoic acid inducible gene I (RIG-I) product (Yoneyama et al.,
2004), the melanoma differentiation-associated gene 5 (mda-5)
product (Andrejeva et al., 2004; Kang et al., 2002), the
dsRNA-dependent protein kinase (PKR) and by the coupled
2V–5V oligoadenylate synthetase (2–5 OAS)/RNaseL system
(Gil and Esteban, 2000; Player and Torrence, 1998).
Although CSFV is generally noncytopathogenic (ncp) in cell
culture and in DC, cytopathogenic (cp) biotypes have been
isolated from infected animals (Aoki et al., 2001; Kosmidou et
al., 1998; Meyers and Thiel, 1995) or arose spontaneously after
multiple passages in cell culture (Meyers and Thiel, 1995;
Mittelholzer et al., 1997). With few exceptions (Aoki et al.,
2004; Gallei et al., 2005; Hulst et al., 1998), the cytopathic
effect (cpe) observed so far with cp CSFV correlated with the
presence of defective interfering particles (DI) associated with
the wild-type helper virus. This DI consists of the 5V NTR, thecodon for methionine, the genes NS3 to NS5B and the 3V NTR.The CSFV DI was shown to function as cp replicon
independently of the helper virus, establishing that the genes
Npro to NS2 were non-essential for pestivirus RNA replication
in cell culture (Behrens et al., 1998; Moser et al., 1999; Tautz et
al., 1999). In addition, cp CSFV could be rescued in cell culture
by transfection of ncp CSFV-infected cells with DI RNA
(Meyers et al., 1996) or by co-transfection of in vitro transcripts
of the cp replicon and the helper virus (Moser et al., 1999). For
CSFV, the significance of the cp biotype in pathogenesis is
probably minor (Aoki et al., 2003; Kosmidou et al., 1998), in
contrast to BVDV for which the cp biotypes are associated with
mucosal disease (Kummerer et al., 2000; Tautz et al., 1998). An
interesting observation is that cp pestiviruses produce a
significantly higher amount of total viral RNA compared with
the corresponding ncp biotype (Kupfermann et al., 1996;
Lackner et al., 2004; Mendez et al., 1998; Mittelholzer et al.,
1997; Vassilev and Donis, 2000). However, the double-stranded
form of the viral RNA has never been specifically analyzed.
The present study aimed to characterize the role of viral
dsRNA and Npro in CSFV-mediated innate immune activation
in vitro. Induction of IFN-a/h was analyzed by comparing the
IFN-a/h-competent PK-15 cell line with the SK-6 cell line that
cannot be stimulated to produce IFN-a/h (Ruggli et al., 2003).
Porcine monocyte-derived DC were employed to analyze the
role of viral dsRNA and Npro in DC activation and maturation.
Both ncp and cp CSFV biotypes were used as models of low
and high dsRNA-producing viruses, and the role played by
Npro was tested with DNpro mutants of ncp and cp CSFV.
Results
Cp CSFV produces higher amounts of dsRNA than ncp CSFV
Viral dsRNA is a well-known trigger of innate immune
activation. Previous studies demonstrated that cp pestiviruses
produced higher amounts of total viral RNAwhen compared to
the corresponding ncp pestivirus. However, the double-stranded
fraction of RNA occurring during replication of ncp and cp
CSFV has never been identified and quantified so far. Therefore,
we analyzed dsRNA in CSFV-infected cells by using the
monoclonal antibody (mAb) J2. Detection of dsRNA in
CSFV-infected SK-6 cells by immunofluorescence provided
first evidence that cp CSFV produced more dsRNA than its ncp
parent (Fig. 1A). While the ncp CSFV-infected cells exhibited
faint dsRNA dots (Fig. 1A, panel 4), the cp CSFV-infected cells
contained a high density of large bright fluorescent spots (Fig.
1A, panel 6). Immunostaining of the viral glycoprotein E2
resulted in a comparable signal for both ncp and cp CSFV (Fig.
1A, panels 3 and 5). The specificity of the mAb J2 for CSFV
dsRNA was assessed in a dot blot assay using in vitro
synthesized nucleic acids. For this purpose, serial dilutions of
plasmid DNA encoding the full-length genome (pA187-1) and
of in vitro transcribed CSFV single-stranded (ss)RNA and
dsRNA were dot-blotted onto a nitrocellulose membrane and
subjected to immunodetection as described. With identical
amounts of nucleic acids, a 400-fold stronger signal was
detected for CSFV dsRNA than for CSFV ssRNA, whereas
plasmid DNA was barely detectable even at the highest
concentration (Fig. 1B). The standard curve obtained with serial
dilutions of in vitro synthesized CSFV dsRNAwas then used to
quantify dsRNA in extracts from mock, ncp and cp CSFV-
infected SK-6 cells. Nucleic acid extracts were serially diluted
and dot-blotted onto a nitrocellulose membrane (Fig. 1C).
Quantification revealed a 5- to 10-fold higher amount of dsRNA
synthesized in cp CSFV-infected cells when compared to the ncp
CSFV. This represents the first demonstration that cp CSFV
produces higher levels of dsRNA than ncp CSFV, which may
result in differential activation of the innate immune response.
The amount of dsRNA produced by cp CSFV is dependent on
the cell type
We showed previously that the porcine cell lines SK-6 and
PK-15 display a different response to stimulation by dsRNA
(Ruggli et al., 2003, in press). Therefore, we analyzed dsRNA
in these two cell lines in comparison with primary porcine
monocyte-derived DC. For this purpose, flow cytometry
(FCM) was evaluated as an alternative to the dot blot assay
for the quantification of dsRNA levels produced upon CSFV
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Fig. 1. Detection of dsRNA in mock- and CSFV-infected cells. (A) SK-6 cells were mock-treated (1, 2) or infected with ncp CSFV (3, 4) or with cp CSFV (5, 6) at an
m.o.i. of 2 TCID50/cell. At 16 h p.i., cells were fixed and analyzed by confocal microscopy using the E2-specific mAb HC/TC26 (1, 3, 5) or the dsRNA-specific
mAb J2 (2, 4, 6). The cytoskeleton was detected with fluorescent-conjugated phalloidin. (B) The specificity of the mAb J2 for dsRNAwas tested in a dot blot assay.
10-fold serial dilutions of dsRNA, ssRNA and plasmid DNAwere dot-blotted onto a nitrocellulose membrane. Detection was performed according to Materials and
methods. (C) dsRNA from mock-, ncp CSFV- or cp CSFV-infected SK-6 cells was quantified in a dot blot assay. Nucleic acid extracts obtained 16 h p.i. were diluted
as indicated and blotted onto a nitrocellulose membrane. dsRNA was detected according to Materials and methods and quantified by comparison with a standard
curve based on in vitro synthesized dsRNA. Amounts of dsRNA are indicated in nanograms for each dot.
O. Bauhofer et al. / Virology 343 (2005) 93–105 95
infection. First, SK-6 cells were either mock-infected or
infected with ncp or cp CSFV and monitored for dsRNA
levels by FCM. The difference of dsRNA fluorescence
intensity between ncp and cp CSFV was comparable to that
observed with the dot blot assay (Fig. 2A). Therefore, all
following analyses were carried out by FCM. When SK-6 cells,
PK-15 cells and DC were compared, the differences in dsRNA
levels between ncp and cp CSFV varied considerably. In SK-6
cells that do not produce IFN-a/h, infection with cp CSFV
resulted in the highest dsRNA production. In PK-15 cells,
which are capable of producing IFN-h, the amount of viral
dsRNA was lower compared to SK-6 cells. In DC, the
difference in dsRNA synthesis between ncp and cp CSFV
was the smallest (Fig. 2A). A statistical analysis of the
geometric mean fluorescence intensity (MFI) of two indepen-
dent experiments using ANOVA revealed a significant differ-
ence for the dsRNA levels between mock-treated cells and cells
infected with either ncp or cp CSFV for all the cell types (P <
0.004). The difference between ncp and cp CSFV was also
significant in SK-6 and PK-15 cells (P < 0.004) and in DC
(P < 0.005) (Fig. 2B). Interestingly, the cpe caused by cp CSFV
was strong in SK-6 cells, less pronounced in PK-15 cells (Fig.
2C) and absent in DC (data not shown). The cpe appeared in
both SK-6 and PK-15 cells 16 h post-infection (p.i.) and
resulted in complete lysis of the SK-6 monolayer 40 h p.i.,
whereas the PK-15 cells recovered (data not shown). No
evidence of cell death could be detected in DC at any time (data
not shown), despite virus replication detected by NS3 expres-
sion (Fig. 2C). Taken together, these observations show cell-
dependent differences in the control of virus replication and of
viral dsRNA levels.
Cp CSFV induces IFN-a/b in PK-15 cells but not in DC
As dsRNA is known to induce synthesis of IFN-a/h, weasked whether the level of dsRNA was related to the degree of
IFN-a/h induction. These experiments were performed with
PK-15 and DC cultures since it was observed previously that
no IFN-a/h could be induced in SK-6 cells (Ruggli et al.,
2003). Supernatants of cells infected with cp CSFV, ncp CSFV
and mock were tested for bioactive IFN-a/h after virus
neutralization, using the MxCAT reporter gene assay (Fig. 3).
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Fig. 3. Analysis of IFN-a/h secretion induced by cp CSFV. PK-15 cells and DC
were treated with mock SK-6 cell extract, ncp CSFV and with 5-fold serial
dilutions of cp CSFV. The undiluted virus and the ncp CSFV corresponded to
an m.o.i. of 2 TCID50/cell for PK-15 and 5 TCID50/cell for DC. Supernatants
were collected 16 h p.i. for analysis of IFN-a/h bioactivity. The error bars
represent the 95% confidence interval (n = 5).
O. Bauhofer et al. / Virology 343 (2005) 93–105 97
In accordance with previous data (Carrasco et al., 2004; Ruggli
et al., 2003), ncp CSFV did not induce any detectable IFN-a/h,neither in PK-15 cells nor in DC. In PK-15 cells, cp CSFV
induced IFN-a/h in a non-linear, multiplicity of infection
(m.o.i.)-dependent manner. Interestingly, the infection of DC
with cp CSFV did not induce any detectable interferon
synthesis, which may relate to the only slight increase of
dsRNA when compared with ncp CSFV (see Fig. 2).
Cp CSFV induces DC maturation
Next, we investigated the maturation level of ncp and cp
CSFV-infected DC in order to determine whether these cells
respond differently to the two biotypes. For this purpose, the
level of CD80/86 and MHC II molecules on the cell surface
was monitored by FCM. Cells infected with cp CSFV
expressed higher levels of MHC II and CD80/86 molecules
(Fig. 4A). The differences of the geometrical MFI values
analyzed using ANOVA were statistically significant (P <
0.004 for CD80/86, P < 0.03 for MHC II). The infection with
ncp CSFV did not result in a statistically significant upregula-
tion of MHC II and CD80/86 when compared to mock,
although the raw data indicated a slight increase of the
expression levels. In conclusion, while viral protein expression
in DC did not differ between cp and ncp CSFV as measured by
the levels of Npro and NS3 (Fig. 4B), cp CSFV induced
maturation of DC without detectable IFN-a/h production.
Maturation of DC and IFN-a/b secretion can be triggered by in
vitro synthesized CSFV dsRNA
Since we demonstrated activation of DC by cp CSFV with a
small increase of dsRNA levels and without detectable
Fig. 2. Quantification of dsRNA in mock-, ncp and cp CSFV-infected SK-6 cells, PK
mock, ncp CSFVor cp CSFVat anm.o.i. of 2 (SK-6 and PK-15) respectively 5 TCID50
by FCM. (B) The geometric mean fluorescence intensity (MFI) values obtained in t
submitted to statistical analysis usingANOVAon ranks. TheP values obtained for the
parallel cultures of SK-6 and PK-15 cells were subjected to immunoperoxidase stain
IFN-a/h secretion, the next experiments were aimed at
determining the activation potential of in vitro synthesized
CSFV dsRNA. CSFV dsRNA obtained in vitro was not
replication-competent (data not shown) and was compared
with in vitro transcribed non-replicating CSFV ssRNA and
with plasmid DNA carrying the full-length CSFV genome
sequence as control. DC were lipofected with CSFV DNA,
ssRNA or dsRNA and monitored by FCM for CD80/86 and
MHC II upregulation. Transfection of DC with dsRNA resulted
in a significant increase in MHC II levels (P < 0.005) (Fig. 4C).
CD80/86 expression levels were not increased with dsRNA
and only slightly increased with plasmid DNA (Fig. 4C). The
survival rate of DC after lipofection of dsRNA was reduced
when compared to transfection with plasmid DNA and with
ssRNA (data not shown). Interestingly, transfection of DC with
synthetic CSFV dsRNA resulted in secretion of IFN-a/h(Fig. 4D), in contrast to the infection with cp CSFV (Fig. 3).
The induction was specific for dsRNA and was not due to the
transfection process since ssRNA and plasmid DNA did not
induce IFN-a/h secretion.
DNpro CSFV induces IFN-a/b secretion and DC maturation
Similar to cp CSFV in PK-15 cells (Fig. 3), DNpro CSFV is
an inducer of IFN-a/h in these cells and in macrophages
(Ruggli et al., 2003). The fact that DNpro CSFV is ncp suggests
that there is no direct link between cytopathogenicity and IFN-
a/h induction. To elaborate on this, we analyzed IFN-a/hsecreted by DC in response to infection with DNpro CSFV and
monitored CD80/86 and MHC class II expression. DC
produced IFN-a/h in response to infection with DNpro CSFV
(Fig. 5A) in absence of any detectable cpe. Furthermore, DNpro
CSFV induced an upregulation of CD80/86 and of MHC class
II molecules on DC (Fig. 5B). The difference in the CD80/86
upregulation between DNpro CSFV and ncp CSFV analyzed
using ANOVA was statistically significant (P < 0.008). The
increased activation in absence of Npro occurred despite slight
reduced levels of viral protein expression as measured by NS3
(Fig. 5C). This confirms that cytopathogenicity of CSFV is not
a prerequisite for DC activation and suggests that Npro is
required for the prevention of such activation.
DNpro CSFV- and ncp CSFV-infected cells produce compara-
ble amounts of dsRNA
We have shown above that IFN-a/h induction in PK-15
cells and activation of DC correlated with increased levels of
dsRNA observed with cp CSFV but that cytopathogenicity was
not a prerequisite for DC activation. Since DNpro CSFV
induced IFN-a/h and activated DC without being cytopatho-
genic, we analyzed whether Npro was involved in the regulation
-15 cells and DC. (A) SK-6 cells, PK-15 cells and DC were infected either with
/cell (DC). At 16 h (SK-6, PK-15) and 48 h (DC) p.i., dsRNA levels were analyzed
wo independent experiments (n = 10 for SK-6 and PK-15, n = 6 for DC) were
differences between each group are indicated. (C) At the time of dsRNAdetection,
ing for viral NS3. For DC, analysis of NS3 expression was performed by FCM.
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Fig. 4. Role of cp CSFVand viral dsRNA in DC activation and IFN-a/h secretion. (A) CD80/86 and MHC II upregulation was measured in DC after infection with
ncp and cp CSFV at an m.o.i. of 5 TCID50/cell. (B) The Npro and NS3 expression levels in mock (lane 1)-, ncp CSFV (lane 2)- and cp CSFV-infected DC (lane 3)
were analyzed by SDS-PAGE and Western blotting. (C) CD80/86 and MHC II upregulation was measured in DC and after transfection with in vitro synthesized ss
and dsRNA of CSFV. Transfection of plasmid DNA coding for the full-length CSFV genome, mock transfection and mock-transfected DC supplemented with 1000
U/ml porcine IFN-a and 20 ng/ml porcine TNF (mock + IFN/TNF) served as controls. (D) IFN-a/h secretion was analyzed 48 h post-transfection. In panel (A), the
asterisk represents a statistical significant upregulation of CD80/86 (P < 0.002, n = 6) and of MHC II (P < 0.035, n = 6). In panel (C), the upregulation of MHC II
expression in DC transfected with dsRNAwas statistically significant as shown by the asterisk (P < 0.005, n = 3) when compared with mock, DNA and ssRNA. For
all the graphs, mean values are depicted. The error bars represent the 95% confidence interval.
O. Bauhofer et al. / Virology 343 (2005) 93–10598
of dsRNA levels during virus infection. For this purpose, we
quantified dsRNA induced by DNpro CSFV in comparison with
ncp CSFV in the three different cell types. There was no
statistical difference in the amounts of dsRNA between ncp and
DNpro CSFV in SK-6 and PK-15 cells (Figs. 6A and 6B). In
DC infected with DNpro CSFV, one population of cells
displayed a dsRNA-dependent fluorescence intensity compa-
rable with ncp CSFV-infected cells, and a second population of
cells showed a decreased dsRNA fluorescence at the level of
non-infected cells (Fig. 6A). This could be explained by the
fact that the replication of DNpro CSFV was impaired in DC
when compared with ncp CSFV. At the same m.o.i., only 40%
of the DC infected with DNpro CSFV expressed NS3, whereas
with ncp CSFV approximately 90% of the cells were NS3
positive at the same time after infection (Fig. 6C). From these
experiments, we conclude that IFN-a/h induction in PK-15
cells and activation of DC by DNpro CSFV are not due to
increased dsRNA accumulation.
The lack of Npro in cp CSFV enhances the cp CSFV-mediated
IFN-a/b production and MHC II upregulation
The observation that DNpro-mediated IFN-a/h production
was apparently not induced by higher levels of viral dsRNA
indicates that Npro regulates IFN-a/h induction downstream
of dsRNA. According to this, cp CSFV lacking the Npro gene
should display enhanced IFN-a/h induction and DC activa-
tion. In order to test this hypothesis, we rescued a cp DNpro
CSFV and analyzed virus replication, DC activation and IFN-
a/h induction. SK-6 cells, PK-15 cells and DC were either
mock-infected or infected with ncp CSFV, cp CSFV, DNpro
CSFV or cp DNpro CSFV. All the viruses replicated with
comparable efficiency in SK-6 cells but differed in IFN-a/h-competent cells such as PK-15 cells and DC. The strongest
reduction of virus replication was observed with cp DNpro
CSFV in both, PK-15 cells and DC (Fig. 7A). When IFN-a/hbioactivity was measured, cp DNpro CSFV infection resulted
in a 1.5-to 5-fold increase in IFN-a/h synthesis in PK-15
cells and in DC respectively, when compared with cp CSFV
(Fig. 7B). This was particularly remarkable with DC, in
which cp DNpro CSFV replication was not detectable when
measured by NS3 expression (Fig. 7A). Analysis of DC
activation did not reveal any significant difference in the
levels of CD80/86 with either virus tested (Fig. 7C).
However, for cp DNpro CSFV, expression of MHC II
molecules was increased by two-fold when compared to
either cp CSFV or DNpro CSFV (Fig. 7C). Taken together,
these experiments show that increased amounts of dsRNA
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Fig. 5. IFN-a/h secretion and DC activation after infection with DNpro CSFV.
DC were treated with mock SK-6 cell extract, ncp CSFV and with 5-fold serial
dilutions of DNpro CSFV. The undiluted DNpro CSFV and the ncp CSFV
corresponded to an m.o.i. of 5 TCID50/cell. (A) At 48 h p.i., supernatants were
collected for analysis of IFN-a/h bioactivity. (B) The respective cells infected
with undiluted virus stocks or mock were analyzed for CD80/86 and MHC
class II expression by FCM. The difference between CD80/86 levels induced
by ncp CSFV and DNpro CSFV was statistically significant as shown by the
asterisk (P < 0.008, n = 6). The error bars represent the 95% confidence
interval. (C) Expression of the viral proteins Npro and NS3 in DC 48 h after
infection with mock (line 1), ncp CSFV (line 2) and DNpro CSFV (line 3) was
analyzed by SDS-PAGE and Western blotting.
O. Bauhofer et al. / Virology 343 (2005) 93–105 99
and the lack of Npro synergistically contribute to the
activation of DC and IFN-a/h induction.
Discussion
For immunological sensing of virus infections, DC play a
major role through PAMP receptors such as the TLRs. The
development of memory responses against antigen has been
demonstrated to depend on TLR stimulation (Iwasaki and
Medzhitov, 2004). Many viruses have successfully evolved
mechanisms to evade detection by the immune system,
including the capacity to prevent immune activation and
permit their replication in IFN-a/h-competent cells (for
selected reviews, see Basler and Garcia-Sastre, 2002; Good-
bourn et al., 2000; Tortorella et al., 2000; Weber et al.,
2004). Recently, we showed that CSFV has the capacity to
replicate in DC without inducing their activation (Carrasco et
al., 2004). In order to further elaborate on this observation,
the present study analyzed the interaction of CSFV with
monocyte-derived DC. Of particular interest were the dsRNA
intermediates formed by RNA viruses during their replica-
tion, clear candidates to trigger IFN-a/h production (for
review, see Goodbourn et al., 2000; Jacobs and Langland,
1996). In this same vein, it is known that cp pestiviruses
induce higher RNA levels than their ncp counterparts
(Kupfermann et al., 1996; Lackner et al., 2004; Mendez et
al., 1998; Mittelholzer et al., 1997; Vassilev and Donis,
2000), but it was not known whether this is also true for the
double-stranded forms of the RNA.
The present work demonstrates that cp CSFV produces
elevated amounts of dsRNA, compared with the ncp biotype,
but with variations dependent on the cell type infected. SK-6
cells accumulated more dsRNA than PK-15 cells and DC. In
fact, the latter showed only a minor difference in dsRNA levels
between the cp and ncp biotypes. With the PK-15 cells, this
might be reflecting the induction of IFN-a/h by the cp CSFV.
In DC, although there was no IFN-a/h detectable in the
supernatant, the increased expression of CD80/86 and MHC
class II after cp CSFV infection demonstrated DC activation,
suggesting that also in these cells an antiviral state controlling
dsRNA accumulation would be induced. Interestingly, cyto-
pathogenicity was observed only in cell cultures where high
amounts of dsRNA were produced, the cp CSFV being
cytopathic for SK-6 and PK-15 cells, but not for DC. Similar
results have been reported in terms of cp BVDV being
noncytopathogenic in bovine DC (Glew et al., 2003), although
these were induced to produce IFN-a/h.Honda et al. demonstrated that IFN-a/h signaling is required
for dsRNA-induced DCmaturation in vitro (Honda et al., 2003).
This would appear to be in contradiction with the observation
that cp CSFV upregulates CD80/86 and MHC II molecules
without detectable IFN-a/h bioactivity. Nevertheless, low levels
of IFN-a/h would quickly become assimilated by the DC and
thus be undetectable. Certainly, dsRNA can induce IFN-a/h in
DC as we demonstrate that in vitro synthesized CSFV-derived
dsRNA transfected into DC induced IFN-a/h. Along this line isour recent report that the activation of DC by transfected
synthetic mRNA molecules was dependent on secondary
dsRNA structures (Ceppi et al., 2005). Furthermore, a recent
study demonstrated that the addition of cationic lipids prolon-
gates the retention time of nucleic acids in the endosomal
compartment in DC and thus enhances the IFN response (Honda
et al., 2005).
DC activation by dsRNA can be mediated via TLR3 (for
review, see Matsumoto et al., 2004), but, with RNA virus
infections, it is necessary to consider TLR7 and TLR8 as
receptors for ssRNA (Barchet et al., 2005; Diebold et al.,
2004; Heil et al., 2004; Lund et al., 2004). However, the
monocyte-derived DC used in the present study do not
respond to in vitro synthesized CSFV ssRNA, supporting that
CSFV dsRNA is the main trigger for myeloid DC activation
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O. Bauhofer et al. / Virology 343 (2005) 93–105100
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Fig. 7. Virus replication, IFN-a/h induction and activation of DC infected with cp DNpro CSFV. SK-6, PK-15 and DC were infected with mock, ncp CSFV, DNpro
CSFV, cp CSFVor cp DNpro CSFVat an m.o.i. of 1 (SK-6 and PK-15) respectively 0.5 TCID50/cell (DC) and analyzed 16 h (SK-6 and PK-15) and 48 h (DC) p.i. (A)
The percentage of infected cells was assessed for the different viruses by measuring the percentage of cells expressing the viral protease NS3 by FCM. (B) The IFN-
a/h bioactivity was measured in the supernatant of infected cells 16 h p.i. For all the graphs (except for PK-15 in panel B, single values), mean values obtained from
2 independent experiments (n = 6) are shown, with error bars representing the 95% confidence interval. (C) At 48 h p.i., DC were harvested and analyzed for CD80/
86 and MHC class II expression.
O. Bauhofer et al. / Virology 343 (2005) 93–105 101
observed with cp CSFV. Human monocyte-derived DC also
lack responsiveness to ssRNA, in contrast to plasmacytoid
DC (reviewed in Liu, 2005).
Having identified the importance of dsRNA in triggering
myeloid DC, the question remained concerning the role of the
viral Npro. Previous work has shown Npro to be important for
counteracting cellular antiviral responses such as IFN-a/hsecretion (Horscroft et al., 2005; La Rocca et al., 2005; Ruggli
et al., 2003, in press). Although the absence of Npro did not
impair viral replication in SK-6 cells, DNpro CSFV was
incapable of efficient replication in DC. These mutants induced
IFN-a/h production by the DC, which would explain the
limitation of the virus replication, similar to that reported with
infected macrophages and PK-15 cells (Ruggli et al., 2003).
Horscroft and co-workers also reported that BVDV replicons
Fig. 6. Quantification of dsRNA in DNpro CSFV-infected cells. (A) SK-6 cells, PK-1
of 2 (SK-6 and PK-15) respectively 5 TCID50/cell (DC). At 16 h (SK-6, PK-15) and
MFI obtained in two independent experiments (n = 6) was submitted to statistica
between each group are indicated. (C) At the time of dsRNA detection, parallel cult
viral NS3. For DC, analysis of NS3 expression was performed by FCM.
lacking the Npro gene failed to establish stable replication in
Huh-7 cells (Horscroft et al., 2005). It is still unclear if this
antiviral state is due to IFN-a/h activity. What is known is that
CSFV replication in DC is impaired by IFN-a treatment (data
not shown).
Recently, Lackner and co-workers showed that the temporal
modulation of NS2–3 processing by the NS2 autoprotease is
crucial in RNA replication control and that the intracellular
level of NS3 strictly correlates with the efficiency of RNA
replication (Lackner et al., 2004). Whether these proteins
regulate the dsRNA levels remains to be established. Although
Npro can be implicated in controlling IFN-a/h induction, our
results show that Npro is not regulating the dsRNA turnover. It
is more likely that Npro is inhibiting the cellular response to
dsRNA. Using a virus engineered to combine elevated dsRNA
5 cells and DC were infected with mock, ncp CSFVor DNpro CSFVat an m.o.i.
48 h (DC) p.i., cells were fixed and dsRNA levels analyzed by FCM. (B) The
l analysis using ANOVA on ranks. The P values obtained for the differences
ures of SK-6 and PK-15 cells were subjected to immunoperoxidase staining for
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O. Bauhofer et al. / Virology 343 (2005) 93–105102
levels in absence of Npro (cp DNpro CSFV), an additive effect
on IFN-a/h induction and DC activation was observed.
The present work has given rise to a model of CSFV–host
interaction governing virus evasion of host cell responses.
Regulation of CSFV RNA turnover with minimal accumulation
of dsRNA on one hand and Npro as a viral inhibitor of innate
immune activation on the other hand are key elements. They
account for a successful initiation of virus replication in the
primary target cells (monocytic cells), allowing dissemination
in the host. This is a particularly critical event for a virus
targeting cells of the immune system, especially highly
endocytic cells such as macrophages and dendritic cells. By
such means, CSFV not only replicates in IFN-producing cells,
but also controls IFN induction and DC maturation. Another
viral protein – Erns – may well be involved due to its proposed
control of dsRNA-mediated induction of IFN-h (Iqbal et al.,
2004). Further studies are required to understand how the
dsRNA turnover is regulated and what mechanisms are
involved in Npro-induced control of innate immune activation.
Such a detailed characterization will permit the understanding
of the early phases of CSFV pathogenesis.
Materials and methods
Cells
The swine kidney cell line SK-6 (Kasza et al., 1972) was
propagated in Earl’s minimal essential medium containing 7%
horse serum (GibcoBRL). The porcine kidney cell line PK-15
(American Type Culture Collection, Manassas, VA, USA) was
maintained in Dulbecco’s modified eagle medium (DMEM)
supplemented with non-essential amino acids, 1 mM Na-
Pyruvat and 5% horse serum (GibcoBRL). Porcine DC were
prepared as described elsewhere (Carrasco et al., 2001).
Briefly, peripheral blood mononuclear cells were isolated from
specific pathogen-free pigs bred at our institute. SWC3 positive
monocytes were sorted using the MACS system (Miltenyi
Biotec) and differentiated to DC by incubation for 4 days at 39
-C in DMEM with 10% porcine serum (Sigma) in the presence
of 100 U/ml recombinant porcine IL-4 and 150 ng/ml
recombinant porcine GM-CSF (kindly provided by Dr. Shigeki
Inumaru, Institute for Animal Health, Ibaraki, Japan). The IL-4
was prepared in our laboratory as described previously
(Carrasco et al., 2001).
Viruses
The viruses vA187-1 and vA187-DNpro were rescued by
electroporation of SK-6 cells with in vitro transcribed RNA
from the plasmid cDNA clones pA187-1 (Ruggli et al., 1996)
and pA187-DNpro (Ruggli et al., 2003) as previously described
(Moser et al., 1999). Cp CSFV was obtained by co-transfection
of SK-6 cells with in vitro transcribed RNA from plasmids
pA187-1 and pA187-D4764iv (Moser et al., 1999) at a ratio of
1:4. For the recovery of cp DNpro CSFV, SK-6 cells were co-
transfected with in vitro transcribed RNA from plasmids
pA187-DNpro and pA187-D4764iv at a ratio of 1:10. All the
virus titers were determined on SK-6 cells by standard end-
point dilution technique and were expressed as 50% tissue
culture infectious doses (TCID50)/ml. The m.o.i. used with SK-
6 and PK-15 cells was calculated from the titer obtained with
the respective cell line. Titration on DC was not performed, and
the m.o.i. was calculated based on the SK-6 titer.
In vitro synthesis of CSFV dsRNA
CSFV dsRNA was obtained by annealing of in vitro
transcribed full-length positive and negative-sense viral
RNA. For this purpose, plasmid pA187-1 was modified to
allow in vitro run-off transcription of negative-sense CSFV
RNA. A phosphorylated DNA cassette obtained by annealing
of primers T7-XhoI-L (5V-TATAGTGAGTCGTATTAC) and
T7-XhoI-R (5V-TCGAGTAATACGACTCACTATA) was ligat-ed between the SrfI and XhoI sites of pA187-1, resulting in
plasmid pA187-2xT7. Then, the SalI to ClaI fragment of
plasmid pA187-2xT7 containing the 5V-terminal T7 promoter
and the 5V end of the genome was replaced by the SalI to ClaI
fragment of plasmid pA187T3-SnaBI (Moser C., unpublished)
containing a unique SnaBI restriction site for linearization at
the precise 5V end of the genome. The new plasmid was called
pA187-5V-SnaBI. In vitro transcription of RNA was performed
with the MEGAscript T7 kit (Ambion) as described elsewhere
(Moser et al., 1999). Full-length run-off transcripts were
synthesized in independent reactions from SrfI-linearized
plasmid pA187-1 for positive-sense RNA and from SnaBI-
linearized plasmid pA187-5V-SnaBI for negative-sense RNA.
Subsequently, equimolar amounts of both RNA strands were
mixed and denatured at 75 -C for 5 min prior to annealing at
room temperature to obtain CSFV dsRNA. The integrity of the
dsRNA was confirmed by digestion experiments with RNase
A1 and T in 10 mM Tris–HCl pH 7.5, 300 mM NaCl and 5
mM EDTA. A non-replicating ssRNA control transcript was
synthesized from a pA187-1-derived construct carrying muta-
tions in the NS5B gene resulting in a Gly–Asp–Asp to Ala–
Ala–Ala substitution at amino acid positions 3627 to 3629 of
the polyprotein (details of the construction can be obtained on
request).
Transfection of DC
DC were transfected as previously described (Ceppi et al.,
2005). Briefly, cells were harvested after 4 days of culture and
washed twice with Opti-MEM (GibcoBRL). Nucleic acids
were diluted in Opti-MEM, and Transfast lipofection agent
(Invitrogen) was added at a ratio of 3 Al Transfast per 1 Agnucleic acid. The mixture was incubated at room temperature
for 15 min prior to addition to the cells. 4 Ag of nucleic acids
was transfected per 106 cells. The lipofection mix was
incubated with the cell suspension at 39 -C for 1 h. The cells
were then washed twice in culture medium supplemented with
recombinant porcine GM-CSF/IL4 (concentrations as indicated
above) and plated in 6-well plates. Two days post-transfection,
the cells were harvested and acquired by FCM for CD80/86
and MHC class II expression. Supernatants were subjected to
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O. Bauhofer et al. / Virology 343 (2005) 93–105 103
analysis of IFN-a/h bioactivity. Lipofection efficacy was
monitored with an mRNA encoding EGFP (Ceppi et al., 2005).
Antibodies and cytokines
Monoclonal antibodies directed against the viral protein
NS3 (C16) (Greiser-Wilke et al., 1992) and the E2 glycoprotein
(HC/TC26) (Greiser-Wilke et al., 1990) (kindly provided by
Dr. Irene Greiser-Wilke, Hannover Veterinary School, Hann-
over, Germany) were used for virus detection by FCM and
immunohistochemistry. For the detection of viral proteins by
Western blotting, polyclonal rabbit sera against Npro (Ruggli
et al., in press) and NS3 (Ruggli N., unpublished) were utilized.
dsRNA was visualized with the dsRNA-specific mAb J2
(English and Scientific Consulting Bt., Szirak, Hungary)
described to detect the A-helix of double-stranded polyribo-
nucleotide complexes larger than 11 base pairs (Schonborn
et al., 1991). Secondary rabbit anti-mouse biotinylated anti-
body (DAKO) was used in combination with IR Dye-labeled
streptavidin (Rockland). The level of CD80/86 molecules
expressed on the cell surface was measured using human
CTLA4 coupled to murine immunoglobulin (Ancell). MHC II
levels were determined with mAb MSA3 (Hammerberg and
Schurig, 1986). Recombinant porcine IFN-a was produced in
HEK293EBNA cells as described elsewhere (Balmelli et al.,
2005). Porcine tumor necrosis factor a (TNF) was prepared
from L929 cells expressing porcine TNF, kindly provided by
Dr. Giuseppe Bertoni, Vetsuisse Faculty, University of Berne,
Switzerland (Von Niederhausern et al., 1993).
Immunostaining and confocal microscopy
SK-6 cells were cultured and infected with CSFV in eight-
well culture slides (Becton Dickinson). Prior to immunostain-
ing, the cells were washed with PBS, fixed with 4% (w/v)
paraformaldehyde for 15 min and washed again with PBS.
The cells were then incubated for 20 min at 4 -C with the
respective monoclonal antibody diluted in PBS/0.3% (w/v)
saponin (Sigma) and washed again three times with PBS
containing 0.1% (w/v) saponin. Detection was performed with
Alexa-488 fluorochrome-labeled anti-mouse secondary anti-
body (Molecular Probes). The cytoskeleton was stained with
Alexa-546-conjugated phalloidin (Molecular Probes). Analysis
was performed with a Leica TCS-SL spectral confocal
microscope and Leica LCS software (Leica).
dsRNA dot blot
A dot blot assay was established for the quantification of
dsRNA extracted from CSFV-infected cells. Eight million cells
were harvested 16 h p.i. and washed twice with PBS. The cells
were resuspended in 900 Al lysis buffer (200 mMNaCl, 100 mM
Tris–HCl pH 8, 2% sodium dodecyl sulfate and 2% h-mercaptoethanol). Nucleic acids were extracted and purified
from the lysate with phenol:chloroform:isoamylalcohol
(25:24:1) and precipitated with isopropanol. The pellet was
washedwith 70% ethanol, dried and resuspended in water. Serial
dilutions of the extracts were dot-blotted with a Minifold II dot
blot device (Schleicher & Schuell) onto a Protran nitrocellulose
membrane (Schleicher & Schuell). Nucleic acids were fixed by
baking the membrane at 80 -C for 1 h. The membrane was then
blocked for 1 h at room temperature in a blocking solution
consisting of Odyssey Blocking Buffer (LI-COR) diluted 1:1
with PBS (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl,
pH 7.3). The primary antibody J2 was diluted to a concentration
of 0.5 Ag/ml (1:2000) in the blocking solution. The secondary
biotinylated rabbit F(abV)2 anti-mouse IgG (DAKO) and the IR
Dye-labeled streptavidin were diluted 1:2000 and 1:2500
respectively in the blocking solution containing 0.1% Tween
20. Between incubation steps, the membrane was washed 4
times for 5 min each with PBS supplemented with 0.1% Tween
20. Two wash steps with PBS lacking detergent were carried out
prior to image acquisition and quantification using the Odyssey
Infrared Imaging System (LI-COR). Quantification of the
nucleic acids was based on a standard curve obtained with in
vitro synthesized CSFV dsRNA.
Western blotting
DC were lysed 48 h p.i. with a hypotonic buffer (20 mM
morpholinepropanesulfonic acid, 10 mM NaCl, 1.5 mM
MgCl2, 1% Triton X-100, pH 6.5). Viral proteins were
separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and analyzed by Western blotting
as previously described (Ruggli et al., in press).
Flow cytometry
FCM was performed as described elsewhere (Summer-
field and McCullough, 1997). For staining of cell surface
molecules, unfixed cells were incubated for 15 min on ice with
the respective primary and secondary antibodies diluted in
CellWash (Becton Dickinson). After each antibody incubation,
cells were washed once with CellWash. For the detection of the
intracellular antigens NS3 and dsRNA, cells were fixed and
permeabilized using a cell fixation and permeabilization kit
(Harlan Sera-Lab Ltd). The primary mAb C16 and J2
respectively were diluted in the permeabilization agent. mAb
J2 was used at a working concentration of 10 Ag/ml (1:100
dilution). The acquisition was done by FCM (FACSCalibur,
Becton Dickinson).
Measurement of IFN-a/b bioactivity
Bioactive IFN-a/h in cell culture supernatant was measured
by using an Mx/CAT reporter gene assay initially developed for
the quantification of bovine IFN-a/h (Fray et al., 2001). This
assay was validated for porcine IFN-a/h and was performed as
previously described (Ruggli et al., 2003). Infectious CSFV
present in the samples to be analyzed was neutralized by
incubation for 1 h at 4 -C with polyclonal pig anti-CSFV serum
600–88 (Ruggli et al., 1995) diluted 1:100. RNAwas removed
by treating the samples for 1 h at 37 -C with a cocktail of 5 U/
ml RNaseA and 200 U/ml RNase T1 (Ambion).
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O. Bauhofer et al. / Virology 343 (2005) 93–105104
Statistical analysis
Statistical and graphical analyses were carried out with the
SigmaStat and SigmaPlot software package (SPSS). If appli-
cable, parametric analysis of variance (ANOVA) combined
with the Student’s t test was used. If the samples of the
populations did not satisfy the normal distribution or the equal
variance conditions, ANOVA on ranks combined with a rank
sum test was used. The P values were adjusted with Bonferroni
correction.
Acknowledgments
This work was funded by the Swiss Federal Veterinary
Office (grant # 1.03.04) and in part by the Swiss National
Science Foundation (grant # 31-68237.02). We thank Christian
Griot and Martin A. Hofmann for continuous support and Jon-
Duri Tratschin for critical reading of the manuscript. We are
also grateful to Markus Gerber, Luzia Liu, Viviane Neuhaus,
Valerie Tache and Heidi Gerber for excellent technical
assistance. The SK-6 cell line was kindly provided by M.
Pensaert. We are also grateful to Martin D. Fray for the Mx/
CAT reporter gene assay, to Shigeki Inumaru for the porcine
GM-CSF and to Giuseppe Bertoni for the porcine TNF. We
thank Irene Greiser-Wilke for providing us with mAbs HC/
TC26 and C16.
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