Download Annexin A2 is involved in the production of classical swine fever

Journal of General Virology (2015), 96, 1027–1032
Short
Communication
DOI 10.1099/vir.0.000048
Annexin A2 is involved in the production of classical
swine fever virus infectious particles
Chun Sheng, Xiaoxiang Liu, Qiuyue Jiang, Bin Xu, Chenhao Zhou,
Yujing Wang, Jun Chen and Ming Xiao
Correspondence
College of Life and Environment Sciences, Shanghai Normal University, Shanghai, PR China
Ming Xiao
[email protected]
Received 16 October 2014
Accepted 9 January 2014
Annexin A2 (ANXA2) is an important host factor regulating several key processes in many viruses.
To evaluate the potential involvement of ANXA2 in the life cycle of classical swine fever virus
(CSFV), an RNA interference (RNAi) approach was utilized. Knockdown of ANXA2 did not impair
CSFV RNA replication but significantly reduced CSFV production. A comparable reduction of
extracellular and intracellular infectivity levels was detected, indicating that ANXA2 might play a role
in CSFV assembly rather than in genome replication and virion release. Furthermore, ANXA2 was
found to bind CSFV NS5A, an essential replicase component. Amino acids R338, N359, G378 of
NS5A were revealed to be pivotal for the ANXA2–NS5A interaction. Substitutions of these amino
acids had no effect on viral RNA replication but substantially reduced CSFV production, which
might partly be due to these mutations destroying the ANXA2–NS5A interaction. These results
suggested that ANXA2 might participate in CSFV production process by binding NS5A.
Classical swine fever virus (CSFV) is an important livestock
pathogen and is a member of the genus Pestivirus, along with
bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, and
border disease virus (BDV). They belong to the family
Flaviviridae, which also contains the closely related human
hepatitis C virus (HCV) (Heinz et al., 2000; Becher & Thiel,
2002). CSFV is an enveloped positive-strand RNA virus. The
virus contains a single-strand, positive-sense RNA genome,
which is composed of a 59UTR, an ORF and a 39UTR. The
39UTR and the 59UTR are thought to regulate pestivirus
genome replication (Isken et al., 2003, 2004; Xiao et al., 2004;
Pankraz et al., 2005). The 59UTR includes an internal
ribosome entry site that is responsible for translation
initiation of viral genomes (Fletcher & Jackson, 2002; Xiao
et al., 2011). The ORF codes for a resulting polyprotein that
is subsequently processed into mature proteins (Npro, C, Erns,
E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) by
cellular and viral proteases (Moennig & Plagemann, 1992).
The CSFV NS5B protein has RNA-dependent RNA polymerase (RdRp) activity and is able to bind its cognate 39UTR
and initiates genome replication (Steffens et al., 1999; Xiao et
al., 2004). NS5A is a component of the replication complex.
CSFV NS5A comprises 497 aa and is able to regulate viral
RNA replication and translation through binding to NS5B
and 39UTR (Chen et al., 2012; Xiao et al., 2009).
The viral life cycle requires various viral non-structural
proteins as well as host cellular proteins. Annexin A2
(ANXA2) is known as a lipid raft-associated scaffold protein.
It is calcium dependent and cytosolic and mediates essential
biological processes, including membrane trafficking, endosome formation, and aggregation of vesicles (Drust &
Creutz, 1988). ANXA2 has been identified as an important
000048 G 2015 The Authors
host factor in the regulation of several key processes in
cytomegalovirus (Wright et al., 1995), influenza virus
(LeBouder et al., 2008), calicivirus (González-Reyes et al.,
2009), bluetongue virus (Beaton et al., 2002), human
immunodeficiency virus (Ryzhova et al., 2006) and HCV
(Backes et al., 2010). Recently, a proteomic analysis revealed
that CSFV infection led to a significant increase of ANXA2
protein in host cells (Sun et al., 2008). Haem oxygenase I,
another cellular protein and ANXA2 were found to be
colocalized with the CSFV E2 protein (Shi et al., 2009). The
expression of annexin A1, another member of the annexin
family, was also elevated in PK-15 cells following CSFV
infection (Sun et al., 2010). However, the role of many
cellular proteins in the life cycle of CSFV remains poorly
understood. In this paper, we show that ANXA2 is involved
in CSFV production.
To evaluate the potential involvement of ANXA2 in the
life cycle of CSFV, an RNA interference (RNAi) approach
and CSFV subgenomic replicons and genomic replicons
(Sheng et al., 2010) (Fig. 1a) were utilized. A small
interfering RNA (siRNA) molecule targeted to porcine
ANXA2 sequence (GenBank accession no. NM_001005726)
and a negative control siRNA molecule that has no matches
either in the viral or the porcine genome were synthesized
(Shanghai Gene Chem). An anti-CSFV siRNA directed to
the CSFV NS5B gene served as a positive control (Xu et al.,
2008). Results showed that the ANXA2-specific siRNA
reduced the amount of ANXA2 without a significant
influence on the cell viability (results not shown) while
the control siRNA, anti-CSFV siRNA and the mock
transfection without siRNA addition did not (Fig. 1b).
Silencing ANXA2 did not reduce viral protein expression
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C. Sheng and others
pro
ms
5′UTR N C E
SM
ms
pro
5′UTR N ΔC E
CSM
pro
SM–
5′UTRN
CSM–
5′UTR N
pro
E1
E1
Ems
C
ΔC Ems
E1
E1
(b)
NS3 NS4A NS4B NS5A
E2 p70 NS2
E2 p70 NS2
NS3
NA
NA
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ts
ou
ith
W
1.2
1.0
0.8
0.6
0.4
0.2
0
ro
nt
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A2
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AN
ti-
An
NA
NA
iR
ls
siR
An
V
SF
C
ti-
NS5B
G9973A C9976T
(d)
RNA CSM RNA–1
β-Tubulin
3′UTR
3′UTR
G9973A C9976T
NS3 NS4ANS4B NS5A NS5B 3′UTR
(c)
ANXA2
NS5B
NS3 NS4A NS4B NS5A
E2 p70 NS2
3′UTR
NS5B
NS3 NS4A NS4B NS5A
E2 p70 NS2
24 48 72 h
**
****
iR
ts
ou
ith
W
iR
ls
ro
nt
Co
NA
NA
NA
siR
ti-
An
A2
X
AN
siR
V
SF
C
ti-
An
NA
Log10 TCID50 ml–1
(a)
8
6
4
2
0
24 48 72 h
** **
iR
ts
ou
ith
W
NA
NA
siR
ro
nt
Co
A2
X
AN
ti-
An
NA
NA
iR
ls
** **
**
**
siR
V
SF
siR
C
ti-
An
Fig. 1. Effect of siRNA-mediated knockdown of ANXA2 on CSFV production. (a) Schematic representation of genomic
replicons SM, SM”, subgenomic replicons CSM and CSM”, used in this study (Sheng et al., 2010). (b, c) The indicated
siRNAs were transfected into PK-15 cells using FuGENE HD Transfection Reagent (Roche) according to the manufacturer’s
protocol. Seventy-two hours after siRNA transfection, the transfected cells were retransfected with CSM” and a second dose
of the same siRNA. Viral particles were not formed and newly synthesized viral RNAs were detected in CSM” transfected cells.
(b) Seventy-two hours after retransfection, cells were harvested. The cell lysates were subjected to 10 % SDS-PAGE. The
amounts of ANXA2 (upper) and CSFV NS3 (below) in the cells transfected with the indicated siRNAs were evaluated by
Western blot analysis (Sheng et al., 2012) with anti-ANXA2 or anti-NS3 antibody. b-Tubulin was used as an internal control and
also for monitoring cytotoxicity (middle). (c) Cells were harvested at 24, 48 or 72 h after CSM” subgenomic replicon and siRNA
transfection. Quantification of viral RNA was determined by using RT-qPCR (Wang et al., 2011). Relative replication
efficiencies are shown. The CSM subgenomic replicon was used to normalize the data. (d) The indicated siRNAs were
transfected into PK-15 cells. The transfected cells were retransfected with SM” and a second dose of the same siRNA. The
retransfected cells were harvested at 24, 48 and 72 h post-transfection, and titres were determined by immunofluorescence
(Xiao et al., 2011). The arrowhead represents the detection limit of the assay. All quantified results are shown as means±SD of
at least three independent experiments. ** P,0.01.
and RNA replication (Fig. 1b, c). In contrast, substantial
reduction in viral protein expression and RNA replication
was observed for anti-CSFV siRNA treatment, indicating
that the RNAi approach is efficient in controlling viral
protein expression and RNA replication. Furthermore, the
anti-ANXA2 siRNA resulted in a significant reduction
of virus titre compared to that with the control siRNA
and the treatment without siRNA (Fig. 1d). These results
showed that knockdown of ANXA2 did not impair CSFV
RNA replication, but significantly reduced infectious CSFV
production, suggesting that ANXA2 is involved in the
production of infectious CSFV particles.
To examine the potential role of ANXA2 in the production
of infectious CSFV particles, we analysed the effect of
siRNA-mediated knockdown of ANXA2 on the extracellular and intracellular amounts of viral Core protein.
Lower levels of extracellular Core protein were detected for
the cells transfected with anti-ANXA2 siRNA, while the
amount of intracellular Core protein is comparable to the
1028
level for control siRNA treatment (Fig. 2a, b). The antiANXA2 siRNA resulted in a significant reduction of
extracellular Core proteins by about 70 % compared to
that with the control siRNA (Fig. 2b). These data indicated
that ANXA2 is important for Core protein release from
CSFV RNA transfected cells. The amount of Core protein
released into cell supernatants is used as a surrogate
measure of the production of infectious virus (Miyanari
et al., 2007), thus, ANXA2 is important for the production
of infectious CSFV particles. This reduction in the amount of
extracellular CSFV particles was due to a block in assembly
or release. To address this question, we determined extracellular and intracellular infectivity levels by a TCID50 assay.
Anti-ANXA2 siRNAs significantly decreased extracellular
and intracellular infectivity levels and a comparable
reduction of extracellular and intracellular infectivity levels
was observed (Fig. 2c). We did not find an accumulation of
intracellular infectious particles in the anti-ANXA2 siRNA
transfected cells. Lower levels of specific infectivity (SI) of
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Journal of General Virology 96
Annexin A2 is involved in CSFV production
(a)
(b) 1.5
Extracellular
Intracellular
Extracellular
core
1.0
0.5
Intracellular
core
NA
A
RN
i
ts
ou
l
tro
0
A
RN
siR
2
XA
n
Co
ith
W
A
RN
si
AN
FV
NA
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ts
si
ou
CS
ith
int
int
W
A
A
(c)
****
3
** **
2
RNA SM RNA–1
Log10 TCID50 ml–1
4
X
Co
AN
NA
FV
siR
CS
int
int
A
1.2
RNA
SI
1.0
**
0.8
0.6
0.6
0.4
i
l
tro
A
RN
siR
n
Co
2
si
A
NX
A
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An
**
0
NA
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RN
0.4
**
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0
W
A2
0.8
1
it
NA
siR
1.0
5
ts
r
nt
1.2
Extracellular
Intracellular
6
NA
iR
s
ol
A
(d)
7
u
ho
** **
**
si
C
ti-
An
V
SF
A
RN
NA
iR
it
W
0.2
0
ts
u
ho
SI SM SI–1
Core SM Core–1
β-Tubulin
A
RN
A
RN
i
ls
ro
t
on
C
2
XA
AN
ti-
An
si
NA
V
F
CS
siR
ti-
An
Fig. 2. Effect of siRNA-mediated knockdown of ANXA2 on the extracellular and intracellular amount of viral Core protein and
infectivity. Seventy-two hours after the indicated siRNA transfection, the siRNA transfected PK-15 cells were retransfected with
a second dose of the same siRNA together with a CSFV genomic replicon, SM- (Sheng et al., 2010). (a) At 72 h posttransfection, the cells were harvested and extracellular and intracellular Core proteins were detected by Western blot analysis
with anti-Core antibody. b-Tubulin serves as an internal control. (b) Signal intensities quantified by Kodak ID 3.5 software were
used to evaluate the levels of extracellular and intracellular Core. The relative values were shown by comparing densitometric
values with the densitometric value for the SM replicon, which was set at 1. (c) Extracellular and intracellular infectivity levels
were determined by TCID50 assay as described in Fig. 1(d). (d) Release of virus RNA from transfected cells 72 h posttransfection was determined by using reverse transcript-qPCR (RT-qPCR) as described in Fig. 1(c). Specific infectivity (SI) was
calculated as TCID50 ml”1/RNA copies. The SM was used to normalize the data. All quantified results are shown as means±SD
of at least three independent experiments. ** P,0.01.
virions made in the presence of anti-ANXA2 siRNA were
detected while release of virus RNA was comparable to
the level for control siRNA treatment (Fig. 2d). Taken
together, these results indicated that a decrease in the
production of infectious CSFV particles might be attributable to defective virion assembly rather than to virus
release or RNA replication. The anti-ANXA2 siRNA
treatment might not impair virus RNA, but result in virions
with lower infectivity.
http://vir.sgmjournals.org
Previous reports showed that HCV NS5A is necessary for
HCV assembly and production and interacts with ANXA2
(Appel et al., 2008; Masaki et al., 2008; Saxena et al., 2012).
To investigate whether ANXA2 interacts with CSFV NS5A,
several NS5A mutants (Fig. 3a) formed in our previous work
(Chen et al., 2012) were used for immunoprecipitation
analysis. As shown in Fig. 3(b), ANXA2 specifically
coprecipitated CSFV NS5A. Mutant D335–389 lost
ANXA2 binding activity but other NS5A mutants retained
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1029
137 173 224 268283335390414444 497
+
+
+
+
+
–
+
–
–
+
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+
–
+
–
+
+
+
–
–
+
+
+
WB:FLAG
+
+
IP: ANXA2 WB:ANXA2
–
+
+
D390–414
D415–413
CD53
+
–33
IP: ANXA2 WB:FLAG
–62
–48
+
–33
348
348
348
349
389
389
390
389
389
*
*
(f)
–62
–48
IP: ANXA2 WB:FLAG
*
(d)
WT
R338A
N359A
G378A
G378L
G378L
301
301
301
302
335
335
336
335
335
N359A
G378A
(e)
HCV 52
HCV 1b
HCV 1a
HCV 64
BDV X818
BVDV1 NADL
BVDV2 C413
CSFV Eystrup
CSFV Shimen
WT
R338A
(c)
–62
–48
–62
–48
WB:FLAG
(g)
IP: ANXA2 WB:ANXA2
RNA SM RNA–1
1
WT
ND136
D137–172
D173–223
D224–268
D269–282
D283–334
D335–389
(b)
D390–414
D415–443
CD53
ANXA2 NS5B 3′UTR
binding binding binding
1.2
1.0
0.8
0.6
0.4
0.2
0
24 48 72 h
CSM-WT CSM-N359A CSM-G378L
CSM-R338A CSM-G378A
-Tubulin
NS3
Log10 TCID50 ml–1
(a)
WT
ND136
D137–172
D173–223
D224–268
D269–282
D283–334
D335–389
C. Sheng and others
CSM-WT CSM-N359A CSM-G378L
CSM-R338A CSM-G378A
8
6
4
2
0
24 48 72 hrs
SM-WT
SM-N359A SM-G378L
CSM-R338A CSM-G378A
Fig. 3. ANXA2 interacts with CSFV NS5A. (a) Schematic of the CSFV NS5A protein and its mutant forms from previous reports
(Chen et al., 2012). D335–389 indicates an internal deletion mutant lacking corresponding amino acids. The ANXA2, NS5B and
39UTR binding activity of these proteins are shown on the right. (b) Immunoprecipitation analysis of the interaction between
ANXA2 and CSFV NS5A. PK-15 cells were transfected with the pcDNA3.1/N-FLAG vector containing NS5A (WT or mutant). At
72 h post-transfection, cell lysates were immunoprecitated using anti-ANXA2 antibody. Immunoprecipitation of NS5A-ANXA2
interaction was determined by Western blot analysis with anti-FLAG antibody (upper) or anti-ANXN A2 antibody (below). Total
lysates were fractionated by 10 % SDS-PAGE and subjected to Western blot analysis with anti-FLAG antibody (middle). (c) The
NS5A protein sequence of CSFV was aligned with that of BDV, BVDV and HCV using CLUSTAL_X (partially shown). The conserved
amino acids are indicated by asterisks. (d) Schematic representation of NS5A and its substitution mutants used in this study. (e)
Immunoprecipitation of the NS5A–ANXA2 interaction was determined by Western blot analysis with anti-FLAG antibody or antiANXN A2 antibody as described in Fig. 3(b). (f) PK-15 cells were transfected with the CSM” subgenomic replicon carrying WT or
mutant NS5A proteins (R338A, N359A, G378A or G378L). Quantification of viral RNA was determined as described in Fig. 1(c).
The amounts of NS3 (below) were evaluated as described in Fig. 1(b). (g) PK-15 cells were transfected with the SM” subgenomic
replicon carrying WT or mutant NS5A proteins (R338A, N359A, G378A or G378L). Titres were determined as described in Fig.
1(d). All quantified results are shown as means±SD of at least three independent experiments. **P,0.01.
the activity (Fig. 3b), indicating that amino acids 335–389
might be important for ANXA2 binding. As a comparison,
we aligned the NS5A sequences of CSFV, BDV, BVDV and
HCV. Three conserved amino acids R338, N359 and G378
were indicated (Fig. 3c), suggesting that these amino acids
might be important for NS5A interactions with ANXA2. To
corroborate this assumption, alanine or leucine substitutions
were introduced into R338, N359 and G378 in CSFV NS5A.
Four CSFV NS5A mutants, R338A, N359A, G378A and
G378L were produced (Fig. 3d). Immunoprecipitation
1030
assays revealed that ANXA2 binding activity was lost for
R338A, N359A and G378L and was substantially reduced for
G378A (Fig. 3e). The results indicated that amino acids
R338, N359 and G378 of CSFV NS5A are pivotal for the
ANXA2–NS5A interaction. Furthermore, the same substitution mutations were incorporated into the CSM2
subgenomic replicon and the SM2 genomic replicon,
respectively. These mutations had no substantial effect on
viral RNA replication and viral protein expression (Fig. 3f)
but substantially reduced CSFV production (Fig. 3g).
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Journal of General Virology 96
Annexin A2 is involved in CSFV production
Host proteins participate in many aspects of viral infection.
Vesicle-associated membrane protein-associated proteins
mediate the formation of HCV RNA placation complex on
lipid rafts (Gao et al., 2004). RNA helicase A, another host
protein, has been found to be involved in the expression
and replication of CSFV and bind CSFV 59UTR and 39UTR
(Sheng et al., 2013). Cellular NFAR proteins associate
specifically with both the termini of the BVDV RNA
genome involving regulatory elements (Isken et al., 2003).
ANXA2 is an important host protein that plays a role in
different steps of viral life cycle (Backes et al., 2010; Beaton
et al., 2002; González-Reyes et al., 2009; LeBouder et al.,
2008; Ryzhova et al., 2006; Wright et al., 1995). In the
report, we identified ANXA2 as an important factor in the
production of the CSFV infectious particles. ANXA2 also is
a key host factor that influences HCV yield (Backes et al.,
2010). For the role of ANXA2 in viral RNA replication,
scientists reported contradictory findings. Some people
showed that ANXA2 knockdown reduced viral RNA
replication (Saxena et al., 2012) while another observed
that ANXA2 knockdown did not have this effect (Backes
et al., 2010). Our present results suggested that ANXA2
plays a role in CSFV assembly rather than in genome
replication and virion release. The discrepancy may partly
be due to the differences in virus species and in siRNA
sequences.
The data for immunoprecipitation experiments showed
that ANXA2 interacts with CSFV NS5A, in agreement with
the previous reports in which ANXA2 binds HCV NS5A
(Saxena et al., 2012). The NS5A protein of CSFV, BVDV
and HCV has been shown to be an essential replicase
component (Masaki et al., 2008; Tellinghuisen et al., 2006;
Sheng et al., 2010), thus, ANXA2 might participate in the
CSFV production process by binding to NS5A protein. In
fact, ANXA2 has been found to be involved in the
formation of HCV replication complex on the lipid raft
(Saxena et al., 2012). Mutation analysis revealed that amino
acids 335–389 of NS5A might be responsible for ANXA2
binding (Fig. 3c, d). NS5A is a multifunctional protein. The
C terminus of CSFV NS5A contains several binding sites,
such as a NS5B binding site and a 39UTR binding site
(Fig. 3a) (Chen et al., 2012). Recently, we have found that
the C-terminal sequence from amino acids 478–487 of
NS5A is important for the interaction between CSFV NS5A
and Core protein, which also might be related to the
production of infectious CSFV particles (Sheng et al.,
2014). The amino acid fragment responsible for ANXA2
binding is adjacent to the NS5B binding site at the C
terminus of CSFV NS5A (Fig. 3a) (Chen et al., 2012).
Furthermore, R338, N359 and G378 of CSFV NS5A were
observed be pivotal for ANXA2–NS5A interactions (Fig. 3c,
e). Substitution of these amino acids had no effect on viral
RNA replication but substantially reduced CSFV production (Fig. 3f, g), which might partly be due to this
substitution destroying ANXA2–NS5A interactions. These
results suggested that ANXA2 might participate in the
CSFV production process by binding NS5A. This is
http://vir.sgmjournals.org
attractive for further investigation of the role of ANXA2
in the life cycle of pestivirus and HCV.
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (30870492, 31070671), the Natural Science Foundation of
Shanghai (12ZR1422100), the Shanghai Municipal Science and
Technology Commission (11JC1409300) and the Shanghai Municipal
Education Commission (13ZZ102, 11YZ87, 14ZZ123).
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