Regulatory role of ASK1 in porcine circovirus type 2-induced apoptosis

Li Wei, Shanshan Zhu, Jing Wang, Chunyan Zhang, Rong Quan, Xv Yan, Jue Liu n

Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, No. 9 Shuguang Garden Middle Road,Haidian District, Beijing 100097, People's Republic of China

a r t i c l e i n f o

Article history:Received 21 July 2013Returned to author for revisions14 August 2013Accepted 12 September 2013Available online 8 October 2013

Keywords:Porcine circovirus type 2 (PCV2)ASK1JNK1/2p38 MAPKPI3K/AktApoptotic responses

a b s t r a c t

Porcine circovirus type 2 (PCV2), a major causative agent of postweaning multisystemic wastingsyndrome which is an emerging and important swine disease, can induce apoptotic responses in vitroand in vivo. However, the molecular mechanism of PCV2-induced apoptosis is not well understood.Apoptosis signal-regulating kinase 1 (ASK1), serves as an upstream enzyme that activates the JNK andp38 MAPK pathways, has been shown to play a target role in the regulation of apoptosis. Here, weshowed that PCV2 infection induced ASK1 phosphorylation which preceded activation of JNK1/2 and p38signaling in the cultured cells, and that the phosphatidylinostol 3-kinase (PI3K)/Akt signaling triggeredby PCV2 infection limited the extent of JNK1/2 and p38 activation and thereby apoptotic cell death.Furthermore, inhibition of JNK and p38 activation is associated with PI3K-dependent negative-regulationof ASK1 in the PCV2-infected cells. These results indicate an important regulatory role of ASK1 in PCV2-induced apoptotic responses.

& 2013 Published by Elsevier Inc.

Introduction

Porcine circovirus (PCV), from the genus Circovirus within thefamily Circoviridae (Todd et al., 2005), is a non-enveloped DNA viruswith an icosahedral capsid and has a single-stranded circular genomeof 1.7 kb (Tischer et al., 1982). PCV has two genotypes: PCV1 andPCV2. PCV1 was identified as a persistent non-cytopathic contami-nant of the continuous porcine kidney cell line PK15, and non-pathogenic for pig. PCV2 was pathogenic and considered to be amajor causative agent inducing postweaning multisystemic wastingsyndrome (PMWS), which was now known as PCV2-associateddiseases (PCVAD) (Allan et al., 1998; Opriessnig et al., 2007). PCVADincludes a range of disorders, such as porcine dermatitis andnephropathy syndrome, reproductive failure, porcine respiratorydisease complex and proliferative and necrotizing pneumonia(Segalés et al., 2005). In addition, severely PCV2-infected pigs maydamage immune system and develop immunosuppression, leading toan increased susceptibility to other infectious diseases as well as poorimmune response to vaccines. PCVAD is now endemic in many pig-rearing countries, and increasingly recognized as serious threats tothe swine industry worldwide (Segalés et al., 2005).

Two major open reading frames (ORFs) have been identified forPCV, ORF1, called rep gene, which encodes a protein of 35.7 kDaassociated with virus replication (Mankertz et al., 1998), and ORF2,called cap gene, which encodes the major immunogenic capsid

protein of 27.8 kDa conferring to good protection ability (Cheung,2003; Nawagitgul et al., 2000). In addition to the ORF1 and ORF2proteins, two more proteins, ORF3 and ORF4, were detected inPCV2 productive infection, which are involved in viral pathogen-esis via an apoptotic function, and suppression of caspase activityas well as regulation of CD4(þ) and CD8(þ) T lymphocytes duringPCV2 infection, respectively (Liu et al., 2005, 2006; He et al., 2013).

PCV2 infection induces apoptosis both in vitro and in vivo(Chang et al., 2007; Galindo-Gardiel et al., 2011; Kiupel et al.,2005; Liu et al., 2005, 2006; Resendes et al., 2011; Seeliger et al.,2007; Sinha et al., 2012) and PCV2-induced apoptosis involvedactivating caspase-8 followed by caspase-3 pathway (Liu et al.,2005). Research data suggested that PCV2 can activate nuclearfactor kappa B, extracellular signal-regulated kinase, c-Jun NH2-terminal kinases (JNK1/2) and p38 mitogen-activated proteinkinase (MAPK) pathways and activation of these cellular signalingis involved in virus-induced apoptotic cell death (Wei et al., 2008;Wei and Liu, 2009; Wei et al., 2009). Also, we have recently shownthat the phosphatidylinositol 3-kinase (PI3K)/Akt pathway isactivated early during PCV2 infection, and activation of the PI3K/Akt pathway suppressed premature apoptosis for improved virusgrowth after infection (Wei et al., 2012).

Apoptosis signal-regulating kinase 1 (ASK1) is a member ofMAPK kinase kinase (MAPKKK) family that activates the SEK1-JNKand MKK3/MKK6–p38 MAPK signaling cascades (Ichijo et al.,1997). ASK1 pathway has been found to participate in the regula-tion of apoptosis in some cases of viral infection, such as humanimmunodeficiency virus type 1 (HIV-1) (Gelezlunas et al., 2001),influenza virus (Lu et al., 2010; Maruoka et al., 2003), hepatitis C

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Virology

0042-6822/$ - see front matter & 2013 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.virol.2013.09.011

n Corresponding author. Fax: þ86 10 88433070.E-mail address: liujue@263.net (J. Liu).

Virology 447 (2013) 285–291

virus, and poliovirus (Autret et al., 2008). To further identify themolecular mechanisms underlying PCV2-induced apoptosis, wewant to investigate the role of ASK1 in PCV2-induced apoptoticresponses.

In the present study, we have shown that Akt can be activatedby PCV2 infection and reduction in ASK1 activity by specificinhibitor treatment was resistant to PCV2-induced JNK and p38MAPK activation and cell death. Furthermore, the results demon-strated that activation of the PI3K/Akt pathway induced by PCV2infection negatively regulated the JNK and p38 MAPK pathway viaASK1 signaling, thereby inhibiting JNK- and p38-dependentapoptosis.

Results

PCV2 infection induces activation of ASK1-JNK/p38 MAPK pathways.

Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitouslyexpressed enzyme that activates JNK and p38 MAPK pathways bydirect site-specific Ser/Thr phosporylation of their upstream tar-gets followed by activating JNK and p38 signaling. In our previousresearch, we found that PCV2 infection induced activation ofJNK1/2 and p38 MAPK signaling pathways (Wei et al., 2009). Tofurther determine whether the activation of ASK1 signalingoccurred in the cultured cells during the course of PCV2 infection,the degree of ASK1 phosphorylation at Ser 83 in the PCV2-infectedcells were examined by Western blotting. PK15 cells were infectedwith PCV2 strain BJW at an MOI of 1 TCID50, and whole-cell lysateswere prepared at the indicated time points after infection. Incuba-tion with phosphate-buffered saline (PBS) served as mock-infectedcontrols. As shown in Fig. 1A, amounts of phosphorylated ASK1 inthe PCV2-infected cells increased at 14 h, were maximal at 24 hpostinfection which decreased thereafter. In contrast, the proteinlevels of total amounts of ASK1 remained unchanged in the PCV2-infected cells at various time points after infection compared tothat in the mock-infected cells. A loading control, β-actin in eachsample, was comparable. Consistent with the previous results(Wei et al., 2009), PCV2 infection led to progressive accumulationof p-JNK1/2 and p-p38 signals over time, and the maximal

induction was seen at 72 h postinfection (Fig. 1B). These resultsindicated that PCV2 infection phosphorylated ASK1 and concomi-tantly phosphorylated JNK1/2 and p38 MAPK in the cultured cellsand further revealed that ASK1 phosphorylation preceded JNK1/2and p38 MAPK phosphorylation.

PCV2-induced JNK and p38 MAPK activation dependent upon ASK1activation

We determined the requirement of ASK1 for PCV2 infection-induced JNK/p38 MAPK activation after inhibition of ASK1 activity.As determined by testing for cell viability by trypan blue exclusionstaining, the concentration (250 ng/ml) of the ASK1 inhibitorthioredoxin had no toxic effect on the treated cells (data notshown). We treated the PCV2-infected cells with the inhibitor anddetermined the levels of phosphorylated JNK1/2 and p38 MAPK byWestern blotting analysis. As shown in Fig. 2, amounts of phos-phorylated JNK1/2 significantly decreased in the PCV2-infectedcells after treatment with the ASK1 inhibitor. Similarly, amounts ofphosphorylated p38 MAPK significantly reduced when blocked byinhibition of ASK1 activity. Together, the phosphorylation ofJNK1/2 and p38 MAPK was depressed after blockage of ASK1activity. These results indicated that PCV2 infection activatesASK1-JNK/p38 MAPK signaling pathway.

Inhibition of ASK1 activation prevents PCV2-mediated apoptoticresponses

Research data suggested that PCV2-induced apoptosis wasthrough the activation of caspase-8 followed by caspase-3 path-way (Liu et al., 2005). To determine the role of ASK1 in apoptosisupon PCV2 infection, we firstly used Western blotting to measurethe cleavage of host proteins associated with characteristic hall-mark features of apoptosis, including poly-ADP ribose polymerase(PARP) and caspase-3. As shown in Fig. 3A, PCV2 infection causedthe cleavage of PARP and caspase-3 at 72 h postinfection. Whenthe ASK1 activity was blocked by thioredoxin, amounts of cleavedPARP and caspase-3 were significantly reduced. We then usedspectrofluorometric assay of proteolytic activity to measure

PCV2

p-ASK1

ASK1

-actin

PCV2

-actin

p38

p-p38

JNK1/2

p-JNK1/2

Fig. 1. PCV2 infection activates ASK1-JNK/p38 MAPK signaling pathways in the cultured cells. Whole-cell lysates from PK15 cells after infection with PCV2 strain BJW at anMOI of 1 TCID50. PCV2-infected cells were harvested at the indicated time points postinfection, and whole-cell lysates were prepared and resolved by SDS-PAGE, transferredto nitrocellulose membranes, and immunoblotted. (A) Membranes were reprobed with antibodies to ASK1, p-ASK1. (B) Membranes were reprobed with antibodies to JNK1/2and p38 and their phosphorylated forms. The amounts of β-actin were assessed to monitor the equal loadings of protein extracts. p, phosphorylated.

L. Wei et al. / Virology 447 (2013) 285–291286

caspase-3 activity in the PCV2-infected cells at 72 h postinfectionafter treatment with the ASK1 inhibitor thioredoxin. As expected,PCV2 alone induced activation of caspase-3 in the infected cells,whereas its activity was significantly reduced in the infected cellswhen treated with the ASK1 inhibitor (Fig. 3B). A basal caspase-3activity was detected in the mock-infected cells. In addition,Ac-DEVD-CHO, a peptide inhibitor of caspase-3 activity, was usedas an internal control to confirm assay validity. To further establishthat the regulatory role of the ASK1 in PCV2-induced apoptosis,a TUNEL assay was used to monitor apoptosis at the cellular level.A brown signal was considered to be a TUNEL-positive cell.As shown in Fig. 3C, there was a significant reduction in TUNEL-positive cells (7.3%) in the PCV2-infected cells at 72 h after thethioredoxin treatment as compared to the 23% TUNEL positivityobserved in the PCV2-infected untreated cells. Only 2.3% positivityat 72 h postinfection was observed in the mock-infected cells.These results suggested that the ASK1 signaling pathway actsupstream of JNK1/2 and p38 MAPK and is involved in theregulation of PCV2-induced apoptotic responses.

Reduction of ASK1 activity does not affect PCV2 replication

Reduction of phosphorylation of JNK and p38 followed bydecreased apoptotic responses were observed in the PCV2-infected cells after inhibition of ASK1 activity. We have previouslyshown that inhibition of JNK and p38 phosphorylation blockedPCV2 replication (Wei et al., 2009). To determine whether

activated ASK1 signaling play any role in the replication of PCV2,we examined the effect of the kinase on progeny virus productionin the PCV2-infected cells by blocking ASK1 activity. We infectedPK15 cells with PCV2 in the presence of the ASK1 inhibitorthioredoxin (250 ng/ml) and determined the virus titers in thecell culture supernatant at 72 h postinfection by using an IFA assay.As expected, no PCV2 growth was seen in the mock-infected cells(Fig. 4A). Virus production after inhibition of ASK1 activity wascomparable to that in PCV2 alone-infected cells (104.5 vs 104.38

TCID50 for thioredoxi-treated PCV2-infected and PCV2 alone-infected cells, respectively) (Fig. 4A), indicating that blockage ofASK1 activity did not affect virus yield. We also examined theeffect of inhibition of ASK1 activity on viral protein synthesis. PK15cells were infected with PCV2 (MOI of 1) in the presence orabsence of thioredoxin (250 ng/ml), and ORF2 protein expressionwas monitored by a fluorescence microscope. The ORF2 proteinsynthesis was not significantly affected when PK15 cells weretreated with thioredoxin, as demonstrated by the similar numberof PCV2-positive cells observed in the DMSO-treated infected cells(Fig. 4B). No significant differences were also seen in the ORF2protein expression between DMSO-treated infected cells anduntreated infected cells (data not shown). No ORF2 proteinexpression was detected in the mock-infected cells (Fig. 4B). Theseresults suggested that ASK1 is not required for PCV2 replication.

Akt downregulates JNK1/2 and p38 MAPK via ASK1

In a recent report, we found that the PI3K/Akt activation playsan important role in regulating viral replication and mediatingantiapoptotic responses in the PCV2-infected cells (Wei et al.,2012). It has been shown that the PI3K/Akt signaling inhibits p38-dependent apoptosis to promote endothelial cell survival (Grattonet al., 2001). Also, the PI3K/Akt pathway has been demonstrated todownregulate JNK activation by negatively regulating ASK1 innon-viral systems (Aikin et al., 2004; Kim et al., 2001) as well asin virus-infected cells, such as poliovirus (Autret et al., 2008) andinfluenza virus (Lu et al., 2010; Maruoka et al., 2003). Therefore,we then investigated whether ASK1 bridges the PI3K/Akt andJNK1/2 as well as p38 MAPK pathways. We infected PK15 cellswith PCV2 in the presence of LY294002 (20 μM), a PI3K-specificinhibitor, as described elsewhere (Wei et al., 2012). As shown inFig. 5, blocking activation of the PI3K/Akt pathway abolished theincrease in ASK1 phosphorylation at Ser83 and upregulated PCV2-induced JNK1/2 and p38 activation in the PCV2-infected cells. Inaddition, blocking PI3K/Akt activation greatly increased PCV2-induced apoptotic responses, as evidenced by caspase-3 cleavage.Altogether, these results indicate that the PI3K/Akt pathwaynegatively regulates activation of JNK and p38 MAPK by phos-phorylating and inactivating ASK1 in the PCV2-infected PK15 cells.

Discussion

In response to varieties of stresses including virus infection, JNKand p38 MAPK cascades were perturbed. ASK1, one of MAPKKK,phosphorylates and activates MKK4/MKK7 and MKK3/MKK6 acti-vation followed by phosphorylation/activation of JNK and p38MAPK, respectively, which are responsible for induction of apop-totic responses (Ichijo et al., 1997; Kyriakis and Avruch, 2001;Sumbayev and Yasinska, 2006; Tobiume et al., 2001; Wang et al.,1996). In the present study, we examined the role of ASK1 inapoptotic cell death upon PCV2 infection in order to clarify themechanism in PCV2-induced apoptosis. The results showed thatASK1 plays a target role in the regulation of PCV2-mediatedapoptotic responses.

- + - +

Mock PCV2

Thoiedoxin

p-JNK1/2

JNK1/2

p-p38

p38

-actin

Fig. 2. PCV2 infection-induced JNK and p38 MAPK phosphorylation are depressedafter inhibition of ASK1 activity. PK15 cells were infected with PCV2 in the absence orpresence of ASK1 inhibitor thioredoxin (250 ng/ml). Cell lysates at 72 h postinfectionwere harvested and resolved by SDS-PAGE, transferred to nitrocellular membranes, andimmunoblotted with antibodies to JNK1/2 and p38 and their phosphorylated forms. β-actin was probed as the loading control. p, phosphorylated.

L. Wei et al. / Virology 447 (2013) 285–291 287

Previous research data have shown that ASK1 and its down-stream pathway play an important role in virus-mediated apopto-tic cell death (Autret et al., 2008; Gelezlunas et al., 2001; Lu et al.,2010; Maruoka et al., 2003). We have previously shown that PCV2infection activates JNK1/2 and p38 MAPK and their activations areinvolved in PCV2-induced apoptosis (Wei et al., 2009). In the

present study, we demonstrated that PCV2 infection could triggerASK1 phosphorylation followed by its downstream kinases, JNKand p38 phosphorylation in the cultured cells (Fig. 1A and B).Blockage of ASK1 activity depressed significantly activation of JNKand p38 MAPK in the PCV2-infected cells (Fig. 2) followed byreduction of PCV2-induced apoptotic responses (Fig. 3A–C).

- + - +

Mock PCV2

Thoiedoxin

c-PARP

c-Caspase-3

-actin

0

5

10

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25

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*

Cas

pase

-3 a

ctiv

ity (n

mol

/h)

0

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Fig. 3. Inhibition of ASK1 activity reduces PCV2-induced apoptotic responses. (A) PK15 cells were treated with the ASK1 inhibitor thioredoxin (250 ng/ml) or equal volumesof the solvent (DMSO) after infection with the PCV2 strain BJW at an MOI of 1 TCID50. At 72 h postinfection, the cell lysates were harvested for Western blotting with specificantibodies to detect the cleavage of PARP and caspase-3. Equal protein loads were verified with β-actin blots. p, phosphorylated; c, cleaved. (B) Whole-cell lysates harvestedfrom PCV2-infected cells at 72 h after treatment with the inhibitor thioredoxin were assayed for DEVDase activity using the caspase-3 colorimetric DEVD-AFC. In addition,cells infected with PCV2 alone were treated with the caspase-3 inhibitor DEVD-CHO. Mock-infected cells were used as a negative control. Values shown are means fromthree independent experiments. (C) PCV2-infected PK15 cells at 72 h after treatment with the ASK1 inhibitor were processed for the TUNEL assay. Decreased TUNELpositivity was observed in the PCV2-infected thioredoxin-treated cells, as compared to the PCV2-infected untreated cells and mock-infected controls. The values shown aremeans from two independent experiments. *, Po0.05 for a comparison of PCV2-infected and ASK1 inhibitor-treated PCV2-infected cells.

100101102103104105106107

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l)

0

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60

80

100

120

Rel

ativ

e vi

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in

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)

Fig. 4. Inhibition of ASK1 activity does not affect PCV2 replication. (A) Supernatants of PCV2-infected PK15 cells at 72 h after treatment with the ASK1 inhibitor thioredoxin(250 ng/ml) were inoculated on monolayers of PK15 cells. Virus production was assayed by the IFA method. (B) PCV2-infected PK15 cells 72 h in the presence of the inhibitorthioredoxin were assayed for the amount of viral protein synthesis by the IFA method. The amounts of PCV2 ORF2 protein expression are shown as percentages of the PCV2-positive signals in the PCV2 alone-infected cells. The values represent the means of results from three independent experiments.

L. Wei et al. / Virology 447 (2013) 285–291288

However, ASK1 activity was not involved in PCV2 replication(Fig. 4A and B) as observed for other viruses, such as influenzavirus (Maruoka et al., 2003). These results indicated that PCV2infection modulated ASK1 phosphorylation and its downstreamkinases, JNK and p38-mediated apoptotic responses in the PCV2-infected cells, and that activation of JNK and p38 MAPK requiresASK1 phosphorylation.

The PI3K/Akt pathway, which serves as an important signalingpathway, modulates diverse cellular activities related to cellgrowth, survival, and apoptotic cell death (Datta et al., 1999; Yaoand Cooper, 1995). We have shown recently that PCV2 infection-induced PI3K/Akt activation during the early stages of infectioncontributes to PCV2-induced cell survival and prevention ofapoptosis for improved virus growth (Wei et al., 2012). The PI3K/Akt-mediated survival pathway has been demonstrated to inhibitp38-mediated apoptosis for endothelial cell survival (Gratton et al.,2001) and to limit JNK activation during poliovirus infection(Autret et al., 2008). This implicates that there is a cross talkbetween the PI3K/Akt and JNK as well as p38 MAPK pathways,which facilitates to maintain the fine balance of cell survival andapoptotic cell death. In the present study, we investigated whetherthe PI3K/Akt and JNK/p38 MAPK pathways cross talk to regulateapoptosis coordinately and found that inhibition of PI3K/Aktactivation downregulated level of p-ASK1 and induced higherlevels of p-JNK1/2 as well as p-p38 MAPK during PCV2 infection(Fig. 5). This indicates that the PI3K/Akt pathway triggered byPCV2 infection may negatively regulate JNK and p38 activity viaASK1 kinase which in turn block JNK- and p38-mediated apoptoticresponses in the PCV2-infected cells as observed for other viruses,such as poliovirus and influenza A virus (Lu et al., 2010). In hostcellular signals, Akt acts upstream of ASK1 kinase and canphosphorylate ASK1, leading to decreased ASK1 activity (Kimet al., 2001). Inhibition of ASK1 activity led to decreased levels ofp-JNK and p38 MAPK in the PCV2-infected cells (Fig. 2). Therefore,these results suggested that the PI3K/Akt pathway may phosphor-ylate ASK1, which leads to decreased ASK1 kinase activity, therebydownregulating activation of JNK and p38 MAPK during PCV2infection.

In conclusion, the results presented here demonstrated thatphosphorylation of ASK1 upon PCV2 infection preceded its down-stream JNK and p38 activation which contributed to JNK- and p38-mediated apoptotic responses in the PCV2-infected cells. Theresults also demonstrated that activation of the PI3K/Akt pathway

triggered by PCV2 infection negatively modulated the JNK and p38MAPK pathway via ASK1 signaling, thereby inhibiting JNK- andp38-dependent apoptosis. Knowledge of the target role of ASK1 inapoptosis upon PCV2 infection will extend our understanding ofthe molecular mechanism of PCV2 pathogenesis.

Materials and methods

Virus and cells

PK15 cells were maintained in minimal essential medium(MEM) supplemented with 5% heat-inactivated fetal bovine serum(FBS), 5% L-glutamine, 100 U of penicillin G/ml, and 100 μl ofstreptomycin/ml at 37 1C in a humidified 5% CO2 incubator. PCV2strain BJW (Liu et al., 2005) was inoculated onto monolayer PK15cells at a multiplicity of infection (MOI) of one 50% tissue cultureinfective dose units (TCID50) per cell. Additionally, the cells weretreated with 300 mM D-glucosamine at 24 h after inoculation asdescribed previously (Tischer et al., 1987).

Reagents and antibodies

A specific PI3K inhibitor LY294002 and ASK1 inhibitor thior-edoxin were obtained from Calbiochem (La Jolla, CA). The cyto-toxicity of these two inhibitors on PK15 cells was determined bytrypan blue exclusion dye staining. It was noted that throughoutall doses of the inhibitors used in the present study, cell viabilityassay showed no detectable cell death in the PK15 cells.

Rabbit, goat, or mouse antibodies against ASK1, phosphorylated(p)-ASK1 (Ser-83), poly-ADP-ribose polymerase (PARP), cleavedcaspase-3, and β-actin were purchased from Santa Cruz Biotech-nology. Antibodies specific for JNK1/2 and p38, as well as phos-phorylated forms of JNK (p-JNK) and p38 (p-p38), were obtainedfrom Cell Signaling Technology. Horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Sigma. Fluor-escein isothiocyanate (FITC)-conjugated secondary antibodieswere purchased from DAKO.

Indirect immunofluorescence assay (IFA)

PK15 monolayer cells seeded in 24-well culture plates wereinfected with PCV2 strain BJW. At 72 h, the cells were washed withPBS and fixed in 4% paraformaldehyde (PFA). After three washes,the cells were incubated with mouse anti-ORF2 antibody dilutedin 3% bovine serum albumin (BSA)–PBS at room temperature (RT)for 1 h. After three further washes, cells were incubated with FITC-conjugated anti-mouse immunoglobulin G (Sigma) at RT for 1 hand washed with PBS three times. The cells were examined undera fluorescence microscope and cells positive for PCV2 viral anti-gens were counted in six fields of view.

Virus infectivity assay

PK15 cells were infected with PCV2 strain BJW at an MOI of1 TCID50 for 72 h in the presence of thioredoxin (250 ng/ml). Cellcultures were harvested at the indicated time point postinfectionby three cycles of freeze-thawing followed by clarification. Virusinfectivity was assayed by IFA as described previously (Wei et al.,2008).

Whole cell lysates

Whole cell lysate extracts from PK15 cells after infection at theindicated time points were prepared with the Nuclear Extract kit(Active Motif) according to the manufacturer's protocol.

- + - +

Mock PCV2

LY294002

p-ASK1

p-JNK1/2

p-p38

β-actin

Fig. 5. The PI3K/Akt pathway limits activation of JNK and p38 MAPK by regulatingASK1 phosphorylation in the PCV2-infected cells. PK15 cells were mock infected orinfected with PCV2 (24 h) in the presence or absence of the PI3K inhibitorLY294002 (20 μM). Phosphorylation of ASK1, JNK1/2, and p38 MAPK, were analyzedin whole-cell lysates by Western blotting with specific antibodies to theirphosphorylated forms. β-actin was probed as the loading control. p,phosphorylated.

L. Wei et al. / Virology 447 (2013) 285–291 289

Western blotting

The whole cell lysates prepared were diluted in 2� samplebuffer and boiled for 5 min. Twenty micrograms of each extractwas resolved on 10–12% sodium dodecyl sulphate-polyacrylamidegel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose(NC) membranes with a semidry transfer cell (Bio-Rad Trans-BlotSD). The membranes were blocked for 2 h at room temperature(RT) in blocking buffer TBST (20 mM Tris–HCl [pH 7.4], 150 mMNaCl, 0.1% Tween-20) containing 5% skim milk powder to preventnonspecific binding, and then incubated with specific primaryantibodies raised against ORF2, ASK1, JNK1/2, p38, p-ASK1,p-JNK1/2, p-p38, PARP, cleaved caspase-3, as well as β-actin atRT for 2 h. The membranes were washed three times with TBSTbuffer, and incubated for 2 h at RT with HRP-conjugated secondaryantibodies diluted in blocking buffer. Immunoreactive bands werevisualized by enhanced chemiluminescence system (Kodak ImageStation 4000R).

TUNEL assay

PK15 monolayer cells were infected with PCV2 at an MOI of1 with or without the inhibitor thioredoxin (250 ng/ml) treatment.A DeadEnd colorimetric TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) system kit(Promega, Madison, WI) was used to detect apoptosis as describedrecently (Wei et al., 2012).

Statistical analysis

Fluorimetric assay of caspase-3 activitySpectrofluorometric assays of proteolytic activity were carried

out using the synthetic fluorogenic substrate 7-amino-4-trifluo-romethyl coumarin (AFC) to measure caspase-3 activity. A BDApoAlert caspase fluorescent assay kit (Clontech) was used todetermine caspase-3 activity of PCV2-infected PK15 cells afterASK1 inhibitor thioredoxin treatment as described previously (Weiet al., 2009).

Statistical analysisResults are presented as averages7the standard deviations or

standard errors of the means, as indicated. Statistical comparisonsare made by using student's t test, and differences between groupswere considered significant if the P value waso0.05.

Acknowledgment

This study was supported by grants from the National ScienceFund for Distinguished Young Scholars (31025028), the NationalBasic Research Program 973 (2010CB1347507), and the NationalNatural Science Fund (30871866), People's Republic of China.

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