Institute of Veterinary Sciences, Academy of Military Medical Sciences, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, 666 Liuying West Road,hangchun 130122, ChinaShanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No. 518, Ziyue Road, Shanghai 200241, ChinaInstitute of Biotechnology, College of Science, Northeastern University, No. 11, 3 Lane, Wenhua Road, Shenyang 110004, China
r t i c l e i n f o
rticle history:eceived 23 July 2012eceived in revised form 15 January 2013ccepted 16 January 2013vailable online 25 January 2013
a b s t r a c t
Heme oxygenase 1 (HO-1) is an inducible enzyme that exerts potent antioxidant and anti-inflammatoryeffects, which also plays a critical role in host defenses against microbial, and particularly viral, infec-tions. In our previous study, up-regulation of HO-1 was observed in peripheral blood leukocytes (PBLs) bygenomic expression profiling, following infection of pigs with virulent classical swine fever virus (CSFV),the causative agent of a highly contagious disease threatening global pig industry (Shi et al., 2009). To
study the potential involvement of HO-1 in CSFV proliferation, the role of its down-regulation in CSFV-infected PK-15 cells was further investigated. Results showed that infection with virulent CSFV strainShimen significantly up-regulated the expression of HO-1 and that its down-regulation by small inter-fering RNA (siRNA) could inhibit CSFV proliferation as measured by genomic replication and productionof infectious virus. The study revealed the involvement of HO-1 in CSFV proliferation, indicating thatHO-1 is a potential target for inhibition of CSFV replication.
. Introduction
Classical swine fever virus (CSFV) is the causative agent of classi-al swine fever (CSF) which is a highly contagious infectious diseasef pigs, featuring high fever, extensive hemorrhages in the skin,ucosa and internal organs, and severe leukopenia with high mor-
idity and mortality (Thiel et al., 1996). A dramatic decrease oferipheral B- and T-cells is the main outcome of CSFV infectionf pigs, due to bystander apoptosis in uninfected cells (Susa et al.,992; Summerfield et al., 1998).
CSFV is a small enveloped virus with a single, positive-tranded RNA genome, a member of the genus Pestivirus withinhe family Flaviviridae (Simmonds et al., 2011). The viral genomes approximately 12.5 kb in size and contains a single largepen reading frame that encodes a 3898 amino acid polypro-ein which is cleaved by cellular and viral proteases into the2 final products: NH2-Npro-C-Erns-E1-E2-p7- NS2-NS3-NS4A-S4B-NS5A-NS5B-COOH (Lindenbach et al., 2007). Protein C and
lycoproteins Erns, E1, and E2 are structural proteins of the virion,he latter three being viral envelope components (Lindenbach et al.,007).
As intracellular pathogens, the life cycle of viruses is completelydependent on the cellular growth environment and recruitment ofcellular components (Viswanathan and Früh, 2007). With regard toCSFV, studies have identified several host factors involved in CSFVreplication and pathogenesis. Glycoprotein Erns interacts with cel-lular membrane-associated heparan sulfate (HS) to facilitate viralentry (Hulst et al., 2001). The interaction of the internal ribosomalentry segment (IRES) within the 5′ nontranslated region of the CSFVgenomic RNA with eukaryotic translation initiation factor 3 (eIF3)enhances the efficiency and accuracy of viral RNA binding to 40Ssubunits in an orientation promoting entry of the initiation codoninto the ribosomal P site (Sizova et al., 1998). Zhao et al. (2011) haveshown that over-expression of MxA, an IFN-induced antiviral pro-tein, can inhibit CSFV replication. The interaction of CSFV proteinNpro with interferon regulatory factor 3 (IRF3) induces proteasomaldegradation of the latter and then suppresses IFN-�/� induction(Bauhofer et al., 2007). Additionally, Npro has also been found tointeract with I�B� (an inhibitor of NF-�B and transcription factorinvolved in apoptosis, the immune response and IFN production)and anti-apoptotic protein HAX-1 (Doceul et al., 2008; Johns et al.,2010). Most recently Gladue et al. (2011) have reported that theinteraction of CSFV core protein with IQGAP1, a regulator of the
cytoskeleton, is essential for completion of virus growth in vitroand for its virulence in vivo. The CSFV core protein also interactswith SUMO-1 (small ubiquitin-like modifier) and its conjugatingenzyme UBC9, and disruption of its binding to SUMO-1 and UBC9
an attenuate its virulence in swine (Gladue et al., 2010). Here, weeport that heme oxygenase 1(HO-1) of PK-15 cells is also involvedn CSFV replication.
HO-1 is an inducible enzyme exerting potent antioxidant andnti-inflammatory effects (Paine et al., 2010; Kapturczak et al.,004; Ryter et al., 2006), also playing a critical role in immunomod-latory functions (Chung et al., 2008; Pamplona et al., 2007; Georget al., 2008). Upon exposure to oxidative stress, the HO-1 path-ay initiates cytoprotection by up-regulating HO-1 expression.ecently HO-1 has attracted major attention since it also plays
critical role in host defense against microbial, and particularlyiral, infections (Chung et al., 2009). Choi et al. (1996) showedhat the HO-1 gene was up-regulated in the lungs of mice infectedith influenza virus H1N1. Hashiba et al. (2001) demonstrated that
verexpression of HO-1 in infected mice with acute lung injuryaused by H1N1 could prevent the death of infected animals viarotection of respiratory epithelium from caspase 8-mediated celleath. Expression of HO-1 was found to remain unaltered fol-
owing infection with HIV and HBV, but induction of HO-1 couldunction as a host defense mechanism to suppress HIV replicationn infected cells (Devadas and Dhawan, 2006). In an acute hep-titis B transgenic mouse model, induction of HO-1 resulted in
pronounced antiviral effect by repressing HBV replication andignificantly alleviating HBV-induced liver injury (Protzer et al.,007). In our previous study, the transcription of HO-1 in PBLs wasound to be increased following CSFV infection of pigs (Shi et al.,009) but the involvement of this cellular protein in CSFV pro-
iferation and pathogenesis was not further studied. The presenttudy investigated the involvement of HO-1 in infection of CSFV andemonstrated that the expression of HO-1 is also up-regulated inK-15 cells following CSFV infection, and that inhibition of this pro-ein significantly reduced the replication and production of CSFV inhe cells.
. Materials and methods
.1. Cell culture and CSFV infection
PK-15 cells (85 passages) in MEM (Sigma, St. Louis, MO, USA)ontaining 10% fetal bovine serum (FBS) free from bovine viral diar-hea virus and its antibody (Gibco-BRL, Gaithersburg, MD, USA),00 U/mL penicillin G, 100 mg/mL streptomycin sulfate and 2 mM l-lutamine (Invitrogen, Carlsbad, CA, USA) were added to 6-well cellulture plates (Corning, NY, USA) and incubated at 37 ◦C in 5% CO2.
hen 80–90% confluent, the cells were infected with 103 TCID50 ofSFV strain Shimen and further cultured in MEM/2% FBS for variousime periods.
.2. Transfection with small interfering RNA (siRNA)
The siRNAs used in this study were designed as detailed below,nd chemically synthesized by Dharmacon Research Inc. (Lafayette,O, USA). To eliminate the possibility of siRNA off-target effects, 2iRNAs, siHO-1 and siHO-1(698), respectively targeting nt 347–365nd nt 698–716 regions of HO-1 transcript (transcript accession #M001004027) were used. Their sequences were GGACAUGGCCU-CUGGUAU and GCUCAACAUUCAGCUGUUU respectively, while
non-targeting control siRNA (siScr) consisted of a scrambledequence of siHO-1 (GCCUGUGUUACAGCGUGUA). Transfectionsith the siRNAs were conducted in 80%-confluent PK-15 cell
onolayers cultured in 6-well plates using FuGENE HD Transfec-
ion Reagent (Roche, Indianapolis, IN, USA). After a wash with MEM,he monolayers were incubated 4–6 h at 37 ◦C with 200 nM siRNAn 1 mL MEM without FBS and antibiotics, following which the
173 (2013) 315– 320
transfection mixture was suctioned off, and the cells were washedwith MEM and reincubated with MEM/2% FBS without antibiotics.
2.3. Western blot analysis
Total cellular proteins were extracted and their concentrationswere determined with the Walterson Protein Assay kit (WaltersonBiotechnology Inc., Beijing, China). The total protein of 20 �g wassubjected to SDS-PAGE, and separated protein bands were electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). Themembranes were blocked overnight with 3% BSA (Sigma) in PBS atpH7.2 and then incubated for 2 h at ambient temperature with theappropriate antibody. After washing 3 times with PBST (PBS/0.5%Tween-20; Sigma), the membranes were further incubated at roomtemperature for 1 h with a second, HRPO-conjugated antibody.Reactive protein bands were visualized using chemiluminescencesubstrates according to the manufacturer’s instructions (ThermoFisher Scientific Pittsburgh, PA, USA). Beta-actin was used as a load-ing control. Statistical analysis using Student’s t-test was performedand all data were obtained from three independent experiments.
2.4. Analysis of cellular viability and apoptosis
Cellular viability was measured by flow cytometry using dou-ble staining with fluorescein isothiocyanate (FITC)-Annexin V andpropidium iodide (PI) (ACTGene, Piscataway, NJ, USA). After trypsintreatment, the cells were washed twice with PBS and resuspendedin binding buffer (ACTGene). FITC-Annexin V and PI were addedaccording to the manufacturer’s directions. The mixtures wereincubated for 10 min in the dark at ambient temperature and thenmeasured using a FACS Vantage flow cytometer and CellQuest soft-ware (Becton Dickinson, San Jose, CA, USA).
2.5. Quantification of the CSFV genome by Real-time RT-PCR
Copy numbers of viral genome in CSFV-infected control andsiRNA-treated PK-15 cells were quantified by real-time RT-PCRin an ABI PRISM 7000 cycler (Applied Biosystems, Carlsbad, CA,USA) according to a protocol described previously (Shi et al., 2009).Briefly, quantification was achieved by relating the viral Ct value tothe Ct value on a standard curve of a measured number of copiesof a plasmid bearing a 362 bp fragment of the CSFV 5′ non-codingregion. A comparative analysis of the viral genome copy numbersin control and treated cells was made using the analysis of varianceand Student’s paired t-test functions of SPSS 11.0 software (SPSSInc. Chicago, IL, USA).
2.6. Titration of virus progeny
At 24, 48 and 72 h p.i., PK-15 cell culture supernatants were col-lected and cells were lysed by three freeze-thaw cycles. The lysateswere 10-fold serially diluted from 10−1 to 10−6, added to 80% con-fluent PK-15 cells in 96-well microtitration plates, and incubatedfor 1 h at 37 ◦C. Each dilution was tested in quadruplicate. Lysateswere then removed, the cells were washed once with PBS, freshMEM was added and incubation continued at 37 ◦C. Wells wereexamined at 72 h p.i. by IFA using a previous method (Sun et al.,2008), and infectious virus in the supernatants was titrated by theKärber method as used by Xu et al. (2008).
3. Results
3.1. Up-regulation of HO-1 in PK-15 cells by CSFV infection
Our previous study showed that transcription of HO-1 mRNAin peripheral blood lymphocytes (PBLs) increased during CSFV
Z. Shi et al. / Virus Research 173 (2013) 315– 320 317
Fig. 1. Western blot analysis of HO-1 and �-actin expression in CSFV-infected andcontrol PK-15 cells. 20 �g samples of protein extracts were loaded per lane andresolved by 12% SDS-PAGE gel. The relative abundance of HO-1 in each group(n = 3) was plotted. Data are expressed as percentage of band intensity relativeto that of 24 h group following normalization to �-actin. Results of a representa-twd
iiHtiu
3
sdnsncstt
Fig. 3. Inhibition of HO-1 in PK-15 cells results in decrease in viral genome replica-tion. Quantitative analysis of viral genome was conducted by real time RT-PCR, viralgenome copies decreased by 1.4- (p = 0.004), 16.6- (p = 0.022) and 1.7-fold (p = 0.006)(mean value of three independent experiment) in HO-1 silencing cells at 24, 48
FswEaoOd
ive experiment of three with similar results are shown. Densitometry analysesere conducted using Quantity One software (Bio-Rad). Error bars indicate standardeviations.
nfection (Shi et al., 2009). To determine if this also occurredn vitro, PK-15 cells were infected with CSFV strain Shimen, andO-1 expression was assessed by western blot analysis at different
imes thereafter. As shown in Fig. 1, expression of HO-1 graduallyncreased from 24 to 72 h p.i., indicating that CSFV infection couldp-regulate HO-1 in PK-15 cells (p < 0.05).
.2. Knock-down of HO-1 by RNAi and cell viability analysis
Compared with the control transfected with the scrambled non-pecific siRNA sequence (siScr) HO-1 expression was significantlyecreased in PK-15 cells following siHO-1 transfection (targetingt 347–365 of HO-1 transcript) as shown by western blot analy-is (Fig. 2). In this circumstance the apoptosis of PK-15 cells wasot detected in the numbers of both dead and definably apoptotic
ells at all tested times by FITC-Annexin V and PI staining followingiHO-1 treatment, as compared with siScr-transfected and mock-ransfected PK-15 cells (p > 0.05, data not shown), indicating thathe knock-down of HO-1 expression had no effect on cell viability.
ig. 2. Inhibition of HO-1 in PK-15 cells results in decrease in viral protein. PK-15 cells wiRNA (siScr) and infected with CSFV at 24 h following transfection. Cells were lysed at 24,
estern blot at the indicated time points after infection. After HO-1 detection, membranes2 expression was monitored by western blot at 24, 48 and 72 h after infection. The levet 48, 72 h p.i. respectively. Data are expressed as a percentage of band intensity relativef HO-1 and E2 in each group was plotted. Results of a representative experiment of threne software (Bio-Rad). Error bars indicate standard deviations. Statistical significance wifferences.
and 72 h p.i. respectively. Data are expressed as the mean ± S.D. from three sepa-rate experiments. Statistical significance was determined by Student’s paired t-test.p < 0.05 and p < 0.01 represent significant differences.
Therefore, the RNAi method was applied to investigate whether thereduced expression of HO-1 had an impact on CSFV proliferation.
3.3. Inhibition of CSFV replication by reduced HO-1 expression
Twenty four hours after siHO-1 transfection, PK-15 cells wereinfected with CSFV and viral replication was analyzed by westernblot analysis of viral E2 protein and quantification of viral genomicRNA. Levels of E2 in siHO-1 treated cells decreased by up to almost2-fold during the test period compared with the controls, in whichexpression of both HO-1 and E2 remained constant (Fig. 2). By48 h post-infection, viral genome copies in siHO-1 treated cellsdecreased by 16.6-fold; by 72 h however, renewed viral replica-tion had reduced this decrease to1.7-fold (Fig. 3). Titers of progenyvirus in siHO-1 treated cells decreased by 10–32-fold over the 72 hpost-infection compared with siScr-transfected controls (Fig. 4A).Yields of infectious virus in culture supernatants of siHO-1 treatedcells decreased by 10–56-fold compared with controls (Fig. 4B).
To eliminate the possibility of off-target effects, the experimentswere repeated using siHO-1(698) targeting nt 698–716 of the HO-1
transcript. Results showed that the knock-down expression of HO-1 was also observed following siHO-1(698) transfection, leading toa corresponding reduction of CSFV E2 expression at 24, 48 and 72 hp.i. (Fig. 5A–C).
ere transfected with HO-1 specific siRNA (siHO-1) or with non-targeting control48 and 72 h postinfection (p.i.). (A–C) The HO-1 level in cell lysates was analyzed by
were stripped and reblotted using �-actin as a control for total protein loads. CSFVl of E2 decreased by 2.1-, 1.5-fold (mean value of three independent experiments)
to that of controls following normalization with �-actin. The relative abundancee with similar results are shown. Densitometry analyses were done using Quantityas determined by Student’s paired t-test. p < 0.05 and p < 0.01 represent significant
318 Z. Shi et al. / Virus Research 173 (2013) 315– 320
Fig. 4. Inhibition of HO-1 in PK-15 cells results in a decrease in viral titer. PK-15 cells were treated with HO-1 specific siRNA (siHO-1) or with non-targeting control siRNA(siScr) and infected with CSFV at 24 h post-transfection. Supernatants as well as cells were harvested at 24 h intervals. Cell-associated virus titer (A) and extracellular virus(culture supernatant) titer (B) were determined. Viral titer in cells of siHO-1 treated cells decreased 32- (p = 0.019), 10- (p = 0.02) and 18-fold (p = 0.013) at 24, 48 and 72 hp d cellst ts. Star
1Yto
4
Hmi
Fswt2oOar
.i. respectively, yields of infectious virus in culture supernatants of siHO-1 treatehat of controls. Data are expressed as the mean ± S.D. of three separate experimenepresent significant differences.
Titers of progeny virus in siHO-1(698)-treated cells decreased25-, 56- and 32-fold at 24, 48 and 72 h p.i. respectively (Fig. 5D).ields of infectious virus in culture supernatants of siHO-1(698)-reated cells decreased by 32-, 56- and 56-fold compared with thosef controls (Fig. 5E).
. Discussion
Our results indicate that CSFV infection induces up-regulation ofO-1 at the protein level in PK-15 cells and that its down-regulationay significantly decrease viral growth, indicating that HO-1 is an
mportant factor in CSFV replication in PK-15 cells.
ig. 5. Knock-down of HO-1 by HO-1-specific siRNA siHO-1(698) results in decrease in viRNA, siHO-1(698) or with non-targeting control siRNA (siScr) and infected with CSFV
estern blot at 24 h (A), 48 h (B) and 72 h (C) p.i. HO-1 expression decreased 2.33- (p =
ransfection. Correspondingly, the E2 expression decreased by 2.86- (p = 0.009), 4.00- (p =4, 48 and 72 h p.i. respectively. Data are expressed as a percentage of band intensity relatf HO-1 and E2 in each group was plotted. Results of a representative experiment of threne software (Bio-Rad). Error bars indicate standard deviations. Titers of progeny viruss indicated. Data are expressed as the mean ± S.D. of three separate experiments. Statiepresent significant differences.
decreased by 10- (p = 0.02), 18- (p = 0.013) and 56-fold (p = 0.012) compared withtistical significance was determined by Student’s paired t-test. p < 0.05 and p < 0.01
It is well established that CSFV replication is related to the cel-lular growth state, and HO-1 is one of the cellular factors involvedin modulation of cell proliferation and apoptosis (Deng et al.,2004; Choi et al., 2004). The question remains as to whether thedecrease in CSFV replication following treatment of PK-15 cellswith siHO-1 was an indirect effect of a decrease in cell viabilitycaused by down-regulation of HO-1 expression. Analysis of cellviability and apoptosis, however, showed that down-regulation of
HO-1 had no influence on cell viability. Inhibition of CSFV repli-cation in siHO-1 treated PK-15 cells, therefore, appears to be dueto down-regulation of HO-1 rather than the result of reduced cellviability.
iral E2 protein expression and viral titer. PK-15 cells were treated with the secondat 24 h post-transfection. HO-1 and CSFV E2 protein expression were detected by0.045), 9.09- (p = 0.001), 5.88-fold (p = 0.001) at 24, 48 and 72 h after siHO-1(698)
0.01) and 2.33-fold (p = 0.016) (mean value of three independent experiments) ative to that of controls following normalization with �-actin. The relative abundancee with similar results are shown. Densitometry analyses were done using Quantity
in siHO-1(698)-treated cells (Fig. 5D) and its culture supernatants (Fig. 5E) werestical significance was determined by Student’s paired t-test. p < 0.05 and p < 0.01
earch
avpitehimur
ttTo(HhdcoeHocwoigEcgrdEbkepstbivpsrHivs
dafptaleu(ih
Z. Shi et al. / Virus Res
HO-1 possesses antioxidant and anti-inflammatory properties,nd it is strongly induced by hemin and various stressors, includingiral infection. Overexpression or induction of HO-1 may reducero-oxidant levels and increase cellular resistance to oxidant-
nduced cytotoxicity, thereby protecting against cell death andhe apoptosis triggered by oxidative stress conditions (Hamedi-Aslt al., 2012). Indeed, there is increasing evidence to suggest thatost cell oxidative stress status plays an important role in regulat-
ng viral replication and infectivity (Cai et al., 2003). Significantly,any reports have shown that expression of HO-1 is usually reg-
lated by viral infection, while the level of HO-1 expression alsoegulates viral replication.
Up-regulation of HO-1 in CSFV-infected cells may indicate thathis virus can induce oxidative stress in host cells and activatehe HO-1 pathway to protect cells against oxidative stress injury.his is in contrast to certain other viruses, such as spring viremiaf carp virus (SVCV), hepatitis C virus (HCV) and enterovirus 71EV71). These viruses inhibit HO-1 expression, and induction ofO-1 expression may repress their replication. Yuan et al. (2012)ave demonstrated that expression of HO-1 was down-regulateduring SVCV infection in epithelioma papulosum cyprini (EPC)ells and in common carp. HCV infection in vivo and expressionf HCV core protein in vitro also inhibit HO-1 expression (Abdallat al., 2004), and up-regulation of HO-1 results in a decrease inCV replication (Zhu et al., 2008). Tung et al. (2011) reported thatverexpression of HO-1 diminished enterovirus 71 (EV71) repli-ation, and that this effect could be abrogated by pre-treatmentith an HO-1 inhibitor, zinc protoporphyrin IX (ZnPP IX). More-
ver, there are studies indicating that viral replication could benhibited by treatment with antioxidants such as epigallocatechinallate (EGCG) and glutathione (GSH). Ho et al. (2009) reported thatGCG strongly inhibited replication of EV71 by suppressing repli-ation of genomic RNA, accompanied by a significant reduction ineneration of reactive oxygen species (ROS) in cells. Likewise, EV71eplication was enhanced in cells deficient in glucose-6-phosphateehydrogenase, and such enhancement was largely reversed byGCG (Ho et al., 2009). Antioxidant GSH has been reported tolock influenza viral infection in cultures of Madin-Darby canineidney cells and human small airway epithelial cells, to inhibitxpression of viral matrix protein, and to inhibit virus-induced cas-ase activation and Fas up-regulation (Cai et al., 2003). Oxidativetress or other conditions that deplete GSH in the epithelium ofhe oral, nasal, and upper airway may therefore enhance suscepti-ility to influenzal infection (Cai et al., 2003). These observations
ndicate that oxidative stress may enhance host susceptibility toirus and benefit viral replication, and that inhibition of antioxidantrotein expression resulting in maintenance of cellular oxidativetress may be a strategy of these viruses for efficient infection andeplication. In contrast, however, CSFV induced up-regulation ofO-1, and down-regulation of HO-1 inhibited CSFV replication,
ndicating that there is an alternative mechanism used by thisirus to modulate antioxidant stress by inducing HO-1 expres-ion.
Normally CSFV does not cause cytopathic effects (CPE) or celleath in cell culture, but can induce proinflammatory cytokinesnd tissue factor expression; and it inhibits apoptosis and inter-eron synthesis during the establishment of long-term infection oforcine vascular endothelial cells (Bensaude et al., 2004). Consis-ent with up-regulation of HO-1, a similar increase of two otherntioxidant proteins, peroxiredoxin-6 (PRDX6) and thioredoxin-ike protein, has been observed in CSFV-infected PK-15 cells (Sunt al., 2008), and PRDX6 is also up-regulated in primary porcine
mbilical vein endothelial cells (PUVECs) following CSFV infectionLi et al., 2010). These observations indicate that CSFV infection cannduce cellular oxidative stress by compromising up-regulation ofost antioxidative stress proteins to relieve oxidative stress and
173 (2013) 315– 320 319
avoid apoptosis; i.e., activating mechanisms contributing to estab-lishment of a persistent CSFV infection.
There have been few reports that down-regulation of HO-1inhibits viral replication, although inhibition by up-regulation ofHO-1 is a well-established observation. Therefore, inhibition CSFVreplication by down-regulation of HO-1 implicates a singular mech-anism of interaction between CSFV and HO-1. Although precisedelineation of this requires additional studies, the present dataidentify HO-1 as a potential target for inhibition of CSFV replication.
Acknowledgement
This work was supported by the Key Project of NSFC to CT (No.31130052).
References
Abdalla, M.Y., Britigan, B.E., Wen, F., Icardi, M., McCormick, M.L., LaBrecque,D.R., Voigt, M., Brown, K.E., Schmidt, W.N., 2004. Down-regulation of hemeoxygenase-1 by hepatitis C virus infection in vivo and by the in vitro expressionof hepatitis C core protein. Journal of Infectious Diseases 190 (6), 1109–1118.
Bauhofer, O., Summerfield, A., Sakoda, Y., Tratschin, J.D., Hofmann, M.A., Ruggli,N., 2007. Classical swine fever virus Npro interacts with interferon regulatoryfactor 3 and induces its proteasomal degradation. Journal of Virology 81 (7),3087–3096.
Bensaude, E., Turner, J.L., Wakeley, P.R., Sweetman, D.A., Pardieu, C., Drew, T.W.,Wileman, T., Powell, P.P., 2004. Classical swine fever virus induces proin-flammatory cytokines and tissue factor expression and inhibits apoptosis andinterferon synthesis during the establishment of long-term infection of porcinevascular endothelial cells. Journal of General Virology 85 (Pt (4)), 1029–1037.
Cai, J., Chen, Y., Seth, S., Furukawa, S., Compans, R.W., Jones, D.P., 2003. Inhibitionof influenza infection by glutathione. Free Radical Biology and Medicine 34 (7),928–936.
Choi, A.M., Knobil, K., Otterbein, S.L., Eastman, D.A., Jacoby, D.B., 1996. Oxidant stressresponses in influenza virus pneumonia: gene expression and transcription fac-tor activation. American Journal of Physiology 271 (3 (Pt (1))), L383–L391.
Choi, B.M., Pae, H.O., Jeong, Y.R., Oh, G.S., Jun, C.D., Kim, B.R., Chung, H.T., 2004.Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1. Free Radical Biologyand Medicine 36 (7), 858–871.
Chung, S.W., Liu, X., Macias, A.A., Baron, R.M., Perrella, M.A., 2008. Heme oxygenase-1-derived carbon monoxide enhances the host defense response to microbialsepsis in mice. Journal of Clinical Investigation 118 (1), 239–247.
Chung, S., Hall, W.S., Perrella, M.A., 2009. Role of heme oxygenase-1 in microbialhost defense. Cell Microbiology 11 (2), 199–207.
Deng, Y.M., Wu, B.J., Witting, P.K., Stocker, R., 2004. Probucol protects against smoothmuscle cell proliferation by upregulating heme oxygenase-1. Circulation 110 (3),1855–1860.
Devadas, K., Dhawan, S., 2006. Hemin activation ameliorates HIV-1 infection viaheme oxygenase-1 induction. Journal of Immunology 176 (7), 4252–4257.
Doceul, V., Charleston, B., Crooke, H., Reid, E., Powell, P.P., Seago, J., 2008. TheNpro product of classical swine fever virus interacts with IkappaBalpha, theNF-kappaB inhibitor. Journal of General Virology 89 (Pt (8)), 1881–1889.
George, J.F., Braun, A., Brusko, T.M., Joseph, R., Bolisetty, S., Wasserfall, C.H., Atkin-son, M.A., Agarwal, A., Kapturczak, M.H., 2008. Suppression by CD4+CD25+regulatory T cells is dependent on expression of heme oxygenase-1 in antigen-presenting cells. American Journal of Pathology 173 (1), 154–160.
Gladue, D.P., Holinka, L.G., Fernandez-Sainz, I.J., Prarat, M.V., O’Donnell, V., Vep-khvadze, N., Lu, Z., Risatti, G.R., Borca, M.V., 2010. Effects of the interactionsof classical swine fever virus Core protein with proteins of the SUMOylationpathway on virulence in swine. Virology 407 (1), 129–136.
Gladue, D.P., Holinka, L.G., Fernandez-Sainz, I.J., Prarat, M.V., O’Donnell, V., Vep-khvadze, N.G., Lu, Z., Risatti, G.R., Borca, M.V., 2011. Interaction between Coreprotein of classical swine fever virus with cellular IQGAP1 protein appears essen-tial for virulence in swine. Virology 412 (1), 68–74.
Hamedi-Asl, P., Halabian, R., Bahamni, P., Mohammadipour, M., Mohammadzadeh,M., Roushandeh, A.M., Jahanian-Najafabadi, A., Kuwahara, Y., Roudkenar, M.H.,2012. Adenovirus-mediated expression of the HO-1 protein within MSCsdecreased cytotoxicity and inhibited apoptosis induced by oxidative stresses.Cell Stress and Chaperones 17 (2), 181–190.
Hashiba, T., Suzuki, M., Nagashima, Y., Suzuki, S., Inoue, S., Tsuburai, T., Matsuse,T., Ishigatubo, Y., 2001. Adenovirus-mediated transfer of heme oxygenase-1cDNA attenuates severe lung injury induced by the influenza virus in mice. GeneTherapy 8 (19), 1499–1507.
Ho, H.Y., Cheng, M.L., Weng, S.F., Leu, Y.L., Chiu, D.T., 2009. Antiviral effect of epigal-locatechin gallate on enterovirus 71. Journal of Agricultural and Food Chemistry
57 (14), 6140–6147.
Hulst, M.M., van Gennip, H.G., Vlot, A.C., Schooten, E., de Smit, A.J., Moormann, R.J.,2001. Interaction of classical swine fever virus with membrane-associated hep-aran sulfate: role for virus replication in vivo and virulence. Journal of Virology75 (20), 9585–9595.
3 earch
J
K
L
L
P
P
P
R
S
S
20 Z. Shi et al. / Virus Res
ohns, H.L., Doceul, V., Everett, H., Crooke, H., Charleston, B., Seago, J., 2010. Theclassical swine fever virus N-terminal protease Npro binds to cellular HAX-1.Journal of General Virology 91 (11), 2677–2686.
apturczak, M.H., Wasserfall, C., Brusko, T., Campbell-Thompson, M., Ellis, T.M.,Atkinson, M.A., Agarwal, A., 2004. Heme oxygenase-1 modulates early inflam-matory responses: evidence from the heme oxygenase-1-deficient mouse.American Journal of Pathology 165 (3), 1045–1053.
i, S., Qu, H., Hao, J., Sun, J., Guo, H., Guo, C., Sun, B., Tu, C., 2010. Proteomic analysis ofprimary porcine endothelial cells after infection by classical swine fever virus.Biochimica et Biophysica Acta. 1804 (9), 1882–1888.
indenbach, B.D., Thiel, H.J., Rice, C.M., 2007. Flaviviridae: The Viruses and theirReplication, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
aine, A., Eiz-Vesper, B., Blasczyk, R., Immenschuh, S., 2010. Signaling to hemeoxygenase-1 and its anti-inflammatory therapeutic potential. Biochemical Phar-macology 80 (12), 1895–1903.
amplona, A., Ferreira, A., Balla, J., Jeney, V., Balla, G., Epiphanio, S., Chora, A.,Rodrigues, C.D., Gregoire, I.P., Cunha-Rodrigues, M., Portugal, S., Soares, M.P.,Mota, M.M., 2007. Heme oxygenase-1 and carbon monoxide suppress the patho-genesis of experimental cerebral malaria. Nature Medicine 13 (6), 703–710.
rotzer, U., Seyfried, S., Quasdorff, M., Sass, G., Svorcova, M., Webb, D., Bohne, F.,Hösel, M., Schirmacher, P., Tiegs, G., 2007. Antiviral activity and hepatoprotec-tion by heme oxygenase-1 in hepatitis B virus infection. Gastroenterology 133(4), 1156–1165.
hi, Z., Sun, J., Guo, H., Tu, C., 2009. Genomic expression profiling of peripheral bloodleukocytes of pigs infected with classical swine fever virus. Journal of GeneralVirology 90 (Pt (7)), 1670–1680.
immonds, P., Becher, P., Collett, M.S., Gould, E.A., Heinz, F.X., Meyers, G., Monath,T., Pletnev, A., Rice, C.M., Stiasny, K., Thiel, H.J., Weiner, A., Bukh, J., 2011. Familyflaviridae. In: King, A.M.Q., Lefkowitz, E., Adams, M.J. (Eds.), Virus Taxonomy.Eighth Report of the International Committee on Taxonomy of Virus. AcademicPress, San Diego, pp. 1002–1020.
173 (2013) 315– 320
Sizova, D.V., Kolupaeva, V.G., Pestova, T.V., Shatsky, I.N., Hellen, C.U., 1998. Specificinteraction of eukaryotic translation initiation factor 3 with the 5′ nontrans-lated regions of hepatitis C virus and classical swine fever virus RNAs. Journal ofVirology 72 (6), 4775–4782.
Tung, W.H., Hsieh, H.L., Lee, I.T., Yang, C.M., 2011. Enterovirus 71 induces integrin�1/EGFR-Rac1-dependent oxidative stress in SK-N-SH cells: role of HO-1/CO inviral replication(1). Journal of Cellular Physiology 226 (12), 3316–3329.
Viswanathan, K., Früh, K., 2007. Viral proteomics: global evaluation of viruses andtheir interaction with the host. Expert Review of Proteomics 4 (6), 815–829.
Xu, X., Guo, H., Xiao, C., Zha, Y., Shi, Z., Xia, X., Tu, C., 2008. In vitro inhibition ofclassical swine fever virus replication by siRNAs targeting Npro and NS5B genes.Antiviral Research 78, 188–193.
Yuan, J., Su, N., Wang, M., Xie, P., Shi, Z., Li, L., 2012. Down-regulation ofheme oxygenase-1 by SVCV infection. Fish and Shellfish Immunology 32 (2),301–306.
Zhao, Y., Pang, D., Wang, T., Yang, X., Wu, R., Ren, L., Yuan, T., Huang, Y., Ouyang, H.,
2011. Human MxA protein inhibits the replication of classical swine fever virus.Virus Research 156 (1/2), 151–155.
Zhu, Z., Wilson, A.T., Mathahs, M.M., Wen, F., Brown, K.E., Luxon, B.A., Schmidt, W.N.,2008. Heme oxygenase-1 suppresses hepatitis C virus replication and increasesresistance of hepatocytes to oxidant injury. Hepatology 48 (5), 1430–1439.
